Diffusible proteins regulate neural development at a variety of\r\nstages. Using a novel neuronal culture assay, I have identified several\r\ncytokines that regulate the expression of neurotransmitters and\r\nneuropeptides in sympathetic neurons. These cytokines fall into two\r\nfamilies. The first group is termed the neuropoietic cytokines, while\r\nincluding CDF/LIF, CNTF, OSM and GPA, induces expression of the same\r\nset of neuropeptide mRNAs in cultured sympathetic neurons. These four\r\nfactors not only exhibit similar biological activities; they also share a\r\npredicted secondary structure and bind to a signal-transducing receptor\r\nsubunit in common with IL-6 and IL-11. The latter two cytokines display a\r\nweaker activity in this assay. In addition, I find that several members of\r\nthe TGF-\u03b2 superfamily, activin A, BMP-2, and BMP-6, have a selective\r\noverlap with the neuropoietic family in the spectrum of neuropeptides\r\nthat these cytokines induce in sympathetic neurons. Different patterns of\r\nneuropeptides induced by the TGF-\u03b2 family members, however,\r\ndemonstrate that the activities of these cytokines are distinct from those of\r\nthe neuropoietic family. Another 30 cytokines are without detectable effect\r\nin this neuronal assay.
\r\n\r\nActivin A induces a set of neurotransmitters and neuropeptides that\r\nis somewhat similar to the phenotype of sympathetic neurons innervating\r\nsweat glands in rat footpads. In situ hybridization and RNase protection\r\nwere carried out to test whether activins were involved in the phenotypic\r\ntransition when sympathetic neurons contact sweat glands. I find that\r\nactivin mRNA is present in both cholinergic and noradrenergic targets.\r\nMoreover, homogenates of footpads do not contain activin-like activity in\r\nthe neuronal assay in vitro. Taken together, these data do not support\r\nactivins as the best candidates for the sweat gland factor.
\r\n\r\nSeveral novel factors that regulate neuropeptide expression exist in\r\nheart cell conditioned medium. I attempted to purify these factors in\r\ncollaboration with Dr. Jane Talvenheimo. Our results suggest that these\r\nfactors are sensitive to the storage conditions used. Several modifications\r\nof purification strategy are discussed.
\r\n", "doi": "10.7907/2rm0-fg46", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7307", "collection": "thesis", "collection_id": "7307", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12062012-091333466", "primary_object_url": { "basename": "Hsu_hs_1993.pdf", "content": "final", "filesize": 17852866, "license": "other", "mime_type": "application/pdf", "url": "/7307/1/Hsu_hs_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Properties of the first genetically engineered neuron.", "author": [ { "family_name": "Hsu", "given_name": "Hsiaolan S.", "clpid": "Hsu-Hsiaolan-S" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na^+ channels, brief pulses (< 1ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3s) depolarizing plateau; this plateau was caused by slow and incomplete Na^+ channel inactivation. In cells expressing both Na^+ and Drosophila Shaker H4 transient K^+ channels, there were neuron-like action potentials. In cells with appropriate Na^+/K^+ current ratios, maintained stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to \"lockup\" of the potential at a positive value after several seconds of stimulation; the latter effect was due primarily to slow inactivation of the K^+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels. This model system provides a direct control of complexity of neuronal behavior, and a tool for studying various forms of neural modulation at molecular and cellular levels.
", "doi": "10.7907/svss-ye57", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7324", "collection": "thesis", "collection_id": "7324", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12112012-085623512", "primary_object_url": { "basename": "Garrity_pa_1993.pdf", "content": "final", "filesize": 36320812, "license": "other", "mime_type": "application/pdf", "url": "/7324/1/Garrity_pa_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The in vivo examination of transcriptional control mechanisms in mammalian cells", "author": [ { "family_name": "Garrity", "given_name": "Paul Allen", "clpid": "Garrity-P-A" } ], "thesis_advisor": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "thesis_committee": [ { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In the investigations described in this thesis I have examined various\r\nmechanisms involved in transcriptional control. The first chapter is a general\r\nexamination of the mechanisms used in transcriptional control. The second\r\nchapter addresses issues associated with the selection of transcriptional start\r\nsite. This work shows that core promoter usage can be altered in a tissue specific\r\nand environmentally-responsive manner. The third chapter of this\r\nwork describes a significant improvement in the technique of ligation mediated\r\nPCR-aided in vivo footprinting and genomic sequencing. This\r\nimprovement in the quality of in vivo footprint data allows the pattern of\r\nprotein : DNA interactions to be obtained with greater signal to noise and for a\r\nlarger group of DNA sequences. The fourth chapter of this thesis uses these in\r\nvivo footprinting techniques to investigate the mechanisms controlling the\r\ntranscription of the mouse interleukin-2 gene upon T cell stimulation. T cell\r\nstimulation was shown to result in the coordinated occupancy of a number of\r\nmajor groove binding proteins to the previously unoccupied IL-2 regulatory\r\nregion in vivo. Finally, the appendix describes in vivo footprints at the\r\ndifferentiated-muscle-specific promoter of the delta-subunit of the nicotinic\r\nacetylcholine receptor. Multiple protein : DNA interactions were seen at the\r\npromoter both before and after muscle cell differentiation, suggesting that\r\ntranscriptional regulation of this gene occurs at the level of protein\r\nreplacement or an alteration of the ability of the assembled proteins to activate\r\ntranscription.", "doi": "10.7907/kt02-n508", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7340", "collection": "thesis", "collection_id": "7340", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12182012-093217041", "primary_object_url": { "basename": "Hunkapiller_t_1993.pdf", "content": "final", "filesize": 95076367, "license": "other", "mime_type": "application/pdf", "url": "/7340/1/Hunkapiller_t_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Diversity and Evolution of the Immunoglobulin Gene Superfamily", "author": [ { "family_name": "Hunkapiller", "given_name": "Tim", "clpid": "Hunkapiller-Tim" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The Immunoglobulin Gene Superfamily is characterized by a common protein\r\nhomology unit that is present in arguably the largest and most diverse set of genes and\r\ngene families of any protein motif. This distribution indicates that the homology unit is\r\na remarkably versatile functional unit. Its central role in defining the complex\r\nphenotypes of the immune and nervous systems, likewise, is testament to the ability of\r\nthe motif to support an amazing and unique degree of diversification. Understanding\r\nmore about the function, structure and evolution of the Immunoglobulin Gene\r\nSuperfamily can provide insights into both the general issues of complex system\r\nevolution as well as the specific nature of the various systems the superfamily plays a\r\ncentral role in. This thesis is a collection of work aimed at a more thorough\r\nunderstanding of these elements. Particularly, these works summarize much of our\r\ncurrent understanding of the members of the Immunoglobulin Gene Superfamily along\r\nwith speculations on their evolutionary history as well as both the evolutionary and\r\nsomatic mechanisms responsible for their diversity. This work includes initial\r\ndescriptions of several features relevant to somatic diversification of rearranging\r\nimmune receptors, including: l) the role of joining imprecision in the generation of\r\njunctional diversity in immunoglobulin kappa chain; 2) the initial description of the T-cell\r\nbeta chain J/C locus; 3) the translation of T-cell beta chain D gene segments in all\r\nthree reading frames; 4) the occurrence of a cryptic rearrangement signal in most\r\nrearranging V families; 5) the first description of the mechanisms of class switching\r\nbetween heavy chain mu and delta genes; 6) the limited diversity of germline T-cell\r\nbeta chains; 7) the shared complementary determining region structure of T-cell beta\r\nchains and immunoglobulin heavy chains. Also, from these efforts, new members of\r\nthe superfamily have been identified including MHC class I molecules, L3T4 and\r\nMyelin Associated Glycoprotein. Various observations concerning the evolutionary\r\nrelationships of these molecules and motifs have been made. Particularly, a variation\r\non the basic homology unit motif has been proposed that probably more nearly\r\nrepresents the primordial sequence and function.
\r\n\r\nAs a result of these discoveries, a new, comprehensive picture of the\r\nimmunoglobulin superfamily is emerging that has implications for interpreting current\r\nfunctional relationships in the context of the evolutionary history of the members.\r\nParticularly, it is suggested from this work that the ability of the homology unit to\r\naccommodate diversity has made possible the evolution of the superfamily. Given the\r\ntremendous diversity within the superfamily, it might be assumed that selective\r\npressures favoring diversity have driven its evolution. However, much of the analysis\r\nwithin this collection suggests that, on the contrary, diversity is an inherent feature of\r\nthe conserved protein and gene structure of the homology unit and that it was the a\r\npriori diversity itself that drove and shaped the evolution of the complex systems that\r\nemploy the homology unit today. This basic diversity is the consequence of three\r\ncharacteristics of the homology unit. First, the tertiary structure of the protein motif is\r\nsuch that homology units tend to interact preferentially to form homo- or heterodimers,\r\nforming the basis of many of the receptors and the receptor/ligand interactions common\r\nwithin the superfamily. These combinatorial associations increase both the somatic and\r\nevolutionary potential for diversification. This can lead to the rather sudden\r\nappearance of new functional associations between existing members of the superfamily\r\npreadapted for otherwise unrelated functions. Second, except for a minimal number of\r\namino acid residues involved in critical intra- and interchain interactions, the primary\r\nstructure of these units can vary dramatically and still provide for essentially the same\r\ntertiary structure. This has been borne out by various crystallographic studies. The\r\nvariability is particularly true of the loop structures normally identified with antigen\r\nspecificity, but seen in other extended families as well. Reduced constraints on\r\nstructural sequences inherently promote the establishment of variation within\r\npopulations. Third, with very few exceptions the genes of the superfamily, the\r\nhomology units are not only encoded by discrete exons, but these exons have a shared\r\n1/2 splicing rule. That is, each is begun with the second 2 bases of a codon and ended\r\nwith the first base. This allows the in-frame splicing of any number of tandem\r\nhomology units, while maintaining functional protein domains. This rule generally\r\napplies to the non-homology unit exons of member genes as well. This allows, through\r\nrelatively simple genetic events, the development of new contexts for homology unit\r\nexpression, both by simple expansion and contraction of homology unit number and\r\nexon shuffling. This is probably at work, as well, in the frequent occurrence and\r\nutilization of alternative transcripts seen throughout the superfamily. Many of the\r\nrecognized occurrences of alternative splicing, such as that between membrane-bound\r\nand secreted forms, indicate that this gene structure provides for a further level of\r\nfunctional diversity and the expansion of the virtual genetic information.
\r\n\r\nBeyond the explicit discussion of the superfamily members, this work also\r\nspeaks to various issues of evolution in general. In particular, the history of the\r\nsuperfamily suggests the importance of canalization and non-gradual episodes of\r\nevolutionary change. It can contribute, as well, to the discussion of adaptive versus\r\nneutral change.
", "doi": "10.7907/mcbv-n026", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:6671", "collection": "thesis", "collection_id": "6671", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09152011-080428062", "primary_object_url": { "basename": "Wang_w_1992.pdf", "content": "final", "filesize": 56468825, "license": "other", "mime_type": "application/pdf", "url": "/6671/1/Wang_w_1992.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Expression, structural and functional studies of fasciclin I", "author": [ { "family_name": "Wang", "given_name": "Wen-Ching", "clpid": "Wang-Wen-Ching" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Fasciclin I is a cell surface glycoprotein thought to be involved in growth cone guidance in the embryonic insect nervous system. It is expressed on the cell surfaces of all\r\nperipheral nervous system (PNS) axons, a subset of central nervous system (CNS) axons and on some nonneuronal cells. Fly embryos bearing mutations eliminating expression of\r\nboth fasciclin I and the Abelson tyrosine kinase exhibit a severe phenotype in which many axon pathways fail to form. Fasciclin I mediates homophilic adhesion in transfected tissue culture cells, suggesting that it may affect growth cone guidance through homophilic interactions. To facilitate structure-function studies of fasciclin I, we have generated mammalian (CHO) cell lines expressing fasciclin I at a high level. The expressed fasciclin I\r\nprotein was released from the cell surface in a soluble form by phospholipase C treatment. Milligram quantities of soluble expressed fasciclin I were purified on an immunoaffinity column. Large single crystals were obtained that diffracted to ~5 \u00c5 resolution which is\r\ninsufficient for a structure determination to atomic resolution by x-ray crystallography. In an effort to produce a form of fasciclin I more amenable to crystallization, we also generated CHO and Drosophila cell (S2) lines that produce a truncated form of fasciclin I.\r\nThe soluble fasciclin I expressed in S2 cells contains significantly less carbohydrate (~15 kDa) as compared to the molecules expressed in CHO cells. Therefore, S2-derived\r\nfasciclin I may be more suitable for crystallization. Biochemical characterization of the expressed fasciclin I indicates that fasciclin I exists as a monomer in solution, an observation consistent with homophilic interaction properties only if the interaction is of low affinity. Electron micrographs of fasciclin I suggest that it has a compact rectangular shape with no obvious flexible linker regions, in contrast to what has been seen in electron\r\nmicroscopic studies of other adhesion molecules. Circular dichroism analysis suggests that fasciclin I contains significant amounts of \u03b1-helical structure, which together with the electron microscopic results, suggests that its structure is substantially different from the \u03b2-sheet structures predicted for adhesion molecules that are members of the immunoglobulin superfamily and/or contain fibronectin III repeats. Future structural and functional studies of fasciclin I will ultimately increase our understanding of neuronal cell surface recognition\r\nand axon guidance.\r\n", "doi": "10.7907/ykbt-jt41", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:6299", "collection": "thesis", "collection_id": "6299", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-142633680", "primary_object_url": { "basename": "Wang_ks_1991.pdf", "content": "final", "filesize": 2963696, "license": "other", "mime_type": "application/pdf", "url": "/6299/1/Wang_ks_1991.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular characterization of a receptor for the Togavirus Sindbis virus", "author": [ { "family_name": "Wang", "given_name": "Kang-Sheng", "clpid": "Wang-Kang-Sheng" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Strauss", "given_name": "Ellen G.", "clpid": "Strauss-E-G" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The first step in any virus entry process is binding to the plasma membrane of the host cell. The nature of this obligatory step depends upon both the viral and cellular components and may be quite diverse among viruses. The entry of Sindbis virus into a host cell is reported to occur via receptor-mediated endocytosis. We have used several approaches to isolate and characterize the receptor(s) for Sindbis virus. In one such approach, we searched for monoclonal antibodies (mAb) that could interfere with Sindbis virus attachment to and infection of baby hamster kidney (BHK) cells. Mice were immunized with multiple injections of whole BHK cells or with BHK cell membranes. Hybridomas were prepared and supernatants from approximately 3600 hybridoma clones were screened by a plaque reduction assay for their ability to interfere with virus infection. One IgM mAb from a mouse immunized with whole BHK cells inhibited Sindbis virus attachment to BHK cells by 80% at 20 \u00b5g/ml, and immunoprecipitated a 68 kDal membrane protein from BHK cells. This mAb also inhibits virus attachment to two other mammalian cell lines tested, Vero cells (monkey) and SW13 cells (human), and also immunoprecipitates a 68 Kd protein from these cells. The mAb does not interfere with virus infection of chicken cells but did immunoprecipitate a 71 kDal protein from chicken cells. This mAb was used to screen 10^6 plaques from a \u03bbgt11 cDNA library from BHK cells, and 15 reactive phages were found. Six of the fifteen were shown by sequence analysis to react with overlapping regions of a protein that was identical in sequence to the mouse high affinity laminin receptor. By rescreening with a probe from one of these reactive phages, other lambda phages containing the remaining regions of the gene were found. The complete sequence of this protein was deduced by sequence analysis of the cDNA clones and was identical to that of the mouse laminin receptor and 99% identical to the human laminin receptor. A full length cDNA clone of the gene was constructed and inserted into a high efficiency expression vector. BHK cell lines stably transfected with vector expressing the plus sense BHK laminin receptor cDNA are 3-5 fold more susceptible to infection by Sindbis virus as measured by plaque assay, and overexpress the receptor protein on their surface as assayed by flow cytometry analysis. Conversely, cell lines transfected with vector expressing antisense laminin receptor cDNA are only about one half as susceptible to infection by Sindbis virus as the nontransformed BHK cells, and expression of laminin receptor on the cell surface is reduced as measured by flow cytometry analysis. In a second study we looked for Sindbis virus receptors on the surface of chicken cells, using specific molecular mimicry to identify receptor molecules. It has been postulated that viral receptors may share structural features (idiotypes) with antibodies directed against the cell attachment protein of virus. Using antiidiotypic antibodies directed against Sindbis-specific neutralization antibodies, we have demonstrated that an antiidiotypic antibody to a neutralizing mAb reactive with the E2 glycoprotein of Sindbis virus specifically interferes with the binding of wild type Sindbis virus to chicken cells. This antiidiotypic antibody also immunoprecipitates a 63 kDal protein from chicken cells and binds to the surface of these cells. This 63 kDal protein is presumably a receptor for Sindbis virus in chicken cells. The relationship between this protein and the laminin receptor used as a Sindbis receptor in mammalian cells remains to be determined. We also wished to determine the domains of the virus envelope proteins that are responsible for attachment to the cell membrane. The Sindbis virus envelope contains two species of integral membrane glycoproteins, El and E2, which assemble into heterodimers. Each spike on the surface of the virion is a trimer of these dimeric units. We attempted to map the neutralization epitopes on the surface of the virus, including epitopes implicated in virus binding to cells by the antiidiotypic antibody results described above. A \u03bbgt11 expression library was constructed containing cDNA inserts 100-300 nucleotides in length obtained by randomly primed synthesis on Sindbis genomic RNA. This library was probed with several neutralizing monoclonal antibodies specific for E2 and one neutralizing antibody specific for El. Four positive clones, all of which contained inserts from the region of the Sindbis genome that encodes amino acids 173 to 220 of glycoprotein E2, were found from the screening with mAb 23. No reactive clones could be identified using any of the other antibodies. We hypothesize that this domain of E2 centered at residue 200 forms part of the virus binding site for attachment to the cell to initiate infection.", "doi": "10.7907/qdr9-p250", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2922", "collection": "thesis", "collection_id": "2922", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07182007-074159", "primary_object_url": { "basename": "McCormack_k_1991.pdf", "content": "final", "filesize": 4889181, "license": "other", "mime_type": "application/pdf", "url": "/2922/1/McCormack_k_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-function studies of Drosophila shaker potassium channels", "author": [ { "family_name": "McCormack", "given_name": "Ken", "clpid": "McCormack-K" } ], "thesis_advisor": [ { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "thesis_committee": [ { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Voltage-dependent ion channels mediate electrical signals in the nervous system; many sodium (Na+), calcium (Ca++) and potassium (K+) selective channels are structurally related, and thus represent a family. These proteins undergo interesting conformational changes in response to alterations in transmembrane potential. However, the functional determinants involved in these transitions are not well understood. Chapters 2A and 2B describe the identification and characterization of an amino acid sequence motif (a leucine-heptad repeat) that is evolutionarily conserved among this family of voltage-dependent ion channels. Conservative, single amino-acid substitutions within this region of Drosophila Shaker (Sh) proteins have substantial effects on the voltage-dependence of activation. The observed alterations suggest that the heptad-repeat region is an important determinant in the conformational transitions leading to channel opening.\n\nNa+ and Ca++ channels are composed of four homologous domains, each of which is equivalent to a single K+ channel subunit. Thus, K+ channels are thought to be functional multimers. Furthermore, there are a large number of different voltage-dependent K+ genes and alternatively spliced products that potentially can be expressed in the same cell. Therefore, the potential number of different K+ channel multimers could be quite extensive. Chapter 3 describes the physiological characteristics of combinations of K+ channels belonging to the Sh family that have been coexpressed in Xenopus oocytes. Members of the same molecular class of Sh channel form heteromultimers with novel functional properties, adding to the diversity of K+ channel function. Members of different molecular classes do not form heteromultimeric channels, suggesting that there are distinct K+ channel systems. The Appendix describes an alternative exon in the \"constant\" region of the Drosophila Sh gene, the existence of which suggests, that the molecular diversity of this gene is greater than previously determined.", "doi": "10.7907/63q7-hw24", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:8615", "collection": "thesis", "collection_id": "8615", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07282014-134702796", "primary_object_url": { "basename": "Mueller_PR_1990.pdf", "content": "final", "filesize": 18868113, "license": "other", "mime_type": "application/pdf", "url": "/8615/1/Mueller_PR_1990.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "In vivo analysis of interactions between trans-acting factors and their target genes", "author": [ { "family_name": "Mueller", "given_name": "Paul R.", "clpid": "Mueller-P-R" } ], "thesis_advisor": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "thesis_committee": [ { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.
\r\n\r\nThe second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.
