The cystic fibrosis transmembrane conductance regulator (CFTR) is a\r\nchloride channel member of the ATP-binding cassette (ABC) superfamily of\r\nmembrane proteins. CFTR has two homologous halves, each consisting of six\r\ntransmembrane spanning domains (TM) followed by a nucleotide binding fold,\r\nconnected by a regulatory (R) domain. This thesis addresses the question of\r\nwhich domains are responsible for Cl^- selectivity, i.e., which domains line the\r\nchannel pore.
\r\n\r\nTo address this question, novel blockers of CFTR were characterized.\r\nCFTR was heterologously expressed in Xenopus oocytes to study the\r\nmechanism of block by two closely related arylaminobenzoates,\r\ndiphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by\r\nboth is voltage-dependent, with a binding site \u2248 40% through the electric field\r\nof the membrane. DPC and FFA can both reach their binding site from either\r\nside of the membrane to produce a flickering block of CFTR single channels.\r\nIn addition, DPC block is influenced by Cl^- concentration, and DPC blocks with\r\na bimolecular forward binding rate and a unimolecular dissociation rate.\r\nTherefore, DPC and FFA are open-channel blockers of CFTR, and a residue of\r\nCFTR whose mutation affects their binding must line the pore.
\r\n\r\nScreening of site-directed mutants for altered DPC binding affinity\r\nreveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6\r\nabolishes most DPC block, greatly reduces single-channel conductance, and\r\nalters the direction of current rectification. Additional residues are found in\r\nTM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC\r\nblock; other mutations move the DPC binding site from TM-6 to TM-12. The\r\nstrengthened block and lower conductance due to mutation T1134F is\r\nquantitated at the single-channel level. The geometry of DPC and of the\r\nresidues mutated suggest \u03b1-helical structures for TM-6 and TM-12. Evidence is\r\npresented that the effects of the mutations are due to direct side-chain\r\ninteraction, and not to allosteric effects propagated through the protein.\r\nMutations are also made in TM-11, including mutation S1118F, which gives\r\nvoltage-dependent current relaxations. The results may guide future studies on\r\npermeation through ABC transporters and through other Cl^- channels.
\r\n", "doi": "10.7907/eyre-8424", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7284", "collection": "thesis", "collection_id": "7284", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11262012-114541543", "primary_object_url": { "basename": "Fujiwara_m_1993.pdf", "content": "final", "filesize": 14388032, "license": "other", "mime_type": "application/pdf", "url": "/7284/1/Fujiwara_m_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Characterization of pH-Dependent Poly(Acrylic Acid)-Vesicle Interaction and its Application in Cellular Drug Delivery", "author": [ { "family_name": "Fujiwara", "given_name": "Mitsuko", "clpid": "Fujiwara-Mitsuko" } ], "thesis_advisor": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "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": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Polymers can be used to control drug targeting and drug release from phospholipid vesicles through changes in pH. Understanding the effect of polymers on vesicle properties is important in obtaining a better control over drug delivery using phospholipid vesicles. The pH-dependent interaction of poly(acrylic acid) (PAA) with phosphatidylcholine (PC) vesicles (bilayers) and monolayers was investigated to elucidate the mechanism of P AA-induced vesicle destabilization. PAA induced aggregation of vesicles below pH 4.6 and lipid intermixing of vesicles below pH 4.1. Mixing of aqueous contents was not observed, indicating that the process is not a true membrane fusion. An increase in the membrane permeability accompanied lipid intermixing of vesicles. Both the hydrocarbon chain and phospholipid headgroup packing was more disordered from\r\npolymer association as evidenced by the increase in the CH_2 asymmetric stretching peakwidth and peak position and by the increase in the binding of potential-sensitive dye to\r\nthe vesicle surface, respectively. Polymer binding to vesicles at pH 3.8 showed a negative cooperativity with a K_b of 1.5 x 10^6 M^(-1).The driving force for PAA adsorption on vesicles is the formation of hydrogen-bonds between the protonated carboxyl groups of the polymer and the phospholipid molecule. The polymer mobility was restricted upon complexation with vesicles as evidenced by the anisotropy measurements of polymerassociated fluorophore. The polymer-associated vesicles exhibited fused vesicles and tubular structures in freeze-fracture images. Formation of nonbihyer phases or phase separation was not detected in freeze-fracture images or ^(31)P-NMR powder spectra of PC vesicles. PAA induced expansion of PC monolayers at low pH, and the extent of expansion was dependent on the monolayer surface pressure and on the pH of the subphase. Polymer-induced expansion became more difficult at increasing monolayer packing density and increasing pH. Therefore PAA-induced destabilization of vesicles is believed to be initiated by partial penetration of PAA on vesicle surface. The stress on the membrane created by PAA association presumably resulted in the rupture of bilayer and lipid intermixing of vesicles.
\r\n", "doi": "10.7907/ky3h-q886", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7275", "collection": "thesis", "collection_id": "7275", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11192012-103302776", "primary_object_url": { "basename": "Clark_sm_1993.pdf", "content": "final", "filesize": 37671547, "license": "other", "mime_type": "application/pdf", "url": "/7275/1/Clark_sm_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Advances in scanning force microscopy of biological structures", "author": [ { "family_name": "Clark", "given_name": "Steven Manning", "clpid": "Clark-Steven-Manning" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A multifacted approach to the imaging of biological structures by scanning force microscopy is described. The major problems addressed are the distortion of biological samples by excessive forces applied by the cantilever stylus and sample motion relative to the imaging substrate.
\r\n\r\nThe first two chapters discuss the design of digital signal processor based scanning force microscope control electronics and a novel microscope head that eliminates the application of excessive forces to the sample caused\r\nby electronic or vibrational noise.
\r\n\r\nThe third chapter presents a novel use of chemical vapor deposition for application of heterofunctional alkoxysilanes to scanning force microscopy imaging subsrates. This technique provides imaging substrates which have chemical groups that can be used for sample immobilization without compromising substrate smoothness. The use of the chemically derivatized substrates for scanning force microscopy is also explored.
\r\n\r\nThe final chapter presents high resolution images of bovine liver catalase micro-crystals. The images of the protein micro-crystals show resolution on the order of 2 to 3 nanometers allowing the visualization of individual catalase tetramers. To our knowledge this is the first report of images of protein micro-crystals taken by scanning force microscopy which have resolution comparable to that of electron microscopy.
\r\n", "doi": "10.7907/0vhp-pk35", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:1057", "collection": "thesis", "collection_id": "1057", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03222005-105400", "primary_object_url": { "basename": "Baselt_dr_1993.pdf", "content": "final", "filesize": 14972657, "license": "other", "mime_type": "application/pdf", "url": "/1057/1/Baselt_dr_1993.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Tip-Sample Interaction in Atomic Force Microscopy and its Implications for Biological Applications", "author": [ { "family_name": "Baselt", "given_name": "David Randall", "clpid": "Baselt-David-Randall" } ], "thesis_advisor": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes the construction of an atomic force microscope and its application to the study of tip-sample interactions, primarily through the use of friction and hardness (elasticity) imaging.\r\n\r\nPart one describes the atomic force microscope, which consists of a scanned-cantilever stage (chapter 2); a versatile digital signal processor-based control system with self-optimizing feedback, lock-in amplifier emulation (for hardness imaging), and macro programmability (chapter 3); and image processing software (chapter 4).\r\n\r\nPart two describes a number of results that have helped to characterize the tip-sample interaction and the contact imaging modes used for its study. Meniscus forces act laterally as well as normally, and that they vary with position (chapter 5). Friction measurements couple with scanner position quid feedback, and the meniscus effects friction images (chapter 6). Sliding of the tip over the sample surface introduces slope-dependence into hardness measurements (chapter 7). Dull tips can create prominent topography artifacts even on very flat surfaces (chapter 8).\r\n\r\nIn an investigation of collagen fibrils, AFM has revealed die characteristic 65 nm banding pattern, a second, minor banding pattern, and microfibrils that run along the fibril axis. The distribution of proteoglycans along the fibrils creates a characteristic pattern in friction images. Although imaging in water reduces interaction forces, water can also make biological samples more sensitive to force. However, for robust biological samples imaged in air, tip shape presents a greater obstacle than tip-sample interaction forces to obtaining high-resolution images. Tip contamination increases tip-sample friction and can occasionally improve resolution (chapter 9).\r\n\r\nFor a separate project I have designed a general-purpose nearfield scanning optical microscope (chapter 10).", "doi": "10.7907/5ZMM-7Q64", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7294", "collection": "thesis", "collection_id": "7294", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11292012-090633873", "primary_object_url": { "basename": "Driscoll_rj_1993.pdf", "content": "final", "filesize": 36345789, "license": "other", "mime_type": "application/pdf", "url": "/7294/1/Driscoll_rj_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Scanning Tunneling Microscopy and Spectroscopy: I. Semimetals and Semiconductors. II. Atom-Resolved Imaging of DNA", "author": [ { "family_name": "Driscoll", "given_name": "Robert James", "clpid": "Driscoll-Robert-James" } ], "thesis_advisor": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The topographic and electronic structure of semiconductor and semimetal surfaces were investigated using scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS), respectively. The longrange morphology and atomic-scale characteristics of cleaved materials such as highly oriented pyrolitic graphite (HOPG), boronated pyrolitic graphite (BPG), titanium disulfide, and gallium arsenide (GaAs) were revealed by STM performed under ultrahigh vacuum (UHV) conditions.
