The yeast mitochondrial genome encodes seven hydrophobic subunits of oxidative phosphorylation enzymes and Var1p, an essential protein in the small ribosomal subunit. Expression of the membrane proteins is dependent on nuclear, mRNA-specific regulatory genes, several of which specify translational activators that recognize sites within the 5$\sp\prime$-untranslated leaders (UTLs) of their target mRNAs. At the onset of this work, it was not known if the expression of the Var1p also requires mRNA-specific regulatory genes. To investigate this and other aspects of Var1p synthesis and function, I developed a novel system in which Var1p is supplied from a recoded gene in the nucleus (VAR1$\sp{u})$ and the VAR1 coding sequence in mtDNA is replaced by a recoded nuclear gene for an arginine biosynthetic enzyme (Arg8p), thus creating a reporter gene designated $var{\it 1\/}{:}{:}ARG{\it 8\/}\sp{m}.$ This system has been used to address the following objectives: (1) Genetic screens were conducted to identify nuclear mutants defective in $var{\it 1\/}{:}{:}ARG{\it 8\/}\sp{m}$ expression. One such gene, SOV1, was identified and cloned. SOV1 is specifically required for the stable accumulation of VAR1 mRNA. (2) To determine whether the targeting information for mitochondrial membrane proteins is contained in UTLs of their mRNAs, I examined the ability of chimeric mRNAs containing the VAR1 UTL to direct expression of COX2 and COX3. Although cells expressing these chimeric mRNAs synthesized both proteins, they were deficient in the accumulation of Cox2p and Cox3p. These data suggest that translation of Var1p is different from that of the membrane proteins, and support the physiological importance of interactions between the translational activators and the 5$\sp\prime$-UTLs of the COX2 and COX3 mRNAs for localizing synthesis of hydrophobic proteins to the inner membrane. (3) Heretofore, it has not been possible to produce respiratory competent $\lbrack rho\sp+\rbrack$ diploids by mating two $\lbrack rho\sp-\rbrack$ haploid petites. An explanation for this lack of functional complementation is that $\lbrack rho\sp-\rbrack$ cells are devoid of small ribosomal subunits and translation cannot be restored without a source of Var1p. I have shown that respiratory competent diploids can be obtained in crosses between two complementing $\lbrack rho\sp-\rbrack$ strains, but only when Var1p is supplied from $VAR{\it 1\/}\sp{u}.$

Genetic and physical mapping of human and animal genomes has been greatly facilitated by the use of chromosome specific DNA libraries. Mapping with libraries specific to a chromosome or chromosomal region increases marker saturation by reducing the gaps resulting from a purely random shotgun approach. This study was undertaken to construct a genetic and physical map of microsatellites on the chicken Z chromosome. This chromosome is the fifth largest in the chicken genome, comprising about 8% of the total, yet very few microsatellites have been mapped to it. DNA originating from the chicken Z chromosome was previously isolated and reported. This was used to construct a small insert library in Lambda ZAP Express, representing 14 chromosome equivalents. This library was screened for microsatellites with an (AC)12 oligo, and positive clones were isolated. Confirmation of the presence of the microsatellite, as well as its approximate location in the insert was accomplished by PCR amplification. Clones with adequate flanking regions were sequenced, and primers for 19 microsatellites were developed. These primers were used to genotype individuals from the East Lansing poultry reference population and a linkage map was constructed. Thirteen markers were scorable and polymorphic in the population. These were combined with 64 existing markers, and the resulting map spans 220 cM with an average spacing of 2.7 cM between markers. The physical location of selected markers were established by fluorescent in situ hybridization (FISH.) Hybridization results enabled the anchoring and orientation of the linkage group along the length of the Z chromosome.