\r\n", "doi": "10.7907/yv7h-9335", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2563", "collection": "thesis", "collection_id": "2563", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06122007-144634", "primary_object_url": { "basename": "Mathers_ph_1990.pdf", "content": "final", "filesize": 10131471, "license": "other", "mime_type": "application/pdf", "url": "/2563/1/Mathers_ph_1990.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Developmental regulation and chromosomal decondensation of the 68C glue gene cluster in Drosophila melanogaster", "author": [ { "family_name": "Mathers", "given_name": "Peter Hiram", "clpid": "Mathers-P-H" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "clpid": "Lipshitz-H-D" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The larval salivary gland secretion genes (Sgs-3, Sgs-7 and Sgs-8) at chromosome position 68C in Drosophila melanogaster, are developmentally regulated and coordinately expressed. Each gene codes for a component of the mucoprotein glue which is synthesized in the third instar salivary glands. Expression of these three genes is associated with the 68C intermolt puff present in the polytene chromosomes of the salivary gland secretory cells. Expression occurs only in the salivary glands of third instar larvae, and requires the steroid hormone ecdysterone. We show that synthesis of the 68C glue gene RNAs is prevented in larvae carrying trans-acting mutations within the Broad-Complex (BR-C), while a puff persists at 68C in these mutant larvae. We use the l(1)su(f)[superscript ts67g] mutation (which has reduced ecdysterone levels and also prevents 68C glue gene expression) and a mutation in the BR-C (nprl[superscript 3]) to study the cis-acting elements responsible for interaction with trans-acting factors by analyzing the protein:DNA contacts which occur upstream of the Sgs-3 glue gene during active synthesis, using in vivo DMS-footprinting. The proximal promoter can direct tissue- and stage-specific expression and is shown to possess three protein-binding domains. Comparison of contacts at these three domains between expressing and non-expressing tissues (including salivary glands from (l)su(f)[superscript ts67g] and nprl[superscript 3] mutant larvae) identifies a single binding domain responsible for controlling developmental expression of Sgs-3.\n\nWe also analyze the cis-acting sequences required for the chromosomal puffing associated with 68C glue gene expression. Examination of various fragments of 68C DNA reintroduced into the Drosophila chromosomes by P-element transformation identifies a region of 152 basepairs between Sgs-7 and Sgs-8 which is necessary, but not sufficient, to promote puffing. Only when this region is accompanied by an adjacent promoter element is there a puff. The insertion sites containing these reintroduced fragments fail to puff in mutant larvae; therefore, formation of the 68C intermolt puff requires the products of the su(f) and BR-C loci, and the puff at 68C in mutant larvae is not the same puff as that associated with expression of Sgs-3, Sgs-7 and Sgs-8.", "doi": "10.7907/qm6d-4135", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:8616", "collection": "thesis", "collection_id": "8616", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07282014-141222100", "primary_object_url": { "basename": "Preugschat_f_1990.pdf", "content": "final", "filesize": 25667020, "license": "other", "mime_type": "application/pdf", "url": "/8616/1/Preugschat_f_1990.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Proteolytic processing of the nonstructural proteins of dengue 2 virus", "author": [ { "family_name": "Preugschat", "given_name": "Frank", "clpid": "Preugschat-F" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Strauss", "given_name": "Ellen G.", "clpid": "Strauss-E-G" } ], "thesis_committee": [ { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The genomes of many positive stranded RNA viruses and of all\r\nretroviruses are translated as large polyproteins which are proteolytically\r\nprocessed by cellular and viral proteases. Viral proteases are structurally\r\nrelated to two families of cellular proteases, the pepsin-like and trypsin-like\r\nproteases. This thesis describes the proteolytic processing of several\r\nnonstructural proteins of dengue 2 virus, a representative member of the\r\nFlaviviridae, and describes methods for transcribing full-length genomic\r\nRNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the\r\nnonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system\r\nthat allows identification of residues within the protease that are directly or\r\nindirectly involved with substrate recognition. Chapter 3 describes\r\nmethods to produce genome length dengue 2 RNA from cDNA templates.
\r\n\r\nThe nonstructural protein NS3 is structurally related to viral trypsinlike\r\nproteases from the alpha-, picorna-, poty-, and pestiviruses. The\r\nhypothesis that the flavivirus nonstructural protein NS3 is a viral\r\nproteinase that generates the termini of several nonstructural proteins was\r\ntested using an efficient in vitro expression system and antisera specific for\r\nthe nonstructural proteins NS2B and NS3. A series of cDNA constructs\r\nwas transcribed using T7 RNA polymerase and the RNA translated in\r\nreticulocyte lysates. Proteolytic processing occurred in vitro to generate\r\nNS2B and NS3. The amino termini of NS2B and NS3 produced in vitro\r\nwere found to be the same as the termini of NS2B and NS3 isolated from\r\ninfected cells. Deletion analysis of cDNA constructs localized the protease\r\ndomain necessary and sufficient for correct cleavage to the first 184 amino\r\nacids of NS3. Kinetic analysis of processing events in vitro and experiments\r\nto examine the sensitivity of processing to dilution suggested that an\r\nintramolecular cleavage between NS2A and NS2B preceded an\r\nintramolecular cleavage between NS2B and NS3. The data from these\r\nexpression experiments confirm that NS3 is the viral proteinase\r\nresponsible for cleavage events generating the amino termini of NS2B and\r\nNS3 and presumably for cleavages generating the termini of NS4A and NS5\r\nas well.
\r\n\r\nBiochemical and genetic experiments using viral proteinases have\r\ndefined the sequence requirements for cleavage site recognition, but have\r\nnot identified residues within proteinases that interact with substrates. A\r\nbiochemical assay was developed that could identify residues which were\r\nimportant for substrate recognition. Chimeric proteases between yellow\r\nfever and dengue 2 were constructed that allowed mapping of regions\r\ninvolved in substrate recognition, and site directed mutagenesis was used\r\nto modulate processing efficiency.
\r\n\r\nExpression in vitro revealed that the dengue protease domain\r\nefficiently processes the yellow fever polyprotein between NS2A and NS2B\r\nand between NS2B and NS3, but that the reciprocal construct is inactive.\r\nThe dengue protease processes yellow fever cleavage sites more efficiently\r\nthan dengue cleavage sites, suggesting that suboptimal cleavage efficiency\r\nmay be used to increase levels of processing intermediates in vivo. By\r\nmutagenizing the putative substrate binding pocket it was possible to\r\nchange the substrate specificity of the yellow fever protease; changing a\r\nminimum of three amino acids in the yellow fever protease enabled it to\r\nrecognize dengue cleavage sites. This system allows identification of\r\nresidues which are directly or indirectly involved with enzyme-substrate\r\ninteraction, does not require a crystal structure, and can define the\r\nsubstrate preferences of individual members of a viral proteinase family.
\r\n\r\nFull-length cDNA clones, from which infectious RNA can be\r\ntranscribed, have been developed for a number of positive strand RNA\r\nviruses, including the flavivirus type virus, yellow fever. The technology\r\nnecessary to transcribe genomic RNA of dengue 2 virus was developed in\r\norder to better understand the molecular biology of the dengue subgroup. A\r\n5' structural region clone was engineered to transcribe authentic dengue\r\nRNA that contains an additional 1 or 2 residues at the 5' end. A 3'\r\nnonstructural region clone was engineered to allow production of run off\r\ntranscripts, and to allow directional ligation with the 5' structural region\r\nclone. In vitro ligation and transcription produces full-length genomic\r\nRNA which is noninfectious when transfected into mammalian tissue\r\nculture cells. Alternative methods for constructing cDNA clones and\r\nrecovering live dengue virus are discussed.
", "doi": "10.7907/nn0p-gt03", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:7936", "collection": "thesis", "collection_id": "7936", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08232013-141734040", "primary_object_url": { "basename": "Sweder 1989.pdf", "content": "final", "filesize": 26958277, "license": "other", "mime_type": "application/pdf", "url": "/7936/1/Sweder 1989.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Isolation and Characterization of Proteins that Bind to Yeast Origins of DNA Replication", "author": [ { "family_name": "Sweder", "given_name": "Kevin Scot", "clpid": "Sweder-Kevin-Scot" } ], "thesis_advisor": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" } ], "thesis_committee": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.
\r\n\r\nWe have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARS1. This same protein binds to and protects a similar size region at the HMR-E ARS.
\r\n\r\nWe also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.
\r\n\r\nTo address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a \u03bbgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.
", "doi": "10.7907/d0tj-k332", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:1948", "collection": "thesis", "collection_id": "1948", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05222007-091103", "type": "thesis", "title": "Regulatory Elements in ColE1 DNA Replication in Escherichia coli. Mutants of Saccharomyces cerevisiae DNA Polymerase I Resistant to Nucleotide Analogs: dNTP Binding Site Definition", "author": [ { "family_name": "Ma", "given_name": "Doreen", "clpid": "Ma-Doreen" } ], "thesis_advisor": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Control of Co1E1-type plasmid DNA replication in Escherichia coli was investigated. The initiation of DNA replication in Co1E 1-type plasmids is regulated by two trans-acting negative control elements: RNA I and the rop protein. RNA I is a transcript 108 nucleotides long made off the L-strand (lagging strand) of the plasmid and is complementary to the 5' portion of the preprimer, RNA II. The direct base-pairing interaction between the two RNA species precludes the formation of an RNA II:template DNA hybrid, which is processed by RNase H at the origin of replication to create the 3'-OH end of the mature primer. Another trans-acting regulatory element is a 63 amino acid plasmid-encoded protein, the rop gene product. By providing the rop gene product in trans on a compatible plasmid, suppression of the runaway replication phenotype of pJN75 was observed. Utilizing this property of the regulatory mechanism, we proceeded to select pJN75 derivatives that are insensitive to rop-mediated suppression. These mutant plasmids were designated pJN75nsr for non-suppressible by rop. Sequence analyses of 7 nsr, showed disruptions of base-pairing within the stem of loop structure III of RNA II and loop structure III' of RNA I, implying that rop mediates its action via the region comprising loop structures III and III'. We were also interested in the presence of a dnaA protein binding site about 90bp downstream of the ori-pBR322. We investigated the role of dnaA protein in Co1E1-type DNA replication by purifying the dnaA protein to homogeneity from an overproducing strain and examining its effect on various mutant DNA templates in an in vitro E. coli DNA replication extract developed in the Campbell lab. We found that the combination of dnaA protein binding at the dnaA consensus sequence can substitute for the lack of primosome assembly site (pas) on the H-strand (leading strand), which is postulated to be the point of transition between DNA polymerase I and polymerase III-dependent DNA synthesis. In the absence of the H-strand pas, dnaA protein may direct other essential proteins to form a replication complex at the dnaA site, functionally acting as proteins i, n, n' and n\" at pas.
\r\n\r\nIn an effort to identify and characterize the nucleotide and/or pyrophosphate binding site(s) of yeast DNA polymerise I, we have attempted to isolate pol1 mutants that are resistant to nucleotide/pyrophosphate analogs. We successfully constructed a Saccharomyces cerevisiae strain that depends on exogenous thymidine for survival and also contains a temperature-sensitive DNA polymerase I allele (pol1-17). Using this strain, 167-poll-17, we screened for pol1 mutants that are resistant to nucleotide/pyrophosphate analogs, e.g., AraT or PAA, which are normally impermeable to the cell wall of yeast. Our strategy was to introduce a plasmid containing a mutagenized pol1 gene into 167-poll-17, which contains a ts DNA polymerase. By screening for survivors at 37\u00b0C (non-permissive temperature of pol1-17 allele) on plates containing 1 mM AraT and 40 mM PAA, we hoped to isolate DNA polymerase I mutants on the transformed plasmid. To our disappointment, we were unable to isolate such DNA pol I mutants. We offer two explanations for the failure of our strategy: 1) the presence of another essential polymerase, CDC2 gene product and 2) perturbation of pyrimidine nucleotide pool. Finally, we propose to conduct site-directed mutagenesis of DNA polymerase I at putative dNTP/PPi binding domains and to analyze the mutant polymerases in vitro for resistance to dNTP/PPi analogs. Site-directed mutagenesis experiments are now in progress, and we are hopeful that these mutants will provide structural and functional information regarding the nucleotide/pyrophosphate binding site(s) of yeast DNA polymerase I.
", "doi": "10.7907/5b4k-wx16", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:332", "collection": "thesis", "collection_id": "332", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01252005-085241", "type": "thesis", "title": "Regulation of Class I Genes by Interferons", "author": [ { "family_name": "Korber", "given_name": "Bette Tina Marie", "orcid": "0000-0002-2026-5757", "clpid": "Korber-Bette-Tina-Marie" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "local_group": [ { "literal": "Caltech Distinguished Alumni Award" }, { "literal": "div_chem" } ], "abstract": "Major histocompatibility (MHC) class I gene expression is increased in response to interferons. In order to identify critical regulatory regions in mouse MHC (H-2) class I genes, the 5' flanking region and the DNA downstream of the transcription initiation site were analyzed separately. The promoters of H-2Dd and H-2Ld were linked to the reporter gene chloramphenicol acetyl transferase (CAT). Conversely, the H-2Ld structural gene was linked to non-interferon regulated promoters. These constructs were transfected into several different cell lines, and their ability to respond to interferons was assessed. Both regions, 5' and 3' of the transcriptional initiation site, were able to independently contribute to the regulation of class I genes by interferons. The basal levels of expression, interferon inducibility. and the relative contributions of the 3' and 5' responses to overall interferon regulation, were cell-type dependent.
\r\n\r\nSequence analysis of the 5' flanking region of class I genes led to the identification of multiple DNA motifs that are highly homologous to regulatory elements found in other genes. The H-2Dd promoter contains a TATA bog, CAAT elements, enhancer regions, and an interferon consensus sequence that is found in the promoters of many genes that are regulated by interferons. Deletion analysis and expression studies of the H-2Dd promoter revealed several interesting regulatory features of the interferon consensus sequence. It was required for both type I (alpha and beta) and type II interferon (gamma) responses. In some cell types an additional sequence was required for a type I interferon response; this sequence is located 5' and adjacent to the interferon consensus sequence. Type II interferon action was independent of this upstream sequence in all cell-types tested. Therefore the promoter controlled response to interferons is complex and the nature of the response depends both on the type of interferon and the cell-type being tested.
\r\n\r\nWe have noted that an interferon consensus sequence homology exists in the promoters of interferon genes. As interferons have a capacity to be auto-regulatory, we propose a model of gene regulation by interferons that incorporates what our studies and others have shown about the regulation of class I genes by interferon, and what is known about the regulation of interferon genes themselves.
\r\n", "doi": "10.7907/FWT2-VC76", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7428", "collection": "thesis", "collection_id": "7428", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01232013-085319311", "primary_object_url": { "basename": "Hu-mc-1988.pdf", "content": "final", "filesize": 39809650, "license": "other", "mime_type": "application/pdf", "url": "/7428/1/Hu-mc-1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "I. The E. coli Lac Operator-Repressor System is Functional for Control of Gene Expression in Animal Cells. II. Molecular Cloning and Characterization of the Mouse Skeletal Muscle (\u03b1) Actin Gene", "author": [ { "family_name": "Hu", "given_name": "Mickey ChienTsung", "clpid": "Hu-Mickey-ChienTsung" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "We have investigated the use of the E. coli lac operator-repressor system to regulate the expression of genes introduced into mammalian cells by gene transfer. We find that the bacterial lac repressor protein encoded in a suitable expression vector is synthesized in mammalian cells in culture, assembles into a tetramer, enters the nucleus to some extent, and represses expression of another gene that has one or several lac operator sequences inserted into any one of several sites in the promoter region of the gene. Derepression can be achieved by exposure of the cells to IPTG. From a practical point of view of an inducible genetic switch, this system confers an induction level of somewhere between 10- and 20-fold in most of the cases we have tested. That is not better than those that have been achieved with heat shock, mouse mammary tumor virus, and metallothionein promoters. There may, however, be some situations and some promoters for which the use of the lac operator system is advantageous. We have also shown that this lac control system can be used to regulate the expression of genes introduced into Xenopus oocytes by micro-injection.
\r\n\r\nAt present, we have been trying to revise this system by using a newly developed promoter containing a symmetric lac operator sequence inserted at the various strategic points within the human metallothionein IIA promoter and enhancer regions, which consist of several positive control elements, in order to achieve induction ratios of a factor of 100 or more. We have also generated a new lacI gene which encodes a repressor containing at its carboxyl terminus the nuclear localization signal of the SV40 large-T antigen. Overall, by combining the newly developed lac control promoters with the new repressor producing cell lines, we hope to generate an inducible expression system with large induction ratios that can be used as a general genetic switch in the future.
\r\n\r\nIn section II, the nucleotide sequence of the mouse skeletal muscle (\u03b1) actin gene is presented and discussed.
\r\n", "doi": "10.7907/2a59-9p43", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7423", "collection": "thesis", "collection_id": "7423", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01222013-140636948", "primary_object_url": { "basename": "Garfinkel_md_1988.pdf", "content": "final", "filesize": 42805044, "license": "other", "mime_type": "application/pdf", "url": "/7423/1/Garfinkel_md_1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structural and Functional Studies of the 68C Glue Protein Gene Cluster of Drosophila melanogaster", "author": [ { "family_name": "Garfinkel", "given_name": "Mark David", "clpid": "Garfinkel-Mark-David" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The 68C locus of the Drosophila melanogaster polytene chromosomes contains the structural genes for three glue polypeptides (sgs-3, sgs-7, and sgs-8) synthesized in the third instar larval salivary glands. The three 68C glue mRNAs are coded in a gene cluster of less than 5000 base-pairs, and are expressed coordinately under the control of the steroid hormone ecdysterone. Neither amplification nor DNA rearrangement of the locus occurs in the salivary gland. The nucleotide sequence of genomic DNA that includes the entire gene cluster was determined, as were the structures of each of the three glue protein mRNAs. Analysis of the sequences revealed that the three glue proteins form a diverged gene family. Each member of the gene family contains an amino-terminal hydrophobic block of amino acids, which is absent in the mature, secreted glue proteins, and a cysteine-rich carboxyl terminal module. sgs-3 differs from sgs-7 and sgs-8 by containing a third module between the other two, comprised largely of tandem repeats of the five amino acids Pro-Thr-Thr-Thr-Lys.
\r\n\r\nTwo of the genes Sgs-7 and Sgs-8 are divergently transcribed with 475 base-pairs separating the two 5' ends. A transcriptional fusion gene was constructed by joining the 5' untranslated region of Sgs-7 to the 5' untranslated region of the D. melanogaster Adh gene. A translational fusion gene was constructed by joining the Sgs-8 gene to the Escherichia coli lacZ gene. When the fusion genes are placed in their normal divergently transcribed arrangement and reintroduced into D. melanogaster using P element gene transfer, third instar larval salivary gland expression of both alcohol dehydrogenase activity and \u03b2-galactosidase activity was observed. Expression of the two fusion genes requires the l(1)npr-1\u207a gene product, which is known to regulate the 68C glue protein genes, supporting the proposal that this trans-acting factor affects glue protein gene transcription. Normal tissue, stage, and quantity of Sgs-7\u2014Adh fusion gene expression is observed when 211 bp of the 5' flanking sequence is present. An Sgs-7\u2014Adh fusion gene with 92 base-pairs upstream is non-functional. Third instar larval salivary gland expression of the Sgs-8\u2014lacZ fusion gene is observed when 432 base-pairs of the intergenic region are present, while 415 base-pairs of 5' flanking sequence permits normal tissue and stage of expression at levels at least twentyfold reduced. The experiments suggest that a single region functioning bidirectionally, located closer to the Sgs-7 gene, is required for expression of both genes.
", "doi": "10.7907/gjtw-k928", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7422", "collection": "thesis", "collection_id": "7422", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01222013-140535393", "primary_object_url": { "basename": "Hann-cs-1988.pdf", "content": "final", "filesize": 36611241, "license": "other", "mime_type": "application/pdf", "url": "/7422/1/Hann-cs-1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-Function Relationships in the Structural Proteins and in the RNAs of Alphaviruses and Flaviviruses", "author": [ { "family_name": "Hahn", "given_name": "Chang Soo", "clpid": "Hahn-Chang-Soo" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "thesis_committee": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The RNA virus families Togaviridae and Flaviviridae were considered one family as recently as 1983. These two families contain more than 100 members, many of which are important pathogens for humans and domestic animals. Studies on members of both families which were undertaken to increase our understanding of the functions of the virus structural proteins and of RNA sequence elements that interact with virus proteins, and of the evolution of these two families of RNA viruses, are presented in this thesis. These investigations include two on the nature and function of a virus-encoded self-protease that functions in the processing of the structural proteins, several studies on the role of the virus structural glycoproteins in assembly of progeny virions and viral virulence, and studies on the evolution of these viruses, including the demonstration that recombination has occurred in the Togaviridae to produce an important new pathogen, and that RNA sequence elements have been conserved during the evolution of the Flaviviridae.
\r\n\r\nAlphavirus structural proteins are translated from a subgenomic messenger RNA as a polyprotein, which is cleaved to the final products by proteolytic processing. This processing was studied by comparative sequence analysis of three temperature sensitive mutants of Sindbis virus (the type alphavirus) which have a defect in processing of the polyprotein at the nonpermissive temperature. These mutations were localized in the C-terminal region of the capsid protein. From the position of these mutations and from sequence similarities between the alphavirus capsid proteins and animal serine proteses, we hypothesized that the capsid protein was a serine autoprotease whose active site is formed by His-141, Asp- 147 and Ser-215. To study this capsid protein protease activity in more detail, we have altered the proposed catalytic triad of the protease by site-directed mutagenesis. We have assayed the protease activity in the mutagenized capsid proteins by in vitro transcription and translation, and attempted to rescue virus from mutagenized full-length \"infectious\" clones. The results supported our hypothesis.
\r\n\r\nSindbis virus matures when preformed nucleocapsids acquire their envelopes by budding through virus-modified areas of the cell surface membrane. ts103 is a mutant of Sindbis virus which has a defect in this late maturation step such that it generates multicored particles, and it has provided a good system for studying structure-function relationships during viral assembly and maturation. Hybrid genomes were constructed that were formed from a full-length cDNA clone of wild type Sindbis in which restriction fragments were replaced with cDNA from ts103. Virus rescued from these constructs were used to determine the protein responsible for the multicored phenotype and to map the mutation. tsl03 was found to have a single amino acid substitution in glycoprotein E2. The implications of this mutation for our understanding of virus assembly are\r\ndiscussed.
\r\n\r\nVirus surface glycoproteins are believed to be important determinants of virulence and tissue tropism. Ne urovirulence of Sindbis virus for mice has been used as an animal model system in which to explore the effects of each individual protein or of a particular domain of a given protein, or even of a single amino acid residue, on neurovirulence. By constructing hybrid genomes among various strains of Sindbis virus at the cDNA level and rescuing virus in vitro using in vitro transcription and transfection, it was possible to evaluate the effect of each protein on neurovirulence. From these studies, we concluded that Sindbis virus glycoproteins are important determinants of neurovirulence, but not the sole determinants. The virulence phenotypes of various recombinant viruses in both weanling mice and suckling mice are discussed, with reference to the role of particular residues in producing the neurovirulent phenotype.
\r\n\r\nWe have also undertaken a study of virulence in flaviviruses. The 17D vaccine strain of yellow fever virus, the type flavivirus, is one of the most reliable and stable live virus vaccines ever developed. By comparison of the nucleotide sequences of the 17D vaccine strain and its parental virulent Asibi virus we have located all of the changes which occurred during the attentuation of yellow fever to produce the 17D vaccine. This comparison led us to the conclusion that changes in the viral envelope protein play an important role in attenuation.