\r\n\r\nAtomic resolution constant-current and current-imaging data, as well as barrier height information obtained from tunneling gap modulation, are presented. Both point and line defects were observed on these surfaces; the origin and role of native and adsorbed surface defects are discussed. Visual evidence of coulombic screening caused by adsorption of charged species on n-type GaAs(110) is provided. The atomic corrugation of the GaAs surface was measured to be as little as 0.03 \u00c5 peak-to-valley, attesting to the stability of the microscope design. The BPG sample used in these studies consisted of up to 0.5% boron; boron is the only known substitutional impurity of graphite. Boron substituent atoms appeared as small protrusions approximately 3 \u00c5 in diameter, with an atom density consistent with the assumed concentration.The BPG surface exhibited frequent line defects, including large-angle grain\r\nboundaries, and monolayer-depth etch pits. Images of BPG in air using graphite tips showed similar results; the validity of the popular \"sliding-planes\" mechanism for graphite imaging is evaluated.
\r\n\r\nThe effects of anisotropic stress on the morphology and reconstruction of a thermally annealed Si(111) wafer were explored. The height and orientation of step bunches, as well as terrace widths, on the (7x7) surface were determined. Electromigration effects were also observed; although the overall surface slope was conserved, the step bunches were \"smeared out\" by reversal of the current direction during heating. Line fault defects at step\r\nkinks were observed; a theory for the origin and structure of these features based on stress relief is proposed. Current imaging tunneling spectroscopy (CITS) and localized STS revealed differences between the adatom sites of the\r\n(7x7) surface. Atom-resolved barrier height images were also obtained;comparison to constant-current images may in fact provide a means of differentiating between defects and adsorbed species on the surface. The local effective barrier height was seen to depend strongly on the \"cleanliness\" of the STM tip. The barrier height increased dramatically following voltage pulsing on the order of ten volts. The large height of the step bunches also provided a good test to evaluate the sharpness of the STM tip; examples of \"tip changes\" affecting image resolution and \"multiple-tipping\" are provided. Silicon samples annealed at temperatures below 1000\u00b0C revealed substantial\r\nsilicon carbide (SiC) contamination which effected step pinning. No SiC islands were observed on samples annealed above 1250\u00b0C.
\r\n\r\nIn addition, atom-resolved STM images of duplex DNA supported on a HOPG surface were obtained in UHV. These images revealed double-helical structure, major and minor groove alternation, base pairs, and atomic-scale substructure. Comparison of the DNA dimensions derived from the STM data were in agreement with those from x-ray crystallography for \"random-sequence\" A-form DNA. Cross sectional profiles of the experimental STM data showed excellent correlation with the atomic contours of the van der Waals surface of A-DNA. Barrier height cross-sections showed general correlation with the atomic-scale topography over the phosphate-sugar backbone but distinct anticorrelation (complementarity) over the base pair region. The problems of aggregation and deposition coverage are discussed in the context of possible future applications of STM to DNA sequencing. The use of alternate techniques of DNA deposition, including electrospray\r\nionization, for increased experimental reproducibility are described. The limitations of HOPG as a substrate for biomolecular adsorbates in STM experiments are evaluated.
\r\n", "doi": "10.7907/fp55-ng98", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:2675", "collection": "thesis", "collection_id": "2675", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-131544", "primary_object_url": { "basename": "Bowman_jl_1991.pdf", "content": "final", "filesize": 73373417, "license": "other", "mime_type": "application/pdf", "url": "/2675/1/Bowman_jl_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular genetics of flower development in Arabidopsis thaliana", "author": [ { "family_name": "Bowman", "given_name": "John L.", "clpid": "Bowman-J-L" } ], "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": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Flowers of Arabidopsis thaliana consist of a precise pattern of organs arranged in four concentric whorls, with each whorl containing a different type of floral organ in characteristic positions and numbers. Arabidopsis flowers begin their development as small outgrowths of cells on the flank of the inflorescence meristem. Cells within each flower primordium must somehow assess their position relative to others and subsequently differentiate accordingly. Homeotic mutations at four loci (AGAMOUS, APETALA2, APETALA3, PISTILLATA) identified in Arabidopsis appear to cause cells in two adjacent whorls of the developing flower primordium to misinterpret their position and differentiate inappropriately. The development of wild-type flowers and that of several alleles of each locus is analyzed as well as the development of double and triple mutant combinations between the homeotic mutations. Based on this genetic data, a model is proposed for how this limited number of floral homeotic genes, by being expressed in overlapping fields of cells in the meristem of the developing flower primordium, and acting alone and in combination, could specify the identity of each of the whorls. AGAMOUS and APETALA2 are proposed to negatively regulate each other's activity with the result that they are expressed in mutually exclusive domains, APETALA2 in the outer two whorls of the flower and AGAMOUS in the inner two whorls. The activities of APETALA3 and PISTILLATA are proposed to be localized to the second and third whorls, with another gene, SUPERMAN, negatively regulating their activities in the fourth whorl. By protein sequence homology to known transcriptions factors, SRF of humans and MCM1 of yeast, AGAMOUS encodes a putative transcription factor. In support of the proposed model, RNA tissue in situ hybridizations to developing flowers show that AGAMOUS RNA is spatially localized to the inner two whorls in developing floral buds. Furthermore, in apetala2 mutant flowers, AGAMOUS RNA is detected in all floral whorls suggesting that APETALA2 negatively regulates AGAMOUS expression in the outer two whorls at the trancriptional level. Expression patterns of AGAMOUS late during flower development suggest that AGAMOUS may also play a role in cell fate specification during cellular differentiation of stamens and carpels.", "doi": "10.7907/VJCE-Z966", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2673", "collection": "thesis", "collection_id": "2673", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-075739", "primary_object_url": { "basename": "Hoh_jh_1991.pdf", "content": "final", "filesize": 16024320, "license": "other", "mime_type": "application/pdf", "url": "/2673/1/Hoh_jh_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies on the structure and molecular diversity of the gap junction", "author": [ { "family_name": "Hoh", "given_name": "Jan Hakan", "clpid": "Hoh-Jan-Hakan" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An improved method for the isolation of hepatic gap junctions that substantially shortens preparation time and improves the yield of previous methods is described. The topology of the 28 kD protein component (connexin-32, Cx32) of gap junctions isolated with this method is examined using proteases and antibodies against specific peptides. These experiments are consistent with the current model for the organization of the protein in the membrane, but reveal that an unexpectedly large part of the carboxy-terminus is protected from proteolytic attack. Together with data from comparisons of the Cx32 protein sequence with other channel proteins, a modified topological model is proposed.\r\n\r\nThe structure of the gap junction is further studied by atomic force microscopy. Using this new technology, high resolution images of a gap junction in phosphate buffered saline are obtained, and after \"force dissection,\" which removes half the plaque, the extracellular domains of individual connexons in a hexagonal array with lattice constant of 9.1 nm are revealed. These are the first images of an ion channel by atomic force microscopy, and the observations open the door for a variety of new experiments not previously possible.\r\n\r\nLow stringency screening of a rat genomic library produced genomic clones for Cx32 and a new member of the gene family, connexin-31 (Cx31) or [beta]3. Cx31 has a unique distribution and is found in the eye, Harderian gland, skin, and placenta. Comparison of the Cx31 with the other known connexins, reveals unique and conserved domains in the protein sequences. This comparison is extended to a phylogenetic analysis of the entire gene family that shows two major branches of connexins that diverged 1.3-1.9 billion years ago. Comparison with other ion channels reveals a short sequence similarity between the connexins and channels such as the voltage activated K+ channel. In K+ channels the sequence has been shown to line the aqueous pore, and the model for connexin organization is modified to account for this possibility. The similarity also suggests that gap junctions are part of a superfamily of ion channels.", "doi": "10.7907/8n7d-2t98", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2549", "collection": "thesis", "collection_id": "2549", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112007-104332", "type": "thesis", "title": "Vacuolar Protein Sorting in Yeast: Characterization of Mutants and Identification of a Protein Required for Vacuole Biogenesis", "author": [ { "family_name": "Banta", "given_name": "Lois Margaret", "clpid": "Banta-Lois-Margaret" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The lysosome-like vacuole of the yeast Saccharomyces cerevisiae is an acidic compartment containing a number of hydrolytic glycoproteins including carboxypeptidase Y (CPY), proteinase A (PrA) and proteinase B (PrB). A gene fusion-based selection scheme was utilized to isolate ~600 mutants defective in the localization and processing of vacuolar proteins. These vacuolar protein sorting (vps) mutants define >33 complementation groups and exhibit hybrid protein-independent defects in the sorting of CPY, PrA, and PrB. Light and electron microscopic analyses of the vacuole morphology revealed three distinct classes of vps mutants. The class A mutants (26 complementation groups) contain 1-3 large vacuoles that resemble those of the parental strain. One class A mutant is sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a role for vacuolar pH in protein sorting, perturbation of vacuole acidification resulted in the missorting and secretion of CPY and PrA in wild-type cells. Mutants in the three class B complementation groups exhibit a fragmented vacuole morphology. The class C vps mutants (four complementation groups) lack any compartment resembling a wild-type vacuole, but accumulate vesicles and other membranous structures. Many class C strains exhibit genetically linked defects including temperature-sensitivity and sensitivity to osmotic stress. Unlike other vps mutants, these mutants secrete up to 50% of a vacuolar membrane marker enzyme. The gene defined by one class C mutant, vps33, has been cloned. The predicted VPS33 gene product is hydrophilic and shares sequence similarity with a family of ATP-binding proteins. Disruption of VPS33 is not lethal but results in temperature-sensitive growth. Vps33p-specific antisera recognize a cytosolic protein of ~75 kD. One temperature-sensitive vps33 mutant carrying a missense mutation contains apparently normal vacuoles at the permissive temperature, but lacks vacuoles specifically in the bud at the nonpermissive temperature. We propose that the abnormalities in vacuole morphology and inheritance in vps33 mutants are a consequence of a primary defect in Golgi-to-vacuole protein delivery. A second VPS gene, VPS28, has also ben cloned. Our data suggest that the VPS28 gene product only indirectly affects vacuole protein sorting, but may function in a late protein modification process.