Homologous recombination is generally considered a major mechanism by which cells repair many types of DNA lesions and damaged replication forks. However, if this process is left unchecked, cells often show a hyper-recombination (hyper-rec) phenotype, and are susceptible to large deletions, duplications, or inversions of important genetic information. This dissertation describes two projects aimed at examining molecular mechanisms by which cells regulate homologous recombination. The first shows several 3'-5' exonucleases prevent RecA-GFP loading by destroying potential substrates. It is shown that two genetic pathways exist: one consisting of ExoIII and another comprised of ExoVII, ExoIX, ExoX, and ExoXI. ExoI acts upstream of both of these pathways. Although xthA cells have an increase in DSBs and recB-dependent loading of RecA-GFP, they are viable with a recB mutation and do not display a large increase in SOS expression. The increase in RecA-GFP is also independent of base excision repair (BER). These experiments uncovered that DNA in a population of wild type cells undergoes DSBs and is often repaired in a RecA-independent manner after processing by ExoI and ExoIII. The second project shows the helicase, UvrD limits the number and intensities of RecA-GFP foci. This activity is due to the ability of UvrD to remove RecA from DNA where it is loaded in a RecF pathway-dependent manner. This activity requires ATP binding by UvrD, suggesting that helicase/translocase activity is important for RecA-removal. The hyper-helicase mutation, uvrD303 confers UV sensitivity to cells. Epistasis analyses showed uvrD303 is defective in the recA pathway of UV repair and not in nucleotide excision repair (NER). Surprisingly, UvrD303 does not directly remove RecA after UV, as new RecA-GFP foci appear like in wild type cells. UvrD303 does, however, slightly inhibit SOS induction, and constitutively activating the SOS response restores UV resistance to these cells in a way that is independent of recA overexpression. Furthermore, uvrD303 was capable of suppressing the constitutive SOS phenotype of recA730. These experiments suggested that UvrD303 antagonizes the ability of RecA filaments to induce the SOS response, rendering cells UV sensitive.

During the course of normal DNA replication, replication forks are constantly encountering "housekeeping" types of routine damage to the DNA template that may cause the forks to stall or collapse. One product of this fork collapse is the induction of the SOS response, a coordinated global response to help pause the growth and replication of a cell while DNA damage is addressed and repaired. In E. coli, this response is activated by the formation of ssDNA, to which the RecA protein binds and forms a nucleoprotein filament, which acts as the activator for autocleavage of the LexA transcriptional repressor, which normally represses expression of SOS genes. Damage responses are crucial to maintaining genomic integrity, and are therefore essential to all forms of life, and this type of regulatory system is highly conserved. However, cells have mechanisms for tightly regulating induction of these responses, and can often repair routine damage to their chromosomes without the need to induce SOS. This is chiefly evidenced by the observation that more than 20% of cells in a population have RecA filaments, but less than 1% are induced for SOS. How cells make this decision to induce SOS is the subject of this work. This dissertation describes three projects aimed at examining molecular mechanisms by which cells regulate RecA filaments, and therefore the decision to induce the SOS response. The first examines the disparity between the formation of RecA filaments, as evidenced by RecA-GFP foci, and the induction of SOS in the absence of damage, using a psulA-gfp reporter system. It is shown that there are three independent factors that repress SOS expression in undamaged E. coli cells. These are radA, the amount of recA in the cell, and in some circumstances recX. The first two limit SOS in wild type cells in the absence of external damage, while the third is an additional factor required in xthA mutants, likely due to the fact there are more RecA loading events in these mutants. These factors are thought to change the character and reduce the half-life and persistence of RecA filaments in the cell. The second project shows that suppression of SOS through the use of recA4162 and uvrD303 mutants is substrate and situation-specific. This specificity is demonstrated by the fact that, while both recA4162 and uvrD303 can suppress SOS in the SOS constitutive mutant recA730, recA4162 can only suppress SOS when the signal occurs at replication forks and not at any other place on the chromosome, while uvrD303 appears to suppress SOS with less specificity, and can suppress after UV (shown previously), at induced DSBs, and other places not directly at the replication fork. Here mutants of different replication factors are used that uncouple the replisome and induce SOS to a high degree. The third project determines the factors necessary for loading RecA filaments at the replication fork versus other locations on the chromosome when SOS is induced in the absence of damage, and helps elucidate further mechanisms for induction of SOS at these substrates. It is shown that the sbcB and recJ exonucleases assist in inappropriate RecA filament formation by substrate processing exclusively at replication forks, but not other substrates, likely through mechanisms that are reliant on the activities of the RecA loading factors RecBCD and RecFOR.