\r\n\r\nThe 25 members of the genus Alphavirus have for the most part diverged by linear descent from a common ancestor. We have now found that Western equine encephalitis virus is an exception to this. Western equine encephalitis virus is a close relative of Sindbis virus as determined by immunological cross-reaction, but it is a New World virus that causes encephalitis in humans and horses, whereas Sindbis virus is an Old World virus not normally associated with encephalitis. The nucleotide sequence and deduced amino acid sequence of the structural proteins of Western equine encephalitis virus reveal that it arose by recombination between Eastern equine encephalitis virus (or a recent ancestor of it) and a virus closely related to Sindbis virus. The importance of recombination in the evolution of RNA viruses and in the generation of new potentially pathogenic virus strains, as well as the implications of the amino acid changes which have occurred in Western equine encephalitis virus (subsequent to the initial recombination event) for our understanding of the interaction between the structural proteins of alphaviruses, are discussed.
\r\n\r\nTo study evolution in flaviviruses, sequences at the 5'and 3' ends of several flaviviruses have been compared. Conserved structures or sequence elements have been identified, one pair of which could result in cyclization of flavivirus RNA. The significance of these sequences is discussed.
", "doi": "10.7907/n3rf-9306", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7507", "collection": "thesis", "collection_id": "7507", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03112013-111743236", "primary_object_url": { "basename": "Reyes-vm-1988.pdf", "content": "", "filesize": 40531838, "license": "other", "mime_type": "application/pdf", "url": "/7507/1/Reyes-vm-1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Transcription and Processing of Transfer RNA in S. cerevisiae", "author": [ { "family_name": "Reyes", "given_name": "Vicente Mendoza", "clpid": "Reyes-Vicente-Mendoza" } ], "thesis_advisor": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A collection of studies on tRNA transcription and splicing in the yeast S. cerevisiae are presented. These studies employ a combination of recombinant DNA, oligonucleotide-directed mutagenesis and in vitro synthetic technologies applied using the reverse genetics approach. Chapter I introduces the reader to these topics. In Chapter II, an attempt to solve the puzzle of tandem tRNA gene transcription in yeast is described. This tandem, the S. cerevisae tRNAArg-tRNAAsp gene pair, is transcribed solely by use of the upstream gene promoter signals, giving rise to a dimeric precursor which is processed into two mature tRNA molecules, much like prokaryotic systems. A collection of specific point and deletion mutations were constructed to answer the question of why the downstream tRNAAsp gene is apparently inactive. Our results show that it is so only in this configuration; the tRNAArg and spacer sequences, which constitute its upstream flanking sequences in this arrangement, seem inhibitory to the independent activity of the tRNAAsp gene. Taken together, these results emphasize the importance of flanking regions in eukaryotic tRNA gene transcription. In Chapter III, the construction and characterization of a heterologous system for the in vitro synthesis of pre-tRNA are presented. A strong bacteriophage T7 promoter was fused to a S. cerevisiae pre-tRNAPhe gene. We show that pre-tRNAPhe is synthesized efficiently from this system by the cognate T7 RNA polymerase, and that this RNA has the correct sequence, mature terminii, and is spliced efficiently and accurately by our in vitro splicing system, which consists of highly purified tRNA splicing endonuclease and ligase enzymes. In Chapter IV, an extensive investigation on tRNA splicing substrate specificity is described. A collection of 15 carefully-designed mutant pretRNAPhe genes were constructed, and then transcribed and analyzed as above. We find that the endonuclease recognizes two highly conserved, surface residues in pre-tRNAPhe-U8 and C56-and probably contacts these bases during the splicing reaction. We also find that splice site selection by this enzyme is a function of the length of the anticodon stem, and thus proceeds by a simple distance measurement mechanism. We have evidence that this measuring process commences in the thoracic region of the pre-tRNA, where the endonuclease probably binds. Finally, we demonstrate that the highly conserved purine residue 3' proximal to the anticodon may be important for cleavage at the nearby 5' splice site.
\r\n", "doi": "10.7907/3md6-cn17", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7408", "collection": "thesis", "collection_id": "7408", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01182013-085552595", "primary_object_url": { "basename": "Chang-c-1988.pdf", "content": "final", "filesize": 27662980, "license": "other", "mime_type": "application/pdf", "url": "/7408/1/Chang-c-1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular Genetic Studies in Arabidopsis thaliana: I. Cloning and Characterization of the Alcohol Dehydrogenase Gene; II. Complementation of an Alcohol Dehydrogenase Mutant; III. A Restriction Fragment Length Polymorphism Map of the Genome\r ", "author": [ { "family_name": "Chang", "given_name": "Caren", "clpid": "Chang-Caren" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis examines several genetic aspects of the flowering plant Arabidopsis thaliana which has previously been shown to possess several attributes for molecular genetic experiments such as a short life cycle, small size, fecundity, and a strong background of classical genetics. In particular, the nuclear genome is remarkably small and consists almost entirely of single copy sequences.
\r\n\r\nTwo approaches are established for the isolation and study of plant genes using Arabidopsis as a model system. One approach demonstrates complementation of a mutant phenotype using the Arabidopsis alcohol dehydrogenase (ADH) gene. The ADH gene was first isolated from a genomic DNA library by cross-hybridization with a maize ADH1 gene probe. The gene structure was studied by DNA sequence analysis and mapping of the transcript. The gene conferred wild-type ADH activity to an Arabidopsis ADH null mutant when introduced by Agrobacterium-mediated transformation. Transformed plants were subjected to genetic and molecular analyses. The ADH gene may have utility as a gene tag in transformation experiments.
\r\n\r\nThe second topic of this thesis is the construction of a genetic linkage map of restriction fragment length polymorphisms (RFLPs). The map provides an approach to cloning genes about which nothing more is known than a mutant phenotype and a map location because the RFLP markers can serve as starting points for the isolation of overlapping clones from a genomic DNA library. The map contains 90 RFLP markers distributed randomly throughout the nuclear genome. Since the genome consists of about 70,000 kilobase pairs, the markers are at an average physical spacing of approximately 780 kilobase pairs. The map is based on meiotic segregation of RFLPs in two different crosses detected with the restriction enzymes EcoRI, BglII, and XbaI. The RFLP linkage groups have been aligned with the standard genetic map of approximately 80 mutation markers.
", "doi": "10.7907/H8DS-A792", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:11841", "collection": "thesis", "collection_id": "11841", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-120541581", "type": "thesis", "title": "Expression of the Genes Encoding the Cytoskeletal Proteins Vimentin and Protein 4.1", "author": [ { "family_name": "Ngai", "given_name": "John J.", "orcid": "0000-0002-1191-8971", "clpid": "Ngai-John-J" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The investigations presented in this thesis represent an effort to understand the regulated expression of cytoskeletal proteins in differentiating cell systems. Vimentin is an intermediate filament protein whose expression is regulated during the differentiation of a variety of cell types. I have isolated DNA probes specific for chicken vimentin and utilized them for the study of vimentin gene regulation. The single chicken vimentin gene encodes multiple mRNAs that differ in the lengths of their 3' untranslated regions. These mRNAs are differentially expressed in a tissue-specific manner. Furthermore, vimentin mRNA increases to high levels during chicken embryonic erythropoiesis, underlying similar changes in vimentin protein accumulation.
\r\n\r\nUnlike nucleated avian erythrocytes, mammalian erythrocytes are devoid of intermediate filaments. I show that cultured murine erythroleukemia (MEL) cells repress the levels of vimentin mRNA during inducer-mediated differentiation, resulting in a subsequent loss of vimentin filaments. The expression of vimentin in these cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. To examine the molecular basis for divergent vimentin gene regulation in avian and mammalian erythropoiesis, I have studied the behavior of chicken and hamster vimentin genes introduced into MEL cells. During MEL cell differentiation, RNA encoded by transfected chicken vimentin genes significantly increases in abundance, whereas RNAs arising from either transfected hamster vimentin genes or the endogenous mouse vimentin gene are repressed. The results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences.
\r\n\r\nProtein 4.1 is an extrinsic membrane protein that facilitates the interaction of spectrin and actin in the erythroid membrane skeleton. Previous studies have shown that chicken protein 4.1 exists as a multiplet of related polypeptides that are differentially expressed during erythropoiesis. I have isolated cloned cDNA probes for chicken protein 4.1, and have found that a single protein 4.1 gene encodes multiple mRNAs by differential processing; the ratios of protein 4.1 mRNAs change during erythroid development. In vitro translation experiments demonstrate that while the expression of protein 4.1 polypeptides is specified initially at the mRNA level by RNA processing, the ultimate expression of protein 4.1 variants is further determined translationally.
", "doi": "10.7907/qh54-ka26", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11877", "collection": "thesis", "collection_id": "11877", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10312019-114338923", "primary_object_url": { "basename": "Yu_L_1987.pdf", "content": "final", "filesize": 51911087, "license": "other", "mime_type": "application/pdf", "url": "/11877/1/Yu_L_1987.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Nicotinic Acetylcholine Receptor: Gene Expression and Ion Channel Function", "author": [ { "family_name": "Yu", "given_name": "Lei", "clpid": "Yu-Lei" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The nicotinic acetylcholine receptor (AChR) is a complex protein, which functions as a ligand-gated ion channel on the postsynaptic membrane at the neuromuscular junction and mediates signal transmission from neuron to muscle. Research on the AChR has had a long history and has benefited from the endeavors of scientists from many disciplines. The intensive, multidisciplinary studies have yielded valuable knowledge about this molecule, which serves as a model for the understanding of many fundamental questions in biological sciences. Chapter 1 presents a review of the AChR.
\r\n\r\nAs a tissue-specific and developmental stage-specific molecule, AChR is under temporal and spatial control for its synthesis. Chapter 2 reports a qualitative and quantitative study of AChR gene activity during muscle cell differentiation, using a cDNA clone isolated from a murine muscle cell line, which codes for the \u03b3 subunit of the mouse AChR. The results indicate that the regulation of mRNA accumulation levels is a major mechanism in the differential synthesis of the AChR.
\r\n\r\nThe marriage between AChR and molecular biology resulted in many cDNA clones which, after being introduced to African frogs, produced the next generation \u2014 Xenopus oocytes with exotic AChRs on them. Chapter 3 describes the attempt to localize \"determinants\" that specify species subunit identity in the AChR by constructing chimeric cDNA clones composed of fragments from different origins, taking advantage of the Xenopus oocyte expression system. The results from surface toxin-binding assay and two-electrode voltage-clamp recording suggested that while the species specificity can be dictated by certain subunits, the determination of subunit identity does not reside at a defined locus in the fragments tested.
\r\n\r\nDoes the complex composition of multisubunits in the AChR bear any functional significance? Chapter 4 addresses this question through the study of mouse-Torpedo AChR hybrids. The complete substitution of AChR subunits between mouse and Torpedo receptors generated all 16 combinations, and a systematic analysis of these hybrids revealed an interesting pattern with respect to the voltage sensitivity in the ACh-induced response: The identity of the \u03b2 subunit determines, while the interaction between the \u03b2 and \u03b4 subunits modulates, the AChR voltage sensitivity. The results, therefore, suggest that different subunits of AChR may play a central role in different functional properties.
\r\n\r\nPatch-clamp technique has offered an opportunity for analyzing transmembrane current flow with the high resolution of single-channel recording. Chapter 5 describes such a study on homologous and hybrid AChRs. Voltage influence on the three parameters were evaluated, and the results indicate that the single channel conductance is independent of membrane potential and that the channel closing and opening rates together constitute the basis for the voltage sensitivity in whole-cell recording with the closing rate making the major contribution. Also investigated were the subunit roles in species specificity of channel-open duration and voltage dependence. The results are in agreement with those reported on channel duration and support the conclusions of our previous work on the subunit involvement in determining the voltage sensitivity of the AChR response.
", "doi": "10.7907/fj5c-4h83", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11394", "collection": "thesis", "collection_id": "11394", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02152019-094650551", "type": "thesis", "title": "Structure and Expression of the Actin Gene Family of Drosophila melanogaster", "author": [ { "family_name": "Matthews", "given_name": "Beverley Bond", "clpid": "Matthews-Beverley-Bond" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "We have isolated the six actin genes of Drosophila melanogaster from a Drosophila genomic DNA library and have compared structural features of the genes by restriction mapping, electron microscopy and DNA sequencing. We found that at least two of the actin genes contain intervening sequences which interrupt the genes at different positions. Several of the genes were shown to be lacking intervening sequences in the analogous positions. This nonconservation of intron position is in striking contrast to the strong conservation of intron positions seen in other gene families. The DNA sequences of the protein coding regions of the genes are highly conserved while the intron and untranslated sequences are not. The primary sequences of all the Drosophila actins resemble mammalian cytoplasmic actins more than mammalian muscle actins.
\r\n\r\nWe studied the distribution of actin mRNAs in different developmental stages and in different dissected body parts with the use of gene specific hybridization probes which we isolated from the 3' untranslated portions of the genes. We found that the genes fall into three main categories with respect to their patterns of expression in Drosophila. Trancripts from two of the genes are found throughout Drosophila development. They are expressed at higher levels in ovaries and embryonic cultured cells than in muscle containing tissue and are thought to be cytoplasmic actins. Two others encode thoracic muscle actins. Their transcripts accumulate predominantly in the thoracic regions of the adult where the flight and jump muscles are found. The other two genes are most active in larvae and in adult abdomens. They are thought to encode actins used in the larval, pupal, and adult intersegmental muscles.
\r\n\r\nWe studied the structure of the cytoplasmic actin gene, act5C, in detail and found that it encodes at least six different mRNAs. At the 5' end there are two nonhomologous leader exons which are alternately spliced to the remainder of the gene. At the 3' end of the gene, three sites of polyadenylation are used. The 3' variation is the principal cause of the transcript length heterogeneity observed in the transcripts. In whole animal RNA, the two leader exons are expressed with the same pattern through development and with all three polyadenylation sites. There is some developmental variability in the use of the three polyadenylation sites.
\r\n\r\nIn order to determine if each exon is preceded by a functional promoter and to identify sequences important for transcription initiation from each exon, we made fusions between act5C promoter fragments and the bacterial chloramphenicol acetyltransferase (CAT) gene and tested these for promoter activity in transient assays in Kc cells. We found that each exon is preceded by a separate, functional promoter. At least two regions of DNA sequences are necessary for optimal expression from exon 1. One of these lies greater than 1.9 kb upstream from the exon 1 cap site. All of the sequences required for exon 2 transcription lie within 450 bases of its cap site. There is evidence from some constructions that transcription initiation from exon 1 may inhibit transcription initiation from ex on 2.
", "doi": "10.7907/05zz-5w73", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11471", "collection": "thesis", "collection_id": "11471", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04162019-152444688", "type": "thesis", "title": "The Expression, Stability, and Enzymatic Activity of Specific Beta-Lactamase Mutants", "author": [ { "family_name": "Neitzel", "given_name": "James Joseph", "clpid": "Neitzel-James-Joseph" } ], "thesis_advisor": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Three catalytic site mutants (ser 70 to thr, thr 71 to ser, and the double mutant ser 70 to thr-thr 71 to ser) of the pBR 322 beta-lactamase were purified to homogeneity. These mutant proteins were unstable and could only be obtained from E. coli at 30\u00b0C, rather than the normal growth temperature of 37\u00b0. The use of the strong tac promoter increased the yield of the thr 71 to ser mutant protein. This promoter was coupled with a kanamycin resistance gene to create a new vector for the expression of inactive beta-lactamase mutants. A new purification protocol for the pBR 322 lactamase was devised. By replacing an early ion-exchange chromatography column with an ammonium sulfate precipitation, it was possible to isolate unstable beta-lactamases in high yield.
\r\n\r\nThe ser 70 to thr mutant and the double mutant show no catalytic activity and do not form an acyl enzyme intermediate. However, they do still retain the ability to bind benzylpenicillin. The thr 71 to ser mutant is active. The Km values for benzylpenicillin and cephalothin hydrolysis are unchanged from the wild-type enzyme, while the kcat values are approximately 15% of the wild-type value. This mutant also cleaves the poor substrate cefoxitin, again with no significant change in Km, but with kcat reduced to 8% of the wild-type value. Examination of the pre-steady state burst during cefoxitin hydrolysis showed that the thr 71 to ser enzyme acylated at the same rate and to at least 80% of the extent of the wild-type enzyme. Direct measurement of the deacylation rate confirmed that a reduction in the deacylation rate is responsible for the lowered reaction rate in this mutant protein. Additionally, this protein lost catalytic activity at elevated temperature more rapidly than the wild-type enzyme.
\r\n\r\nThe denaturation of the active thr 71 to ser mutant was observed in more detail. This enzyme thermally denatures at 45\u00b0, a temperature 10 to 15\u00b0 lower than that required to denature the wild-type enzyme. This mutant is also more susceptible to digestion by thermolysin, trypsin, and those proteases present in vivo in the periplasmic space of E. coli. The enzyme also loses activity at a urea concentration of 2 M, whereas the wild-type enzyme is still active at urea concentrations greater than 4 M. The inactive mutants ser 70 to thr and ser 70 to thr-thr 71 to ser are even more susceptible to proteolytic attack by E. coli proteases in vivo.
\r\n\r\nA mutant pBR 322 beta-lactamase lacking the disulfide bond found in the wild-type enzyme was also purified to homogeneity. This protein showed no alterations in catalytic activity relative to the wild-type enzyme at temperatures below 40\u00b0. Above this temperature, this enzyme rapidly lost activity. This enzyme was also more susceptible to proteolytic attack at elevated temperature. However, this enzyme is more resistant to thermal and proteolytic denaturation than the thr 71 to ser mutant betalactamase.
", "doi": "10.7907/E6R6-4J57", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11834", "collection": "thesis", "collection_id": "11834", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10212019-143142376", "type": "thesis", "title": "The Molecular Structure of the Beadex and Heldup-A Loci of Drosophila melanogaster", "author": [ { "family_name": "Mattox", "given_name": "William Wayne", "clpid": "Mattox-William-Wayne" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Genetic studies indicate that excess-of-function Beadex mutations and loss-of-function heldup-a mutations affect different parts of a single bipartite genetic unit. In order to investigate the molecular nature of the Beadex and heldup-a mutations we isolated DNA from a 49 kilobase region surrounding the sites at which these mutations occur. We found gross structural alterations associated with one heldup-a mutation and each of the 13 different Beadex mutations that we examined. As expected from previous genetic studies these loci are separated by only a short molecular distance which is at most 1.5 kilobases. The structural alterations associated with the thirteen Beadex excess-of-function mutations examined are clustered within a three kilobase region. Several of these mutations are found to be associated with the deletion of part of an 800 bp region. This indicates that an element that normally represses gene activity is located within this region: A 200 base pair segment which is required for the function of the wild-type heldup-a locus is defined using a heldup-a mutation which results from a small deletion.
\r\n\r\nTwo RNA transcripts have been found which span this 200 base pair segment. One of these two transcripts, a four kilobase RNA, is expressed during stages in which the Beadex structural gene product is expected to be active. The structure of this transcript is affected by one heldup-a mutation and each of five Beadex mutations that were examined. However, only one of the Beadex mutant alleles expresses significantly elevated levels of this RNA. Other RNA species in the region surrounding the the Beadex and heldup-a loci were not affected either in amount or size by Beadex mutations.
\r\n\r\nWe have also reintroduced a wild-type 10.4 kilobase fragment, which includes the region m which Beadex and heldup-a mutations map, into the Drosophila genome by P element mediated transformation. This introduced fragment fails both to complement loss-of-function heldup-a mutations and to enhance an excess-of-function phenotype. This result indicates that some sequences required for the normal heldup-a function must be located outside this 10.4 kilobase region.
", "doi": "10.7907/c2k4-a231", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:7479", "collection": "thesis", "collection_id": "7479", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02152013-085205353", "primary_object_url": { "basename": "Crosby 1986.pdf", "content": "final", "filesize": 19221355, "license": "other", "mime_type": "application/pdf", "url": "/7479/1/Crosby 1986.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Regulation of the Drosophila Glue Gene SGS-3: Sequences Required for Puffing and Transcription", "author": [ { "family_name": "Crosby", "given_name": "Madeline Anne", "clpid": "Crosby-Madeline-Anne" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The 68C intermolt puff of Drosophila melanogaster contains a cluster of\r\nthree glue protein genes, Sgs-3, Sgs-7, and Sgs-8. Analyses of chromosomal\r\nrearrangments which break near the glue gene cluster show that a region of no\r\nmore than 20 kilobase-pairs (kb) is required for expression of the genes and for\r\nformation of the 68C puff. This result is supported by P-element-mediated-transformation\r\nexperiments in which defined segments of the 68C region are\r\nintroduced back into the fly genome. Based on the criteria of correct tissue- and\r\nstage-specific expression, transcription of an RNA of appropriate size and\r\nabundance, and production of an sgs-3 protein, the correctly regulated expression\r\nof the Sgs-3 gene requires less than 3.4 kb of total flanking sequences,\r\napproximately 2.3 kb 5' and 1.1 kb 3'. When upstream sequences are truncated at\r\n130 base-pairs, low levels of Sgs-3 expression are observed in some cases, with\r\nnormal tissue- and stage-specific expression retained. Formation of a new\r\nintermolt puff at the site of insertion is observed for transformants in which the\r\nintroduced DNA contains all three 68C glue genes, but not for those which contain\r\nonly an introduced Sgs-3 gene, even for cases in which the gene is abundantly\r\nexpressed.
\r\n\r\n\r\nAn attempt was made to recover lethal mutations in genes closely flanking\r\nthe 68C glue protein genes by screening mutagenized chromosomes over large\r\ndeficiencies which delete the 68AC region. Although a large number of lethal and\r\nsemi-lethal mutations were recovered, including many which define new\r\ncomplementation groups, none maps close to the region of the 68C glue gene\r\ncluster.
\r\n", "doi": "10.7907/5HDH-D305", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11858", "collection": "thesis", "collection_id": "11858", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10242019-102816056", "type": "thesis", "title": "Antigen Receptors on Lymphocytes", "author": [ { "family_name": "Siu", "given_name": "Gerald", "clpid": "Siu-Gerald" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The structure and evolution of a small VH gene family called the T15 family was analyzed. It was determined that although selection pressure appeared to be operating to maintain the coding region sequence of these VH gene segments, these gene segments were diverging from one another very rapidly. Sequences were identified in the 5' flanking region that were conserved between all VH gene segments and were hypothesized to be important for immunoglobulin heavy-chain gene transcription. Related sequences were identified in immunoglobulin VL gene segment and histone H2B 5' flanking regions, implying coordinate expression between these genes and the immunoglobulin heavy chain genes.
