<p> <i>myo</i>-Inositoloxygenase (MIOX) catalyzes the first step in <i>myo</i>-inositol catabolism. MIOX has not been annotated in <i> Drosophila melanogaster</i>, but the protein encoded by the <i> CG6910</i> gene is similar to the mouse MIOX protein. <i>CG6910 </i> "knocked-down" expression was explored using RNAi. "Knock-down" flies did not survive on inositol defined media, indicating that <i> CG6910</i> encodes MIOX. Survival of these flies on sucrose defined media suggest that MIOX is not essential for development. Biochemical assays demonstrated that <i>D. melanogaster</i> has MIOX activity. Computational analyses revealed potential miRNA sites, and that a number of essential components are conserved. <i>MIOX</i> genes found in other drosopholids are highly similar to <i>D. melanogaster MIOX</i>, and analyses of the syntenic regions concur with established evolution. Western blot analyses showed differential expression amongst <i>D. melanogaster</i> from different geographic locations and between species. These studies may contribute to understanding the role of inositol catabolism in fruit fly development and diabetes.</p>

There is increasing evidence that CUX1 can act as an oncogene in various cell types. First of all, it was found to be overexpressed in different human tumors, such as in uterine leiomyomas, breast tumors and pancreatic tumors. Both p75 and p110 were also found to be overexpressed in tumor cell lines of different origins. My research project has focused on characterizing the oncogenic potential of short CUX1 isoforms through the study of transgenic mice. The first model we used was to overexpress p75 CUX1 under the control of the cytomegalovirus enhancer-chicken β-actin promoter. Interestingly, these mice developed polycystic kidneys at variable penetrance and severity, correlating with transgene expression levels. Cystic kidneys were found to display enhanced proliferation and also showed an upregulation of c-myc and downregulation of p27KIP1. The second model we used was to overexpress p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat in the hypoxanthine phosphorybosyltransferase locus. The first phenotype we observed was that p75 CUX1 transgenic virgin female mice of the first backcross generations developed a myeloproliferative-disease-like myeloid leukemia. These results indicate that overexpression of p75 CUX1 can alter the proliferation and differentiation of some hematopoietic cells. The second phenotype we observed in these mice was that both p75 CUX1 and p110 CUX1 mice of pure FVB background developed mammary tumors of various histopathologies. Metastasis to the lung was observed in the p75 CUX1 transgenic mice only. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested CUX1 DNA binding was increased. Interestingly, higher expression of erbB2 mRNA was seen in most solid carcinomas, while adenosquamous carcinomas displayed higher expression of various Wnt / De plus en plus d'études suggèrent que le facteur de transcription CUX1 soit un oncogène. Premièrement, CUX1 est surexprimé dans certaines tumeurs humaines, dont les léiomyomes utérins, les tumeurs du sein et les tumeurs du pancréas. Les isoformes courtes CUX1 p75 et p110 sont de plus surexprimées dans plusieurs lignées cellulaires provenant de différents types de tumeurs humaines. L'objectif de mon projet de recherche était de vérifier le potentiel oncogénique des isoformes courtes de CUX1 dans des souris transgéniques. Dans le premier modèle de souris, CUX1 p75 était exprimé sous le contrôle du promoteur du Cytomégalovirus. Ces souris ont développé des reins cystiques avec un degré de pénétrance et de sévérité directement proportionnel aux niveaux d'expression du transgène. Les cellules épithéliales des reins cystiques proliféraient davantage que les cellules normales et montraient une expression élevée de c-myc mais réduite de p27KIP1. Dans le second modèle de souris, CUX1 p75 ou p110 était exprimé sous le contrôle du promoteur du virus MMTV (Mouse Mammary Tumor Virus), spécifiquement intégré dans le locus hprt (hypoxanthine phosphoribosyltransferase). Des rétrocroisements successifs dans la souche FVB ont permis d'uniformiser le bagage génétique des souris transgéniques. Les souris CUX1 p75 des premières générations, donc de bagage génétique mixte, ont développé des leucémies caractérisées par une hyperprolifération de cellules myéloïdes neutrophiles. Ces résultats suggèrent que CUX1 p75 peut affecter la prolifération et la différenciation de certaines cellules hématopoïétiques. Dans la souche FVB pure, les souris CUX1 p75 et p110 ont développé des tumeurs mammaires de diverses histopathologies. Des métastases aux poumons ont été observées dans trois cas de souris CUX1 p75 avec des carcinomes solides mammaires. Les transgènes é