CSE1, an essential Saccharomyces cerevisiae gene was initially isolated in a screen for genes important for accurate chromosome segregation. cse1 mutants have pleiotropic phenotypes including defects in the ubiquitin-mediated degradation of mitotic cyclins and in cell cycle progression. SRP1, encodes a nuclear localization signal (NLS) receptor protein involved in nuclear protein import that is an allele specific dosage suppressor of cse1-1. CSE1 can rescue certain srp1 mutant phenotypes, indicating that Cse1p and Srp1p are functionally related or have roles in similar pathways. New mutant alleles of CSE1 were generated by linker insertion mutagenesis, including a temperature sensitive allele, cse1-2, that causes arrest in G2/M, chromosome missegregation and defective mitotic cyclin degradation. Analysis of CSE1 mRNA and Cse1p indicate that their levels do not change significantly during the cell cycle and that Cse1p is not phosphorylated. Cse1p is located in the nucleus and concentrated at the nuclear periphery, probably in association with nuclear pores. Current evidence supports the model that Cse1p is required for the export of Srp1p from the nucleus. Srp1p improperly accumulates in the nuclei of both cse1-1 and cse1-2 cells. Reporter proteins that contain NLS sequences accumulate in the cytoplasm of cse1-1 and cse1-2 cells indicating that Cse1p function is also necessary for nuclear protein import. Cse1p binds the nuclear transport protein Ran/Gsp1p-GTP via a conserved amino-terminal motif. In addition, the human protein CAS, which is an export factor for importin-$\alpha,$ is 60% similar to Cse1p. Although a physical interaction between Srp1p and Cse1p has not been shown, a two-hybrid screen identified two potential Cse1p-interacting proteins, Scj1p and Yma5p. Scj1p is a DnaJ homologue involved in protein translocation, folding, and complex assembly. Yma5p is a novel non-essential protein with an as yet unknown role in these important nuclear processes.