\r\n\r\nThe structure, organization, evolution, and the generation of diversity in the genes encoding the T-cell antigen receptor were analyzed. The T-cell antigen receptor consists of two chains, referred to as the \u03b1 and \u03b2 chains. Each chain consists of two regions, a variable region and a constant region, that are encoded by two different genes. The gene that encodes the variable region of the \u03b2 chain was found to consist of three gene segments, denoted V\u03b2, D\u03b2, and J\u03b2. The V\u03b2 gene segment encodes the first 280-300 bp, the D\u03b2 gene segment encodes the next 10-15 bp, and the J\u03b2 gene segment encodes the final 50 bp of the variable region gene. The rearrangement event that juxtaposes these gene segments during lymphocyte differentiation appears to be mediated by the same recognition signals that mediate immunoglobulin V gene rearrangement.
\r\n\r\nDiversity was found to be generated in at least three different manners in the V\u03b2 gene. Combinatorial joining permits the rearrangement of different V, D and J gene segments to each other to provide different V gene sequences. Deletion of nucleotides from the ends of the germline gene segments and the random addition of nucleotides at the junction of the rearrangement event are two other mechanisms for generating diversity. A comparison of a rearranged V gene with the corresponding germline gene segments showed that with the exception of the junctions, the sequences were identical. Therefore, there is no evidence that somatic hypermutation, the random addition of point mutations to the V gene during late stages of B lymphocyte differentiation, is utilized by the T-cell antigen receptor as it is by immunoglobulins.
\r\n\r\nThe initial stage of V\u03b2 gene formation was found to be the rearrangement of the D\u03b2 gene segment to the J\u03b2 gene segment. Both germ line D\u03b2 gene segments appear to have promoters in the 5' flanking regions that can often result in the transcription of a 1.0 kb mRNA containing D\u03b2-J\u03b2-C\u03b2 sequences after D\u03b2-J\u03b2 rearrangement. This 1.0 kb mRNA message is present at a high level in the thymus but at lower levels in the spleen, lymph nodes, and in mature T cells, implying that this message or a protein product encoded by this message may be important in T cell ontogeny.
\r\n\r\nAnalysis of the protein sequences of the variable regions of the \u03b1 and \u03b2 chains revealed conserved amino acids that are found in all variable region genes. Many of these amino acids were found to be important for V domain structure in immunoglobulins and may be important for the structure of the V\u03b1-V\u03b2 domain as well. In addition, analyses of the \u03b2-strand forming potential and the relative hydrophobicity of the side chains of the amino acids that make up the T-cell antigen receptor variable regions have indicated that these properties are very similar to those of the immunoglobulin variable regions. These analyses indicate that the immunoglobulin and T-cell receptor antigen-binding regions may be very similar in structure to each other.
", "doi": "10.7907/p1zd-zh51", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11862", "collection": "thesis", "collection_id": "11862", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10252019-115550207", "type": "thesis", "title": "Organization and Evolution of the Class I Genes in the Murine Major Histocompatibility Complex", "author": [ { "family_name": "Sun", "given_name": "Yi Henry", "orcid": "0000-0001-8279-5270", "clpid": "Sun-Yi-Henry" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis contains studies of the organization and evolution of the class I gene family in the murine major histocompatibility complex (the H-2 complex).
\r\n\r\nThe first chapter presents the molecular characterization of the H-2dm1 mutation. The mutant gene is shown to be formed by the fusion of the 5' part of the Dd gene and the 3' part of the Ld gene, with the region in between deleted.
\r\n\r\nChapter Two describes the results of chromosome walking experiments and presents a molecular map of 500 kb of cloned DNA, which links the H-2D and Q\u03b1 regions and contains five D region and eight Q\u03b1 region class I genes.
\r\n\r\nChapter Three presents the DNA sequences of the transmembrane exon from 20 class I genes, and the use of 23 low copy-number flanking-region probes to detect homology between the regions containing each gene. The sequence comparison and the hybridization patterns indicate that multiple recombinational events, notably gene duplication and gene conversion, have occurred during the evolution of this large gene family.
\r\n\r\nChapter Four presents a rapid method of restriction site mapping of cosmids and plasmids. The method was developed due to the need of mapping a large number of clones during the course of this study.
", "doi": "10.7907/qfw5-kk48", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11455", "collection": "thesis", "collection_id": "11455", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04122019-155653613", "type": "thesis", "title": "The Structure and Expression of the Gene Coding for Bindin, a Species Specific Sea Urchin Protein", "author": [ { "family_name": "Gao", "given_name": "Boning", "orcid": "0000-0002-1382-395X", "clpid": "Gao-Boning" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Bindin is a major protein of the sea urchin sperm acrosome granule which mediates the species-specific adhesion and binding of sperm to the egg. Bindin protein has been purified from the sperm of the sea urchin Strongylocentrotus purpuratus (S. purpuratus) and the protein has been partially sequenced. The work presented in this thesis is the isolation and the sequence analysis of bindin cDNA and gene, and the study of the expression of the bindin gene.
\r\n\r\nA \u03bbgt10 cDNA library was constructed from S. purpuratus testes poly(A)+ RNA. The library which was screened with a synthetic DNA probe prepared according to the known protein sequence yielded clones representing bindin cDNA. One of these clones containing an 1873 base pair (bp) insert was sequenced and found to code for a bindin precursor (prebindin) which is twice as large as the mature bindin protein. Upon immunoprecipitation with bindin antibody, the testes poly(A)+ RNA in vitro translation product yields a larger precursor. The bind in cDNA was used to study the tissue specificity of expression. The results show that bindin is a sperm specific protein -- its messenger RNA is not detected in the eggs, ovaries, early embryos, coelomocytes, tubefeet, lantern tissue or intestine that we tested.
\r\n\r\nSperm DNA isolated from several individuals was probed with bindin cDNA and reveals that there is one bindin gene per haploid genome. Haploid female coelomocyte DNA also possesses one bindin gene.
\r\n\r\nBindin cDNA was used to screen an EcoRI partially digested sea urchin S. purpuratus DNA charon 4A library. A clone containing a 14 kilobase (Kb) insert hybridized to the 3' end of the bindin cDNA. It also has overlapping restriction enzyme recognition sites with the 3' end of the bindin cDNA. There is an intron in the genomic clone upstream of this overlapping region since it did not hybridize with bindin cDNA. A \u03bbgt10 mini-genomic library was made and a 1.3 Kb genomic DNA clone which hybridized with bindin cDNA has been characterized and partially sequenced. It contains a 219 bp exon in which the 5' end lies 2 bp upstream of the AUG translation initiation codon. This exon is flanked by introns on either side. Thus, there are at least three introns in the bindin gene.
", "doi": "10.7907/h0pw-fh17", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11810", "collection": "thesis", "collection_id": "11810", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10102019-163948204", "type": "thesis", "title": "Class I Genes of the Major Histocompatibility Complex: Structural Studies on Genes of the Tla Locus", "author": [ { "family_name": "Fisher", "given_name": "Douglas Arthur", "clpid": "Fisher-Douglas-Arthur" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis contains investigations into the structure of molecules encoded within the mouse major histocompatibility complex. The first chapter [Steinmetz, M., J. G. Frelinger, D. Fisher, T. Hunkapiller, D. Pereira, S. M. Weissman, S. G. Nathenson, and L. Hood, Cell 24: 125] describes the isolation and characterization of the first cDNA clones encoding murine transplantation (H-2) antigens. This study showed that H-2 antigens contain DNA and protein sequences related to immunoglobulin (Ig) molecules, but that the similarity does not include the great somatic diversity characteristic of Ig molecules.
\r\n\r\nThe second chapter contains methods for cloning and sequencing in M13 bacteriophage vectors. Included is a novel method of generating overlapping subclones for DNA sequencing by making a family of deletions in a DNA insert cloned in M13.
\r\n\r\nIn the third chapter [Fisher, D. A., S. W. Hunt, and L. Hood J. Exp. Med. 162: 528], the complete structure of a gene encoding a serologically defined thymus leukemia (TL) antigen is elucidated. TL antigen is encoded in a gene, gene Tl3c, closely related to H-2 antigens, and appears to have undergone a gene conversion event with an H-2 gene. Tla-specific probes subcloned from T13C enabled us to examine the organization of the eighteen cross hybridizing class I genes of the Tla region.
\r\n\r\nThe last chapter contains the sequence of another gene, gene T1C, previously identified as encoding TL antigen. However, the T1C gene is a non-functional pseudogene, and was probably mis-identified. There is an apparent site of recombination in the T1C gene that occurs precisely at a B2 Alu repeat sequence.
", "doi": "10.7907/ymba-8s86", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11366", "collection": "thesis", "collection_id": "11366", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01302019-123859876", "primary_object_url": { "basename": "Levy_DE_1985.pdf", "content": "final", "filesize": 64210096, "license": "other", "mime_type": "application/pdf", "url": "/11366/1/Levy_DE_1985.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Expression of Endogenous Retroviruses in Inbred Mice : Coordinate Regulation and Structure of Multiple Transcription Units", "author": [ { "family_name": "Levy", "given_name": "David E.", "orcid": "0000-0002-7320-7788", "clpid": "Levy-David-E" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Lerner", "given_name": "R.", "clpid": "Lerner-R" }, { "family_name": "Wilson", "given_name": "M.", "clpid": "Wilson-M" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The control of expression of the murine antigen Gix and of other products of endogenous retroviruses, in strain 129 mice and in its congeneic partner strain 129 Gix-, is an example of the coordinate expression of a dispersed family of independent transcription units. In order to provide a molecular description of the Gix phenotype, evidence is presented, from DNA and RNA hybridization analyses using heterologous viral probes, indicating that this phenotype is specified by a distinct regulatory gene, defined genetically, that acts in trans to control the levels of accumulation of specific mRNA species. The steady state levels of several, structurally distinct polyadenylated RNA species are reduced in Gix- mice, and a major reduction in transcription of these sequences accompanies this drop in abundance. Tissue specific patterns of accumulation of different sized RNA species were detected in numerous organs of the mouse, and the majority of these distinct transcripts were collectively regulated.
\r\n\r\nThe isolation and characterization of cDNA copies of these endogenous retroviral transcripts demonstrated that they were derived from multiple, distinct transcription units. Differences among these RNA species were detected by S1 nuclease protection analyses , which confirmed the tissue specific patterns of RNA accumulation. The nucleotide sequences of endogenous virus cDNA clones fully documented the expression of distinct genes, the nature of the sequence heterogeneity, and the relationship of these normal cellular constituents to exogenous, infectious virus. The polymorphism was found to result from both single nucleotide changes and from deletions of different lengths of coding and non-coding information. Comparison of these sequences with exogenous virus demonstrated that the endogenous transcripts are closely related to the recombinant sequences of eukemogenic, mink cell focus forming viruses.
\r\n\r\n", "doi": "10.7907/165r-9v89", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11372", "collection": "thesis", "collection_id": "11372", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-142248860", "type": "thesis", "title": "The Organization and Expression of the Human Dihydrofolate Reductase Gene in Methotrexate-Resistant and Methotrexate-Sensitive Cell Lines", "author": [ { "family_name": "Masters", "given_name": "Jeffrey Nelson", "clpid": "Masters-Jeffrey-Nelson" } ], "thesis_advisor": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The analysis of several derivatives of the human cell lines HeLa and VA2-B, selected for resistance to methotrexate (MTX), shows a striking variability in the dihydrofolate reductase (DHFR) enzyme levels, chromosome constitution and growth characteristics in the absence of MTX. In contrast to the mouse system, the number of double minutes of the human cells does not correlate with either the increased DHFR levels or the instability of the amplified phenotype.
\r\n\r\nThe isolation of human DHFR eDNA by differential hybridization or by phenotypic expression in E. coli, facilitates the characterization of the human DHFR coding region and its multiple mRNAs. Comparison of the DNA sequences of several DHFR cDNAs shows a high degree of homology between the coding regions of the human and mouse genes (89%) with no obvious identity in the 3'-untranslated region. The analysis also demonstrates that cDNAs of the 3 identified mRNAs are colinear in the 3'-end sequence, and that polyadenylation occurs at different sites.
\r\n\r\nHybridization of EcoRI digested nuclear DNA from the MTX-resistant 6A3 cells with DHFR cDNAs shows EcoRI fragments that are either unamplified or amplified relative to the same fragments of human sperm, HeLa cell, and VA2-B cell DNA. Two of the unamplified fragments were isolated from a cosmid library made from human sperm DNA. The DNA sequence analysis shows that these two fragments contain a DHFR intronless pseudogene including the EcoRI site found in the DHFR cDNAs. If an RNA intermediate directs the formation of this pseudogene, an RNA larger than the 1.0 kb DHFR mRNA must be involved. In contrast to the unamplified DNA fragments, the amplified fragments contain the exons of the human DHFR gene. The gene is about 29 kb in length, with five introns interrupting the DHFR coding region in the same positions found in the mouse gene. The DHFR mRNAs were mapped as a major 5'-end at position -71 of the human DHFR gene. In addition, six minor 5'-ends of DHFR-specific polysomal RNA were mapped from positions -449 to -480 and represent about 1% of the major transcripts. The upstream transcripts are relatively enriched in the nuclear RNA fraction, indicating a different regulation of expression for these minor transcripts.
", "doi": "10.7907/jaa8-jx74", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11374", "collection": "thesis", "collection_id": "11374", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-165301208", "type": "thesis", "title": "Studies of Class I Genes in the Major Histocompatibility Complex of the BALB/c Mouse", "author": [ { "family_name": "Sher", "given_name": "Beverly Taylor", "clpid": "Sher-Beverly-Taylor" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis contains the results of investigations into the structure and organization of Class I genes in the major histocompatibility complex of the BALB/c mouse.
\r\n\r\nIn the body of the thesis, the sequence of the BALB/c H-2Dd transplantation antigen gene is presented. This is the first complete sequence of an H-2Dd gene and is the only genomic sequence to be in full agreement with the available protein sequence. The H-2Dd gene sequence has been used to predict the protein sequence of the H-2Dd molecule, which has been compared to the protein sequences of other Class I molecules. The H-2Dd protein sequence is no more related to that of its closely linked partner, H-2Ld, than it is to the sequence of its presumptive allele, H-2Db, or to the sequence of the H-2Kb molecule, which is from not only another H-2 haplotype but another genetic subregion. The sequence differences between these transplantation antigens are spread throughout the molecules in a mosaic pattern that may have arisen,in part, from small gene conversion events. No obvious evidence of any recent gene conversion event affecting the H-2Dd gene was observed, however.
\r\n\r\nThree Class I genes have been cloned and mapped to the H-2D subregion in BALB/c. These include gene 16.1, whose product has not been identified; the H-2Dd gene; and the H-2Ld gene. There is serological evidence for the existence of additional H-2D-encoded transplantation antigen molecules in BALB/c, but no genes encoding these products have been identified. The sequence of the H-2Dd gene contains potential alternative splice sites in and around the exon encoding the first external domain. use of these splice sites could generate a transplantation antigen molecule with different serological determinants, and might help to resolve the discrepancy between the number of H-2D-subregion Class I genes and the number of serologically defined H-2D-subregion transplantation antigens.
\r\n\r\nThe appendices contain the results of a number of studies related to Class I gene organization and function. Appendix A contains the sequence of gene 27.1. This gene, also known as the Q8 gene, was identified as a Qa pseudogene based on the presence of termination codons in inappropriate locations in its sequence. Appendix B contains the sequence of the H-2Ld gene, which was the first transplantation antigen gene to be sequenced. Appendix C contains the results of DNA-mediated gene transfer experiments that Identified genomic clones containing the H-2Kd, H-2Ld, and H-2Dd transplantation antigen genes as well as Class I genes encoding the Qa2,3 molecule and two different TL differentiation antigen genes. Appendix D contains the results of calculations of protein sequence homology between different Class I molecules.
", "doi": "10.7907/4pj0-z147", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11389", "collection": "thesis", "collection_id": "11389", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02112019-112435563", "primary_object_url": { "basename": "Falke_JJ_1985.pdf", "content": "final", "filesize": 108586280, "license": "other", "mime_type": "application/pdf", "url": "/11389/1/Falke_JJ_1985.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Behavior and Structure of the Band 3 Anion Transport Site: a \u00b3\u2075Cl and \u00b3\u2077Cl NMR Study", "author": [ { "family_name": "Falke", "given_name": "Joseph John", "orcid": "0000-0002-3704-4694", "clpid": "Falke-Joseph-John" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The transport of ions across cellular and organellar membranes is a widespread and fundamental process in biology. The goal of the present work is a molecular picture describing the ion translocation event in band 3, the most heavily used ion transport protein in typical vertebrate systems. The strategy employed involves 35Cl NMR, which is shown both theoretically and experimentally to be a sensitive probe of two microscopic events: 1) the migration of Cl- from solution to the vicinity of a macromolecular binding site, and 2) the binding of Cl- to the site. The technique reveals 35Cl- linebroadening due to two classes of Cl- binding sites on isolated, native red cell membranes. One class is composed solely of low affinity Cl- binding sites of unknown function (KD > > 0.5 M), while the other class is composed solely of band 3 transport sites (KD = 80 \u00b1 20 mM) which are identified by their affinity for substrate (Cl-), competing substrates (HCO-3, F-, Br-, I-) and inhibitor (4,4'-dinitrostilbene-2,2'-disulfonate, or DNDS). A 35Cl NMR method is developed to ascertain the sidedness of Cl- binding sites relative to a compartment barrier such as a membrane: this approach shows that the low-affinity and transport sites are each found on both surfaces of the membrane.
\r\n\r\nThe sequence of events in the Cl- transport cycle is investigated by monitoring the behavior of the transport sites when the concentration of DNDS, p-nitrobenzenesulfonate (pNBS), Cl-, Br-, or H+ is varied. DNDS and pNBS, which are known to bind to outward-facing transport sites, each recruit all of the transport sites on both sides of the membrane to the inhibited outward-facing conformation, indicating that the inward- and outward-facing transport sites observed in the absence of inhibitor are merely different conformations of a single site. In addition, the transport sites on both sides of the membrane together behave like a homogeneous population of sites when [Cl-], [Br-], or pH is varied. These results are quantitatively consistent with the pingpong model for the transport cycle (Gunn and Frolich (1979) J. Gen. Physiol. 74, 351-374), in which a single transport site alternates between the inward- and outward-facing states and can only change states when occupied by bound anion. The rates of Cl- binding and dissociation at both inward- and outward-facing transport sites are investigated with 35Cl and 37Cl NMR, and it is shown that each of these rates exceeds 105 events sec-1 site-1 -- much faster than the known turnover rate of the chloride transport cycle (430 events sec-1 site-1, 0\u00b0C). Assuming that the rates of the influx and efflux half-turnovers of the transport cycle differ by 102 or less, it follows that the translocation of the chloride*transport site complex is the rate-limiting step in both half-turnovers (see Figure).
\r\n\r\nThe structure of the transport machinery is investigated using transport inhibitors and proteases. The reversible inhibitor niflumic acid (NIF) has no effect on the transport site linebroadening: this inhibitor slows the translocation of bound Cl- in both the influx and efflux half-turnovers. The covalent, arginine-specific reagents phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) each eliminate the transport site linebroadening: PG modifies an essential residue in the transport site and CHD slows the migration of Cl- between the site and solution. The observed PG-sensitivity and pH-dependence of the transport site linebroadening (pKA = 11.1 \u00b1 0.1, [Cl-] = 250 mM) indicate that an arginine residue provides the positive charge in at least one conformation of the transport site. A search is conducted for the minimal structure containing the intact transport site: this search begins with the removal of an innessential part of the transport domain, followed by monitoring of the transport site linebroadening for change. A variety of treatments leave some or all of the transport site linebroadening intact, including: 1) removal of the red cell membrane nonintegral proteins, 2) proteolytic removal of the soluble N-terminal domain of band 3, or 3) extensive proteolysis of band 3 by papain, which reduces band 3 to its transmembrane peptides (3-9 kDa). These results indicate that the essential arginine, as well as all other residues essential for Cl- migration and binding to the transport site, are located on the papain-generated transmembrane peptides. The structural data presented here strongly support a picture in which the transport site, including the essential arginine, is buried in the membrane where it is resistant to proteolysis; and access of the buried site to solution Cl- is provided by a channel that can be blocked by CHD. In summary, the minimal sequence of events in the Cl- transport cycle can be schematically illustrated by
\r\n\r\n[Illustration. See abstract in scanned thesis for details]
\r\n\r\nA model is also presented that describes the molecular details of the ion translocation event: the translocation is proposed to begin when the transport site positive charge is neutralized by anion binding, so that a sliding hydrophobic barrier can move past the site and thereby expose the site to the opposite solution, as illustrated by
\r\n\r\n[Illustration. See abstract in scanned thesis for details]
\r\n\r\nA sliding barrier model could explain the translocation event in many other membrane transport systems as well.
", "doi": "10.7907/j15r-rw02", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11356", "collection": "thesis", "collection_id": "11356", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01222019-155306456", "primary_object_url": { "basename": "Parker_VP_1985.pdf", "content": "final", "filesize": 31373314, "license": "other", "mime_type": "application/pdf", "url": "/11356/1/Parker_VP_1985.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies of Gene Structure: I. Expression of Human \u03b1-Globin Genes in COS Cells. II. Isolation and Characterization of the Myosin Light Chain Genes from Drosophila melanogaster", "author": [ { "family_name": "Parker", "given_name": "Vann Phillips", "clpid": "Parker-Vann-Phillips" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The first part of this thesis describes the establishment of COS cells as an effective host for the replication and expression of genes cloned into plasmids containing the SV40 viral replication origin. The human \u03b1-globin gene was cloned into a pBR322 derivative which contained a 311 base pair SV40 fragment. These plasmids were replicated to high copy number. The \u03b1-globin gene was transcribed to produce high levels of RNA which was indistinguishable from human \u03b1-globin mRNA. Deletion mutants were generated which defined the region from 55 to 87 base pairs upstream from the mRNA cap site as necessary for high levels of transcription of this gene. This region includes the conserved sequence CCAAT found in a similar position upstream from many eukaryotic genes.
