Carbamoylphosphate synthetase (CPS) catalyses the formation of carbamoylphosphate from CO$\sb2$, ATP, and glutamine; the first committed step in the arginine and pyrimidine biosynthetic pathways. Initial interest in the CPS enzyme of Neisseria gonorrhoeae stemmed from the natural occurrence of CPS deficiencies in 10-20% of clinical isolates. This property, causing a concurrent citrulline and uracil auxotrophy, is employed in a typing scheme and there have been some indications that this auxotrophy may be beneficial to the gonococcus. In all of the prokaryotes examined, CPS is a heterodimer encoded by two genes commonly called carA and carB. I have now determined the complete sequence of the carA and car B genes from N. gonorrhoeae CH811. carA (1125 bp) and carB (3237 bp) are similar in size to other prokaryotic CPS genes, and encode all the highly conserved regions present in other CPS's. However, these genes in strain CH811 are separated by a 3287 bp intergenic sequence. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was typical of gonococcal isolates, the sequence between carA and carB was PCR amplified from isolates of N. gonorrhoeae, N. meningitidus, and 9 other Neisseria species. The intervening sequence was found to vary in size, from approximately 2.2 to 3.7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS. The presence of a variable sequence near carA and carB, and the numerous repetitive sequences found in this area in strain CH811, suggests that this region may be a hotspot for recombination and may in part explain the high frequency of occurrence of CPS mutants. PCR amplification of carA and carB from CPS deficient isolates indicate that more than one mutation is responsible for this phenotype. Transformation of a gonococcal CPS mutant with the cloned gonococcal CPS genes has demonstrated that other mutations are present in the arginine and pyrimidine biosynthetic pathways of this isolate, which are hidden by the CPS deficiency. This study also suggests that isolates of auxotype CUH and OUH are closely related. I also report an analysis of the first CPS gene sequence from an archaeon (Sulfolobus solfataricus P2; supplied by R. L. Charlebois). This analysis indicates that the archaeal CPS is similar in function, size and gene organization to other prokaryotic CPS's. A comprehensive phylogenetic analysis of all known CPS genes, including the gonococcal and archaeal sequences, was performed. Using the internal duplication as a root for phylogenetic tree construction, I was able to determine a branching order for the tree of life and provide support for the theory that this archaeon is more related to eukaryotes than bacteria. This is the first report of confirmation of this theory using a metabolic gene and is the first use of an internal duplication within a gene to root the tree of life. This analysis also showed that at least two, and possibly even three, separate gene duplication events led to the formation of two CPS enzymes present in Gram-positive bacteria and most eukaryotes. Comparisons of the branching order with the organization of CPS genes in different organisms further indicated that the evolution of CPS has been complex, involving multiple deletions and insertions. (Abstract shortened by UMI.)

Work in our laboratory has demonstrated that stimulation of bovine chromaffin cells with nicotine elicits as a prelude to exocytosis, (a) redistribution of scinderin (Sc), a novel 80 kD Ca$\sp{2+}$-dependent actin filament severing protein and (b) cortical F-actin disassembly. The work presented in this thesis shows that the second messenger, cyclic AMP (cAMP) modulates Sc redistribution, F-actin disassembly and catecholamine (CA) secretion. Here we show that forskolin (F) produces a concentration-related (10 $\mu$M-50$\mu$M) inhibition of Sc redistribution, F-actin disassembly and CA release in response to 10 $\mu$M nicotine. F also induces a concentration-dependent (10 $\mu$M-50 $\mu$M) increase in cAMP levels. Increasing intracellular cAMP with 50 $\mu$M F or incubation with the F analogs 6-acetyl-7-deacetylforskolin or deacetylforskolin (100 $\mu$M) as well as the cAMP membrane permeant analog, 8-Bromo cAMP (2.5mM) for 40 s also inhibits Sc redistribution, F-actin disassembly and CA secretion. The inhibitory effect of F on nicotine-induced Sc redistribution and F-actin disassembly is observed even upon 5 s of incubation while inhibition of CA secretion cannot be detected until 20 s of incubation with F. These effects are accompanied by an increase in cAMP. The discrepancy in timing between inhibition of both Sc redistribution and F-actin disassembly in relation to CA secretion may be explained by the fact that Sc redistribution and F-actin disassembly seem to occur simultaneously and to precede secretion. Although work on cAMP by others has yielded conflicting results, our findings suggest that cAMP may play a role in modulating cytoskeleton dynamics during secretion. This also implies that this second messenger can attenuate the secretory response either by preventing disassembly of F-actin or activation of Sc thus, denying the secretory vesicles access to exocytotic sites. Alternatively, the observed effects may represent a modulation of nicotinic receptor activity by cAMP.

