I. Electron Microscope Heteroduplex Analysis of DNA Sequences in F-Prime Factors. II. Electron Microscope Studies of λ and Mu Prophages. III. An Electron Microscope Study of Sindbis Virus RNA

Year: 1974 DOI: 10.7907/c22c-kg15
This thesis is composed of three parts. Part I is concerned with studies of the DNA sequences of several F-prime factors and the sequence relations among them using electron microscope heteroduplex methods. It was found that the DNA sequence of the F factor can be roughly divided into three regions: 1) a region about one-fourth of the molecule which is concerned with the fertility functions of the F factor. 2) a region about one-third of the molecule which is rich in A+T sequence and contains the sequences used to interact with the bacterial chromosome for the integration or excision of F factor DNA. J) a region which contains the genes for autonomous replication and female phage resistance and the structural element for conjugal transfer.

The structure of the bacterial DNA carried in several classical episomes, F100, F152, F8 and some of their derivatives was studied extensively. Bacterial markers between fep and uvrB were analyzed both genetically and physically. A method was developed to reconstruct the original episomes from their deletion variants. The results confirm the history that F100 and F152 were derived from the same Hfr. The formation of a new episome, F80, from F8 suggested that there is a hot spot in the E. coli chromosome for the recombination with F sequence between 93.2 and 94.5/0.0F.

In part II, the structures of λ and Mu prophages and Mu phage DNA were studied. The λ prophage carried in an F-prime factor was found by electron microscope heteroduplex analysis to be circularly permuted relative to the vegetative viral DNA. On the other hand, Mu prophage DNA was shown to be collinear with the viral DNA. The integrated Mu prophage DNA was used as a marker for physical mapping of bacterial genes in E. coli.

Sequence heterogeneity in Mu phage and prophage DNA's was also studied. The G loop heterogeneity was found to be present in both phage and prophage DNA.'s and was shown to be due to sequence inversion. The heterogeneous split ends sequences were found to be absent in the several prophages studied.

Part III contains an electron microscope study of viral RNA of Sindbis virus and a method for mapping poly A sequences in RNA molecules. Under weak denaturing conditions Sindbis virus RNA appears in circular form with a double stranded "handle" of about 250 nucleotides long. This implies that Sindbis RNA contains complementary sequences at or near the ends of the molecule.

A technique using glyoxal as a denaturing agent for mapping polyA sequences in RNA was developed. Glyoxal attaches to guanine base irreversibly and thus removes the secondary structure of RNA without inhibiting the renaturation capacity of polyA sequences in the molecule. A polyA sequence was found at or near one end of Sindbis RNA by this method.