\r\n\r\nThe second part of this thesis describes the cloning of muscle specific genes from Drosophila melanogaster and a detailed analysis of the two myosin light chain genes. A genomic library was screened with RNA isolated from the late pupal stage of development. Recombinant DNA clones which mapped to 24 independent locations were identified. The clones containing the myosin light chain genes were identified from within this group. A cDNA library was generated from late pupal RNA. Clones homologous to each of the myosin light chains were identified and their cloned inserts were sequenced. Their classification as Drosophila myosin light chain genes was established by comparing the derived amino acid sequence to the protein sequence of the chicken myosin light chains. Each myosin light chain is single copy. The alkali myosin light chain maps to the chromosomal locus 98B. It hybrid selects RNA which may be translated in vitro to yeild several polypeptides of molecular weight 18,000 to 19,000 daltons. The gene for myosin light chain-2 maps to the chromosomal locus 99E and is shown to encode two proteins of apparent molecular weights 26,000 and 17,000 daltons. Each protein has a putative divalent cation binding domain.
", "doi": "10.7907/msv6-0s82", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11361", "collection": "thesis", "collection_id": "11361", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01242019-174640334", "primary_object_url": { "basename": "Livant_DL_1985.pdf", "content": "final", "filesize": 36438786, "license": "other", "mime_type": "application/pdf", "url": "/11361/1/Livant_DL_1985.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Size of a Murine Heavy Chain Variable Region Gene Family: Implications for the Magnitude and Evolution of the V\u2095 Locus in Mouse", "author": [ { "family_name": "Livant", "given_name": "Donna Lucy", "orcid": "0000-0002-6164-6580", "clpid": "Livant-Donna-Lucy" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The problem of how much antibody diversity is encoded in the germline as variable region genes has long been of interest to immunologists. We have measured the size of the J558 VH family in the BALB/c mouse by a probe excess titration method, and found that the family contains approximately 1000 members. As a control for systematic error, we used the same method to measure the number of class I MHC genes in BALB/c. We found that the third domain of the class I Dd gene detects 36-40 class I genes. Dot blots and genome blots with copy number controls give results consistent with a J558 family size of 500-1000 VH genes. We note that each band evident on genomic blots of DNA from several mouse strains contains multiple VH genes, and that a significant fraction of these bands are polymorphic among the mouse strains tested. We discuss the implications of this result for both the size and evolution of the VH locus in mouse.
", "doi": "10.7907/22vp-vt51", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11370", "collection": "thesis", "collection_id": "11370", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-124340304", "type": "thesis", "title": "Antibody Genes, Oncogenes and Antisense Genes", "author": [ { "family_name": "Kim", "given_name": "Stuart Kilsu", "clpid": "Kim-Stuart-Kilsu" } ], "thesis_advisor": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "thesis_committee": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "There are a number of mechanisms involved in producing a diversity of antibodies including multiple germline genes, somatic gene rearrangement, somatic hypermutation and combinatorial association. By the process of somatic hypermutation, one immunoglobulin gene in the germline can be mutated to produce many different genes in B cells. In chapter 2, this process is characterized. It was found that phosphorylcholine binding antibodies are encoded by one germline VH gene segment. In B cells, this VH gene segment may have extensive point mutations, many of which are silent, indicating the presence of some somatic hypermutational mechanism. Only the VH gene was found to be mutated indicating that the mutational mechanism was specific for VH genes.
\r\n\r\nOne way to study somatic immunoglobulin gene rearrangements, presented in chapter 3, might be to characterize rearrangements which are not easily explained. Immunoglobulin gene rearrangement was thought to exclusively involve immunoglobulin genes. However, some immunoglobulin genes can reproducibly rearrange with other DNA sequences. Insight into the basis of these rearrangements was uncovered by identifying the chromosomal origin of the nonimmunoglobulin rearranging DNA. This DNA originated on chromosome 15 whereas the immunoglobulin gene originated on chromosome 12. The juxtaposition of these sequences is common in plasmacytomas but rare or absent in normal B cells suggesting that it is involved in tumorigenesis. For example, it may be that aberrant immunoglobulin rearrangements can activate a cellular oncogene resulting in a plasmacytoma. This possibility was supported by results from other laboratories when it was found that the non-immunoglobulin rearranging DNA contained the cellular homologue of the myc oncogene.
\r\n\r\nTo understand lymphocyte tumorigenesis, it would be useful to understand the function of the c-myc gene product in normal and transformed cells. One way to begin is to determine which types of cells express the c-myc gene. This approach was employed in chapter 5 and it was found that the c-myc gene is expressed in dividing, but not resting, lymphocytes. One possible function for the c-myc gene product is that it functions in cellular proliferation.
\r\n\r\nAnother way to study the function of the c-myc gene product would be to prevent expression of the rearranged c-myc gene in plasmacytomas. For example, it may be possible to inhibit the synthesis of the c-myc gene product by antisense c-myc RNA. If the antisense RNA can hybridize to the c-myc RNA in vivo, synthesis of myc protein may be prevented. A test case, in which antisense TK RNA is used to inhibit TK expression, is presented in chapter 6. In L cells, high levels of antisense TK RNA expression were capable of inhibiting TK activity. The mechanism of inhibition involves RNA:RNA hybridization since double stranded RNA was formed. If this test case can be applied to other instances, it may be possible to use antisense RNA to inhibit the synthesis of a particular gene product and thus study its cellular function.
", "doi": "10.7907/xsdv-c915", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11253", "collection": "thesis", "collection_id": "11253", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10302018-171502394", "type": "thesis", "title": "Functions and Regulation of RNAs Transcribed from the Drosophila melanogaster 68C Puff", "author": [ { "family_name": "Crowley", "given_name": "Thomas Edward", "clpid": "Crowley-Thomas-Edward" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Puffs appear and disappear on Drosophila salivary gland polytene chromosomes at specific developmental timepoints or in response to external stimuli. This thesis is an analysis of the functions and regulation of three RNAs transcribed from the puff at 68C on the left arm of the third chromosome.
\r\n\r\nThe nucleotide sequence of the DNA encoding these RNAs was used to predict the physical and chemical properties expected of their protein products. Analysis of radiolabeled salivary glands revealed polypeptides having the characteristics predicted for the products of the 68C RNAs. Amino acid sequencing of these proteins showed that they are in fact encoded by the 68C RNAs. All three polypeptides were found to be part of the salivary gland glue: one is the previously described sgs-3, the others the newly identified glue proteins sgs-7 and sgs-8.
\r\n\r\nThe effect of the steroid hormone ecdysterone on the 68C RNAs was examined by culturing salivary glands in vitro in the presence or absence of the hormone. The presence of the steroid caused the RNAs to disappear more rapidly than they would in its absence. Pulse-labeling experiments demonstrated that the effect of ecdysterone is on an early step in RNA production, probably transcription. The effect on the 68C RNAs is very rapid, more rapid than puff regression. The three RNAs appear to be coordinately regulated.
\r\n\r\nThe expression of glue protein genes in a non-pupariating mutant strain of Drosophila has been studied. Although a puff is present at position 68C on the third chromosome in the mutants, the Sgs-3, Sgs-7 and Sgs-8 genes are not expressed. Pulse-labeling experiments indicate that the mutation affects transcription of these genes. Other researchers have mapped the non-pupariating mutation to position 2B on the X chromosome. It appears that a product of a gene at 2B or a product whose synthesis is induced by a gene at 2B is necessary for transcription of the 68C glue protein genes. This trans-acting regulatory element produces its effect by interacting with DNA sequences within or very close to the glue genes at 68C. These results along with transcription autoradiograms show that the puff at 68C is not caused by transcription of Sgs-3, Sgs-7, Sgs-8 or any\r\nother genes located within the puff region.
", "doi": "10.7907/7vtr-zc75", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11251", "collection": "thesis", "collection_id": "11251", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10292018-120530235", "primary_object_url": { "basename": "Banerjee_U_1984.pdf", "content": "final", "filesize": 63119892, "license": "other", "mime_type": "application/pdf", "url": "/11251/1/Banerjee_U_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Alamethicin: Secondary Structure in Solution and Interactions with Phospholipid Membranes", "author": [ { "family_name": "Banerjee", "given_name": "Utpal", "clpid": "Banerjee-Utpal" } ], "thesis_advisor": [ { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Kuppermann", "given_name": "Aron", "clpid": "Kuppermann-A" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The icosapeptide alamethicin [sequence included in scanned thesis' abstract, p. v] isolated from the fungus Trichoderma viride induces voltage-gated ionic conductance in black lipid film membranes (Latorre, R. & Alvarez, D. (1981) Physiol. Rev. 61, 77). Single-channel measurements have indicated that passive ion transport across membranes is mediated by alamethicin channels that fluctuate between several conduction states. While these studies provide phenomenological description of the nature of alamethicin-assisted ionic conduction, very few studies probed the molecular structure of this peptide and the nature of its interaction with lipid membranes. These issues are addressed in the present investigation.
\r\n\r\nAn analysis of the proton magnetic resonance spectrum is undertaken. Two-dimensional NMR is employed to achieve a complete assignment of the protons in the molecule to NMR resonances. The spectral assignment is a necessary first step towards molecular interpretations.
\r\n\r\nMeasurement of coupling constants and two-dimensional NOE's suggest a half-helical, half-extended dimeric structure for the molecule in methanol. This proposed model for the secondary structure, consistent with the NMR data as well as a line of other experimental observations erstwhile published, predicts that (a) the amide protons of residues 15 through 20 are intermolecularly hydrogen-bonded with the corresponding residues of the opposing molecule to create a rigid, extended parallel \u03b2-pleated structure for the C-terminal end of the molecule; (b) the proline at position 14 breaks the continuity of this structure, and amino acids 10 through 14 are forced into an open, non-hydrogen-bonded conformation, and (c) amino acids 3 through 9 are folded into an \u03b1-helix, with Gln-7 side chains from the two strands in the right juxtaposition to facilitate a hydrogen bond between them. The resultant structure is highly amphipathic: one face is completely hydrophobic with the aliphatic side chains exposed, whereas the other face is primarily hydrophilic with polar side chains and peptide groups lining the extended \u03b2-sheet region.
\r\n\r\nThe dimeric structure is further supported by relaxation measurements that indicate that the N-acetyl methyl groups at the N-termini of the two helices in the dimer have distinct proton spin-spin relaxation times. This difference is eliminated once the dimers are dissociated with urea.
\r\n\r\nSpectral assignments in water are complicated by broadened NMR signals due to aggregation. Standard two-dimensional and decoupling techniques for assignments are inadequate for this case. A successful assignment is achieved by solvent titration from methanol. No changes in coupling constants are noted during the titration, and it is expected that the conformation in water is similar if not identical, to that in methanol. Relaxation measurements in water are consistent with a tightly bound dimeric unit that micellises to larger aggregates.
\r\n\r\nThe interaction of alamethicin with multilayers is inferred from a spectroscopic investigation of the phospholipid bilayer prepared from dimyristoyllecithin (DML) in the presence and absence of alamethicin, and, for contrast, in the presence of other membrane active molecules. The dynamics and conformation of phospholipid head group and chains are examined by P31 and H2 NMR. A P31 line shape calculation has helped identify the dependence of the spectrum on various motional, relaxation and conformational parameters.
\r\n\r\nAs part of the investigation of lipid packing and dynamics in membranes, small bilayer vesicles are also studied. Proton NMR indicates that the outside-facing and inside-facing leaflets of the bilayer in small vesicles have lipids packed in different densities. This is due to the differences in the extent and sign of curvature of the two leaflets. At the high field at which this NMR study is undertaken, the differences in packing show up as distinct proton peaks from the inside and outside chain methylene and methyl groups nthat differ in width and in chemical shift.
\r\n\r\nFinally, the interaction of alamethicin with DML multilayers is characterized by P31, H2 and H1 NMR and Raman spectroscopy. The reduction in chemical shift anisotropy (\u0394\u03c3) of the P31 signal is interpreted in terms of an interaction of the peptide at the water-membrane interface that causes a change in the average head group orientation. Deuterium NMR shows no changes in quadrupolar splittings (and hence C-D order parameters) of the chain deuterons, and Raman spectroscopy shows no change in the gauche-trans ratio of methylene segments in the chain. These results are contrasted with P31 and H2 NMR of the gramicidin S/DML system that shows polymorphism due to partial disruption of the multilayer structure and the chlorophyll A/DML system that exhibits a 7\u00b0 C change in phase transition temperature as a clear indication of incorporation of the phytol chain into the bilayer.
\r\n\r\nTaken together, these experiments unequivocally indicate that the peptide interacts with lipid bilayers at the lipid-water interface. The proposed amphiphilic aggregated solution structure for the peptide is ideally suited for such an interaction. Inasmuch as the conductance characteristics of alamethicin are only explained in terms of transmembrane pore formation, it is proposed that the large dipole moment of this aggregate facilitates the transfer of the peptide into the bilayer once a gradient of field is applied.
\r\n", "doi": "10.7907/9y1x-zh89", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11255", "collection": "thesis", "collection_id": "11255", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10302018-173746907", "type": "thesis", "title": "Molecular Studies on the Alcohol Dehydrogenase Gene of Drosophila melanogaster", "author": [ { "family_name": "Goldberg", "given_name": "David Alan", "clpid": "Goldberg-David-Alan" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Maniatis", "given_name": "Thomas P.", "clpid": "Maniatis-T-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In this thesis, I describe the isolation of the alcohol dehydrogenase (Adh) gene of Drosophila melaogaster and some preliminary biochemical characterizations of the gene and its expression. The isolation of the Adh gene was accomplished by screening a bacteriophage \u03bb library containing inserts of Drosophila DNA with eDNA probe made from size selected mRNA. One clone which showed hybridization in the initial scr\u2022en was shown to contain Adh sequences by virtue of its lack of hybridization to Adh deficiency DNA, in situ hybridization, translation of ADH protein by mRNA selected by hybridization to the clone, and by partial DNA sequence analysis. Using the clone, approximately 35 kb of the Adh chromosomal region was isolated. This region was found to be composed largely of single-copy sequences, showed limited polymorphism between strains, and encoded only one RNA transcript prevalent in larvae and adults - the Adh mRNA. Two intervening sequences within the Adh coding region were demonstrated by S1 nuclease mapping.
\r\n\r\nIn order to identify sequences important in Adh expression, the cloned Adh gene was transformed into the Drosophila germ line by utilizing the hybrid dysgenesis P element vector of Spradling and Rubin. The correct developmental expression of the Adh gene was retained by the transformed gene, even though it had integrated into many locations. These results delimit the sequences and chromoscmal enviroiiilent necessary for correct developmental expression of the Adh gene. In addition, the 'transient' expression of cloned DNA in larvae and adults directly grown from injected embryos was investigated. In most instances, ADH activity was found only in tissues that normally express ADH, although low level of activity was observed in some cells which do not normally produce detectable levels of Adh. Together, these results form the basis for assay systems that may be combined with in vitro mutagenesis in order to determine in detail which sequences are necessary for correct developmental expression of the Adh gene.
", "doi": "10.7907/skx9-0t33", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11258", "collection": "thesis", "collection_id": "11258", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10302018-183325232", "type": "thesis", "title": "Regulation of ColEl Plasmid DNA Replication in Escherichia coli", "author": [ { "family_name": "Moser", "given_name": "David Randall", "clpid": "Moser-David-Randall" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A number of plasmid copy-number mutants have been characterized in order to gain insight into the mechanism by which initiation of plasmid DNA synthesis is regulated. One nutant, pFHllB, which has a 12-fold higher copy number than the homologous wildtype plasmid, RSF1050, contains 16 additional base pairs within a region of the plasmid that has been shown to be essa1tial for norrral replication. The insertion nutation lies approximately 500 base tairs upstream of the origin of DNA synthesis within the coding region of a small untranslated RNA, RNA I. The copy-number nutation is recessive and can be complemented both in cis and in trans by the wild-type RNA I gene. The results of the complementation studies reveal that replication of the ColE1-type plasmids is regulated by a mechanism of negative control in which RNA I functions as a repressor of plasmid DNA synthesis.
\r\n\r\nThe target of RNA I inhibition is believed to be near the 5' end of a second plasmid transcript, RNA II. RNA II transcription begins 555 base pairs upstream of the replication origin and terminates several hundred base pairs downstream of the origin. RNA II can be processed by RNase H to generate a primer for the initiation of DNA synthesis by DNA polymerase I. Purified RNA I has been shown to inhibit primer formation in vitro. Dominant high copy-number mutants of a specially-constructed plasmid, pDM247, contain single base-pair changes within a seven base-pair sequence of the RNA II coding region. The altered nucleotides in RNA II are located within the single-stranded loop of a prominent hairpin structure that can form near the 5' end of the transcript. A romplenentary hairpin structure exists in RNA I. A model for RNA I-RNA II interaction involving base pairing of loop structures has been proposed.
\r\n\r\nA single base-pair mutation has been identified in a plasmid which has a temperature-sensitive high ropy-number phenotype. The ts mutant exhibits normal copy control in cells growing at the permissive temperature of 30\u00b0C but replicates uncontrollably in cells shifted to the nonpermissive temperature, 37\u00b0C. Runaway replication of plasmid DNA is lethal to the host cells within 3-4 hours after the temperature shift. The orp mutation (for over-replication) affects both the RNA II transcript and the promoter for RNA I. RNA I promoter-galK fusion studies indicate that the mutation does not create a temperature-sensitive promoter. It has been proposed that the orp mutation creates a thermosensitive secondary structure in RNA II. The breakdown of this structure at the higher temperature renders the plasmid insensitive to replication inhibition by RNA I. Secondary mutations which suppress the temperature-sensitive phenotype have been isolated. These mutations also affect nucleotides within RNA II.
\r\n\r\nA small plasmid-encoded polypeptide also plays a role in plasmid copy number control. Although this protein, referred to as the rop gene product, is not essential for regulation of plasmid DNA replication (the deletion of the rop gene causes only a three to five-fold increase in plasmid copy number), its presence in the cell can suppress lethal DNA over-replication of the temperature-sensitive copy number mutant. This suppression was used as the basis of a selection for rop insensitive mutants. The identification of point mutations within the RNA I coding region of these mutants is consistent with the hypothesis that the product of the rop gene modulates the interaction between RNA I and the primer precursor.
", "doi": "10.7907/bf0f-4378", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11279", "collection": "thesis", "collection_id": "11279", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11212018-100547724", "primary_object_url": { "basename": "Kuo_C-L_1984.pdf", "content": "final", "filesize": 28474761, "license": "other", "mime_type": "application/pdf", "url": "/11279/1/Kuo_C-L_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Use of Temperature Sensitive Mutants to Study Yeast DNA Replication", "author": [ { "family_name": "Kuo", "given_name": "Chia-lam", "clpid": "Kuo-Chia-lam" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Raftery", "given_name": "Michael A.", "clpid": "Raftery-M-A" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "An improved in vitro DNA replication system in Brij-treated Saccharomyces cerevisiae has been used to screen a random population of temperature-sensitive strains for mutants specifically defective in DNA synthesis. Twenty mutants defective in in vitro DNA synthesis have been isolated. Seven of them fall into three complementation groups -- cdc2, cdc8, and cdc16 -- involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in in vivo at the time of permeabilization, our findings that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23\u00b0c but not at 37\u00b0c supports the conclusion that these two mutations directly affect DNA synthesis. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. The usefulness of our screening procedure is further demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.
\r\n\r\nThe in vitro system has also been used to purify the CDC8 protein. cdc8 mutant strains are temperature-sensitive for DNA chain elongation and the CDC8 gene product is required for DNA synthesis in vitro in permeabilized yeast cells. Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8 mutant. A small, heat-stable protein responsible for this complementation has been partially purified from wild-type cells.
\r\n\r\nThe CDC8 gene has been isolated on recombinant plasmids. The yeast-E. coli shuttle vector YCp50 was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping and DNA sequencing. This fragment lies 1 kilobase downstream from the well characterized sup4 gene, a gene known to be genetically linked to CDC8 thus confirming the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the cdc8-1 mutation.
\r\n\r\nBy the following criteria, we have shown that thymidylate kinase, which catalyzes the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-diphosphate in the pathway of synthesis of dTTP from dTMP, is the product of the CDC8 gene. First, transformed strains carrying the CDC8 gene on a stable high-copy-number plasmid express higher levels of both the gene transcript and the kinase activity than does wild type. Secondly, extracts of strains bearing different alleles of cdc8 show no detectable thymidylate kinase activity. Third, the DNA sequence of CDC8 gene reveals an open reading frame that encodes a protein of 216 amino acids with the same amino terminal sequence as thymidylate kinase purified from yeast.
", "doi": "10.7907/gekb-yq20", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11317", "collection": "thesis", "collection_id": "11317", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12142018-113612227", "primary_object_url": { "basename": "Pearson_LT_1984.pdf", "content": "final", "filesize": 38709673, "license": "other", "mime_type": "application/pdf", "url": "/11317/1/Pearson_LT_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "A Model for the Lateral Organization of Protein Molecules in Lipid Bilayers", "author": [ { "family_name": "Pearson", "given_name": "Laurence Timothy", "clpid": "Pearson-Laurence-Timothy" } ], "thesis_advisor": [ { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Interactions between membrane bound proteins are examined using freeze-fracture etch electron microscopy. The pair distribution functions (PDF's) of protein particles in natural and synthetic membrane systems are examined, and compared with PDF's that are calculated from potential energy functions. In particular, PDF's calculated from the hard-disc only interaction between particles serve as a useful reference for determining whether particle interactions are attractive or repulsive. Of particular interest is the possibility that the lipid bilayer membrane mediates protein interactions. A model is presented for a lipid-mediated interaction that predicts that if protein molecules perturb the bilayer membrane away from its equilibrium (protein free) configuration and that if the perturbation is propagated laterally through the membrane over a sufficient distance, then an attractive interaction is the result.
\r\n\r\nThe model is tested on recombinants of cytochrome c oxidase with dimyristoyl phosphatidyl choline and glycerol and with cardiolipin. Each recombinant is frozen from above the phase transition temperature of the lipid, so the membranes are expected to be fluid. Aggregation of protein into patches is seen, but all PDF's are indicative of a long-ranged repulsion. The model must be modified to account for the repulsion, a modification that would explain the observations in the inclusion of a vector membrane order parameter, namely lipid tilt, into the model. Tilt perturbation can be described by using the formalism already developed for describing nematic and smectic liquid crystals. A repulsive interaction between protein particles that is analogous to that seen between Schlieren textures in liquid crystals can be shown to occur if protein molecules induce tilt deformations in the bilayer around their boundaries.