Glucocorticoid receptor (GR) is a ligand activated sequence specific transcription factor that belongs to the nuclear receptor superfamily. Most often GR regulates transcription through binding to glucocorticoid DNA response elements (GREs) found in the regulatory regions of target genes. Regulation of transcription by GR usually occurs in a combination with other heterologous transcription factors whose DNA response elements are found in proximity of GREs in the gene regulatory regions. The Herpes Simplex Virus thymidine kinase (HSV tk) proximal promoter contains two DNA binding sites for Sp1, and one "CCAAT" DNA motif. Surprisingly, when GR was transiently co-transfected with a CAT reporter gene driven by the HSV tk promoter, it induced HSV tk transcription 5 fold in response to dexamethasone in the absence of any apparent GREs. Activation of HSV tk transcription was dexamethasone dependent, as RU 486, a GR antagonist was unable to activate transcription. Activation of HSV tk was also GR specific as androgen receptor was unable to activate HSV tk transcription following DHT treatment. To map the region of GR responsible for GRE independent activation of HSV tk transcription, several different GR expression constructs were used. (Abstract shortened by UMI.)

Breast cancer remains one of the leading malignancies in women of the developed world despite the extra efforts that have been deployed in the past decade or so to the study of this disease. It is well known that the degree of aggressivity of the tumor is related to the stage of differentiation. Transformation of a cell having a high proliferation potential will lead to a more aggressive tumor than transformation of a more differentiated cell. For this reason it is important to have a good understanding of the process of mammary differentiation. The work in this thesis was directed at finding novel genes that are involved in the normal development of the breast with the hope that these may open new avenues for breast cancer therapies. The approach used was to look for genes that are expressed during breast development in the living mouse. A promoter-trap retrovirus containing on oncogene as the promoterless indicator gene was introduced locally in the mammary glands of pubescent female mice. Integration of proviruses near an active cellular promoter lead to the expression of the oncogene from a cellular promoter which was detected by the formation of a tumor. To identify genes that are specifically active during differentiation of the breast tissue we looked for the appearance of tumors after the animals were mated. Two promoter-trap retroviruses were tested. The first one (COPT) was replication deficient and although it did work in vitro it did not give a high enough level of infection to be used in vivo. The second virus (PyT) was replication competent and was found to be a more suitable promoter-trap to be used in vivo. This was demonstrated by the isolation of a novel murine gene named TRAP1.

Genomics is a rapidly expanding field of research that seeks to study the structure, function and evolution of an organism's genome. Genomic investigations were conducted on three species of haloarchaea, a monophyletic group of prokaryotes belonging to the kingdom Euryarchaeota of the domain Archaea that are adapted to high-salt environments. A physical and genetic map of the genome of Halobacterium salinarum GRB is described. This map and the previously published map of the genome of Haloferax volcanii DS2 were compared with the object of detecting any conservation in the order or spacing of homologous loci between the two genomes. A computer program--COMPAGEN--was developed to aid in the analysis of the data generated by this comparison. No map order conservation could be detected at the 15 kbp average resolution of this comparison between genomes estimated to have diverged 600 million years ago. A second comparison was performed between the chromosomes of Haloferax volcanii DS2 and Haloferax mediterranei ATCC 33500 (R-4). Extensive conservation was found between these two genomes which diverged approximately 80 million years ago showing only three rearrangements: two inversions and a transposition. Conclusions drawn from an analysis of the comparisons include: (1) that higher resolution is required to deal with distantly related genomes, likely making use of sequence data, and (2) that it is important to compare genomes that have diverged at different times if one wishes to investigate the dynamics of genomic evolution within a phylogenetic group. The local context model was developed in an effort to explain the pattern of conservation and divergence seen in these and other prokaryotic genome comparisons. This model states that since the expression of genes is affected by flanking genetic elements, genes will resist changing their position relative to one another so long as this change is likely to alter gene expression in a way deleterious to the cell. The local context model thus provides a force promoting the conservation of genomic map order. The implications of this model for the evolution of the haloarchaea is discussed and future directions of prokaryotic genomics in general is explored.

The goal of this thesis is to study cellular senescence with the use of the determinate nodule of soybean as the model system. The determinate nodule is an appropriate model system for the study of senescence due to the homogeneity of infected cells of various developmental stages within the organ. In this model system consisting of Glycine max (L.) Merrill, Maple Arrow, infected with Bradyrhizobium japonicum strain 61A76, we showed that nodule senescence occurs late in nodulation, at the time of flowering and pod filling. We also demonstrated that nodule senescence can be induced with the addition of 20mM ammonium nitrate. The strategy to study the molecular mechanisms of nodule senescence involves the isolation of senescence-associated nodulin (SAN) cDNAs by the differential screening of a cDNA library representing mRNA from senescent nodules. This library constructed in the $\lambda$gt11 vector is screened with two bulk probes made from the PCR amplification of the cDNA inserts of libraries in the $\lambda$gt10 vector: one representing mRNA of healthy nodules that are actively fixing nitrogen and the other of senescent nodules. In this thesis, one SAN cDNA, clone 8-4 was characterized through a Northern analysis to demonstrate the up-regulation of the corresponding gene with senescence. In the natural senescent system, SAN8-4 expression was demonstrated to increase with nodule senescence. A search through nucleotide and protein sequence banks demonstrates that SAN 8-4 may be a novel F3H related protein. (Abstract shortened by UMI.)

The functional significance of Class III $\beta$-tubulin during neurogenesis has been investigated. A combination of northern blotting, immunofluorescence microscopy, enzyme-linked immunosorbent assay, and isoelectric focusing was used to characterize the expression of $\beta$-III tubulin in P19 embryonal carcinoma cells induced to differentiate along a neuronal pathway retinoic acid. Following 48 hours differentiation, $\beta$-III tubulin mRNA is evident and $\beta$-III tubulin appears in the mitotic spindle of neuronal precursors. Neurite outgrowth is obvious by day and $\beta$-III tubulin and mRNA levels increase concurrently until approximately day 8, when $\beta$-III mRNA levels begin to decrease while protein levels remain high. In addition, increasingly acidic $\beta$-III isoforms appear with days of differentiation. The accumulation of these isoelectric variants occurs concomitant with a temporal increase in the incorporation of $\beta$-III tubulin into colchicine-stable microtubules. P19 cells were grown on coverslips and then treated with taxol concentrations from 10$\sp{-6}$ M to 10$\sp{-9}$ M for 24 hours. The microtubule cytoskeleton was examined after double-immunofluorescence labelling with a monoclonal antibody to $\alpha$-tubulin (YOL 1/34) and a monoclonal neuron-specific $\beta$-III tubulin antibody (TuJ1). Treatment of undifferentiated P19 cells with concentrations of taxol greater than 4 X 10$\sp{-8}$ M causes microtubule bundling and multiple aster formation and promotes polymerization the low levels of $\beta$-III tubulin found in these cells. In neurons, at 2 X 10$\sp{-8}$ M taxol, bundling of microtubules at the base the neurite is apparent. At taxol concentrations greater than 10$\sp{-7}$ M enhanced assembly of $\beta$-III tubulin is noted, although long neurites are not observed. An additional isoform of $\beta$-III tubulin is detected following treatment with 10$\sp{-6}$ M taxol. These results indicate that taxol treatment alters the normal subcellular sorting tubulin isotypes, promotes the polymerization and translational modification of $\beta$-III tubulin, and interferes with neurite outgrowth. The coding sequence of mouse $\beta$-III tubulin was transiently expressed in undifferentiated P19 cells and its assembly and resistance to colchicine-depolymerization examined by immunofluorescence microscopy and isoelectric focusing. Two $\beta$-III tubulin isoforms are detected on isoelectric focusing immunoblots. Microtubule disassembly by colchicine appears identical in the transfected and adjacent non-transfected cells. The results reveal that Class III $\beta$-tubulin is assembly competent and can undergo at least one posttranslational modification in non-neuronal cells. Several posttranslational modifications of $\beta$-III tubulin have been identified, including phosphorylation of Ser$\sp{444}$ and/or Tyr$\sp{437}$, and glutamylation of Glu$\sp{438}$. Of these, phosphorylation of Ser$\sp{444}$ is unique to $\beta$-III tubulin in brain. To investigate the role of Ser$\sp{444}$ phosphorylation of $\beta$-III tubulin, site-directed mutagenesis was used to generate two mouse $\beta$-III tubulin mutants. In one, Ser$\sp{444}$ was replaced by Ala$\sp{444}$ to provide a $\beta$-III tubulin molecule which could not undergo phosphorylation at that site. In the second mutant, Ser$\sp{444}$ was replaced by Glu$\sp{444}$ to mimic a constitutively phosphorylated protein. When $\beta$-III tubulin Ser$\sp{444}$ mutant expression vectors are transiently expressed in P19 cells, examination reveals that both mutant $\beta$-III tubulins yield labelling patterns which are similar to the wild-type $\beta$-III tubulin. Neither $\beta$-III tubulin mutation confers resistance to 45 min colchicine-induced depolymerization in non-neuronal cells. (Abstract shortened by UMI.)

A 1213 base pair region of mitochondrial DNA corresponding to the cytochrome oxidase subunit I gene (COI) from 7 pleuronectid flatfishes (Order Pleuronectiformes) and one outgroup (Paralichthys dentatus) was PCR-cloned and sequenced. Pleuronectid taxa were chosen so that the main branching points in a cladogram of almost all species of Pleuronectidae (Cooper 1996) could be tested using an independent data set. Our results show that the COI sequence is highly conserved and that most of the substitutions that occur between species are synonymous changes at the third codon position. Analyses of the substitutional spectrum reveals a bias favouring the occurrence of transitions over transversions. Furthermore, transitions between C and T and transversions between A and C or T are more frequent than transitions or transversions involving G. COI sequence composition shows a bias against the occurrence of G and a preference for A at the third position of fourfold degenerate codons. Phylogenetic analyses based on all substitutions and on transversions only using parsimony and distance methods were congruent in most respects but were unable to resolve the branching order of Pseudopleuronectes americanus and Limanda ferruginea due either to a shortage of characters or homoplasy. The topology of the molecular trees provide support for the subfamily relationships in Cooper's classification (1996). However, the basal position of Microstomus pacificus in the molecular trees is largely incongruent with the morphology tree. This result may question the monophyletic status of the Pleuronectinae (sensu Cooper 1996) or may be an indication of mitochondrial introgression involving Microstomus pacificus.

The ability of the c-fos gene to respond to a variety of stimuli reflects the complexity of its regulation. Indeed, aberrant regulation can lead to cellular transformation. The fine regulation of the gene relies on the interaction of protein factors interacting with a set of regulatory elements present within the promoter of the gene in a cooperative fashion. In these studies, multiprotein complexes capable of binding to the serum response element, the cAMP response element, and sis-conditioned medium response element, were identified in liver cell nuclei. A subset of these proteins was able to interact with multiple regulatory elements and might be able to direct functional cooperation between them. These multiple factors could be targeted by different signalling pathways. The behavior of these multiprotein complexes was analyzed under physiological conditions of transient c-fos expression in regenerating rat liver and under pathological conditions of chemically induced hepatocarcinogenesis and in solid 5123tc Morris hepatomas whereby constitutive expression of the gene occurs. Elevated expression of the c-fos gene in both normal and in transformed cells correlated with the loss and/or complete absence of the 47 kDa CRE-binding activity. The 47 kDa CRE-binding protein was characterized as highly CRE-specific and distinct from the other members of the CREB/CREM/ATF family of proteins. The 47 kDa CREB factor displayed DNA-binding activity in the dephosphorylated form. The functional importance of the c-fos promoter binding proteins was assessed by in vitro transcription which suggested the presence of a transcriptional block in quiescent liver cells which was relieved in proliferating hepatocytes. The results are consistent with the identification of the 47 kDa CRE-specific DNA-binding protein as a transcriptional repressor in quiescent liver cells.

The regulation of the proliferation of non-neoplastic T51B rat liver epitheloid cells involves a complex interplay between the epidermal growth factor (EGF) and the protein kinase C (PKC) signal-transduction pathways. T51B cells can be made proliferatively quiescent upon prolonged serum deprivation, which results in their growth-arrest at the G1 phase of the cell cycle. Addition of EGF (1.5$\eta$M) or, re-addition of serum (10% BCS) to the conditioned medium initiates DNA synthesis, within 8 hours, which reaches a maximum by 18-24 hours, when a significant number of cells are in the S phase. The DNA synthetic responses of EGF and serum are additive. Growth factor or, serum-stimulated DNA synthesis is followed by an increase in cell number. The mitogenic response of EGF can be blocked by 15-20$\mu$M genistein, a tyrosine kinase inhibitor. EGF activates particulate PKC within 15 minutes, and the stimulation reaches a maximum by 35-45 minutes. PKC activity then remains elevated for up to 24 hours. The tumour promoting phorbol ester, TPA, which is known to activate PKC, is not mitogenic to T51B cells. TPA modulates the responses of EGF or, serum depending upon the culture conditions. When TPA is added to serum-deprived cells in the presence of EGF or serum, it potentiates DNA synthesis within 18 hours, during which time it down-regulates PKC. On the other hand, if TPA is present in the medium during serum-deprivation, the response to EGF in these PKC-inactivated cells is as mitogenic as that caused by serum. However, TPA pretreatment makes the cells less responsive to EGF or serum in that, the net DNA synthesis is significantly reduced ($>$30%) in control, and in treated cells. The inhibitory response of genistein is slightly attenuated in the presence of EGF and TPA. (Abstract shortened by UMI.)