", "doi": "10.7907/TZHF-3J73", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2503", "collection": "thesis", "collection_id": "2503", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-091301", "type": "thesis", "title": "Modification of Membrane Surfaces with Carbohydrates: An Approach for Stabilization During Freezing and Drying", "author": [ { "family_name": "Goodrich", "given_name": "Raymond Paul, Jr.", "orcid": "0000-0002-5945-4571", "clpid": "Goodrich-Raymond-Paul-Jr" } ], "thesis_advisor": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A new class of molecules possessing amphipathic character was prepared. These compounds possessed a hydrophobic region capable of intercalation into a lipid bilayer, a hydrophilic linker group capable of extending beyond the surface of a membrane, and a carbohydrate attached at the end of this linker group. These features of this class of compounds permitted their direct incorporation into vesicle formulations and hence the direct examination of interactions occuring in the dry state between carbohydrates and lipid groups in such vesicle membrane systems.
\r\n\r\nSamples of treated vesicle preparations were subjected to freezing and thawing as well as to direct dehydration via lyophilization. Under these conditions, the stability and integrity of the membrane was examined via several spectroscopic techniques.
\r\n\r\nThrough these studies of systems in which a carbohydrate is directly bound to a membrane surface, it was possible to determine a defined ratio, independent of solution and concentration effects, at which carbohydrates can afford protection to dehydrated membranes. In addition, the interactions responsible for conferring the protection were determined. It was found that direct intercalation of the carbohydrates into a membrane interface preserves the membrane structure and organization that is normally observed in the presence of water. This behavior prevents the phase transitions, lipid phase separations, and fusion phenomena that normally compromise dehydrated membrane systems. This phenomena is directly related to the amount of carbohydrate that is present and the structure of the carbohydrate that is used. These results indicate that the partitioning behavior of the carbohydrates at the interface is of prime importance in determining the effectiveness in this regard.
", "doi": "10.7907/ff9e-th51", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2312", "collection": "thesis", "collection_id": "2312", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302007-153631", "type": "thesis", "title": "Characterization of the SEC18 Gene of S. cerevisiae: Identification of a Protein Involved in Yeast Secretion", "author": [ { "family_name": "Eakle", "given_name": "Kurt Andrew", "clpid": "Eakle-Kurt-Andrew" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "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": "SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum and the Golgi complex. We have cloned the SEC18 gene by complementation of the secl8-1 mutation. Deletion/disruption of this gene has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2271 by open reading frame which would code for a protein of 83.9 kd. The predicted protein sequence showed no significant homology to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kd. Antisera raised against a Sec18-?-galactosidase fusion protein, detects two proteins from in vivo 35S labeled yeast cells identical in size to those seen by in vitro translation. Although potential sites for N-linked glycosylation are present in the Sec 18p sequence, the sizes of the in vivo SEC18 gene products are unaffected by the drug tunicamycin. Hydrophobicity analysis indicated that the protein is hydrophilic in nature and lacks any region that would be predicted to serve as a signal sequence or transmembrane anchor. These results suggest that the Secl8p resides in the cell cytoplasm. Pulse-chase experiments indicate that the two forms of Sec 18 protein are not the result of post-translational processing. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec 18 protein do not arise from mRNAs with different 5' ends. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of the Sec 18 protein. While cell fractionation studies show that the Sec 18p are not associated with ER or Golgi compartments, association with a 100,000 x g pellet fraction has been observed suggesting that Sec 18p may bind transiently to small vesicles such as those presumed to participate in ER to Golgi transport.
", "doi": "10.7907/6aqb-xd83", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:7489", "collection": "thesis", "collection_id": "7489", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02212013-094740326", "primary_object_url": { "basename": "King 1988.pdf", "content": "final", "filesize": 16926957, "license": "other", "mime_type": "application/pdf", "url": "/7489/1/King 1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies of Human Mitochondria. I. Steady-State Levels and Metabolic Properties of the Mitochondrial tRNAs. II. Injection of Mitochondria into Human Cells Leads to a Rapid Replacement of the Endogenous Mitochondrial DNA", "author": [ { "family_name": "King", "given_name": "Michael Paul", "clpid": "King-Michael-Paul" } ], "thesis_advisor": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "thesis_committee": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The steady-state levels and metabolic properties of mitochondrial tRNAs have been analyzed in Hela cells and correlated with the function of the tRNAs for organelle-specific protein synthesis. DNA excess hybridization experiments utilizing separated strands of mitochondrial DNA (mtDNA) and purified tRNA samples from exponential cells long-term labeled with [\u00b3\u00b2P] orthophosphate have revealed a steady-state level of 6 x 10\u2075 tRNA molecules per cell, with three-fourths being encoded in the heavy (H)-strand and one-fourth in the light (L)-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNAF and tRNAV are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs from cells labeled with very short pulses of [5-\u00b3H] uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady state amounts. Therefore, the 15-fold to 60-fold higher rate of transcription of the tRNAV and tRNAF genes (transcribed with the rDNA transcription unit) relative to the other H-strand tRNA genes (transcribed with the whole H-strand transcription unit) and the 13-fold to 20-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes (other than tRNAV and tRNAF genes) are not reflected in the rates of formation of the corresponding tRNAs. The available data indicate that the majority of tRNAV and tRNAF transcribed from the rDNA transcription unit are degraded as they are excised from the primary transcripts. It also seems likely that the majority of the L-strand-encoded tRNAs are degraded before they are excised from the short-lived polycistronic transcripts. Furthermore, a role of the aminoacyl tRNA synthetases in stabilizing the different tRNA species at relatively uniform levels is suggested. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.
\r\n\r\nIn other experiments, isolated human mitochondria containing a mitochondrial DNA (mtDNA)-coded chloramphenicol resistance marker were injected at an average dose of less than one into sensitive human cells partially depleted of their mtDNA by ethidium bromide treatment. Under selective conditions, the mitochondria became established in the recipient cells with a frequency greater than 2 to 3 x 10\u207b\u00b3). A rapid and, in some cases, complete replacement of the resident mtDNA by the exogenous mtDNA took place in the transformants, as shown by multiple mtDNA and nuclear DNA polymorphisms. Intracellular mtDNA selection played a crucial role in this replacement, with significant implications for mitochondrial genetics.
\r\n", "doi": "10.7907/45zh-5315", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7493", "collection": "thesis", "collection_id": "7493", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02222013-111607349", "type": "thesis", "title": "Models for the Avoidance Response in Phycomyces", "author": [ { "family_name": "Matus Bloch", "given_name": "Ivan J.", "clpid": "Matus-Bloch-Ivan-J" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The sporangiophore of the fungus Phycomyces is able to avoid obstacles placed a few millimeters from its growing zone. The work described in this thesis presents evidence that the avoidance response is mediated by gases. Avoidance occurs in still air in the diffusion limit, even at relative humidities close to 100%. It is shown that the effect of the surfaces of the obstacles cannot only be to reflect these gases: the surfaces must play a more active role. Models in which the surfaces adsorb growth-inhibitors or adsorb an inert precursor that is re-emitted as a growth-promoter that decays are in agreement with our experimental observations.
", "doi": "10.7907/c5e3-v765", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:11468", "collection": "thesis", "collection_id": "11468", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152019-165931960", "type": "thesis", "title": "Regional Distribution and Subcellular Associations of Type II Calcium and Calmodulin-Dependent Protein Kinase in Rat Brain", "author": [ { "family_name": "Erondu", "given_name": "Ngozi Emmanuel", "clpid": "Erondu-Ngozi-Emmanuel" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Four monoclonal antibodies generated against the Type II CaM kinase have been characterized. Two of these antibodies were used to confirm that both alpha and beta subunits were part of the holoenzyme complex. I also developed liquid phase and solid phase radioimmunoassays for the kinase.
\r\n\r\nWith the solid phase radioimmunoassay, the distribution of the kinase in rat brain was examined. This study revealed that the concentration of the kinase varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of total hippocampal protein, 1.3% of cortical protein and 0.7% of striatal protein. It is less concentrated in lower brain regions ranging from 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The unusually high concentration of the kinase in telencephalic regions may confer upon their neurons specialized responses to calcium that are different from those of neurons in lower brain regions.