", "doi": "10.7907/k7m9-xb92", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11885", "collection": "thesis", "collection_id": "11885", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11042019-115006064", "primary_object_url": { "basename": "snyder-mp-1983.pdf", "content": "final", "filesize": 5671154, "license": "other", "mime_type": "application/pdf", "url": "/11885/1/snyder-mp-1983.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Organization and Expression of a Cluster of Drosophila Cuticle Genes", "author": [ { "family_name": "Snyder", "given_name": "Michael Paul", "orcid": "0000-0003-0784-7987", "clpid": "Snyder-Michael-Paul" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A 50 kb DNA segment of the Drosophila genome has been cloned and characterized. This segment lies at chromosomal location 44D and contains two small gene families. One family is comprised of four related cuticle genes clustered within 7.9 kb of DNA. The four genes encode four of the five major third instar larval cuticle proteins. These cuticle genes are coordinately expressed in the integument of third instar larvae, and they are not abundantly expressed in other developmental stages. A fifth cuticle-like gene lies within this gene cluster. It is judged to be a pseudogene, because several features of its structure and the absence of transcripts suggest that it is nonfunctional. Sequence comparisons indicate it arose by an unequal crossing over event involving two closely related and adjacent cuticle genes.
\r\n\r\nEleven kb away from the cuticle gene cluster lies another gene family. This family is comprised of three genes that are 55-60% homologous in DNA sequence and clustered within 8 kb of DNA. The three genes are expressed together in larval stages and adults but show a different pattern of developmental expression from the third instar larval cuticle protein genes. Thus two small gene families can lie adjacent on the chromosome and exhibit different patterns of developmental expression, even though individual genes within a clustered family are coordinately expressed.
\r\n\r\nAdditionally, a Drosophila strain has been studied which fails to synthesize one of the cuticle proteins. A molecular characterization of this strain is reported, which includes the finding of a transposable element in the promoter region of the unexpressed gene.
", "doi": "10.7907/mfee-te69", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:3464", "collection": "thesis", "collection_id": "3464", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09112006-153134", "primary_object_url": { "basename": "Mitchell_ma_1983.pdf", "content": "final", "filesize": 12004261, "license": "other", "mime_type": "application/pdf", "url": "/3464/1/Mitchell_ma_1983.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Interhelical DNA-DNA Crosslinking of Bacteriophage Lambda: Bis(monoazidomethidium)octaoxahexacosanediamine and Bis(psoralen)nonaethyleneoxy ether, Probes of Packaged Nucleic Acid", "author": [ { "family_name": "Mitchell", "given_name": "Mark Allen", "clpid": "Mitchell-Mark-Allen" } ], "thesis_advisor": [ { "family_name": "Evans", "given_name": "David A.", "clpid": "Evans-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Evans", "given_name": "David A.", "clpid": "Evans-David-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A strategy for determining the structure of packaged bacteriophage DNA is developed. The synthesis and characterization of two new interhelical crosslinking reagents, bis(monoazidomethidium)-octaoxahexacosanediamine (BAMO) (13) and bis(psoralen)nonaethyleneoxy ether (BPNE) (7) are reported. Both BAMO and BPNE are capable of crosslinking neighboring DNA helices within intact bacteriophages \u03bb and T7. BAMO crosslinks packaged bacteriophage \u03bb DNA at low binding levels (r = 0.01 BAMO/bp) and can be photolyzed quickly (0.5-1 hr) with light of low energy (\u03bb > 400 nm) to yield 17% crosslinked structures as observed under the electron microscope after isolation and restriction cleavage of the crosslinked DNA. BPNE yields 10% crosslinked structures when incubated for three hours with bacteriophage \u03bb at a binding ratio of r = 0.28 BPNE/bp, photolysed for five hours (\u03bb = 365 nm), followed by isolation and restriction digestion of the crosslinked DNA.
\r\n\r\nMethodology necessary to uniquely locate the site of crosslinking along the bacteriophage genome has also been developed. Directional measurements of restriction fragments have been carried out on photomicrographs of fragments labeled specifically at one end with electron dense avidin spheres. The avidin spheres are attached to the restriction fragments by incorporating a biotin containing nucleotide (Bio-dUTP) into the restriction fragment using T4 polymerase under standard labeling conditions. Subsequent restriction cleavage generates fragments labeled at one specific end. Incubation of avidin spheres with these biotinylated restriction fragments results in avidin sphere binding specifically to the labeled end of the fragments.
\r\n\r\nWe have begun to generate interhelical nearest neighbors maps from the measured crosslink positions for bacteriophages \u03bb and T7. Models have been constructed for packaged T7 DNA according to the coaxial solenoid and coil of coils models discussed in the literature. Hypothetical interhelical nearest neighbors maps from these models have been prepared.
", "doi": "10.7907/mtd8-tb22", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11783", "collection": "thesis", "collection_id": "11783", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08302019-152439482", "primary_object_url": { "basename": "Crews_ST_1983.pdf", "content": "final", "filesize": 42807254, "license": "other", "mime_type": "application/pdf", "url": "/11783/1/Crews_ST_1983.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Structure of Mammalian Genes: (1) Antibody Heavy Chain Variable Region Genes: Organization, Diversity, and Somatic Mutation. (2) Structure and Transcription of the DNA Encompassing the Origin of Replication of Human Mitochondrial DNA", "author": [ { "family_name": "Crews", "given_name": "Stephen Thomas", "orcid": "0000-0002-1432-401X", "clpid": "Crews-Stephen-Thomas" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis describes two experimental systems utilized to study mammalian gene structure and expression: (1) antibody heavy chain variable region genes and (2) mitochondrial DNA.
\r\n\r\nIn order to study the organization and structure of antibody genes and the relative germline and somatic contributions towards antibody diversity, we have analyzed the germline genes encoding the murine immune response to phosphorylcholine. Molecular cloning studies were undertaken and conclusively show that there is only one germline VH gene segment encoding the immune response to phosphorylcholine. Protein sequencing work on monoclonal antibodies that bind phosphorylcholine reveals many different protein sequences related to one predominant sequence. We are able to conclude that these variant sequences are the result of somatic diversification operating on one germline gene segment. We are further able to show that this diversification is mutational and not recombinational. Finally, somatic mutation is correlated with the class of the antibody; IgG and IgA antibodies undergo somatic mutation, IgM antibodies do not.
\r\n\r\nWe have isolated and sequenced a family of four closely related VH gene segments designated V1, V3, V11 and V13. Their function varies: V1 encodes the immune response to phosphorylcholine, V3 is a pseudogene, V11 encodes the immune response to influenza hemagglutinin, and V13 has an unknown function but is not obviously a pseudogene. Structural analysis of recombinant clones containing this family of related VH gene segments and other VH gene segments reveals several important points about the organization of VH gene segments. First, closely related VH gene segments can be clustered together within the VH gene locus. Second, the spacing distance between adjacent VH gene segments is variable; it may be as short as 5 kb and greater than 30 kb. Finally, the average spacing distance between VH gene segments is large, at least 23 kb. Assuming a minimum of 200 germline VH gene segments, the size of the VH gene locus may be greater than 5 million base pairs.
\r\n\r\nThe human mitochondrial genome is the second system that has been chosen to study gene structure and expression, and to accomplish this, we have applied both DNA and RNA sequencing technologies. We sequenced the DNA encompassing the origin of DNA replication and then localized the origin at the nucleotide level. The human mitochondrial origin of DNA replication shares structural characteristics with other known origins of DNA replication; in particular, the presence of extensive secondary structure in the form of a stem-loop structure. In order to precisely localize mitochondrial transcripts to the DNA, we developed techniques that allowed the isolation and sequencing of the 5'-ends of mitochondrial transcripts. This technology was utilized to precisely localize the 5'-end of the mitocohndrial 12S rRNA species 457 nucleotide pairs 5'-to the origin of DNA replication. Analysis of the DNA sequence in this region revealed a phenylalanine tRNA gene whose 3'-end was joined end-to-end with the 5'-end of the 12S rRNA. This analysis first demonstrated the extreme enconomy of genetic material in mammalian mitochondrial DNA.
", "doi": "10.7907/4dgr-za66", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11787", "collection": "thesis", "collection_id": "11787", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09032019-120820356", "primary_object_url": { "basename": "Ellison_JW_1983.pdf", "content": "final", "filesize": 37939184, "license": "other", "mime_type": "application/pdf", "url": "/11787/1/Ellison_JW_1983.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure and Evolution of Human Immunoglobulin C\u03b3 Genes", "author": [ { "family_name": "Ellison", "given_name": "Jay William", "clpid": "Ellison-Jay-William" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In order to learn about the evolution of the human immunoglobulin C\u03b3 gene family, the structural features of individual C\u03b3 genes were examined. The complete nucleotide sequences were determined for three members of the gene family-the C\u03b31, C\u03b32, and C\u03b34 genes. A comparison of these sequences with those of the three reported mouse C\u03b3 genes (C\u03b31, C\u03b32a, C\u03b32b) fails to reveal any pairs of corresponding genes in the two species. Moreover, the sequence homology shared by human C\u03b3 genes in both coding and noncoding regions (about 95%) is significantly greater than that seen within the mouse C\u03b3 family (about 70-80%). The presumably neutral mutations accumulated in the noncoding regions of the human genes have been used to estimate that approximately 6-8 million years have elapsed since the divergence of these genes from a common ancestral sequence. This divergence is considerably more recent than inferred for the mouse C\u03b3 genes, and suggests that gene duplication or gene correction events have occurred more recently in humans than in mice.
\r\n\r\nIn contrast to the CH domain exons and adjacent noncoding regions, the hinge exons of human C\u03b3 genes are quite divergent both in length and sequence. This coding sequence variability is seen to extend into the regions of CH domains which border the hinge in the polypeptide chain. This divergence is interpreted as being the result of natural selection for particular hinge structures in the IgG subclasses. The implication is that these polypeptide regions are important for immunologic effector functions carried out by IgG molecules.
\r\n\r\nThe arrangement of the C\u03b32 and C\u03b34 genes in human chromosomal DNA has been determined to be 5'-C\u03b32-17 kilobase pairs-C\u03b34-3'. The genetic processes generating hybrid IgG molecules from these two genes are discussed, along with the relationship of these processes to gene duplication and gene correction.
", "doi": "10.7907/9crt-qq78", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11826", "collection": "thesis", "collection_id": "11826", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-143201215", "type": "thesis", "title": "Gene Expression in B and T Lymphocytes: (1) Evolution of Rat C\u03ba Alleles (2) The T-cell Receptor Problem", "author": [ { "family_name": "Kronenberg", "given_name": "Mitchell", "orcid": "0000-0001-6318-6445", "clpid": "Kronenberg-Mitchell" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The amino acid sequence of the two kappa chain constant region allotypes found in inbred rat strains indicated that these alleles are very different and therefore may have had an unusual evolutionary history. To understand the evolution of these genes, serologic tests were performed to determine if inbred rats express latent or unexpected C\u03ba alleles. They apparently do not do so. Wild Norway rats were tested, and it was found that the laboratory strains do not represent a subset of the rat C\u03ba polymorphism. Further tests indicated that only one of the two serologic specificities could be found in related rodent species.
\r\n\r\nThe structure of the T cell antigen-binding receptor is a major controversial issue in immunology. It has been asserted that the T cell antigen-receptor is homologous to immunoglobulins, and one popular theory contends that VH genes are responsible for the specificity of the receptor. We tested these theories by hybridizing immunoglobulin DNA probes to RNA and DNA from cloned T cells. First, we determined that the C\u03bb, J\u03ba, C\u03ba, JH, C\u00b5 and C\u03b1 genes and the sequences involved in heavy chain class switching are not rearranged in a T helper, a cytotoxic T cell and a T lymphoma. These cells also do not transcribe C\u03ba, C\u03bb, JH, C\u00b5 and C\u03b1 RNA. Second, a cDNA clone encoding heavy chain variable region characteristic of most B cells which respond to the antigen GAT was isolated and sequenced. Poly(A)+ RNA was prepared from 12 cloned T lymphocytes specific for GAT. While six of these T cells display antigenic determinants present on immunoglobulins that bind GAT, none of them contained a transcript homologous to the cDNA probe. Finally, using a random primer, large cDNA libraries (105-106 colonies) were constructed from three T-cell hybridomas. These libraries were screened by two separate, well-characterized methods which should permit the detection of all or most VH gene segments. No VH cDNA colonies were found by these methods. Therefore immunoglobulin gene segments are not likely to be part of the T cell antigen receptor.
\r\n\r\nThe I-J serologic specificity has been reported to be present on T cell-derived antigen-binding molecules. Cosmid clones have been previously obtained containing all the sequences between the I-A and I-E subregions of the murine major histocompatibility complex, where I-J has been genetically mapped. The putative I-J DNA does not, however, hybridize to RNA from I-J positive suppressor T cells. Also, suppressor T lymphocytes do not rearrange this DNA. Therefore the I-J coding sequences must map elsewhere.
", "doi": "10.7907/p24z-a733", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:6148", "collection": "thesis", "collection_id": "6148", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10202010-084119498", "primary_object_url": { "basename": "Begley_tp_1983.pdf", "content": "final", "filesize": 30091847, "license": "other", "mime_type": "application/pdf", "url": "/6148/1/Begley_tp_1983.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Bispecific, Cleavable, Protein DNA Crosslinker, Psoralen-Diol-Nitroveratrole. A Probe of Bacteriophage Structure", "author": [ { "family_name": "Begley", "given_name": "Tadhg Pulcarious", "orcid": "0000-0001-5134-2623", "clpid": "Begley-Tadhg-Pulcarious" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The synthesis of a cleavable, photochemically activated, bispecific protein-DNA crosslinking reagent, psoralen-diol-nitroveratrole (PDN) is described. The reagent crosslinks the capsid proteins to the packaged DNA in bacteriophage T7. The SDS dissociated crosslinked phage appears under the electron microscope as a rosette with the phage head at the center. DNA from the crosslinked phage does not enter agarose gels in the absence of SDS. Treatment of the crosslinked phage with proteinase K or cleavage of the crosslink with sodium periodate restores the gel mobility of the DNA to that of the non-crosslinked phage DNA. No evidence for protein-protein crosslinking was obtained when the protein composition of crosslinked and non-crosslinked phage was compared by polyacrylamide gel electrophoresis. No evidence for DNA-DNA crosslinking was obtained when the frequency of crossed BglI restriction fragments for the crosslinked and non-crosslinked phage was compared by electron microscopy. An attempt was made to analyze the distribution of protein crosslinked to intraphage DNA. It was not possible to carry out this analysis by electron microscopy as the non-crosslinked phage gave too high a background of phage heads attached to the DNA. Sodium periodate treatment of the crosslinked DNA-protein complex failed to give detectable levels of protein on a silver stained polyacrylamide gel.
\r\n\r\nThe phage head of bacteriophage \u03bb was also crosslinked to the DNA by treatment with PDN and irradiation at long wavelength (> 360 nm). The crosslink was cleaved by sodium periodate. However, proteinase K treatment did not remove the protein from the DNA. Evidence is presented indicating that the \u03bb phage head is exceptionally stable to dissociation. Preliminary crosslinking results are presented for simian virus 40.
", "doi": "10.7907/BPQM-KP51", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:10870", "collection": "thesis", "collection_id": "10870", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05092018-121105492", "primary_object_url": { "basename": "Ou_J-HJ_1982.pdf", "content": "final", "filesize": 73941511, "license": "other", "mime_type": "application/pdf", "url": "/10870/1/Ou_J-HJ_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure and Replication of Alphavirus RNAs", "author": [ { "family_name": "Ou", "given_name": "Jing-hsiung James", "clpid": "Ou-Jing-hsiung-James" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "thesis_committee": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Both ends of the alphavirus genomic RNA are potentially important in its replication. The region preceding and including the 5'-end of the subgenomic 26S RNA in genomic RNA might also be involved in 26S RNA transcription. Sequences of these regions of up to 10 alphaviruses were determined by using strategies including enzymatic, chain-termination and cDNA sequencing methods.
\r\n\r\nComparison of the nucleotide sequences reveals three highly conserved sequences. The first conserved sequence is 19 nucleotides in length and is located at the extreme 3'-end next to the poly(A) tail. The second conserved sequence, which is 21 nucleotides in length, precedes the 5'-end of 26S RNA and includes the first two nucleotides of it. The third conserved sequence is 51 nucleotides in length and is located at a position of about 130 to 150 nucleotides from the 5'-end, depending on the virus. The last conserved sequence in all alphaviruses examined is capable of forming two stable hairpin structures and could also base-pair stably with the 3'-terminal sequences to cyclize genomic RNAs. Besides these three conserved sequences, a highly conserved stem and loop structure could also be formed at the extreme 5'-end of genomic RNA.
\r\n\r\nDefective interfering (DI) RNAs of alphaviruses are mutated genomic RNAs which often contain deleted, repeated and translocated sequences, but yet retain all elements essential for their replication. By studying the sequence organization of alphavirus DI RNAs, and the 3'-terminal sequences of the genomic RNAs of two alphavirus variants and their replication, the importance of these conserved sequences and secondary structures in alphavirus replication are discussed.
\r\n\r\nBoth the 3'- and 5'-terminal sequences of several alphavirus 26S RNAs were also determined. Results show that 26S and genomic RNAs are coterminal. Together with the results previously published, the total length of the 26S RNAs of two alphaviruses, Sindbis virus and Semliki Forest virus, were determined to be 4102 and 4074 nucleotides, respectively.
\r\n\r\nThe NH2- and COOH-terminal sequences of the precursors of nonstructural proteins (translated from genomic RNA) and structural proteins (translated from 26S RNA) of several alphaviruses were deduced from the nucleotide sequences determined. The initiation codons of most alphavirus genomic and 26S RNAs are preceded by the sequence CANN. To determine the importance of these tetranucleotides, their sequences in 65 eucaryotic mRNAs were surveyed. Results show that the sequence distribution of these tetranucleotides are non-random and they might be involved in initiation of translation.
\r\n\r\nThe 3'-noncoding regions of alphavirus genomic RNAs contain AU rich sequences. Sequence organization in the 3'-noncoding regions is similar to those in alphavirus DI RNAs. Mechanisms for the generation of these sequence rearrangements are discussed.
", "doi": "10.7907/2sr4-6m60", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:11123", "collection": "thesis", "collection_id": "11123", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07132018-112110004", "type": "thesis", "title": "Studies of the Organization and Expression of Individual Repetitive Sequence Families of the Sea Urchin Genome", "author": [ { "family_name": "Posakony", "given_name": "James William", "clpid": "Posakony-James-William" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Individual repetitive DNA sequence families of the sea urchin Strongylocentrotus purpuratus were investigated with regard to their genomic organization, the internal structure of their members, and the structural and developmental characteristics of their RN A transcripts.
\r\n\r\nAnalysis by gel blot hybridization and reassociation kinetics of cloned genomic DNA fragments containing members of three specific repeat families reveals a different pattern of organization in each case. One family is organized into long regions of repeated DNA, usually containing several members of the family in a tandem or clustered arrangement. A second family exists as long repeated elements occurring only once in a local genomic region. The third family consists of short repetitive sequence elements which are generally flanked on either side by single-copy sequences.
\r\n\r\nThe internal structure of eight cloned repetitive sequence elements was examined by determination of their nucleotide sequences. The lack of sequence homology among the eight elements indicates that they are representative of distinct repeat families. For the most part they consist of complex sequence internally, with a minor fraction of the length of five of the eight occupied by direct or inverse sequence repetitions. Six of the eight sequences are not translatable. Comparison of the nucleotide sequences of three different members of the same repeat family reveals that they are not simply colinear sequence variants, but that they differ in the presence and/or arrangement of small sequence subelements.
\r\n\r\nHybridization with cloned repetitive sequence elements was used to demonstrate that the level of representation of specific repeat sequences is quantitatively similar in the egg RNA of two sea urchin species, S. purpuratus and S. franciscanus.
\r\n\r\nEgg and embryo polyadenylated RNAs bearing specific repetitive sequences were analyzed by cDNA cloning, DNA and RNA gel blot hybridization, and DNA sequencing. It was found that the two complements of a given repeat are carried on different sets of polyadenylated transcripts, which are generally quite long (> 3 kilobases, with an estimated number average length of 5-6 kilobases). Within these transcripts, specific short repetitive sequence elements are found interspersed either with single-copy sequences or with other repeat sequences. It is demonstrated by sequencing that one such repeat-containing region is not translatable. The sets of polyadenylated transcripts deriving from several individual repeat families undergo substantial quantitative and probably qualitative modulation during early sea urchin development. Analysis of specific transcripts with single-copy probes from repeat-containing cDNA clones indicates that the embryo genome is transcribed to produce at least some of the same interspersed RNAs as are stored in the oocyte during oogenesis. Finally, the transcripts bearing specific repeat sequences in the polyadenylated egg RNA of two related sea urchin species were found to be qualitatively dissimilar.