Mouse biliary glycoproteins (Bgp) are members of the carcinoembryonic antigen (CEA) family, a subfamily of the immunoglobulin superfamily. Nine similar but distinct mouse Bgp cDNA clones have been isolated and characterized. These Bgp isoforms encode polypeptides that contain multiple Ig domains, a single transmembrane domain and a short or long intracytoplasmic domain. Sequence analyses have demonstrated that Bgp isoforms are generated through complex alternative splicing of a single gene and allelic variation. Mouse Bgp proteins are highly expressed in the epithelial cells of the liver and the intestine and are implicated in diverse cellular functions including cell adhesion and bile acid transport, and are postulated to be involved in signal transduction. Investigation into the expression of mouse Bgp isoforms in normal and malignant tissues revealed that Bgp expression is downregulated in tumors. Furthermore, Bgp expression is influenced by transcriptional control mechanisms involving DNA methylation of the Bgp gene upstream regulatory regions.

Bacteriophage D108 is a temperate, transposable, mutator phage similar to phage Mu. These heteroimmune phages have 37 kb linear, double-stranded DNA genomes which are over 90% homologous. / We have isolated four independent insertions of an entire thermoinducible D108 c ts10 phage genome in the low-copy plasmid pSC101. We also characterized a previously isolated Mu c ts62 insertion in the same plasmid replicon. Fine-structure analysis of these insertions showed that lytically transposing Mu and D108 genomes caused 5 bp duplications of the target site. In addition, we cloned and sequenced the terminal 800 bp of the right end of D108, a region important for the transposition of the phage genome. This analysis has delineated the critical DNA sequences required for D108 transposition, and has also indicated that these sequences are not entirely homologous to those of phage Mu, despite the fact that the phages can transpose each other's DNA. Moreover, a 520 bp insertion of DNA found in the right end of D108, but not Mu, has also been characterized, and has begins 72 bp from the right end of D108, immediately adjacent to the cis -acting sequences required for D108 transposition. This insertion does not appear to be an insertion element, but rather an extra gene in the right end of D108 whose function is apparently non-essential for D108 growth. The sequence differences and rearrangements between Mu and D108 in this region indicate a complex evolutionary relationship between these phages at their right extremities. / In addition to characterizing the DNA differences between Mu and D108, we have also examined a difference occurring at the protein level. The L gene products of Mu and D108 have different molecular weights, but are encoded from a region of homology between the two phages. We cloned a region of D108 DNA that was able to rescue amber mutations in the L gene of Mu by homologous recombination, but not complement Mu L amber phages in vivo. This region produced a gene product which was truncated at the carboxy terminus, but was able to cross-react with anti-Mu phage serum. The production of this truncated protein was lethal to growing Escherichia coli cells, apparently interfering with cell wall biosynthesis.

In this study, we describe the isolation and characterization of a functionally signaling-deficient Galpha16-negative somatic mutant (hereafter referred to clone 43) derived from the wildtype Jurkat T cell line by means of an activation-induced cell death resistance-based selection strategy. The catalytic activity of Lck in abrogated in clone 43 most likely due to a lack of autophosphorylation at the stimulatory site within the kinase domain of Lck, Y394. Based on Galpha16 cDNA sequencing data, the deficiency presented in clone 43 is most likely due to one or two point mutations located at amino acid residues 26 and 300 of the Galpha16 subunit. Importantly, these signaling defects were fully reversed by re-introduction of wildtype Galpha16 version into clone 43. Indeed, our findings are the first of their kind to validate the proposed model by which the Galpha subunit modulates the activity of Src family of protein tyrosine kinases. These studies underscore the integral role played by Galpha 16 in TCR-mediated signaling and offer a powerful genetic tool to strengthen our knowledge about the regulation and function of G proteins in T lymphocytes. We also describe the phenotype of two other signaling-deficient, Jurkat-derived clones, named clone 15 and clone 21. (Abstract shortened by UMI.)