\r\n\r\nThe association of the kinase with elements of the cytoskeleton was also investigated. The results of this study showed that autophosphorylation causes an increase in the association of the enzyme with taxol-polymerized microtubules and F-actin. This increase in association was reversed by dephosphorylating phosphokinase with protein phosphatase. These results suggest that autophosphorylation could constitute a mechanism for the regulation of the subcellular associations of the Type II CaM kinase by neuronal activity.
", "doi": "10.7907/30tg-qa85", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11472", "collection": "thesis", "collection_id": "11472", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04162019-154502622", "type": "thesis", "title": "The Avoidance Response of Phycomyces in a Controlled Environment", "author": [ { "family_name": "Meyer", "given_name": "Paul Wells", "clpid": "Meyer-Paul-Wells" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "If an object is placed 1 mm away from the growing zone of a Phycomyces sporangiophore growing in air, then after 2 to 6 min the sporangiophore bends away from the object, without ever touching it, at a rate of about 1\u00b0/min. The sporangiophore stops bending after about 30 min. This is called the avoidance response of Phycomyces.
\r\n\r\nHow does the sporangiophore detect the object? It seems likely that a chemical mechanism is involved, since other physical stimuli (light, electric and magnetic fields) have already been ruled out.
\r\n\r\nA simple mechanism was proposed 10 years ago, in which the ambient air currents near the surface of an object modify the distribution of a hypothetical, short-lived effector gas emitted by the sporangiophore. However, the avoidance response occurs at its usual rate in the complete absence of ambient air currents. Thus, the suppression of air currents near the surface of a solid object cannot provide the signal for the response.
\r\n\r\nThe avoidance rate depends significantly on the recent history of the experimental chamber, on the length of time the sporangiophore has spent inside the experimental chamber, and on other factors. By carefully controlling environmental variables, the variation in avoidance rate of different sporangiophores in successive experiments can be held to less than \u00b110%. This allows accurate determination of the distance dependence of the response, and accurate comparison of different types of barriers.
\r\n\r\nThe rate of the response falls off above 90 % relative humidity - but does not fall to zero. Surprisingly, the sporangiophore avoids a thin, 120 \u00b5m diameter parallel wire placed 0.5 mm away at about the same rate as it avoids another sporangiophore placed at the same distance. Also, the distance dependence of the avoidance response appears to be much weaker than previously reported, and the response may depend on the chemical composition of the object, in contrast to previous reports. These findings, combined with the results of calculations presented in Appendix 3, argue strongly against the hypothesis that the barrier acts merely by reflecting a diffusible substance emitted by the sporangiophore.
\r\n\r\nThe only remaining viable chemical mechanism for the avoidance response requires that the signal molecule emitted by the sporangiophore be adsorbed by the surface of the avoided object for a nonzero length of time, and not just be reflected by it. Three new versions of this hypothesis are presented which are consistent with the experimental results.
", "doi": "10.7907/s6zv-r568", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11288", "collection": "thesis", "collection_id": "11288", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11282018-124040200", "type": "thesis", "title": "The Post-Translational Processing of Sindbis Virus Glycoproteins", "author": [ { "family_name": "Mayne", "given_name": "Jeffrey Terrell", "clpid": "Mayane-Jeffrry-Terrell" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "thesis_committee": [ { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "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": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus infected chicken cells and shown to be produced from the cleavage of PE2 to produce E2. The N-terminal sequence of E3 is identical to that of PE2. The first 19 amino acids are hydrophobic and presumably serve as the signal sequence for PE2. This sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at position 14. Labeling studies imply that the PE2 \u2192 E2 + E3 cleavage is not closely coupled to budding. E3 is cleaved and released into the culture fluid under conditions where no virions bud, and the kinetics of appearance of E3 in the culture fluid and E2 in virions are dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane glycoproteins.
\r\n\r\nHybridomas were selected by the fusion of NSI/1 myeloma cells with spleen cells from mice inoculated with Sindbis specific antigens. Ten stable hybridomas were obtained, seven producing E1-specific antibodies and three producing capsid-specific antibodies. The seven E1 specific antibodies were divided into two classes, which reacted with different E1 antigenic domains. The two classes of antibodies differed in several tested properties. Two E1 clones inhibited viral infectivity, and one of these precipitated E2 along with E1 in Triton-treated preparations. These properties are discussed with regard to the known relationships between the viral structural proteins.
\r\n\r\nThe tryptic glycopeptides of E1 and E2 grown in BHK or chick cells were purified and analyzed by N-terminal sequencing, pronase digestions and labeling with various radioactive sugars. We found that the glycosylation patterns for the two proteins were essentially identical in the two hosts. E2 contains exclusively complex chains attached to Asn196 and simple chains attached to Asn398. In E1, the Asn135 glycosylation site contained only complex chains, but the Asn245 site contained a mixture of simple and complex chains. A prediction as to the relative importance of the different glycosylation sites to protein function is offered.
", "doi": "10.7907/jvbk-es85", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11202", "collection": "thesis", "collection_id": "11202", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09262018-173300466", "type": "thesis", "title": "Chemotactic Responses of Tethered Bacteria", "author": [ { "family_name": "Block", "given_name": "Steven Michael", "clpid": "Block-Steven-Michael" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Fender", "given_name": "Derek H.", "clpid": "Fender-D-H" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Escherichia coli swim in a three-dimensional random walk of alternating runs and tumbles, using their flagella for propulsion. When moving in a gradient of an attractant or repellent, they bias the walk in such a way as to migrate into a favorable region; this is a basis for chemotaxis. Bacteria may be tethered to a glass surface by means of a single flagellum. When tethered, cell bodies spin alternately clockwise (CW) and counterclockwise (CCW) under the influence of the rotary motor that drives the flagellum. The CCW state corresponds to the run mode and the CW state to the tumble mode. Tethered bacteria remain fixed in place, thereby providing an opportunity to study chemotactic behavior by direct manipulation of attractant or repellent concentration near the cells. Two experimental approaches have been used to exploit this opportunity. In the first, a mixing device that provides programmable concentration changes was used to stimulate tethered cells with exponential temporal gradients or exponentiated sine waves of the attractant \u03b1-methyl-D,L-aspartate. Such changes cause chemoreceptor occupancy to be changed linearly or sinusoidally, respectively. Exponential temporal gradients (both positive and negative) were found to shift the rotational bias (defined as the fraction of time spent spinning CCW) by a fixed amount related to the steepness of the gradient. The bias shifts produced indicate that cells are exquisitely sensitive to small changes in chemoreceptor occupancy. Distributions of CW and CCW intervals remained exponential during such gradients. This result is inconsistent with a response regulator model in which rotational transitions are associated with level-crossings of a fluctuating, hypothetical intermediate. It is consistent with a model in which transitions occur at random between rotational states, the transition probabilities being governed by chemotactic signals. In the second approach, short bursts of an attractant or repellent were delivered iontophoretically, producing an impulse response in the tethered bacteria. Properties of the impulse response show both adaptive and integrative behavior, and imply that cells respond maximally to changes in concentration which occur over times comparable with the length of a run. The impulse response can be used to predict the behavior of cells towards an arbitrary stimulus in the linear domain. Impulse responses from a series of chemotaxis mutants showed that some were defective in adaptation but not excitation; others were defective in both. Taken together, the experiments provide information about the spectral response of bacteria to concentration changes with frequencies ranging from 10-3 Hz up to almost 10 Hz. Both sets of data are consistent with the notion of a cellular \"bias regulator\" signal that sets the transition probabilities between two states; one representing CCW and the other representing CW rotation.
", "doi": "10.7907/2fzz-tb74", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11873", "collection": "thesis", "collection_id": "11873", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10292019-140253277", "primary_object_url": { "basename": "Wang_C_1983.pdf", "content": "final", "filesize": 47575481, "license": "other", "mime_type": "application/pdf", "url": "/11873/1/Wang_C_1983.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Induction and Methylation of Heat Shock Proteins in Cultured Vertebrate Cells", "author": [ { "family_name": "Wang", "given_name": "Chung", "clpid": "Wang-Chung" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Clarke", "given_name": "Steven G.", "clpid": "Clarke-Steven-G." } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "When vertebrate cells are exposed to sodium arsenite, they respond by increased synthesis of a small number of polypeptides similar to the heat shock proteins, which are induced by exposure to 40-45\u00b0C. The 70,000 dalton inducible protein (hsp70) is the most commonly induced species and this protein is methylated at both lysyl and arginyl residues. In chicken fibroblasts, the hsp70 is composed of two major distinct isoelectric variants, as well as several minor components. The more acidic one (hsp70A; pI 5.6) is highly conserved and found in all vertebrate tissues and cultured cells examined, while the more basic one (hsp70B; pI 6.0) at present has only been found in avian cells.
\r\n\r\nThe hsp70 is a prominent cytoplasmic constituent under normal growth conditions. In chicken fibroblasts, the induction by arsenite not only increases the synthesis of hsp70 but also results in an accumulation of this protein. In contrast, the induction in 3T3 and SR-RSV 3T3 cells results in increased synthesis, but no accumulation of this polypeptide.
\r\n\r\nIn chicken cells, \u03b5-N-trimethyl-lysine has been identified as the major component of methyl-lysine species in hsp70, but the contribution of \u03b5-N-monomethyl-lysine and \u03b5-N-dimethyl-lysine is evident. The methyl-arginine of hsp70 is exclusively NG-monomethyl-arginine; these methylations appear to be stoichiometric. Furthermore, these methylations can be modulated by arsenite. In particular, in hsp70A the amount of \u03b5-N-trimethyl-lysine decreases and the \u03b5-N-dimethyl-lysine significantly increases, while in hsp70B the quantity of NG-monomethyl-arginine is reduce fivefold in the presence of sodium arsenite. In 3T3 and SR-RSV 3T3 cells, \u03b5-N-trimethyl-lysine appears to be the only methylated lysine species; both NG-monomethyl-arginine and NG,NG-dimethyl-arginine have been identified as the methylated arginyl residues. Similar to the homologous polypeptide in chicken fibroblasts, the level of arginyl methylation of hsp70 of 3T3 cells can be reduced in the presence of arsenite. This arsenite-induced reduction in arginyl methylation of hsp70 appears to be restricted to the polypeptides synthesized during the arsenite incubation. However, the arginyl methylation level of hsp70 of SR-RSV 3T3 is constitutively lower than their untransformed counterpart (3T3 cells), and the methylation cannot be reduced further by arsenite. In addition, the level of lysyl methylation of hsp70 remains the same after arsenite treatment and transformation. Since the basic amino acid methylation in total cellular proteins remains unchanged after arsenite incubation and Rous sarcoma virus transformation, the reduction in arginyl methylation of hsp70 in chicken fibroblasts and 3T3 cells by these treatments appears to be specific.
", "doi": "10.7907/mpys-2s35", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11842", "collection": "thesis", "collection_id": "11842", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-124810283", "type": "thesis", "title": "Biochemistry and Diversity of the Gap Junction Protein: A Study of Liver, Heart and Lens", "author": [ { "family_name": "Nicholson", "given_name": "Bruce John", "orcid": "0000-0003-1649-7173", "clpid": "Nicholson-Bruce-John" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Fractions highly enriched for gap junctions by morphological criteria have been isolated from rat liver, heart and eye lens, although some question exists as to the nature of the structures from lens. The junctions from each tissue are comprised of a single major protein of Mr 28,000 in the liver, Mr 30,000 in the heart, and Mr 26,000 (MIP 26) in the lens. The polypeptide profile of the liver fraction is complicated by endogenous proteolysis and aggregation in SDS of the gap junction protein and the presence of about 20% non-junctional material. Heart and lens junction proteins are also found to aggregate in SDS, while endogenous proteolysis typically reduces the cardiac gap junction protein to Mr 28,000 during isolation.
\r\n\r\nComparisons of two-dimensional peptide maps of the junctional proteins from these tissues, and the use, where necessary, of a third dimension of resolution (HPLC), demonstrates the three proteins to be very different in terms of their primary structures. The protein of each tissue, however, seems well conserved between mammalian species. For liver and lens, this finding has been confirmed in amino acid analyses and partial NH2-terminal sequences (to 58 and 33 residues, respectively). Cleavage products of these two proteins have also been produced to allow further sequence analysis in the future. In spite of the differences in primary structure, some conservation of the tertiary structures of these proteins is suggested by proteolysis of intact junctions (likely restricted to the cytoplasmic surfaces). Liver and heart gap junction proteins are reduced by trypsin to two fragments of Mr 10,000, while a single Mr 21,000 fragment is produced from lens MIP 26. Sequence analysis (liver and lens only) indicates that most of the protein removed by tryptic hydrolysis is from the carboxy-terminus, although an additional loop of 4,000 daltons is excised from the center of the liver polypeptide and five residues are lost from the NH2-terminus of the lens protein.
\r\n\r\nThe extent and possible significance of this surprising tissue specificity of the gap junction protein are discussed in the light of these findings.
", "doi": "10.7907/bhjv-8053", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11830", "collection": "thesis", "collection_id": "11830", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-171906142", "type": "thesis", "title": "Identification and Characterization of Glial Growth Factor", "author": [ { "family_name": "Lemke", "given_name": "Greg Erwin", "clpid": "Lemke-Greg-Erwin" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" } ], "thesis_committee": [ { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A combination of biochemical, cell biological and immunological techniques have been employed to identify a novel and potent polypeptide mitogen of the brain and pituitary. This molecule, named glial growth factor (GGF), stimulates DNA synthesis and cell division in cultured rat Schwann cells, astrocytes, and fibroblasts.
\r\n\r\nThree independent lines of evidence indicate that GGF activity resides in a basic protein of molecular weight 3.1 x 104. (a) When partially purified preparations are analyzed by native gel electrophoresis at pH 4.5, mitogenic activity migrates with a protein of this molecular weight, as revealed by bioassay coupled with a second dimension of SDS gel electrophoresis. (b) A set of monoclonal antibodies which deplete growth factor activity from heterogeneous solutions specifically recognize a 31,000 dalton protein antigen, as determined by gel immunoautoradiography. (c) GGF activity is recovered at a molecular weight of 3.1 x 104 after denaturing polyacrylamide gel electrophoresis in SDS.
\r\n\r\nThree large-scale purifications of GGF, employing a combination of column chromatography steps and preparative electrophoreses, are described. The molecule has been purified to apparent homogeneity from anterior lobes of the bovine pituitary.
\r\n\r\nThrough the use of nucleic acid precursor incorporation assays, GGF has been shown to be markedly mitogenic for rat Schwann cells, astrocytes and fibroblasts, but inactive when assayed on oligodendrocytes or microglia. Electrophoretic analyses suggest that all responsive cell types are stimulated by a single (the same) molecular species. GGF is the only defined mitogen to which rat Schwann cells respond.
\r\n\r\nGlial growth factor from bovine brain has been found to be indistinguishable from bovine pituitary GGF, as determined by biochemical, immunological and bioactivity criteria. GGF is non-uniformly distributed among bovine brain regions. It is present in brain extracts prepared from a wide variety of vertebrate species.
\r\n\r\nPurified human platelet-derived growth factor (PDGF) shares many important properties with GGF. PDGF has been shown to be unable to significantly stimulate the division of rat Schwann cells, however, and therefore appears to be distinct.
\r\n\r\nObservations made in vitro suggest several possible biological roles for GGF in vivo. These are discussed.
\r\n", "doi": "10.7907/774r-7520", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11812", "collection": "thesis", "collection_id": "11812", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10112019-091636747", "type": "thesis", "title": "Biochemical and Genetic Studies of Peripheral Myelination in Normal Development and in the Mouse Mutant Trembler", "author": [ { "family_name": "Fryxell", "given_name": "Karl Joseph", "orcid": "0000-0002-2975-7897", "clpid": "Fryxell-Karl-Joseph" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "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": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "I developed a radioimmunoassay for P0, the major peripheral myelin protein, and adapted an existing radioimmunoassay for myelin basic protein. Results from these assays showed that Schwann cells do not make either protein before myelination begins, and accumulate P0 and myelin basic protein with the same time course during development. Schwann cells cultured in the absence of neurons do not express detectable levels of either protein, but they do continue to synthesize sulfatide, a myelin sulfolipid, apparently indefinitely, as shown by biosynthetic labeling.
\r\n\r\nThe trembler (Tr/+) mouse mutant has a pronounced reduction in peripheral myelin, while central myelin and peripheral unmyelinated nerves appear normal. This myelin deficiency is caused by an autonomous Schwann cell defect. Since the Trembler phenotype is expressed in heterozygotes, most previous studies were confined to Tr/+ mice. Using two alleles, I show here that the six possible genotypes can be ordered as follows: +/+ > Trj/+ > Tr/+ > Trj/Trj > Trj/Tr > Tr/Tr, based on myelin basic protein radioimmunoassays of sciatic nerve extracts. The amount of compact myelin in each genotype, as shown by electron microscopy, is in good agreement with the radioimmunoassay results, with two important provisos. First, Tr/Tr mice have 1% of wild-type myelin basic protein levels but essentially no compact myelin. Secondly, the first steps in the above genetic series reduce both the average myelin sheath area and the number of myelin sheaths by similar amounts, while the last steps primarily reduce the number of myelin sheaths. These results suggest that, if the activity of the Tr+ gene product is reduced below a certain point, myelin protein synthesis can be induced without forming a myelin sheath. Visualization of P0 by indirect immunofluorescence shows that Schwann cells without compact myelin express much lower levels of P0 than adjacent Schwann cells, associated with the same axon, that do form compact myelin. Finally, although Trembler Schwann cells proliferate excessively in vivo, their proliferation is not affected by Tr gene dose. In vitro Trj/Trj Schwann cell proliferation is apparently normal. Thus, it is likely that the function of the Tr+ gene product is not directly concerned with Schwann cell proliferation.