", "doi": "10.7907/4e58-pq58", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:1544", "collection": "thesis", "collection_id": "1544", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04292005-082735", "type": "thesis", "title": "Programmed DNA Rearrangements During Differentiation: Immunoglobulin Class Switching", "author": [ { "family_name": "Davis", "given_name": "Mark Morris", "orcid": "0000-0001-6868-657X", "clpid": "Davis-Mark-Morris" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Delbruck", "given_name": "Max", "clpid": "Delbr\u00fcck-M" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Maniatis", "given_name": "Thomas P.", "clpid": "Maniatis-T-P" } ], "local_group": [ { "literal": "Caltech Distinguished Alumni Award" }, { "literal": "div_biol" } ], "abstract": "The events of B-lymphocyte differentiation can be reconstructed in part\r\nthrough an analysis of the organization of heavy-chain genes isolated from B-cell\r\ntumors (myelomas). A mouse immunoglobulin alpha heavy-chain gene is shown to be\r\ncomposed of at least three non-contiguous segments of germline DNA -- a VH gene\r\nsegment, a JH gene segment adjacent to the C\u03bc coding region, and a C\u03b1 gene\r\nsegment. These gene segments are joined together by two distinct types of DNA\r\nrearrangements: variable region formation and immunoglobulin class switching.\r\nThree examples of IgM \u2192 IgA elass switching were examined and in each case a\r\ndifferent site adjacent to C\u03bc and a different site adjacent to C\u03b1 were joined together\r\nin the process of switching. Two of the three C\u03bc sites shared significant homology to\r\neach other (15/25 nucleotides) and all three of C\u03b1 sites were highly homologous (22/30\r\nnucleotides). We believe these sequences serve as recognition sites for class\r\nswitching. Furthermore, the lack of homology between the C\u03b1 consensus sequence\r\nand sequences reported for C\u03b31 and c\u03b32b recombination sites suggests that this\r\nprocess is mediated by class-specific recognition sequences and, presumably, class-specific\r\nregulatory mechanisms. A number of predictions and possible explanations of\r\nimmune phenomena result from this observation. Apparently nonproductive DNA\r\nrearrangements, occurring in the same tumor lines, seem also to utilize some of the\r\n_same regulatory apparati. In addition, it appears that in one example, MClOl, class\r\nswitching has progressed from C\u03bc \u2192 C\u03b1 \u2192 C\u03b31. This switching pathway presents\r\ndifficulties for the simple deletional model of CH switching.", "doi": "10.7907/KFJS-G857", "publication_date": "1981", "thesis_type": "phd", "thesis_year": "1981" }, { "id": "thesis:4032", "collection": "thesis", "collection_id": "4032", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10112005-110000", "type": "thesis", "title": "Studies on the Structural Proteins of Sindbis Virus", "author": [ { "family_name": "Rice", "given_name": "Charles Moen, III", "orcid": "0000-0003-3087-8079", "clpid": "Rice-Charles-Moen-III" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Maniatis", "given_name": "Thomas P.", "clpid": "Maniatis-T-P" } ], "local_group": [ { "literal": "Nobel Prize" }, { "literal": "Caltech Distinguished Alumni Award" }, { "literal": "div_biol" } ], "abstract": "Conditions are described for the synthesis of long cDNA transcripts of Sindbis virus 26S and 49S RNA in high yield. This single-stranded cDNA could be cut with Type II restriction endonucleases including Hae III, Hha I, Rsa I, or Taq I to give reproducible patterns of discrete, virus-specific fragments which were suitable for subsequent end-labeling and direct sequence analysis. Using these methods, the strategy used for obtaining nearly the entire 26S RNA sequence from cDNA synthesized in vitro is presented. The 26S RNA is approximately 4.2 kb in length, and from the AUG codon initiating synthesis of the capsid protein, contains an open reading frame for 3735 nucleotides. From this sequence, the amino acid sequences of the encoded virus structural proteins, which include a basic capsid protein and two integral membrane glycoproteins (El and E2), as well as the sequences of two nonstructural polypeptides have been deduced. Features of the primary structure of these proteins and the proteolytic cleavage sites involved in their processing are discussed.
\r\n\r\nThe orientation of the virion glycoproteins with respect to the lipid bilayer was studied by digesting intact Sindbis virus with \u03b1-chymotripsin. A single membrane-associated peptide is produced from each of the two virion glycoproteins. These peptides contain covalently attached palmitic acid, are rich in hydrophobic amino acids and are located at the extreme COOH-terminal end of each glycoprotein. Both peptides contain uninterrupted sequences of uncharged amino acids of sufficient length to span the lipid bilayer, and it is suggested that they serve to anchor the viral glycoproteins in the membrane. The properties of these and other well-characterized transmembrane segments are discussed.
\r\n\r\nSpecific antisera to each of the virus structural proteins was produced and used to study the association of the virion glycoproteins and their precursors. E1 and E2 could be cross-linked into heterodimers using bifunctional amino-reactive imidates. This association is present both in intact virions and infected cells and is stable after solubilization of the virion envelope by Triton X-100. Cross-linking data of pulse-labeled monolayers and cells infected with ts mutants are summarized. These data suggest that PE2 (the precursor to E2) and E2 are in different conformations with respect to E1, and that the glycoprotein precursors synthesized at elevated temperatures have an increased tendency to undergo intracellular aggregation.
", "doi": "10.7907/9aan-kg88", "publication_date": "1981", "thesis_type": "phd", "thesis_year": "1981" }, { "id": "thesis:8080", "collection": "thesis", "collection_id": "8080", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02182014-134518249", "primary_object_url": { "basename": "Stumph_we_1979.pdf", "content": "final", "filesize": 19895118, "license": "other", "mime_type": "application/pdf", "url": "/8080/1/Stumph_we_1979.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Gene Enrichment Using Antibodies to DNA/RNA Hybrids: Mapping the Ribosomal DNA of Slime Mold and Rat", "author": [ { "family_name": "Stumph", "given_name": "William Edward", "clpid": "Stumph-William-Edward" } ], "thesis_advisor": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" } ], "thesis_committee": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A novel method for gene enrichment has been developed and applied\r\nto mapping the rRNA genes of two eucaryotic organisms. The method makes\r\nuse of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the\r\nsynthetic hybrid poly(rA)\u2022poly(dT). Antibodies which cross-react with non-hybrid\r\nnucleic acids were removed from the purified IgG fraction by adsorption on columns\r\nof DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent\r\npurification of the specific DNA/RNA hybrid antibody was carried out on a column\r\nof oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these\r\nantibodies to CNBr-activated Sepharose produced an affinity resin which specifically\r\nbinds DNA/RNA hybrids.
\r\n\r\nIn order to map the rDNA of the slime mold Dictyostelium discoideum,\r\nR-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs\r\nof this organism. This mixture was passed through a column containing the affinity\r\nresin, and bound molecules containing R- loops were eluted by high salt. This purified\r\nrDN A was observed directly in the electron microscope. Evidence was obtained\r\nthat there is a physical end to Dictyostelium rDN A molecules approximately\r\n10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding\r\nis consistent with reports of other investigators that the rRNA genes exist as\r\ninverse repeats on extra-chromosomal molecules of DNA unattached to the remainder\r\nof the nuclear DNA in this organism.
\r\n\r\nThe same general procedure was used to map the rRNA genes of the rat.\r\nMolecules of DNA which contained R-loops formed with the 188 and 288 rRNAs\r\nwere enriched approximately 150- fold from total genomal rat DNA by two cycles\r\nof purification on the affinity column. Electron microscopic measurements of\r\nthese molecules enabled the construction of an R-loop map of rat rDNA. Eleven\r\nof the observed molecules contained three or four R-loops or else two R-loops\r\nseparated by a long spacer. These observations indicated that the rat rRNA genes\r\nare arranged as tandem repeats. The mean length of the repeating units was\r\n37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent\r\nrepeating units of exactly the same length within the errors of the measurements,\r\nalthough a certain degree of length heterogeneity cannot be ruled out.\r\nIf significantly shorter or longer repeating units exist, they are probably much\r\nless common than the 37.2 kbp unit.
\r\n\r\nThe last section of the thesis describes the production of antibodies to\r\nnon-histone chromosomal proteins which have been exposed to the ionic detergent\r\nsodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did\r\nnot seem to affect either production of antibodies or their general specificity.\r\nAlso, a technique is described for the in situ immunofluorescent detection of protein\r\nantigens in polyacrylamide gels.
", "doi": "10.7907/9V3M-QH52", "publication_date": "1979", "thesis_type": "phd", "thesis_year": "1979" }, { "id": "thesis:18405", "collection": "thesis", "collection_id": "18405", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03062026-161324227", "primary_object_url": { "basename": "Conrad _SE_1979.pdf", "content": "final", "filesize": 47510662, "license": "other", "mime_type": "application/pdf", "url": "/18405/1/Conrad _SE_1979.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "I. Sequence Organization of Drosophila melanogaster 5S rRNA and 4S RNA Genes. II. In Vitro Studies on Replication of Plasmid DNAs", "author": [ { "family_name": "Conrad", "given_name": "Susan Ellen", "clpid": "Conrad-Susan-Ellen" } ], "thesis_advisor": [ { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Maniatis", "given_name": "Thomas P.", "clpid": "Maniatis-T-P" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "I. The sequence organizations of Drosophila melanogaster 4S RNA (tRNA)\r\nand 5S rRN A genes have been investigated. Segments of Drosophila DNA containing\r\nthese genes have been propagated in recombinant plasmids using Escherichia coli\r\nas a host and Co1E1 as a vector.
\r\n\r\nElectron microscope partial denaturation mapping, mapping by ferritin\r\nlabeling and restriction enzyme-gel electrophoresis analysis all indicate that the\r\nDrosophila DNA inserts of the 5S rRNA gene containing plasmids consist of tandem\r\nrepeats of 5S genes and spacer regions. The repeat length is approximately 380\r\nnucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer\r\nof length 260 ntp. Little heterogeneity in the lengths of the repeats has been\r\ndetected.
\r\n\r\nA tRNA gene containing clone has been characterized by electron microscopic\r\nmethods and by restriction endonuclease mapping. Four tRNA genes were\r\ndetected on a 9.34 kb fragment of DNA. Three of these genes appear to be\r\nidentical and different from the fourth. No evidence was found for extensive\r\nsequence homology in the sequences surrounding the genes. In situ hybridization\r\nwith cRNA transcribed from the plasmid showed localization at region 42A of\r\nchromosome 2R.
\r\n\r\nII. An improved system for in vitro replication of Escherichia coli plasmid\r\nDNAs has been developed and characterized. Endogenous DNA is removed from\r\nthe extracts, making replication dependent upon exogenous DNA even when plasmid\r\ncontaining cells are used as a source of extracts. Replication in this system requires\r\nE. coli DNA polymerases I and III, RNA polymerase, DNA gyrase, and the products\r\nof at least five genes required for E. coli replication (dnaB, dnaC, dnaG, dnaP, dnaZ).
\r\n\r\nThis system has been used to study the replication properties of the\r\nampicillin resistant plasmid RSF1030. We have found that, like Co1E1, RSF1030\r\nreplicates unidirectionally from a unique origin. We have also investigated the\r\nreplication of pFH118, a high copy number mutant derived from Co1E1. The\r\nmutation in pFH118 is due to the insertion of 20-30 base pairs into the coding\r\nsequence for a 100 nucleotide RNA that is transcribed during DNA replication.\r\nResults of reversion studies suggest that this RN A plays a role in determining\r\nplasmid copy number. Furthermore, the RN As transcribed from Co1E1 and RSF1030\r\nhave significant sequence homology, although the plasmids were isolated independently\r\nand have been thought to have no sequence homology.
", "doi": "10.7907/d94z-c749", "publication_date": "1979", "thesis_type": "phd", "thesis_year": "1979" }, { "id": "thesis:17762", "collection": "thesis", "collection_id": "17762", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11142025-230228251", "primary_object_url": { "basename": "Gottesfeld_JM_1976.pdf", "content": "final", "filesize": 38991788, "license": "other", "mime_type": "application/pdf", "url": "/17762/1/Gottesfeld_JM_1976.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Chromatin Structure and Gene Expression", "author": [ { "family_name": "Gottesfeld", "given_name": "Joel M.", "orcid": "0000-0002-4643-5777", "clpid": "Gottesfeld-Joel-M" } ], "thesis_advisor": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" } ], "thesis_committee": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" }, { "family_name": "Britten", "given_name": "Roy", "clpid": "Britten-Roy" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Rat-liver chromatin has been separated into nuclease-sensitive\r\nand resistant fractions after mild digestion with DNAase II. The\r\nnuclease-sensitive material is further fractionated into Mg++-soluble\r\nand insoluble chromatin fractions. The kinetics of production of\r\nthese chromatin fractions have been investigated. After a brief\r\nenzyme treatment (5 min under standard conditions), 11% of the input\r\nchromatin DNA is found in the Mg++-soluble fraction. This DNA has a\r\nweight-average single strand length of about 400 nucleotides and, as\r\ndetermined by renaturation kinetics, comprises a subset of middle\r\nrepetitive and nonrepetitive DNA sequences of the rat genome. Cross-reassociation\r\nexperiments show that a fractionation of whole genomal\r\nDNA sequences has been achieved. Moreover, the Mg++-soluble fraction\r\nof liver chromatin is enriched in nonrepeated sequences coding for\r\nliver RNA but not for brain RNA. Fractionation does not depend on\r\nsome general property of chromatin but is specific with regard to the\r\ntemplate activity of the tissue from which the chromatin was obtained.\r\nThe Mg++-soluble, template-active fraction is enriched five-fold in\r\nDNA sequences complementary to RNA.
\r\n\r\nThe Mg++-soluble fraction is enriched in nonhistone chromosomal\r\nproteins and depleted in histone protein. Histone I (fl) is absent\r\nfrom the Mg++-soluble active fraction. About half of the DNA of both\r\nMg++-soluble and Mg++-insoluble fractions is resistant to prolonged\r\ndigestion with DNAase II or staphylococcal nuclease. The nuclease-\r\nresistant structures of inactive (Mg++-insoluble) chromatin are DNAhistone\r\ncomplexes which sediment at 11-13S. Two nuclease-resistant\r\nspecies are present in active chromatin. These particles sediment\r\nat 15 and 20S, respectively, and contain DNA, RNA, histone and nonhistone\r\nproteins. Thermal denaturation studies suggest that the\u00b7\r\nDNA of active chromatin is complexed primarily with nonhistone proteins.
", "doi": "10.7907/3ayy-cg55", "publication_date": "1976", "thesis_type": "phd", "thesis_year": "1976" }, { "id": "thesis:8192", "collection": "thesis", "collection_id": "8192", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04182014-094111781", "type": "thesis", "title": "The Flocculation of E. coli with Polyethyleneimine", "author": [ { "family_name": "Treweek", "given_name": "Gordon Paul", "clpid": "Treweek-Gordon-Paul" } ], "thesis_advisor": [ { "family_name": "Morgan", "given_name": "James J.", "clpid": "Morgan-J-J" } ], "thesis_committee": [ { "family_name": "McKee", "given_name": "Jack E.", "clpid": "McKee-J-E" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Friedlander", "given_name": "Sheldon K.", "clpid": "Friedlander-S-K" }, { "family_name": "Morgan", "given_name": "James J.", "clpid": "Morgan-J-J" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "A comprehensive study was made of the flocculation of dispersed E. coli bacterial cells by the cationic polymer polyethyleneimine (PEI). The three objectives of this study were to determine the primary mechanism involved in the flocculation of a colloid with an oppositely charged polymer, to determine quantitative correlations between four commonly-used measurements of the extent of flocculation, and to record the effect of varying selected system parameters on the degree of flocculation. The quantitative relationships derived for the four measurements of the extent of flocculation should be of direct assistance to the sanitary engineer in evaluating the effectiveness of specific coagulation processes.
\r\n\r\nA review of prior statistical mechanical treatments of absorbed polymer configuration revealed that at low degrees of surface site coverage, an oppositely- charged polymer molecule is strongly adsorbed to the colloidal surface, with only short loops or end sequences extending into the solution phase. Even for high molecular weight PEI species, these extensions from the surface are theorized to be less than 50 \u00c5 in length. Although the radii of gyration of the five PEI species investigated were found to be large enough to form interparticle bridges, the low surface site coverage at optimum flocculation doses indicates that the predominant mechanism of flocculation is adsorption\r\ncoagulation.
\r\n\r\nThe effectiveness of the high-molecular weight PEI species 1n producing rapid flocculation at small doses is attributed to the formation of a charge mosaic on the oppositely-charged E. coli surfaces. The large adsorbed PEI molecules not only neutralize the surface charge at the adsorption sites, but also cause charge reversal with excess cationic segments. The alignment of these positive surface patches with negative patches on approaching cells results in strong electrostatic attraction in addition to a reduction of the double-layer interaction energies. The comparative ineffectiveness of low-molecular weight PEI species in producing E. coli flocculation is caused by the size of the individual molecules, which is insufficient to both neutralize and reverse the negative E.coli surface charge. Consequently, coagulation produced by low molecular weight species is attributed solely to the reduction of double-layer interaction energies via adsorption.
\r\n\r\nElectrophoretic mobility experiments supported the above conclusions, since only the high-molecular weight species were able to reverse the mobility of the E. coli cells. In addition, electron microscope examination of the seam of agglutination between E. coli cells flocculation by PEI revealed tightly- bound cells, with intercellular\r\nseparation distances of less than 100-200 \u00c5 in most instances. This intercellular separation is partially due to cell shrinkage in preparation of the electron micrographs.
\r\n\r\nThe extent of flocculation was measured as a function of PEl molecular weight, PEl dose, and the intensity of reactor chamber mixing. Neither the intensity of mixing, within the common treatment practice limits, nor the time of mixing for up to four hours appeared to play any significant role in either the size or number of E.coli aggregates formed. The extent of flocculation was highly molecular weight dependent: the high-molecular-weight PEl species produce the larger aggregates, the greater turbidity reductions, and the higher filtration flow rates. The PEl dose required for optimum flocculation decreased as the species molecular weight increased. At large doses\r\nof high-molecular-weight species, redispersion of the macroflocs occurred, caused by excess adsorption of cationic molecules. The excess adsorption reversed the surface charge on the E.coli cells, as recorded by electrophoretic mobility measurements.
\r\n\r\nSuccessful quantitative comparisons were made between changes in suspension turbidity with flocculation and corresponding changes in aggregate size distribution. E. coli aggregates were treated as coalesced spheres, with Mie scattering coefficients determined for spheres in the anomalous diffraction regime. Good quantitative comparisons were also found to exist between the reduction in refiltration time and the reduction of the total colloid surface area caused by flocculation. As with turbidity measurements, a coalesced sphere model was used since the equivalent spherical volume is the only information available from the Coulter particle counter. However, the coalesced sphere model was not applicable to electrophoretic mobility measurements. The aggregates produced at each PEl dose moved at approximately the same vlocity, almost independently of particle size.
\r\n\r\nPEl was found to be an effective flocculant of E. coli cells at weight ratios of 1 mg PEl: 100 mg E. coli. While PEl itself is toxic to E.coli at these levels, similar cationic polymers could be effectively applied to water and wastewater treatment facilities to enhance sedimentation and filtration characteristics.
\r\n", "doi": "10.7907/Y732-V890", "publication_date": "1975", "thesis_type": "phd", "thesis_year": "1975" }, { "id": "thesis:3686", "collection": "thesis", "collection_id": "3686", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09222004-140527", "primary_object_url": { "basename": "Rosenberg_sto_1975.pdf", "content": "final", "filesize": 8859841, "license": "other", "mime_type": "application/pdf", "url": "/3686/1/Rosenberg_sto_1975.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Studies of Bovine Blood Cell Surfaces", "author": [ { "family_name": "Rosenberg", "given_name": "Suzanne Thelma Ostrand", "clpid": "Rosenberg-Suzanne-Thelma-Ostrand" } ], "thesis_advisor": [ { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "thesis_committee": [ { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The cell surface expression of genetically defined bovine red cell antigens has been studied by electron microscopic and serological techniques. Electron microscopic cell surface localization of specific antigens on bovine red cells has been achieved with the use of an indirect labeling reagent: hemocyanin glutaraldehyde coupled to rabbit-antibovineimmunoglobulin (Hcy-RABI). When the specific antigenic sites on the cell surface are combined with their corresponding antibodies, the secondary application of Hcy-RABI serves to visualize these sites for electron microscopy.\r\n\r\nSerological dosage reagents for the Z antigen are known to differentiate Z homozygotes (Z/Z) from heterozygotes (Z/-) in terms of the kinetics of complement mediated hemolysis. In the present study, cells homozygous for the Z antigen and saturated with anti-Z antibody were found to take up approximately twice as much Hcy-RABI as cells heterozygous for Z; cells negative for Z showed only background labeling values. Cells possessing the J antigen, a soluble serum substance secondarily absorbed to the red cell surface, were also examined for their quantitative uptake of hemocyanin. Many intergrades of J positive cells exist, ranging from cells which require large amounts of anti-J antibody for complement mediated lysis to cells which are lysed by minute quantities of specific antibody. The quantity of label taken up by a sampling of cells was found to be inversely related to the amount of antibody necessary to lyse those cells.\r\n\r\nSequential double labeling studies were conducted to characterize the cell surface steric configurations of antigens whose genes reside in 1) the same blood group system; 2) different blood group systems; and 3) cis versus trans conformations within a system. In no case was steric hindrance found. This result indicates that each antigenic determinant examined is spacially distant from others; it suggests that the determinants may be coded for by distinct genes, and that the antigens labeled are not a series of determinants on a common backbone macromolecule. Sequential double labeling of one set of antigens gave a value which was twice the sum of the two single label values. This phenomenon was noted only for one particular pair of antigens, and only on cells treated initially with one of the antisera. The increased uptake of the second antibody was highly specific. This observation suggests that new antigenic sites are revealed in the presence of bound antibody directed against another specificity, on cells labeled for this particular pair of antigens.\r\n\r\nConcanavalin A (Con A) binding experiments on trypsinized and nontrypsinized cells strongly indicate that the Con A receptor and the A antigen are molecular unique cell surface entities. Trypsinization of A positive cells caused increased binding of anti-A, accompanied by clustering of the A antigen sites and of the intramembranous particles seen in freeze-fracture experiments. These phenomena were accompanied by cell agglutination.\r\n\r\nUsing bovine red cell blood typing reagents in a leukocyte microcytotoxicity system, bovine leukocytes were found to have specific surface antigens. In this preliminary study there is no obvious association between leukocyte antigens and red cell antigens of any individual animal. The leukocyte and erythrocyte antigenic systems appear to be distinct from each other.", "doi": "10.7907/SCPF-E841", "publication_date": "1975", "thesis_type": "phd", "thesis_year": "1975" }, { "id": "thesis:14120", "collection": "thesis", "collection_id": "14120", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04132021-203938881", "primary_object_url": { "basename": "Simmons_DT_1974.pdf", "content": "final", "filesize": 57119564, "license": "other", "mime_type": "application/pdf", "url": "/14120/1/Simmons_DT_1974.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Function and Replication of Sindbis Virus-Specific RNA's in Infected Cells", "author": [ { "family_name": "Simmons", "given_name": "Daniel Tawil", "clpid": "Simmons-Daniel-Tawil" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "thesis_committee": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Russell", "given_name": "Richard L.", "clpid": "Russell-R-L" }, { "family_name": "Wood", "given_name": "William Barry", "clpid": "Wood-W-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "During infection with Sindbis virus, two species of Sindbis-specific single-stranded RNA are synthesized. One of them, 49S RNA is the RNA of the vinis and has a molecular weight of 4.3 \u00b1 0.3 x 106 daltons. This molecular weight was estimated by a variety of methods, including polyacrylamide gel electrophoresis, sedimentation after reaction with fonnaldehyde and analysis of the molecular weight of its double-stranded form. The other species of single-stranded RNA, 26S RNA, was found only in infected cells and has a molecular weight of 1.6 x 106 daltons, determined by sedimentation in dimethylsulfoxide. Hybridization-competition experiments showed that 26S RNA is a specific one-third of the 49S RNA genome.