Most eukaryotic mRNAs possess a 5' cap structure which is important for their translation. The eukaryotic translation initiation factor 4E (eIF4E) binds this structure directly and is repressed by the eIF4E-binbing proteins (4E-BPs), which in turn are regulated through the FRAP/mTOR pathway by a multitude of extracellular stimuli. The focus of this thesis is to investigate the function of the 4E-BPs in the cell by the use of mouse embryonic fibroblasts (MEFs) in which both the genes for 4E-BP1 (Eif4ebp1) and 4E-BP2 (Eif4ebp2) were disrupted. We first examined the role of 4E-BPs in encephalomyocarditis virus (EMCV) infection. 35S-methionine metabolic labeling of MEF proteins revealed that 4E-BPs are not essential for EMCV replication. We then decided to identify mRNAs regulated by 4E-BPs. This was achieved by polyribosomal analysis of specific mRNAs. Our results showed that translation efficiency of c-myc and ornithine decarboxylase (ODC) mRNAs is enhanced in double knockout cells. We also tested MEFs for their sensitivity to rapamycin, a drug known to inhibit FRAP/mTOR. Fluorescence-activated cell sorting (FACS) analysis demonstrated that 4E-BPs contribute to rapamycin-induced growth arrest. Finally, we studied Eif4ebpl-/- ;Eif4ebp2-/- mice in order to test whether they show a phenotype similar to Eif4ebp1 -/- which are known to be smaller and display a lower blood glucose compared with their wild type counterparts. Surprisingly, Eif4ebp1-/-;Eif4ebp2 -/- mice were neither small nor hypoglycemic. The above results emphasize the importance of the 4E-BPs in the cell, thus encouraging further investigations in order to better understand their function.

The activation of death receptor Fas (CD95/APO1) triggers apoptosis inducing caspase activation by direct (extrinsic) and mitochondria dependent (intrinsic) pathways. Mitogen activated protein kinases (MAPK), which include p38, JNK and ERK kinases, are also activated during Fas signaling and are known to either positively or negatively regulate apoptosis. Chemical inhibition of JNK (SP600125) and p38 (PD169316) sensitize tumor cells to Fas mediated apoptosis. I studied Fas mediated apoptosis in Jurkat cells and in some experiments HeLa cells. PD169316 is less potent than SP600125 and attenuates the effect of the later when present together. PD169316 inhibits two isoforms of p38 which either promote (p38alpha) or inhibit (p38beta) apoptosis; I investigated their relative regulatory influences on Fas signaling. I show that p38alpha is essential for Fas mediated caspase-8 activation: it promotes the dephosphorylation and exclusion of c-FLIPS but not c-FLIPL from the death inducing signal complex (DISC). Distally both p38 isoforms positively influenced the intrinsic pathway by common and selective effects on pro-apoptotic Bcl-2 proteins. The sensitizing effects of p38 and JNK inhibition were ablated by Bcl-2 localized at the endoplasmic reticulum. In HeLa cells, the proapoptotic effects of p38alpha are overridden by the anti-apoptotic effects of ERK.