To study polyhydroxyalkanoate (PHA) accumulation and PHA regulation in Bacillus megaterium, transposon mutagenesis with Tn917-LTV1 was carried out and seventy two mutants that produce more or less PHA than normal were isolated and partially characterized. The chromosomal regions of B. megaterium flanking the Tn917-LTV1 insertions were cloned and sequenced from two PHA over-producing and six leaky mutants. The results showed that Tn917-LTV1 was less than ideal for generating a mutant bank due to the unavoidable occurrence of sibling transposants. Furthermore, the sequence data revealed that chromosomal deletions mediated by Tn917-LTV1 insertions were common. An alternative method was developed using a direct plating procedure and it was shown to circumvent these two problems. One PHA leaky mutant and one overproducing mutant were studied in more detail. The PHA leaky mutant, T4, had significantly reduced levels of PHA accumulation in all media tested and could not form spores. Chromosomal DNA, contiguous with the transposon was cloned and sequenced. Analysis of the sequence data showed Tn917-LTV1 inserted 24-bp upstream of an operon encoding a putative RNA helicase (deaD) gene and a nuclease (nucP) gene. A chromosomal target repeat 5$\sp\prime$-TATTT-3$\sp\prime$ was found on both sides of the insertion which indicated that no deletion was involved in the Tn917-LTV1 transposition. A plasmid carrying the intact RNA helicase operon was constructed and transformed into mutant T4. The transformant recovered the ability to form spores and accumulate wild-type levels of PHA in minimal media. Deletions in either DeaD or NucP did not complement the T4 mutant to the phenotype of VT1660. The results show that both DeaD and NucP are involved in PHA accumulation and are required for spore formation. The DeaD protein of B. megaterium is the first protein in the DEAD-box helicase family that is not essential for the organism. The fact that the growth rate of mutant T4 and VT1660 were similar in both LB and minimal media indicates that this DeaD protein may target specific mRNA, such as those of pha and spo genes. B001S, a PHA over-producing mutant, unlike its parental strain VT1660, produced large quantities of PHA in rich media. The chromosomal DNA flanking the transposon was cloned from both sides of the insertion. The 8.4 kilobase pairs of chromosomal sequence from left end (IR-L side) of Tn917-LTV1 coded for sixteen open reading frames (ORFs). Ten putative products of the sixteen ORFs shared sequence homology with known gas vesicle proteins (Gvp). The 8.4-kb fragment and its deletion derivatives were cloned into pBluescriptIISK and the plasmids were transformed into E. coli. Gas vesicles were formed and observed by phase contrast microscope, differential interference contrast microscopy and electron microscopy. The deletion analysis and sequence comparison with known gas vesicle proteins suggested 14 out of the 16 ORFs formed the gvp operon of B. megaterium VT1660. The deletion analysis showed that 11 genes are the maximum required for the gas vesicle formation in E. coli. The E. coli cells containing gas vesicles showed increased buoyancy. This is the first time that a functional organelle has been transferred to E. coli.

Cse4p is a centromere-specific chromatin protein with a histone-fold domain that is greater than 60% identical to histone H3 and the mammalian centromere protein, CENP-A. Cse4p has similar biochemical properties to H3 and is believed to replace H3 in centromere-specific nucleosomes in yeast. To identify residues in the histone-fold domain of Cse4p that function in specifying centromere structure and function, amino adds that differ between Cse4p and H3 were systematically changed to analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The severity of the phenotypes exhibited by the histone-fold domain mutants correlates directly with the number of putative DNA contact sites changed or alterations in regions implicated in histone interactions. These results indicate that the histone-fold domain of Cse4p functions through a cooperative mechanism in which residues throughout the histone-fold domain recognize centromere DNA. To investigate the relationship between the histone-fold domain of Cse4p and centromere DNA, chromosome loss rates were measured in double mutants carrying both a mutation in Cse4p and chromosomes with mutant centromere DNA. The Cse4p mutants had conserved changes in which analogously positioned residues in H3 were substituted for Cse4p residues at sites in the histone-fold domain that are in close proximity to the DNA. Mutations throughout the histone-fold domain caused significant increases in chromosome loss rates of chromosomes carrying conserved centromere DNA elements CDE I and II but showed no effect with CDE III mutant centromeres. This genetic evidence strongly supports direct interactions between Cse4p and CDE I and CDE II centromere DNA elements. The results are discussed in the context of the known structure of H3 and models are proposed which best describe the path of the centromere DNA around a Cse4p variant nucleosome that relies on critical contacts between CDE I and II, but not CDE III.