\r\n\r\nIn infected cells, 26S RNA was primarily associated with ribosomes, and was found to be the predominant species of viral messenger RNA. A small amount (10 % by weight) of the messenger RNA in the cells was Sindbis 49S RNA. No other unique and separate species of Sindbis messenger RNA could be detected in infected cells.
\r\n\r\nThe two species of Sindbis single-stranded RNA were translated in lysates of rabbit reticulocytes. Sindbis 26S RNA was translated primarily into the nucleocapsid protein and into a protein shown by others to be the precursor of the two glycoproteins of the virus. These results indicated that Sindbis 26S RNA codes solely for the structural proteins of the virus.
\r\n\r\nSindbis 49S RNA was translated in vitro into 8 or 9 polypeptides ranging in molecular weight from 60,000 to 180,000 daltons. None of these polypeptides coincided with any known Sindbis proteins.
\r\n\r\nThe replication of Sindbis-specific RNA was studied by analyzing the forms of double-stranded RNA (or replicative forms) in infected cells. When RNA from infected cells was treated with ribonuclease, three species of Sindbis-specific double-stranded RNA (RF's I, II, and III) were isolated. Their molecular weights were determined to be 8.8 x 106 daltons for RFI, 5.6 x 106 daltons for RFII, and 2.9 x 106 daltons for RFIII.
\r\n\r\nBy hybridization-competition experiments, it was shown that RFI is the double-stranded form of 49S RNA, RFIII, the double-stranded form of 26S RNA, and RFII, the double-stranded form of a species of RNA with molecular weight of 2.8 x 106 daltons and identical to two-thirds of the genome.
\r\n\r\nThe size and structure of Sindbis replicative intermediates (RI's) were studied and found to consist of a double-stranded region the size of RFI and of various lengths of single-stranded tails. Our model for the replication of Sindbis-spccific RNA predicts that Sindbis RI's exist in two classes, called RIa and RIb. RIa is the template for the synthesis of 49S RNA and is reduced to RFI when treated with ribonuclease. When Rib is treated with ribonuclease, it is reduced to RF's II and III due to a single-stranded gap in the \"plus\" strand of the RI (the virus RNA is \"plus\"-stranded). The portion of RIb corresponding to RFIII is the template for the synthesis of 26S RNA, and the portion corresponding to RFII is the template for synthesis of a species of RNA of 2.8 x 106 daltons, which has not been detected in its single-stranded form. We hypothesize that there must be two different regions on RIb where chain synthesis is initiated since 26S RNA is synthesized at a much faster rate than the product of RFII.
", "doi": "10.7907/grvs-e385", "publication_date": "1974", "thesis_type": "phd", "thesis_year": "1974" }, { "id": "thesis:11101", "collection": "thesis", "collection_id": "11101", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07062018-115304742", "primary_object_url": { "basename": "Froehner_SC_1973.pdf", "content": "final", "filesize": 53875734, "license": "other", "mime_type": "application/pdf", "url": "/11101/1/Froehner_SC_1973.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Isolation, Purification and Characterization of Three RNA Polymerases from Novikoff Hepatoma Ascites Tumor", "author": [ { "family_name": "Froehner", "given_name": "Stanley Charles", "clpid": "Froehner-Stanley-Charles" } ], "thesis_advisor": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" } ], "thesis_committee": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Dreyer", "given_name": "William J.", "clpid": "Dreyer-W-J" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "DNA-dependent RNA polymerase has been isolated from nuclei of Novikoff hepatoma ascites tumor cells and resolved into three activities, designated Ia, Ib, and II, by a combination of phosphocellulose and DEAE cellulose chromatography. Ia and Ib have been further purified by sucrose density centrifugation. Both gradient profiles exhibit coincidence of the polymerase activity and protein peaks, suggesting that the two may be homogeneous enzymes. Ia migrates as a single species on non-denaturing polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis indicates that Ia contains subunits of 170,000, 125,000, 69,000, 49,000, 44,000 and 37,000 molecular weights in equimolar ratios except for the\r\n69,000 and 37,000 dalton subunits which may be present in two copies per enzyme molecule. A molecular weight of\r\n600,000 for the enzyme calculated from the molecular weights of the subunits is in good agreement with that determined by exclusion chromatography. The probable molecular structure of Ib is subunits of 190,000 and 135,000 daltons, each present twice per enzyme molecule. The enzymological characterization of these three enzymes suggests that Ia and Ib are the nucleolar polymerases while II is nucleoplasmic. Ia and Ib are most active at low ionic strength with Mg++ on native DNA and are insensitive to \u03b1-amanitin. II prefers Mn++, high ionic strength, a denatured template and is inhibited by low concentrations of \u03b1-amanitin. A factor present in the material which does not bind to the DEAE cellulose column used in the purification scheme, stimulates the activity of all three of the enzymes. Ia and Ib are inactive at low enzyme concentrations in the absence of this factor. The active agent in the factor is probably a protein, since it is heat sensitive, and may be a subunit of the enzyme.
\r\n", "doi": "10.7907/M8Q4-DB66", "publication_date": "1973", "thesis_type": "phd", "thesis_year": "1973" }, { "id": "thesis:11126", "collection": "thesis", "collection_id": "11126", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07192018-114123511", "primary_object_url": { "basename": "Holmes_DS_1973.pdf", "content": "final", "filesize": 33629564, "license": "other", "mime_type": "application/pdf", "url": "/11126/1/Holmes_DS_1973.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Studies on Nuclear RNA", "author": [ { "family_name": "Holmes", "given_name": "David Salway", "clpid": "Holmes-David-Salway" } ], "thesis_advisor": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" } ], "thesis_committee": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Russell", "given_name": "Richard L.", "clpid": "Russell-R-L" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The isolation of giant nuclear RNA (HnRNA) from rat ascites cells \r\nis described. By the criteria of sedimentation through sucrose,\r\nformaldehyde and dimethyl sulfoxide, it is estimated that the\r\nmajority of the radioactivity of giant HnRNA after a 30 minute pulse\r\nof 3H-uridine is associated with molecules in the range 5-10 x 106\r\ndaltons. In the electron microscope, under denaturing conditions,\r\n84% (mass %) of giant HnRNA has a contour length of 4-9\u00b5 corresponding\r\nto a molecular weight of about 5-10 x 106 daltons.
\r\n\r\nGiant HnRNA has a \"DNA-like\" base composition (G+C = 46-54%) and\r\nhas considerable secondary structure (ca. 60% helix conformation) as\r\njudged by its melting profile and reactivity with formaldehyde.
\r\n\r\nRat nuclear DNA is characterized by its reassociation profile\r\n((Na+) = 0.18 at 62\u00b0, Tm - 23\u00b0) as judged by chromatography on\r\nhydroxyapatite. Single-copy DNA (Cot 1/2 observed = 1.5 x 103)\r\ncomprises 65% of the genome and 19% of the genome consists of sequences\r\nrepeated an average 1,800 times (middle repetitive DNA, Cot 1/2\r\nobserved = 1.0). 9% of the genome (highly repetitive DNA)\r\nreassociates faster than is measured in these experiments (Cot 1/2\r\nobserved < 2 x 10-2).
\r\n\r\nMiddle repetitive and single-copy DNA are isolated and \r\ncharacterized with respect to their reassociation kinetics and melting\r\nprofiles. They reassociate with kinetics similar to the kinetics\r\ndescribing these components when they are present in total\r\nDNA. The reassociated single-copy DNA has a high thermal stability\r\nindicative of fidelity of base pairing; the reassociated middle\r\nrepetitive DNA has a lower thermal stability which is probably\r\nattributable, in part, to base-pair mismatch.
\r\n\r\nRat giant nuclear RNA (HnRNA, 5-10 x 106 daltons) is hybridized\r\nto isolated single copy or middle repetitive DNA ((Na+) = 0.18 at 62\u00b0)\r\nHnRNA hydbridizes to about 4.5% of the single-copy and 9.4% of the\r\nmiddle repetitive DNA. The Tms of single-copy and middle repetitive\r\nhybrids are 1-2\u00b0 lower than those of the reassociated single-copy\r\nand middle repetitive DNA respectively. The DNA isolated from the\r\nsingle-copy or middle repetitive hybrids reassociates with kinetics\r\nsimilar to the input single-copy or middle repetitive DNA respectively.\r\nHnRNA is hybridized to total genomic DNA present in excess. 37% of the\r\nHnRNA hybridizes with kinetics (Cot 1/2 = 2.0 x 103) similar to\r\nsingle-copy DNa and 12% hybridizes with kinetics (Cot 1/2 = 5.6), a\r\nlittle more slowly than the major reassociating component of middle\r\nrepetitive DNA.
\r\n\r\nA chromatin-associated RNA (cRNA) prepared from rat ascites cells\r\nhybridizes to about 16% of isolated middle repetitive and 1% of\r\nisolated single copy rat DNA. In a hybridization reaction to total\r\nDNA, present in excess, at least 50% of the cRNA hybridizes at an\r\naverage rate similar to the major component of the middle repetitive\r\nDNA. These experiments indicate that the majority of cRNA consists\r\nof repetitive transcripts. Under conditions which assay essentially\r\nonly repetitive transcripts cRNA hybridizes to about 4.7% and giant\r\nnuclear RNA (HnRNA) hybridizes to about 4.6% of total nuclear rat\r\nDNA immobilized on filters. The Tm of cRNA hybrids (73.5\u00b0) and HnRNA\r\nhybrids (75.5\u00b0) are considerably lower than the Tm of native rat DNA\r\n(85.5\u00b0). This lowering of Tm is probably attributable, at least in\r\npart, to base-pair mismatch. Under the same conditions of hybridization\r\nthere is some hybridization competition for complementary DNA sites\r\nbetween cRNA and HnRNA, presumably between repetitive transcripts.\r\nDue to probable base-pair mismatch it is possible to infer only that\r\nthere is a similarity between HnRNA and cRNA transcripts and not\r\nnecessarily an identity.
", "doi": "10.7907/XDYW-S154", "publication_date": "1973", "thesis_type": "phd", "thesis_year": "1973" }, { "id": "thesis:3318", "collection": "thesis", "collection_id": "3318", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09032002-103837", "type": "thesis", "title": "Part I. Properties of Helical Polycytidylic Acid. Part II. Interactions of Purine with Proteins and Amino Acids. Part III. Binding of Basic Proteins to DNA", "author": [ { "family_name": "Akinrimisi", "given_name": "E. Olabisi", "clpid": "Akinrimisi-E-Olabisi" } ], "thesis_advisor": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" } ], "thesis_committee": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Horowitz", "given_name": "Norman", "clpid": "Horowitz-N" }, { "family_name": "Ts'o", "given_name": "Paul O. P.", "clpid": "Ts'o-Paul-O-P" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis is divided into three sections. The first part deals with the secondary structure of poly C in acid solution, as revealed by several of its physical-chemical properties in solution. The interpretation of the data was based on previous knowledge of the general properties of polynucleotides in solution and on the properties of poly C monomer.
\r\n\r\nThe second part of the thesis deals with the interaction of purine with the proteins. Conformation changes in the proteins are easily measurable in terms of changes in optical rotation even at the visible wavelength regions. Full use was made of this fact to study the nature of interaction of purine with the proteins. The most significant aspect of the studies is not the theoretical speculation on the mechanism of the interaction, but rather the possible practical applications of the findings. This is briefly considered in the discussion. The mechanism of urea. interaction with the proteins cannot be regarded as solved in spite of the voluminous literature on the subject. Similarly, the mechanism of purine interaction with proteins cannot be regarded as solved. The speculations presented at length on the mechanism of the interactions of purine or urea with the proteins, therefore, merely represent a simplified deduction from available data -- a deduction intended to stimulate more experiments and perhaps a modified interpretation of the nature of the interactions.
\r\n\r\nThe third part of the thesis presents initial findings on a very complex problem -- the relationship of polyvalent polymer-polymer interactions to some of their other physical-chemical and biochemical properties. The conclusions drawn from the data are not new or peculiar to current thinking about the nature of interaction of the histones or protamines to DNA. However, certain satisfactions are derived from the fact that it has been possible to carry out reliable physical-chemical measurements on the nucleohistone and nucleoprotamine systems by means of simple standard techniques. Such data are rare in the nucleohistone or nucleoprotamine literature. No doubt, the confirmation of more complex findings on these very important systems will often require these simple physical-chemical data.
\r\n", "doi": "10.7907/EFG9-2C60", "publication_date": "1964", "thesis_type": "phd", "thesis_year": "1964" }, { "id": "thesis:1032", "collection": "thesis", "collection_id": "1032", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03202006-085153", "type": "thesis", "title": "I. Steric and Electrostatic Repulsions in the Inhibition of \u03b1-Chymotrypsin Catalysed Hydrolyses by Indole Derivatives. II. Steric Requirements for Substrates of \u03b1-Chymotrypsin", "author": [ { "family_name": "Abrash", "given_name": "Henry Ivan", "clpid": "Abrash-Henry-Ivan" } ], "thesis_advisor": [ { "family_name": "Niemann", "given_name": "Carl G.", "clpid": "Niemann-C-G" } ], "thesis_committee": [ { "family_name": "Roberts", "given_name": "John D.", "clpid": "Roberts-J-D" }, { "family_name": "Buchman", "given_name": "Edwin R.", "clpid": "Buchman-E-R" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Dean", "given_name": "Richard A.", "clpid": "Dean-Richard-A" }, { "family_name": "Niemann", "given_name": "Carl G.", "clpid": "Niemann-C-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The enzyme-inhibitor dissociation constants, i.e., KI's, were evaluated for the six isomeric pairs of C-substituted indolecarboxylate ions and carboxamides. The variation of KI with the position and nature of the substituent indicates that the enzyme-indole complex exhibits a high degree of steric hindrance near the 4 position of the indole ring and electrostatic repulsion due to a negative group near the indole nitrogen.
\r\n\r\nThe synthesis of D,L-\u03b2,\u03b2-dimethylphenylalanine was modified by use of air oxidation of 4, 6-di-(\u03b1,\u03b1-dimethylbenzyl)pyrogallol to 3,5-di-(\u03b1,\u03b1-dimethylbenzyl)coumalic acid and permanganate oxidation of this product to obtain \u03b1-keto-\u03b2-phenylisovaleric acid. The by-products of the air oxidation were investigated.
\r\n\r\nD,L-2,6-Dimethyltyrosine, a previously unreported amino acid, and several of its derivatives were synthesized.
\r\n\r\nStudies on the rates of \u03b1-chymotrypsin catalysed hydrolyses of N-acetyl-D,L-t-leucine methyl ester, N-acetyl-D,L-\u03b2,\u03b2;-dimethyl-phenylalanine methyl ester and N-acetyl-D,L-2,6-dimethyltyrosine methyl ester indicate the presence of a strong \u03b2 steric effect.
\r\n\r\nMethods of resolution of D,L-\u03b2,\u03b2-dimethylphenylalanine and D,L-2,6-dimethyltyrosine derivatives were investigated.
\r\n\r\nMethyl indole-2-carboxylate is not a substrate of \u03b1-chymotrypsin.
\r\n", "doi": "10.7907/YTFM-K212", "publication_date": "1961", "thesis_type": "phd", "thesis_year": "1961" }, { "id": "thesis:1072", "collection": "thesis", "collection_id": "1072", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03232006-153024", "type": "thesis", "title": "Infrared Absorption Associated with Strong Hydrogen Bonds", "author": [ { "family_name": "Albert", "given_name": "Norman Edward", "clpid": "Albert-Norman-Edward" } ], "thesis_advisor": [ { "family_name": "Badger", "given_name": "Richard McLean", "clpid": "Badger-R-M" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Badger", "given_name": "Richard M.", "clpid": "Badger-R-M" }, { "family_name": "King", "given_name": "Robert Burnett", "clpid": "King-R-B" }, { "family_name": "Robinson", "given_name": "G. Wilse", "clpid": "Robinson-G-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The hydrogen bond systems in potassium hydrogen fluoride, acetamide hemihydrochloride, nickel dimethylglyoxime, potassium hydrogen bis-phenylacetate and maleic acid were studied via infrared spectroscopy between 5000 cm-1 and 400 cm-1. Each compound was studied in detail by the potassium bromide pressed disc technique.
\r\n\r\nThe infrared active fundamentals of the bifluoride ion were found to be shifted from the values reported for the pure, crystalline compound. Integral absorption intensities of the two infrared active fundamentals in the KBr solid solution were evaluated and interpreted in terms of the effective charge motion.
\r\n\r\nSpectra of acetamide hemihydrochloride, nickel dimethylglyoxime and potassium hydrogen bis-phenylacetate exhibited unusual features: an intense, extremely broad background absorption between 2000 cm-1 and 400 cm-1 and a deep \"window\" in the broad background. Maleic acid, however, was found to give a normal, well-behaved spectrum. These unusual features have been interpreted in terms of the symmetry of the hydrogen bond and the complexity of the hydrogen bonded molecules. The broad background is regarded as the superposition of several hydrogen bond vibrational modes, each of which is anharmonically coupled with low frequency intermolecular vibrational modes. The \"windows\" have been explained on the basis of a special perturbation between certain vibrational energy levels of the hydrogen bond modes and the molecular group modes in a small fraction of the molecules; in a majority of the molecules the perturbation is either absent or is very weak. It is suggested that these features are characteristic of short symmetric hydrogen bonds in complex molecules.
", "doi": "10.7907/JACR-N881", "publication_date": "1961", "thesis_type": "phd", "thesis_year": "1961" }, { "id": "thesis:517", "collection": "thesis", "collection_id": "517", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02062006-102644", "primary_object_url": { "basename": "Engleman_r_1959.pdf", "content": "final", "filesize": 8243061, "license": "other", "mime_type": "application/pdf", "url": "/517/1/Engleman_r_1959.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "On the Recombination Rate of Iodine Atoms in the Presence of Various Gases", "author": [ { "family_name": "Engleman", "given_name": "Rolf", "clpid": "Engleman-Rolf" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Wulf", "given_name": "Oliver Reynolds", "clpid": "Wulf-O-R" }, { "family_name": "Buchman", "given_name": "Edwin R.", "clpid": "Buchman-E-R" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lauritsen", "given_name": "Thomas", "clpid": "Lauritsen-T" }, { "family_name": "Mazo", "given_name": "Robert Marc", "clpid": "Mazo-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The rate of iodine atom recombination in the presence of various gases has been studied by the flash photolysis technique. The temperature dependence of the rate constant has been determined for recombination in helium, hydrogen, benzene, and methyl iodide. The negative temperature dependences are fitted by the form, k∝1/Tn, where n(He) = 0.80, n(H2 = 1.48, n(C6H6 = 2.53, and n(CH3I = 3.24.
\r\n\r\nThe iodine atom recombination rate constant was also measured at 50\u00b0C. for ethyl iodide, hydrogen iodide, carbon monoxide, and nitric oxide. The rate constants relative to helium at 50\u00b0C. are:
\r\n
\r\nHe 1.00 C2H5I 179.
\r\nH2 3.33 NI 21.1
\r\nC6H6 61.1 CO 4.07
\r\nCH3I 88.8 NO >1.4 x 104
\r\n
\r\nNitric oxide appears to be a very efficient third body gas and only a lower limit on the rate constant could be established.
Calculations have been made for some possible mechanisms of iodine atom recombination. A classical three body collision model is proposed for atom recombinations. The classical equations of motion were programmed to be solved numerically on a digital computer and some trajectories were examined.
", "doi": "10.7907/KAVE-WG75", "publication_date": "1959", "thesis_type": "phd", "thesis_year": "1959" }, { "id": "thesis:10480", "collection": "thesis", "collection_id": "10480", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10032017-141351791", "primary_object_url": { "basename": "Alder_bj_1952.pdf", "content": "final", "filesize": 53344761, "license": "other", "mime_type": "application/pdf", "url": "/10480/1/Alder_bj_1952.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Radial Distribution Function and the Thermodynamic Properties of Monatomic Liquids. A Statistical Mechanical Theory of the Coefficient of Thermal Conductivity of Monatomic Liquids", "author": [ { "family_name": "Alder", "given_name": "Berni Julian", "clpid": "Alder-Berni-Julian" } ], "thesis_advisor": [ { "family_name": "Kirkwood", "given_name": "John Gamble", "clpid": "Kirkwood-J-G" } ], "thesis_committee": [ { "family_name": "Kirkwood", "given_name": "John Gamble", "clpid": "Kirkwood-J-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Epstein", "given_name": "Paul Sophus", "clpid": "Epstein-P-S" }, { "family_name": "Niemann", "given_name": "Carl G.", "clpid": "Niemann-C-G" }, { "family_name": "Pauling", "given_name": "Linus", "clpid": "Pauling-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A fluid consisting of molecules interacting with the Lennard-Jones intermolecular potential but with rigid cores is treated by the Kirkwood and the Born-Green statistical\u00ad mechanical formulations. The integral equation for the radial distribution function of this fluid is solved numeri\u00adcally by a series expansion of all temperature dependent quantities in the reciprocal of the temperature. The first three terms of this series for the radial distribution func\u00adtion have been evaluated over a wide range of densities for the Born-Green integral equation.
\r\n\r\nThe distribution functions so obtained have been used to calculate the equation of state, the excess internal energy, and the excess entropy of this fluid. The two phase region of this equation of state is determined. For reason\u00adable values of the parameters in the potential, these calcu\u00adlated quantities agree within 10% to 20% with experimental data available for argon.
\r\n\r\nAt one density a comparison between the Kirkwood and the Born-Green theories shows that the two formulations agree closely.
\r\n\r\nA molecular theory of the coefficient of heat conduc\u00adtivity of monatomic liquids is developed on the basis of the general theory of transport processes presented by Kirkwood in 1946. The coefficient is expressed in terms of the inter\u00admolecular force and the equilibrium radial distribution func\u00adtion. Substituting for these, respectively, the Lennard-Jones potential and a reasonable analytic approximation to the experimental radial distribution function, the product of the thermal conductivity and the friction constant has been eval\u00aduated, for liquid argon at 89\u00b0K. With a preliminary estimate of the friction constant, the value of the coefficient of thermal conductivity is then given.
", "doi": "10.7907/22ER-1E05", "publication_date": "1952", "thesis_type": "phd", "thesis_year": "1952" } ]