During HIV-1 assembly tRNALys isoacceptors are selectively packaged into HIV-1. One of the tRNALys isoacceptors, tRNA Lys3 serves as a primer for the reverse transcriptase-catalyzed synthesis of minus strand, strong stop cDNA. Lysyl-tRNA synthetase (LysRS) a tRNA Lys-binding protein is also selectively package into HIV-1, making it a candidate for being the signal which targets tRNALys for incorporation into viruses. We have tested whether the cytoplasmic tRNA Lys/LysRS interaction is required for selective packaging of tRNA Lys into HIV-1 by constructing anticodon mutations within the anticodon domain of tRNALys3. The anticodon is the major binding domain for tRNALys/LysRS interaction. We found that the ability of tRNALys3 to bind to LysRS, and be aminoacylated was directly correlated with its ability to be incorporated into HIV-1. / However it was not clear from these results if in addition to the tRNALys/LysRS interaction, tRNA aminoacylation was required for selective packaging of tRNA Lys3. To investigate this, we constructed and expressed two mutant LysRS species. One has lost its aminoacylation activity because of a deletion within the catalytic core. The other mutant LysRS has a reduced ability to bind to tRNALys, because it contains a deletion within tRNA Lys binding site. This mutant LysRS does not have the ability to facilitate tRNALys packaging into the virus, while the mutant LysRS which binds to tRNALys, but fails to aminoacylate it, does facilitate tRNALys packaging, indicating that tRNALys aminoacylation is not required for its packaging into viruses. / LysRS is carried into the viruses by its interaction with Gag. We have mapped the sites of interaction between HIV-1 Gag and LysRS using in vivo and in vitro techniques. We find that Gag sequences within the C-terminal domain of CA which contains the CA dimerization site, interact with LysRS sequences which include motif 1, which contains the LysRS dimerization site.

The initiation of protein synthesis consists in the recruitment of a ribosome·initiator tRNA complex to the initiation codon of a messenger RNA. In eukaryotes, this process is facilitated by eukaryotic translation initiation factor 4E (eIF4E), which recognizes the cap structure present at the 5' end of all nuclear-transcribed mRNAs. The eIF4E-binding proteins (4E-BPs) inhibit cap-dependent translation by binding to eIF4E, and preventing ribosomal recruitment to the mRNA. The interaction of 4E-BPs with eIF4E is reversible, and it is regulated by the 4E-BPs phosphorylation state. Here I report the isolation of a third member of the 4E-BP family, 4E-BP3, and I demonstrate that it shares all the basic structural and functional characteristics of 4E-BP1 and 4E-BP2. I also analyzed the genomic locus for human 4E-BP3 (EIF4EBP3). I established that EIF4EBP3 can be fused with the gene located immediately upstream, and which encodes the human homologue of Drosophila MASK (Multiple Ankyrin Repeats Single KH domain). Interestingly, the open reading frame of the MASK-BP3 fusion transcript is different from that of 4E-BP3, indicating that the EIF4EBP3 locus encodes two different proteins. Finally, I investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice are remarkably lean, and the males' white adipose tissue contains cells exhibiting the distinctive multilocular appearance of brown adipocytes, and it expresses the uncoupling protein 1. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1) is increased in white adipose tissue from Eif4ebp1-/- mice. PGC1 is a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis. These findings demonstrate that 4E-BP1 is a novel regulator of metabolism in mammals.

Members of the zinc cluster (or binuclear cluster) protein family are characterized by a Zn(II)Cys6 zinc finger involved in DNA recognition and binding. These fungal proteins are transcriptional regulators of genes involved in a wide variety of cellular processes. One member, Gal4p, is involved in galactose metabolism, while others play a major role in primary and secondary metabolism, control of meiosis, and multidrug resistance. Sequencing of the Saccharomyces cerevisiae genome has revealed that 55 genes encoding putative zinc cluster proteins are present in budding yeast. However, the roles of many of these zinc cluster proteins are unknown. In order to better understand their functions, we have performed a phenotypic analysis of these putative zinc cluster proteins. We have implicated a number of them in a variety of processes in the cell, including multidrug resistance. Stb5p has been shown to be a major player in regulating the expression of multidrug resistance genes. Other zinc cluster activators of multidrug resistance genes include Pdr1p, Pdr3p, and Yrr1p. These regulators of multidrug resistance appear to interact with each other, forming many different sub-populations of homo- and heterodimers. Stb5p is found predominantly as a heterodimer with Pdr1p. It also appears that Pdr1p is a master regulator able to interact with many different partners, enabling it to mediate control over multidrug resistance genes.