During the course of DNA replication, replication forks often stall or collapse as they proceed from oriC to the terminus due to housekeeping types of DNA damage or proteins bound to DNA. If the DNA is not repaired or if the replication forks do not restart, viability of the cell then becomes compromised. In Escherichia coli, if DNA damage is detected, approximately 40 genes are expressed to repair the offending DNA lesion. This is known the SOS response. Two proteins RecA and LexA regulate the SOS response, where RecA (when bound to ssDNA), serves as the sensor for DNA damage and LexA serves the repressor for SOS expression. When the RecA nucleoprotein filament forms, this complex will accelerate autocleavage of LexA inducing the response. Recently it has been observed that in a population of cells approximately 15% of the population had RecA bound to DNA, however at any given time approximately 0.3% of the population is induced for SOS expression suggesting that the cell can decide whether induce the SOS response or not. The aim of this work is to understand how the cell decides whether or not to express the SOS response at housekeeping types of DNA damage. Regulation is important because the cell would not want to express the SOS response every time replication forks encounter housekeeping types of DNA damage. The first component of this work looks at SOS expression in populations of cells during log phase growth using the fluorecense microscopy and the transcriptional fusion sulA-gfp. Results show that a SOS expression is stochastic and occurs in a small population of wild type cells. The second component of my work focuses on how the cell decides when to express the SOS response by using recA constitutive mutants that are defective in this regulation. Results show that the concentration and conformation of the RecA nucleoprotein filament is crucial for this to occur. Lastly novel recA mutants were created and examined for their role in suppressing constitutive SOS expression. It is observed that suppression of constitutive SOS expression could be seen when these mutations were supplied in cis and in trans, suggesting multiple levels of SOS regulation.

p53 protein is considered a major player in maintaining the genomic integrity. It regulates cell cycle, recognizes damaged DNA, and promotes apoptosis of cells that are defective or developmentally programmed for removal. Most cancers, including breast cancer, lose genomic integrity and have impaired p53. Li-Fraumeni patients and BALB/c mice bearing germline mutations in one allele of the p53 tumor suppressor gene develop mammary tumors. The p53 pathway is also required for the protective effect of pregnancy on breast cancer. Therefore, identifying the cellular pathways that regulate p53 will provide improvements in assessing breast cancer risk in individuals as well as therapeutic targets. The function of p53 could be regulated directly by Mdm2 and Mdm4. While Mdm2 is clearly a negative regulator of p53, the roles of Mdm4 on p53 are still not fully defined. Transgenic mice which over-express Mdm4 specifically in mammary gland were used to investigate the effects of Mdm4 on p53 function. Ovarian hormones also regulate p53 activity, but through indirect mechanisms. Oligonucleotide-based transcriptional profiling was conducted to identify mechanisms, by which estrogen and progesterone enhance p53 activity in mammary epithelial cells. The results from these studies showed that Mdm4 is unlikely to be a primary regulator of p53 functions in mammary epithelium. In contrast, expression profiling revealed groups of genes that are associated with the sensitization of p53 by estrogen and progesterone as well as tamoxifen and progesterone. These results implicate a common pathway used to sensitize p53 which involves proteins in the extracellular matrix.

Loss of p53 function can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53 +/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c-Trp53 +/−females, but are rare in C57BL/6-Trp53 +/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) is significantly more common among tumors arising in BALB/c-Trp53+/− mice compared to C57BL/6J-Trp53+/− mice. This increased LOH in BALB/c-Trp53+/− tumors was shown to be due to recombination events. The BALB/c strain has been shown to have a deficiency in non-homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis was that BALB/c-Trp53 +/− mice are more susceptible to mammary tumors due to impaired Homologous Recombination Repair (HRR) leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c-Trp53 +/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J- Trp53+/− MEFs. Similarly, co-localization of H2AX and the homologous recombination protein RAD51 remain at dsbs longer in BALB/c-Trp53+/− MEFs compared to C57BL/6-Trp53+/− MEFs. Palb2 , a gene that lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer, was chosen as an initial candidate gene. No coding SNPs or expression differences of Palb2 were found in the mammary glands between the two strains. Additional fine mapping and use of a filtering criteria in the SuprMam1 region yielded an additional 34 candidate genes. We demonstrate no significant differences in any of these genes in whole mammary glands and primary mammary epithelial cells between the two strains. Finally, using a congenic mouse strain, we demonstrate the lack of irradiation (IR) sensitivity alleles within the SuprMam1 region. These results suggest a possible defect in HRR in the BALB/c strain that is unlikely related to Palb2. The gene or genes responsible for increased mammary tumor incidence in the BALB/c-Trp53+/− remain to be identified.