", "doi": "10.7907/eahp-bx93", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:5095", "collection": "thesis", "collection_id": "5095", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12212004-095323", "type": "thesis", "title": "The Role of Filamin in the Morphogenesis of the Skeletal Muscle Sarcomere", "author": [ { "family_name": "Gomer", "given_name": "Richard Hans", "orcid": "0000-0003-2361-4307", "clpid": "Gomer-Richard-Hans" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "During chicken skeletal myogenesis in tissue culture, filamin is found on stress fibers in myoblasts and early myotubes. Approximately one day after fusion and shortly before \u03b1-actinin transits to Z lines, filamin disappears from the cells. The disappearance of filamin is correlated with a cessation of its synthesis. Approximately six days after fusion, filamin reappears at the Z lines of myogenic cells, shortly before desmin and vimentin transit to the Z line. In adult muscle, filamin is found at the periphery of the Z disk, along with desmin and vimentin. Peptide map analysis of the various filamins shows that gizzard and fibroblast filamins are identical while myoblast filamin is quite similar to these two filamins. Cultured myotube and adult myofibril filamins are virtually identical to each other and are quite different polypeptides when compared to gizzard, fibroblast and myoblast filamins. Analysis of terminally differentiated slow and fast muscle shows that both muscle types contain identical, skeletal muscle type filamins although in the slow muscle, filamin is distributed additionally on the I band. The molar filamin to actin ratio is 1:25 in gizzard and fibroblast, 1:54 in myoblasts, 1:820 in fast skeletal myofibrils and 1:82 in slow skeletal myofibrils.
\r\n\r\nThese results offer several new insights into eucaryotic molecular morphogenesis. From the disappearance of filamin during myogenesis, we see that a morphogenetic process may involve the temporary removal of a family of proteins. The different distributions of identical filamin polypeptides in slow and fast muscle indicates that filamin may be synthesized at different times and rates in the two myogenic processes. It appears that, at least in the case of the skeletal muscle sarcomere, temporal control of protein synthesis may be an important part of eucaryotic molecular morphogenesis.
", "doi": "10.7907/97M8-B306", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:10888", "collection": "thesis", "collection_id": "10888", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152018-105719665", "primary_object_url": { "basename": "Granger_BL_1982.pdf", "content": "final", "filesize": 113586082, "license": "other", "mime_type": "application/pdf", "url": "/10888/1/Granger_BL_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Composition and Function of Intermediate Filaments in Avian Muscle Cells and Erythrocytes", "author": [ { "family_name": "Granger", "given_name": "Bruce Leslie", "clpid": "Granger-Bruce-Leslie" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Intermediate filaments comprise a family of morphologically similar cytoplasmic structures whose individual subunits are biochemically and immunologically distinguishable, and largely cell-type specific. This thesis is an investigation of the intermediate filaments in avian smooth and skeletal muscle, and avian erythrocytes.
\r\n\r\nConditions have been found under which sheets of interconnected Z-discs can be generated from skeletal muscle. The stability of these sheets demonstrates that the Z-discs of adjacent myofibrils are firmly linked to one another. Desmin, the intermediate filament subunit characteristic of muscle, encircles each Z-disc and thereby forms an insoluble, two-dimensional scaffold at right angles to the fiber axis within each Z-plane. Desmin may thus be responsible for maintaining the cross-striated appearance of skeletal muscle fibers, and may function to mechanically integrate the contractile actions of their constituent myofibrils. Vimentin, the intermediate filament subunit characteristic of mesenchymal cells, coexists with desmin at the periphery of the Z-disc, and demonstrates that a terminally differentiated cell can possess more than one class of intermediate filament subunit.
\r\n\r\nA 230,000 dalton polypeptide, named synemin, copurifies with desmin from smooth muscle. It coexists and colocalizes with desmin and vimentin in skeletal muscle at all stages of differentiation, from fusing myoblasts to mature fibers. Nondenaturing conditions under which synemin can be separated from desmin and vimentin have not been found.
\r\n\r\nDetection of synemin in avian erythrocytes has led to the realization that this cell might be a relatively simple model system for the study of intermediate filaments. A fraction of the intermediate filaments in avian erythrocytes is stably associated with the plasma membrane, but can be selectively removed from it with water; this results in preparations consisting predominantly of vimentin and synemin. Immunoelectron microscopy reveals that vimentin forms the bulk of the core filament in these cells, and synemin exists at regular intervals along this core. The axial periodicity of synemin appears to change during erythropoiesis, perhaps in accordance with some structural or functional change in the filaments. Synemin appears to crosslink the filaments through self-association, and may thus regulate the rigidity or dispersion of the intermediate filament network in erythrocytes as well as in muscle cells.
", "doi": "10.7907/z9x1-xs25", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:10895", "collection": "thesis", "collection_id": "10895", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-092739245", "primary_object_url": { "basename": "Green_SH_1982.pdf", "content": "final", "filesize": 92489877, "license": "other", "mime_type": "application/pdf", "url": "/10895/1/Green_SH_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Genetic Studies of Neuronal Development in Drosophila melanogaster", "author": [ { "family_name": "Green", "given_name": "Steven Haym", "clpid": "Green-Steven-Haym" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Konopka", "given_name": "Ronald J.", "clpid": "Konopka-Ronald-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The projections into the central nervous system (CNS) of several wild-type and genetically ectopic sensory structures were studied by cobalt filling or silver staining and compared for the purpose of determining what factors guide the growth of the sensory axons. The head bristles all arborize in a similar fashion in the subesophageal ganglion although they reach the target by three different routes depending on their position on the head. This arborization is L-shaped with a longitudinal branch and a medially directed branch that crosses the midline. The antennal projection consists of an olfactory lobe component, organized into glomeruli, and an antennal mechanosensory component which can be further subdivided into three branches, the anteriormost of which is identical to the head bristle projection. The tarsi all have similar U-shaped projections into their segment's neuromere with no ascending, descending or contralateral branches.
\r\n\r\nAxons of ectopic thoracic bristles on the head may enter the brain or the optic lobes. The routes into the brain taken by the ectopic bristles were initially like those of the normal head bristles but were followed for greater or lesser distances and the region of the subesophageal ganglion that is the target of the head bristles was seldom reached. The terminal arborizations of the ectopic bristle axons were generally irregular regardless of where they were: in the subesophageal ganglion, brain or optic lobes. They resembled neither their normal arborizations in the ventral ganglion nor those of the local head sensilla in the brain.
\r\n\r\nAxons from antennal legs have a pattern of projection grossly similar to that of wild-type antennae in that the same regions of neuropil were innervated. The non-olfactory lobe components of the antennal leg projection were like those of the antenna. However, the arborization in the olfactory lobe was chaotic and there were adventitious projections from the lobe into adjacent neuropil, particularly the subesophageal ganglion. Some elements of these adventitious projections in the subesophageal ganglion were found consistently in almost every preparation. No element of the projection resembled the leg projection in the ventral ganglion.
\r\n\r\nThe axons of ectopic sensilla can reach a normal target if the distance to it from the new location is sufficiently small: axons from abdominal legs in bxd mutants terminate in normal metathoracic leg sensory neuropil and the axons of antennae misplaced as a result of the mutation ant can enter normal antennal targets.
\r\n\r\nIn summary, axons of ectopic sensilla can't reach their normal targets if they enter the CNS far from those targets which suggests that there are no long range cues for guidance of sensory axons. In the \"foreign\" part of the CNS the axons of ectopic sensilla do not make projections that resemble their normal ones. They initially take routes characteristic of sensilla in their new location but do not follow them consistently. The exception, antennal leg mechanosensory projections, is likely to be a result of a homology between antennal and leg mechanosensory sensilla. These results suggest the following: insect sensory neurons reach their targets mainly by following local and not long-range cues. The growth of these axons is constrained to specific tracts and it is by these that they are guided over long distances to their targets. Tracts recognized by the axon can be recognized at any point and, as the present study shows, this recognition is required not only at the point of entry but continuously, all along the tract, for guidance of the axon. Guidance by the tract appears to depend on an affinity between the axon and the tract that may also exist between axons and tracts of their segmental or functional homologues. Since axons in foreign neuropil have irregular arborizations characteristic neither of their normal ones nor of those of the local sensilla, the arborization pattern is not a result of an internal branching program alone nor of the axon's milieu directing the branching but must depend on a specific interaction between the axon and its target.
\r\n\r\nThe leg motorneurons were identified and described after HRP backfilling from cut legs. The pattern of their positions differs from segment to segment. The bithorax mutations transform the metathoracic pattern into a mesothoracic pattern, paralleling their effect on the epidermis.
", "doi": "10.7907/3h8z-9176", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:10886", "collection": "thesis", "collection_id": "10886", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142018-174410594", "primary_object_url": { "basename": "Gard_DL_1982.pdf", "content": "final", "filesize": 118360636, "license": "other", "mime_type": "application/pdf", "url": "/10886/1/Gard_DL_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Intermediate Filaments and Myogenesis in vitro", "author": [ { "family_name": "Gard", "given_name": "David Lynn", "clpid": "Gard-David-Lynn" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "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": "This thesis describes my investigations into the composition and function of intermediate filaments (IF) during myogenesis in vitro. I have found that avian embryonic myotubes cultured in vitro contain two intermediate filament subunits, desmin and vimentin. Prior to myoblast fusion vimentin is the sole IF subunit protein detectable by electrophoretic and immunological techniques. The onset of desmin synthesis and its cytoplasmic accumulation appear to coincide with fusion of myoblasts into multinucleate myotubes. Immunofluorescence reveals dense networks of desmin- and vimentin-containing filaments in the sarcoplasm of immature myotubes; however, late in myogenesis antisera to both desmin and vimentin are observed to stain the Z-lines of myofibrils. Double immunofluorescence microscopy using antisera to \u03b1-actinin and desmin revealed that this association occurs after the assembly of \u03b1-actinin into Z-lines, at a time when individual myofibrils are being organized into bundles. Phosphorylation of intermediate filament proteins in muscle has been previously reported (O'Connor et al., Proc. Natl. Acad. Sci. U.S.A. 76: 819-823, 1979). Using two-dimensional tryptic analysis I have found that desmin from embryonic myotubes is phosphorylated at multiple sites which correspond to sites phosphorylated by cAMP-dependent protein kinase in vitro. I have observed phosphorylation of desmin and vimentin in intact myotubes at all stages of myogenesis. However, treatment of mature (7 day and older) myotubes with 8-BrcAMP or isoproterenol results in a specific 2-3 fold increase in phosphorylation of these proteins, with a corresponding increase in 32PO4 incorporation into one major phosphopeptide of desmin. Preliminary evidence indicates that treatment of 6-8 day myotubes with 8-BrcAMP or isoproterenol significantly inhibits the transition of intermediate filaments to the Z-line which occurs during normal myogenesis. These observations suggest that intermediate filaments containing desmin and vimentin are responsible for the organization of skeletal muscle myofibrils into an integral contractile machinery, and that cAMP-dependent phosphorylation of desmin and vimentin plays an important role in the regulation of this function.
