This report describes the development and use of an expression vector system based on the single-stranded DNA bacteriophage M13. A derivative of M13mp8, designated M13mp8/P, was prepared in which the promoter and N-terminal codons of bacterial genes may be fused to a portion of β-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes, and to determine the mutational changes by dideoxy sequence analysis. The feasibility of this method was demonstrated by identification of a large number of mutations in the regulatory regions of two genes, recA and lexA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. My results suggest that this method will be generally useful for mutational analysis of transcriptional and translational regulatory elements. The mutants that were isolated by the above approach were used to investigate the specificity of LexA protein binding by quantifying the repressibility of a several mutant recA and lexA operator/promoter regions fused to the E. coli galactokinase (galK) gene. The results of this analysis indicated that two sets of four nucleotides (terminal nucleotide contacts), one set at each extreme end of the operator, are most critical for repressor binding. In addition, our results indicate that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator had comparable effects on repressibility. The inferred symmetry of the interaction justified the reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus: (5') CTGTATAT. Although the first four positions of this half-site sequence have the greatest effect on LexA repressor binding, the last four are well conserved among binding sites and appear to modulate repressor affinity. The role of the terminal nucleotide contacts and the mechanism by which the internal sequences affect repressor binding is discussed.

Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity from bacteria that were defective in uracil-DNA glycosylase (Ung). After cloning the dug gene, recombinant Dug was overexpressed, purified, and characterized with respect to activity, substrate specificity, product DNA binding, and mechanism of action. Purified Dug excised both uracil and ethenocytosine specifically from double-stranded DNA substrates. One distinctive characteristic of Dug was that the purified enzyme removed a near stoichiometric amount of uracil from DNA containing U/G mispairs. The observed lack of turnover was attributed to tight binding of Dug to the apyrimidinic-site (AP) contained in the DNA reaction product. Catalytic activity was stimulated in the presence of E. coli endonuclease IV that caused AP-site incision and dissociation of Dug. By using enzyme complementation experiments, Dug was shown to initiate uracil-initiated base excision repair (BER) in E. coli (ung) cell-free extracts. The relative rate of repair of uracil- and ethenocytosine-containing DNA in isogenic E. coli cells that were proficient or deficient in Ung and/or Dug was measured using a novel competition assay. Complete ethenocytosine-initiated BER displayed an absolute requirement for Dug and occurred at the same rate as uracil-initiated BER in the presence of both Ung and Dug. However, the rate of Dug-mediated ethenocytosine-DNA repair was 8-fold faster than that of uracil-DNA mediated by Dug. The distribution of BER patch sizes associated with both uracil- and ethenocytosine-containing DNA showed similar results. In both cases, DNA repair synthesis utilized predominantly a long patch BER mechanism involving the incorporation of 2-20 nucleotides. A previously unidentified "very long patch" mechanism of BER involving the incorporation of more than 200 nucleotides was identified and shown to be mediated by DNA polymerase I. The rate-limiting step associated with uracil-initiated BER was found to involve DNA ligase and the distribution of BER patch size was modulated by the ratio of DNA polymerase I and DNA ligase. The fidelity of DNA repair synthesis associated with complete uracil-DNA BER was measured using E. coli cell-free extracts that were proficient or deficient in Ung activity and determined to be 5.5 x 10������ and 19.7 x 10������, respectively. / Graduation date: 2003

Graduation date: 2000

Graduation date: 2000

Graduation date: 1996

The translocation of the T7 genome into the cell is a multistep process. Following adsorption, approximately 850bp of the 40kb linear genome is internalized to expose host-specific promoters in the leading end to transcription components. There are three strong early promoters, PA1, PA2, and PA3 on this leading 850 bp. Further T7 genome internalization is coupled to transcription and I have measured internalization rates to characterize the rate of transcription by E. coli RNA polymerase in vivo. E.coli RNAP internalizes the entire 40kb and distal parts of the genome are internalized nearly as efficiently and at the same rate as the leading end. I have shown that processivity is dependent on the antitermination element boxA, located 63 bp downstream from PA3, and on only one of the three early promoters. However, when any one of boxA, PA3, or the host antitermination factor nusB is mutated the efficiency, rate, and apparent processivity of transcription -- and thus the efficiency of genome internalization are all significantly reduced. The PA3 promoter, boxA, and E. coli nusB are all non-essential for T7 growth, but they confer a fitness benefit to wild-type phage by increasing the rate of genome internalization. In T7, the minimal requirement for antitermination is promoter PA3 and the boxA sequence. I have found that transcripts initiating at PA1 and PA2 are not effectively antiterminated by boxA, however those from PA3 alone do. Upon further investigation it was shown that there is a requirement for sequences upstream of the -35 hexamer of PA3 to confer full antitermination. After T7 expresses its own single-subunit RNA polymerase, bacteriophage T7 must shutoff host transcription via the phage proteins gp0.7 and gp2. In the absence of host RNAP shutoff, T7 DNA is degraded and the infection fails. I have found that the absence of either promoter PA3 or boxA,gene 2 is unnecessary for growth. These results argue the target for shutoff is actually antiterminating transcription. / text

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein), which binds the intron RNA to promote RNA splicing and intron mobility. Mobility occurs by intron RNA reverse splicing directly into a DNA strand and reverse transcription by LtrA. I used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to the cellular poles in both Escherichia coli and L. lactis. This polar localization occurs with or without co-expression of the intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three non-overlapping LtrA subsegments, reflecting dependence on common redundant signals and/or protein physiochemical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. In E. coli, the Ll.LtrB intron inserts preferentially into the chromosomal ori and ter regions, which are pole localized during much of the cell cycle. Thus, the polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in these regions. I established a high throughput method using cellular array and automated fluorescence microscopy for screening transposon-induced mutants, and identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) in which disruptions result in increased proportion of cells having diffuse LtrA distribution. This altered localization is correlated with a more uniform distribution of Ll.LtrB insertion sites throughout the E. coli genome. Finally, I find that altered LtrA localization in all five disruptants is correlated with accumulation and more diffuse intracellular distribution of polyphosphate, and that a ppx disruptant, which also results in polyphosphate accumulation, shows similar LtrA mislocalization. These findings may reflect interaction between LtrA and intracellular polyphosphate. My findings support the hypothesis that the intracellular localization of LtrA is a major determinant of Ll.LtrB insertion site preference in the E. coli genome. Further, they show that alterations in polyphosphate metabolism can lead to protein mislocalization, and suggest that polyphosphate is an important factor affecting intracellular protein localization.

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy