In the course of thymocyte development, progenitor T cells from the bone marrow migrate to the thymus where they are positively or negatively selected depending on specific molecular interactions. This selection process takes place at a developmental stage where immature thymocytes are exquisitely sensitive to glucocorticoid exposure and they readily succumb in a process referred to as apoptosis. Glucocorticoid-induced thymocyte apoptosis has been shown to require RNA and protein synthesis, which indicates an active death-inducing process. One molecular event that occurs in immature thymocytes during this process is the rapid and high induction of a glucocorticoid-induced gene called GIG18. Within this dissertation, I identify a GIG18 open reading frame (ORF), that encodes a ∼50KDa protein, that is 93% conserved between mouse and human. By expressing this ORF with a 6X-Histidine tag, I was able to purify the GIG18 protein on a nickel affinity column to generate a rabbit polyclonal antibody. The western blot showed glucocorticoid-regulated induction of two protein bands, a mildly induced band at the predicted size (50KDa) and a highly induced band at ∼68KDa. Further analysis of mouse and human expressed sequence tags (ESTs) indicated that alternate transcripts produced from this gene, are likely account for the 68KDa band. The mouse GIG18 ORF identified in our lab maps to human chromosome 7 and contains 8 exons covering ∼250 Kb. This region of human chromosome 7 corresponds to homologous sequence on mouse chromosomes 5 and 6. I also show that GIG18 belongs to family of three genes encoded in the human genome with two extensive regions of conserved sequence. Expression analysis of the GIG18 ORF in thymocytes and HeLa cells did not reveal any obvious cellular morphological variations or functions induced by this gene. I conclude this dissertation with a discussion of potential roles that GIG18 may play within the context of thymocyte development.

Herpes Simplex Virus type 1 is an important organism not only because it is a member of a family of disease-causing organisms, but it also serves as a model organism for the study of eukaryotic DNA replication. Here I use HSV-1 to investigate two aspects of DNA replication: initiation and proofreading. Cellular factors have been shown to be involved in DNA replication, and especially in initiation, in a variety of viral systems. Previous studies have identified the first cellular factor implicated in initiation of HSV-1 replication, OF-1. In this study, I have purified OF-1 and investigated its composition, binding properties and interactions with the viral origin binding protein UL9. I show that OF-1 is composed of two subunits, one of which contains DNA binding activity. I also found that OF-1 binds specifically to both single- and double-stranded origin DNA, that OF-1 binds most tightly to single-stranded DNA, and that OF-1 shows a preference for which strand is bound. I have demonstrated that, in the presence of UL9, OF-1 exhibits a higher affinity for its target DNA and that OF-1 inhibits the ATPase activity of UL9. I propose that UL9 binds to the origin of replication, loads OF-1 to the origin, and then is displaced by OF-1. Further implications for this model are discussed. I go on to investigate several aspects of error control in the wild type and a 3'-5' proofreading exonuclease mutant DNA polymerase from HSV-1. Proofreading is a primary factor influencing the fidelity of DNA replication. Previous studies in our lab have shown that exonuclease deficient polymerases are incapable of supporting viral growth in vivo. In these studies, I have expressed and purified both wild type and mutant polymerases and investigated their biochemical properties as well as the mechanism of lethality of the mutant. I have found that the mutant polymerase exhibits substantially elevated rates of nucleotide misincorporation as compared to the wild type. In addition, the mutant polymerase is seen to stall at a misincorporation, exhibiting a reduced ability to replicate past a mismatch. Based on these findings, I suggest that the inability of the mutant polymerase to replicate past a misinsertion is the primary cause of the reduced viability of viruses carrying the mutant enzyme.

The Euglena gracilis chloroplast genome contains over 150 introns, including the only known examples of group III introns. Group III introns may belong to the intron super-family consisting of group II and nuclear pre-mRNA introns. It has been suggested that group III intron evolution may have paralleled that of nuclear pre-mRNA introns through loss of domains from a group II-like ancestor. Homologs of psbC14, a group III twintron containing the coding locus for the mat1 polypeptide, have been identified in 7 Euglenoids. All of the species examined contain both the group III twintron and the mat1 locus. As determined by comparative phylogenetic analysis, group III introns contain a structural homolog of group II intron domain VI. The mat1 loci encode peptide motifs characteristic of group II intron maturases. A group III intron-encoded protein whose predicted sequence is similar to group II intron-encoded maturases, and a bona fide domain VI within group III introns, are compelling evidence for a common ancestor of group II and group III introns. A novel mixed operon, psbK-ycf12-psaM-trnR, has been identified in the photosynthetic protist, E. gracilis. The tetracistronic transcripts are processed through endonucleolytic cleavage of the intergenic spacers and splicing of two group III introns and a group III twintron to produce three monocistronic mRNAs and a tRNA. The psbK operon was cloned by PCR amplification from 9 related Euglenoid species. In each species, gene order and content is conserved. The psbK operons contain phylogenetically conserved eubacterial promoter, translational, and 3' processing elements. Based on comparison of homologous intron content to phylogenetic analysis, group III intron evolution within the Euglenoid lineage is more complex than previously believed. Surprisingly, based on psbCi4 and psbK intron distribution data, group III introns may pre-date group II introns in the Euglenoid lineage. An aadA transformation cassette was adapted for expression in Euglena gracilis chloroplasts. Transgenic DNA was introduced into E. gracilis chloroplast by biolisitc transformation and stably maintained as an episomal element during continuous antibiotic selection. When a truncated E. stellata psbK operon was introduced into E. gracilis chloroplasts, transgenic DNA was transcribed and the resultant pre-mRNA accurately spliced.

Regulation of mRNA stability and mRNA translation are important components of gene expression within the eukaryotic cell. Multiple observations have suggested that the processes of translation and mRNA turnover are interrelated. Based on these observations, and the fact that the translation initiation machinery and the decapping enzyme both utilize the cap structure as a substrate, I hypothesized that these processes might be linked due to a competition at the cap between the cap binding complex and the decapping enzyme. Since disruption of translation using translational inhibitors or insertion of strong secondary structure within the 5' UTR affects the stability of mRNAs, I asked whether mutations within the translation initiation machinery itself would have a similar effect. I found that mutations in many different translation initiation factors led to an increase in the rate of mRNA turnover within the yeast cell. It was found that when the process of translation initiation is impaired in this manner that the rates of both deadenylation and decapping are increased. These results imply that the nature of the translation initiation complex bound to the 5' end of the mRNA is a critical component in determining mRNA half-life. One of the translation initiation factors, the cap binding protein eIF4E, is a logical candidate for a protein that might compete with the decapping enzyme for the cap structure. I purified the decapping enzyme and the cap binding protein from yeast and showed that addition of eIF4E could block decapping by the Dcp1p in an in vitro decapping assay. In addition, this inhibition was dependent on eIEF4E's ability to bind to the cap structure. This observation was then recapitulated in vivo by showing that an allele of eIF4E, which is unable to bind the cap structure, could suppress the decapping defect of the partially functional dcpl-1 allele. This same allele could not suppress the decapping defects of a lsm1Delta or pat1Delta, other mutations which affect decapping. These results argue that the translation initiation machinery acts as a physical block to the decapping enzyme and that decapping is composed of at least two genetically separable steps.

Apoptosis is characterized by a series of well defined morphological and biochemical features that allow cells to initiate self-destruction in response to a variety of stimuli. CD4⁺CD8⁺ is a sub-population of immature thymocytes that are especially prone to the action of apoptosis-inducing agents and are sensitive to glucocorticoid-induced apoptosis, an event that plays a critical role in eliciting the antigen-specific thymocyte repertoire. Glucocorticoids induce apoptosis through activation of the GR, a ligand-induced transcription factor that transduces the hormonal signals into the regulated expression of target genes. While much is known about the structure and function of GR, key steroid-regulated genes believed to be required for thymocyte apoptosis have not been found. Based on the transcriptional-regulation of apoptosis by ecdysone-mediated induction of reaper gene expression in Drosophila, and p53-mediated transcriptional-activation of Bax gene expression in mammalian cells, our laboratory has set out to identify GR-regulated genes in a glucocorticoid-sensitive mouse thymocyte cell line called WEHI 7.2. Identification of such genes involved in this process, and elucidation of the underlying mechanisms by which they mediate apoptosis, have prime importance because malfunctioning of this system has been shown to cause severe human diseases and developmental abnormalities. I have isolated and characterized the full-length nucleotide sequence of mGIG1, a novel gene product that had previously been shown to be up-regulated in thymocytes undergoing glucocorticoid-induced apoptosis. The human homolog of the mouse GIG1 was also identified and mapped to chromosome 8, band q21. A peptide segment of GIG1 protein was expressed in bacteria and used to raise antibodies in rabbits in order to dissect its role in thymocyte apoptosis. Results of DNA sequence analyses suggest that mGIG1 may encode a DNA-binding protein that could function as a transcription factor in thymocytes. In a second project, I established a transient expression cloning system in mouse thymoma cells and isolated a cDNA sequence called mArg-2 on the basis of a functional cloning strategy. However, determination of the possible involvement of both GIG1 and Arg-2 in thymocyte apoptosis will require further experimentation. Finally, spatial and temporal coordination of abrupt changes in mismatch repair (MMR) gene expression with occurrence of apoptosis in mouse thymocytes prompted me to study apoptosis in MMR-deficient mice. My findings indicate that glucocorticoid and anti-Fas antibody-induced cell death machinery is intact and functional in MMR⁻/⁻ mice.

To investigate the regulation of calpastatin gene expression, bovine heart calpastatin cDNAs and 5' regions of the calpastatin gene were isolated. Analysis of 5' cDNA sequence identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site. Conceptual translation from this upstream AUG produces a protein containing 68 additional N-terminal amino acids. This "XL" region contains three potential PKA phosphorylation sites but shares no homology with other regions of calpastatin or with any known protein. Immunoblot studies demonstrated that heart and liver contain a calpastatin protein of 145 kDa on SDS PAGE that comigrates with full length bacterially-expressed calpastatin and calpastatin produced by coupled in vitro transcription-translation from the upstream AUG. An antibody raised against the XL region recognized the 145 kDa band, demonstrating that the upstream AUG is utilized and that the 145 kDa band represents full length calpastatin protein in vivo. The organization of the calpastatin 5' genomic region was determined by comparing calpastatin cDNA and genomic sequences. The region encompassing exon 1-4 contains large introns and spans at least 60 kb. Calpastatin promoter sequence analysis revealed that it belong to the family of "house keeping" genes which lack TATA box and are GC rich at the proximal promoter regions. Transient transfection assays demonstrated that sequence within 272 nucleotides upstream of transcription initiation of the calpastatin gene is sufficient to direct moderate level transcription. Promoter sequences further upstream act to inhibit and stimulate transcriptional activity. Exposure of transfected cells to dibutryl cAMP resulted in a seven to twenty fold increase in calpastatin promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion and mutation analyses identified a cAMP responsive element at nt-76. These findings demonstrate that calpastatin gene and protein are both targets for cAMP-dependent kinase activity. beta-Agonist treatment can induce both calpastatin gene transcription and protein phosphorylation.

Control of mRNA degradation is an important step in the regulation of gene expression. In Saccharomyces cerevisiae, pathways of mRNA decay have been determined and have provided a framework for understanding how mRNA decay is controlled. I have studied how the process of translation affects the decay mechanism of a yeast transcript and I have isolated and characterized yeast mutants that exhibit reduced rates of mRNA decay. The process of translation has been shown to affect mRNA decay rates in eukaryotes. However, using a MFA2 mRNA that cannot be translated due to insertion of secondary structure in its 5' untranslated region, I have determined that translation of the MFA2 mRNA is not required for its degradation. This observation demonstrates that translation of an mRNA, per se, is not required for the normal kinetics or mechanism of mRNA decay. Additionally, I have demonstrated that the translational inhibitor, cycloheximide, reduces the rate at which the MFA2 transcript is decapped. Inhibition of decapping occurs even on MFA2 transcripts that cannot be translated due to insertion of secondary structure. This result suggests that the general stabilizing effects of translational inhibitors on mRNAs may not be due to the inhibition of translation of these transcripts. The identification of mRNA decay pathways in yeast, deadenylation-dependent decapping and deadenylation-independent decapping, provided a basis by which gene products required for mRNA decay through these pathways could be identified. To this end, a screen of mutant yeast strains was undertaken. I have isolated and characterized two mutants, mrt1 and mrt3, that exhibit reduced rates of deadenylation-dependent decapping on several yeast transcripts. This result suggests that the MRT1 and MRT3 gene products promote deadenylation-dependent mRNA decapping. A third mutant, dcp1, was also isolated, and the wild-type DCP1 gene was identified. Characterization of dcp1/ mutants by myself and others revealed that the DCP1 gene encodes the decapping enzyme, or an essential component of the decapping enzyme, required for both deadenylation-dependent and deadenylation-independent mRNA decapping. This result demonstrates that the DCP1 gene product, Dcp1p, is required for all known mRNA decapping in yeast.

The process of mRNA turnover is a critical component of the regulation of gene expression. In the past few years, a discrete set of pathways for the degradation of polyadenylated mRNAs, in eukaryotic cells have been described. The major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which primarily leads to a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic degradation. A critical step in the primary pathway is decapping, since it effectively terminates the mRNA's existence and is the site of numerous control inputs. I discuss the properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover. The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. In this work, I demonstrate by several criteria that CCR4 and CAF1 encode critical components of the major cytoplasmic deadenylase in yeast. First, both Ccr4p and Caf1p are required for normal mRNA deadenylation in vivo. Second, both proteins localize to the cytoplasm. Third, Caf1p co-purifies with poly(A) specific exonuclease activity, and this activity is dependent on the presence of Ccr4p. Interestingly, because Ccr4p and Caf1p have been shown previously to interact with transcription factors, these results suggest an unexpected link between mRNA synthesis and turnover. Both the Ccr4 and Caf1 proteins have significant homology to known exonucleases, in this work I demonstrate by several criteria that CCR4 encodes the catalytic subunit of the deadenylase. First, over-expression of Ccr4p rescues the deadenylation defects of a caf1Δ strain, indicating that Caf1p is not essential for deadenylation. Second, purification of Ccr4p co-purifies with poly(A) specific exonuclease activity, and this activity is not dependent on the presence of Caf1p. Third, point mutants in predicted catalytic residues of the Ccr4p exonuclease domain result in deadenylation defects in vivo and in vitro. The strong conservation of Ccr4p and Caf1p in other eukaryotes suggests that they will function in the process of deadenylation in other organisms.

Until now, CI tetramer cooperativity has been theorized to be critical for the wild type behavior of λ. Specifically, it was believed that a CI cooperativity mutant phage would not be able to grow lysogenically. Further, if it could be altered in some way to grow lysogenically, it was thought that the lysogen would not have the ability to switch to and/or complete lytic growth after induction. However, the work on which these theories were based was done with uncoupled systems. Then, inferences were drawn as to the role cooperativity played in the intact switch. While this research was groundbreaking, CI levels were unregulated since the regulatory network was disconnected from the action of CI. This is unrealistic since within the lysogen, CI levels are extensively coupled to CI activity via feedback. In this work, I study the role of CI tetramer cooperativity in an intact phage. In this way, CI levels remain coupled to the extensive regulation which controls them. The CI level is regulated by its own activity. To test the role of CI cooperativity in λ physiology, specifically in the genetic switch, three CI cooperativity deficient phage were made. None could form stable, single lysogens. Therefore, the CI Y210X mutations were combined with mutations in the O(R) region that should confer increased occupancy of O(R)2, allowing stable lysogeny. A CI cooperativity deficient phage was isolated that could successfully complete all three aspects of λ physiology: lytic growth, lysogenic growth, and a threshold response to the switch between the two. Therefore, CI cooperativity is not required for wild type λ physiology. In addition, a CI Y210F phage was isolated. CI Y210F has wild type cooperativity. However, phenylalanine cannot make the contacts described in the CI structure papers, indicating that the proposed details of the cooperativity contacts shown in the papers are likely to be unnecessary or possibly incorrect.

Our first aim was to generate transgenic mice to express beta-galactosidase reporter gene under the control of the two promoters. The model would allow us to study the temporal and spatial expression of PTH1R during the onset of embryonic endochondral ossification, and in the adult. Our transgenic animals would allow us to identify regulatory elements that are essential for tissue specific PTH1R expression. We have cloned 11 kilobases of mouse PTH1R gene promoter sequence containing four untranslated exons U1, U2, U3 and SS, and fused this to a Lac Z reporter gene, which was in turn fused to a 250 by fragment containing the A-rich polyadenylylation signal. Three additional constructs were made with deletion of transcription start sites in exon U1 (DeltaU1), U3 (DeltaU3) and both (DeltaU1DeltaU3). Both the control and transgenic adult littermates showed high levels of beta-galactosidase-like activity in epiphyseal growth plate and kidney medulla. However, beta-galactosidase activity was not observed for fetuses aged post coital 14.5 and 15.5 days. We were unable to show tissue specific reporter activity in our transgenic animals. / In other study, we found that P2 is the predominant promoter controlling PTH1R gene expression in both bone and cartilage. (Abstract shortened by UMI.)