", "doi": "10.7907/e5gk-4c77", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:929", "collection": "thesis", "collection_id": "929", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03112009-074718", "primary_object_url": { "basename": "Pankow_jf_1979.pdf", "content": "final", "filesize": 5776308, "license": "other", "mime_type": "application/pdf", "url": "/929/1/Pankow_jf_1979.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Dissolution Rates and Mechanisms of Tetragonal Ferrous Sulfide (Mackinawite) in Anoxic Aqueous Systems", "author": [ { "family_name": "Pankow", "given_name": "James Frederick", "clpid": "Pankow-James-Frederick" } ], "thesis_advisor": [ { "family_name": "Morgan", "given_name": "James J.", "clpid": "Morgan-J-J" } ], "thesis_committee": [ { "family_name": "Morgan", "given_name": "James J.", "clpid": "Morgan-J-J" }, { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Duwez", "given_name": "Pol E.", "clpid": "Duwez-P-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nAn experimental study was carried out on the rates and mechanisms of the non-oxidative dissolution of mackinawite (tetragonal FeS) in anoxic aqueous systems of varying pH (3-7), T (5-35\u00b0C), and ionic strength (0.05-0.60 M [M is underscored]). A special glass and teflon dissolution reactor was constructed. The main design criterion was the need to keep the reaction medium absolutely free of oxygen.\r\n\r\nThe flux F[subscript S] (moles/cm[superscript 2]-min) from the surfaces of pellets pressed from [...]l\u03bc FeS particles was found to obey the rate equation\r\n\r\nF[subscript S] = k[subscript 1]a[subscript H superscript +] + k[subscript 2]\r\n\r\nk[subscript 1] and k[subscript 2] are rate constants and a[subscript H superscript +] is the activity of the hydrogen ion At 25\u00b0C, k[subscript 1] = 0.22 cm/min with a relative standard deviation of 18%. The value of k[subscript 2] was measured to be 1.9 moles/cm[superscript 2]-min with a relative standard deviation of 22%. The actual exposed FeS surface area A[...] was related to the projected area A by the expression A[...] = 1.7A. The factor of 1.7 accounts for the roughness of the surface. The actual rate constants k[...] and k[...] were therefore obtained by dividing k[subscript 1] and k[subscript 2] by 1.7: k[...] = 0.13/min and k[...] = 1.1 x 10[superscript -9] moles/cm[superscript 2]-min. The k[...] term dominates at pH < 4.3 and k[...]dominates at pH > 5.6. pH 4.3-5.6 is a transition region.\r\n\r\nIt is argued that these rate constants are relatable to dissolution mechanisms at the FeS crystal surface. k[...] is envisioned to arise from an attack at the surface by H[superscript +]. k[...] is linked to a mechanism which relies upon normal thermal vibrations and H20 solvation effects to liberate lattice constituents. Both k[\u2026] and k[\u2026] are thought to reflect a rate-limiting departure of surficial S(-II) lattice constituents. The activation entropies [...] for these two processes are calculated accordingly. The activation enthalpies [\u2026] were determined from the E[subscript a] data. Concentrations of Cu(II) in the ppm range were found to strongly inhibit the dissolution rate.\r\n\r\nThe k[...] term would dominate at pH's typical of natural waters. This term was used to determine how quickly FeS dissolves when anaerobic sediments are disturbed. When anoxic and oxic sediments are adjacent to one another, the dissolution of FeS in the anoxic portion takes place primarily near the oxic front.\r\n\r\nIf FeS-containing anaerobic sewage sludge is discharged to anoxic receiving waters, the particles of FeS may be expected to dissolve. Expressions were developed by which the lifetimes of such particles may be calculated. Both transport and non-transport controlled situations are analyzed. The effects which the agglomeration of such FeS with other sewage particles would have on the dissolution rate were considered. The implications which this work holds for sediment pyritization rates were also discussed.\r\n", "doi": "10.7907/31FS-0Q87", "publication_date": "1979", "thesis_type": "phd", "thesis_year": "1979" }, { "id": "thesis:17854", "collection": "thesis", "collection_id": "17854", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02032026-160531236", "primary_object_url": { "basename": "Claudio_AR_1979.pdf", "content": "final", "filesize": 29656937, "license": "other", "mime_type": "application/pdf", "url": "/17854/1/Claudio_AR_1979.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Acetylcholine Receptor and its Role in Induction of Experimental Autoimmune Myasthenia Gravis", "author": [ { "family_name": "Claudio", "given_name": "Antonia R.", "clpid": "Claudio-Antonia-R" } ], "thesis_advisor": [ { "family_name": "Raftery", "given_name": "Michael A.", "clpid": "Raftery-M-A" } ], "thesis_committee": [ { "family_name": "Raftery", "given_name": "Michael A.", "clpid": "Raftery-M-A" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "I. Rabbit antibodies were produced against purified acetylcholine receptor\r\n(AcChR) and each of the four acetylcholine receptor subunits from Torpedo\r\ncalifornica. Using the technique of double immunodiffusion in agar, specific- and\r\ncross-reactivities were observed between these antibodies and purified acetylcholine\r\nreceptor and receptor subunits from Torpedo californica, Torpedo marmorata,\r\nTorpedo nobiliana, and Narcine brasiliensis. The specificity of each of the four\r\nanti-subunit antibodies was shown, suggesting the lower molecular weight polypeptide\r\nchains of the AcChR were not degradation products of the higher molecular\r\nweight polypeptide chains. The study also demonstrated conservation of AcChR\r\nand AcChR subunit antigenic determinants in the four electric rays investigated.
\r\n\r\nII. Experimental autoimmune myasthenia gravis (EAMG) has been induced\r\nin a wide variety of animals using AcChR purified from a variety of electric organ\r\nand muscle sources. Electrophoresis of sodium dodecyl sulfate (SDS) polyacrylamide\r\ngels heavily loaded with purified AcChR often reveals the presence of minor\r\ncontaminants. To test if these contaminants or any other components present\r\nin Torpedo californica AcChR preparations could induce EAMG, solubilized T.\r\ncalifornica membrane fragments were depleted of AcChR by passage over an\r\na-bungarotoxin resin and then injected into Lewis rats in an attempt to induce\r\nEAMG. The results demonstrated that some of the minor contaminants present\r\nin purified AcChR preparations were antigenic but EAMG could not be induced\r\nwith preparations enriched in these contaminants or containing other T. californica\r\nnon-AcChR components.
\r\n\r\nIII. Antisera prepared in rabbits and Lewis rats against Torpedo californica\r\nAcChR (purified and denatured to various degrees) were tested for the ability\r\nto inhibit [125I] \u03b1-bungarotoxin (\u03b1-BuTx) binding to native and detergent solubilized\r\nT. californica AcChR. Similar inhibition studies were performed using antisera\r\nand antigen-binding fragments (Fabs) directed against each of the four isolated\r\nAcChR subunits. None of these antisera or Fabs could inhibit \u03b1-BuTx binding\r\nto native AcChR. Antisera and Fabs directed against AcChR could inhibit a\r\nmaximum of 50% \u03b1-BuTx binding to solubilized AcChR. The results using Fabs\r\nindicated the inhibition was not due to antibody-mediated aggregation of AcChR\r\nmolecules. A strong correlation was seen between animals with EAMG and the\r\nability of their antisera to inhibit 5096 of \u03b1-BuTx binding to AcChRs. The results\r\nindicated that particular antigenic determinants on AcChRs could induce EAMG\r\nand that these determinants were lost with SDS denaturation of AcChR.
", "doi": "10.7907/pm26-ex97", "publication_date": "1979", "thesis_type": "phd", "thesis_year": "1979" }, { "id": "thesis:3043", "collection": "thesis", "collection_id": "3043", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-082425", "primary_object_url": { "basename": "West_cm_1978.pdf", "content": "final", "filesize": 11011063, "license": "other", "mime_type": "application/pdf", "url": "/3043/1/West_cm_1978.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "(1) Glycoproteins and Development in the Cellular Slime Mold Dictyostelium discoideum. (2) Separation of Cells Using Isopycnic Centrifugation in Linear Density Gradients of Colloidal Silica", "author": [ { "family_name": "West", "given_name": "Christopher Mark", "clpid": "West-Christopher-Mark" } ], "thesis_advisor": [ { "family_name": "McMahon", "given_name": "Daniel", "clpid": "McMahon-D" }, { "family_name": "Dreyer", "given_name": "William J.", "clpid": "Dreyer-W-J" } ], "thesis_committee": [ { "family_name": "McMahon", "given_name": "Daniel", "clpid": "McMahon-D" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Delbruck", "given_name": "Max", "clpid": "Delbr\u00fcck-M" }, { "family_name": "Dreyer", "given_name": "William J.", "clpid": "Dreyer-W-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "I: Two methods were adapted for the study of glycoproteins in Dictyostelium discoideum. Both techniques relied upon prior separation of glycoproteins on one- or two-dimensional SDS-polyacrylamide gels. The first method was a modified form of crossed immunoelectrophoresis which substituted the carbohydrate-binding protein (lectin) concanavalin A (Con A) as the precipitating agent. In the second method, polyacrylamide gels were simply fixed and incubated in the presence of fluorescein-tagged lectins. After washing away free lectin, the gels were photographed and stained for protein. Identified glycoproteins were defined by their apparent molecular weights, identity and, anomeric linkage of some of their monosaccharides and in two-dimensional gels, their apparent isoelectric points. The ability of these techniques to specifically identify authentic glycoproteins was confirmed using a defined membrane system, the erythrocyte ghost, other known proteins, and hapten inhibitors. Of the two techniques, lectin diffusion was capable of higher resolution but did not give information about receptor multivalency. Both methods were more sensitive than the commonly-used periodic acid-schiff's base stain.\r\n\r\nMore than fifty different glycoproteins were detected in vegetative cells on the basis of their labeling with Con A or wheat germ agglutinin (WGA) and their sensitivity to proteolysis. These glycoproteins were distributed throughout the cell, giving each of several subfractions, including the plasma membrane, its own profile of glycoproteins. WGA receptors were apparently membrane bound and predominantly localized in the plasma membrane. A small number of glycoproteins were also detected with a galactose-binding protein, but these glycoproteins were not present in the plasma membrane or on the cell surface as determined by an independent technique. Receptors for L-fucose binding proteins were also absent from the plasma membrane of these cells. In contrast, another eukaryotic cell plasma membrane, the erythrocyte ghost, contained receptors to all lectins tested.\r\n\r\nDuring the formation of the pseudoplasmodium from vegetative cells, many glycoproteins were lost, modified, or many new ones were synthesized in all cell subfractions, including the plasma membrane. In addition, there was an asymmetry of distribution of glycoproteins in the pseudoplasmodium. There were three prestalk-cell specific glycoconjugates, which were all restricted to the plasma membrane, and two prespore-specific glycoproteins, which were present in plasma membranes as well as in a potential plasma membrane precursor. These plasma membrane differences occurred prior to overt differentiation of any stalk or spore cells. The region-specific glycoproteins were also present in the plasma membranes of cells which had not yet formed pseudoplasmodia, but were absent from vegetative cells.\r\n\r\nIn order to reinforce the temporal evidence that these region-specific glycoproteins were involved in the creation of the pseudoplasmodium, dissociated pseudoplasmodial cells were treated with the lectin which originally identified the region-specific molecules (WGA). Although dissociated cells typically reformed pseudoplasmodia, in the presence of WGA, they did not. This effect of WGA was blocked by a hapten inhibitor.\r\n\r\nII: In other work, separation of cells with different densities was attempted. When cells of Dictyostelium discoideum were centrifuged to density equilibrium in linear gradients of colloidal silica (Ludox), approximately 40 discrete bands appeared. A similar result was found for formalinized red blood cells and plastic beads. Isolated bands of cells rebanded faithfully in new gradients and band spacing depended upon gradient steepness. It was found that cell bands resulted from microscopic discontinuities in the linear gradients caused by centrifugation. When the gradients were analyzed in the analytical ultracentrifuge, absorbance scans revealed that cell bands coincided with \"bands\" of Ludox, which formed even without cells. Evidence ruling out other possible causes for cell bands is presented and procedures which avoid this condition are described.", "doi": "10.7907/NM0K-3F42", "publication_date": "1978", "thesis_type": "phd", "thesis_year": "1978" }, { "id": "thesis:17789", "collection": "thesis", "collection_id": "17789", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12072025-095838407", "primary_object_url": { "basename": "Pearson_WR_1977.pdf", "content": "final", "filesize": 58159711, "license": "other", "mime_type": "application/pdf", "url": "/17789/1/Pearson_WR_1977.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Studies on the Arrangement of Repeated Sequences in DNA", "author": [ { "family_name": "Pearson", "given_name": "William Raymond", "orcid": "0000-0002-0727-3680", "clpid": "Pearson-William-Raymond" } ], "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": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Parameters of repetitive DNA sequence organization have been\r\nmeasured in the rat, Drosophila and the pea genomes.
\r\n\r\nExperiments using melting, hydroxyapatite binding and single strand\r\nnuclease digestion have been used to measure the number length and\r\narrangement of repeated DNA sequences in the rat. About 20% of rat DNA\r\nis repeated 3000 fold. Half of the sequences are 200 - 400 nucleotides\r\nlong while the remainder are longer than 1500 nucleotides. Rat repeated\r\nDNA sequences are interspersed among 2500 nucleotide long single copy\r\nsequences.
\r\n\r\nStudies on the long and short repeated DNA sequences of the rat\r\nshow that the long repeated sequences are also 3000-fold repeated.\r\nCross-hybridization of isolated long and short repeated sequences and\r\nhydroxyapatite binding interspersion experiments indicate long and short\r\nrepeated DNA may share sequences, although this may be due to\r\ncross-contamination. Self-renaturation, melting and electron microscopy\r\nof long repeated DNA fragments suggest some long fragments may be\r\ncomposed of arrays of shorter repeated sequences.
\r\n\r\nA similar sensitive search has been made in Drosophila melanogaster\r\nfor short repetitive sequences interspersed with single copy sequences.\r\nFive kinds of measurements all yield the conclusion that there are few\r\nshort repetitive sequences in this genome: 1) Comparison of long and\r\nshort fragment reassociation kinetics; 2) reassociation kinetics of\r\nlong fragments driven by an excess of short fragments; 3) measurement\r\nof the size of repeated fragments after S-1 nuclease digestion; 4)\r\nmeasurement of the hyperchromicity of repeat sequence bearing fragments\r\nof different lengths; 5) direct assay by kinetics of reassociation of\r\nthe amount of single copy sequence present on 1200 nucleotide long\r\nfragments which also contain repetitive sequences.
\r\n\r\nRenaturation of pea DNA has been used to estimate the size of the\r\npea genome and the fraction of pea DNA containing repeated DNA\r\nsequences. Pea DNA renaturation and single copy tracer hybridization\r\nindicate the size of the pea genome is 0.45 pg. More than 70% of pea\r\nDNA is repeated from 100 to 5000 times.
", "doi": "10.7907/5v4r-gs82", "publication_date": "1977", "thesis_type": "phd", "thesis_year": "1977" }, { "id": "thesis:17772", "collection": "thesis", "collection_id": "17772", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11242025-222023123", "primary_object_url": { "basename": "Ready_DF_1977.pdf", "content": "final", "filesize": 32535867, "license": "other", "mime_type": "application/pdf", "url": "/17772/1/Ready_DF_1977.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Development of the Drosophila Retina", "author": [ { "family_name": "Ready", "given_name": "Donald Furner", "orcid": "0000-0003-3316-4207", "clpid": "Ready-Donald-Furner" } ], "thesis_advisor": [ { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" } ], "thesis_committee": [ { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Russell", "given_name": "Richard L.", "clpid": "Russell-R-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Pattern formation in the Drosophila retina proceeds\r\nby the recruitment of cells, along a morphogenetic front,\r\ninto a lattice. At the advancing front, marked by a dorso-ventral\r\nfurrow in the eye imaginal disc, cells are organized\r\ninto ommatidial precursors, each containing cells destined\r\nto become photoreceptors 2, 3, 4, 5, and 8. Behind the\r\nfront, a mitotic wave produces photoreceptors 1, 6, and 7,\r\nplus the remaining cells needed to complete the ommatidia.\r\nDuring the third larval instar, the front sweeps anteriorly\r\nacross the eye disc, leaving a highly ordered pattern in\r\nits wake. Preceding the dorso-ventral furrow is a groove\r\nthat bisects the eye disc into dorsal and ventral halves\r\nand presumably plays a role in establishing the equatorial\r\nsymmetry line.
\r\n\r\nCell lineage plays little role in pattern formation in\r\nthe eye. Genetic mosaics show that the cells of each ommatidium\r\nare not derived from a single mother cell; the\r\ncells appear to be recruited at random at the morphogenetic\r\nfront. Similarly, the mirror symmetry above and below the\r\nequator is not established by a clonal mechanism; a single\r\nclone can contribute cells to ommatidia on both sides of\r\nthe equator.
", "doi": "10.7907/2n5e-5q95", "publication_date": "1977", "thesis_type": "phd", "thesis_year": "1977" }, { "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" } ]