eIF-4A is a eukaryotic translation initiation factor and DEAD box RNA helicase that is thought to be responsible for the melting of secondary structure in the 5$ sp prime$ untranslated region of messenger RNAs to facilitate ribosome binding. A mutational analysis of eIF-4A revealed that the ATPase A motif (AXXXXGKT) is involved in ATP binding, the ATPase B motif (DEAD) is implicated in ATP hydrolysis, the SAT region is essential for RNA unwinding, and the HRIGRXXR region is required for ATP hydrolysis-dependent RNA binding. Furthermore, defective eIF-4A mutants exhibit a strong dominant negative effect on in vitro translation of several mRNAs, including those translated by a cap-independent internal initiation mechanism. It is demonstrated that eIF-4A functions primarily as a subunit of eIF-4F, and singular eIF-4A is required to recycle through the eIF-4F during translation. Accordingly, eIF-4F appears to be required for cap-dependent and internal initiation of translation.

In the yeast Saccharomyces cerevisiae, HEM13 codes for coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway. Coproporphyrinogen oxidase synthesis has been shown to be regulated negatively by heme and oxygen at the transcriptional level. We are interested in defining the HEM13 cis-acting upstream sequences that mediate regulation by heme. HEM13-lacZ and HEM13-CYC1-lacZ hybrid fusions were made and expression of the resulting $ beta$-galactosidase activity was determined in vivo, under conditions of varying levels of heme. Deletion analysis of the HEM13 promoter revealed the existence of 3 upstream regulatory elements designated as UAS1, UAS2, and URS that span a region of 365 pb from nucleotides $-$640 to $-$275 with respect to the most upstream HEM13 transcriptional start site. The trans-acting factor HAP1 previously identified to activate expression of CYC1 and CTT1 genes in the presence of heme and oxygen, was found to be involved in the regulation of HEM13 expression. Measurements of HEM13-CYC1-lacZ fusions in a hap1 mutant strain indicated that HAP1 was required to fully induce and repress expression of the fusions under heme-deficient and heme-sufficient conditions respectively.

Desmosterolosis is an autosomal recessive condition in which affected individuals lack expression of the final enzyme in the cholesterol biosynthetic pathway, 3β-hydroxyΔ²4cholesterol reductase (DHCR24). The enzyme is responsible for converting desmosterol to cholesterol. It is characterized by several congenital abnormalities, as well as high circulating levels of desmosterol accompanied by hypocholesterolemia. This study was intended to examine the molecular disruptions involved in desmosterolosis and determine the potential impact on steroidogenesis using an in vitro approach. Due to the importance of cholesterol in cell-signaling and cell membrane integrity, it was found that desmosterol was a poor substitute for cholesterol in cell membranes. Cells deprived of cholesterol for 45 minutes rapidly underwent morphological changes and presumably became apoptotic. This effect was potentiated in cells with DHCR24 knock-down. Additionally, cells responded by increasing levels of cholesterol biosynthetic factors such as, SREBP1, SREBP2, SCAP, S1P, as well as the enzyme HMG Co A reductase all of which were measured using real-time PCR. The increase in these transcripts indicates that desmosterol cannot be sensed by the sterol sensing domain (SSD). Following cholesterol repletion, cells were able to restore a certain level normalcy in terms of morphology and the production of the transcripts listed above. Cholesterol is required for steroidogenesis in the adrenal cortex. We examined whether or not desmosterol could be used as a steroidogenic substrate. We measured levels of steroidogenic acute regulatory protein (StAR) as well as measured cortisol production with DHCR24 knock-down following cholesterol depletion and repletion with and without trophic stimulation. We found that in cells stimulated with vasoactive intestinal polypeptide, devoid of cholesterol and with endogenous synthesis impaired at the leve / La desmostérolose est une maladie autosomale récessive dans laquelle les individus atteints ont un déficit de l'expression de l'enzyme terminale du processus de biosynthèse du cholestérol, connue sous le nom de 3β-hydroxyΔ ² 4cholesterol réductase (DHCR24). Cette enzyme est chargée de convertir le desmostérol en cholestérol. Cette maladie est caractérisée par plusieurs anomalies congénitales, ainsi que par des taux élevés de desmostérol dans le sang accompagnés d'hypocholestérolemie.L'objectif de cette étude a été d'examiner les anomalies provoquées par la desmostérolose au niveau moléculaire ainsi que son impact sur la stéroïdogenèse en utilisant une approche in vitro. Nous avons, pour cela, utilisé la technique d'interférence par des ARN pour diminuer l'expression de l'enzyme DHCR24 reproduisant ainsi les conditions de la desmostérolose dans notre modèle cellulaire.Le cholestérol participe à de nombreux processus de signalisation cellulaire et joue un rôle important dans la stabilité des membranes constituant les cellules. Au cours de notre étude nous avons constaté que le desmostérol du fait de sa structure n'est pas capable de se substituer au cholestérol pour remplir ces fonctions. En effet, les cellules dépourvues de cholestérol pendant 45 minutes ont systématiquement subi un changement de morphologie et ont par la suite présenté les caractéristiques de cellules en apoptose. Ce phénomène était encore plus prononcé dans les cellules où l'expression de DHCR24 avait été diminuée expérimentalement. Nous avons également examiné si le desmostérol pouvait être reconnu par la protéine dite 'domaine de reconnaissance des stérols' (SSD) qui in vivo stimule la synthèse endogène de cholestérol quand ses niveaux dans la circulation sanguine sont trop bas. Pour cela, nous avons mesuré les taux d'expression des protéines suivantes : 'sterol regula

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is an essential regulatory protein which acts post-transcriptionally to mediate nuclear export of the incompletely spliced mRNAs encoding the viral structural proteins. Rev mediates its effect, in part, through interaction with a cis-acting RNA target sequence, the Rev responsive element (RRE), located within the env region of the viral structural mRNAs. There exist transdominant Rev mutants that are capable of binding to the RRE yet are unable to carry out wild-type Rev function, strongly suggesting that cellular cofactors are involved in the Rev response. / Towards the isolation of such factors and the elucidation of the mechanism of Rev action, we have developed a highly efficient, one-step, non-denaturing purification protocol for a recombinant form of Rev (H6Rev) and a transdominant mutant of Rev (H6AC4). These recombinant proteins contain six histidine residues at the N-terminus which enable their purification using nickel-affinity chromatography and which can also serve as a means to isolate Rev-host factor complexes reconstituted in vitro. Both purified proteins form stable complexes with the RRE in vitro while only H6Rev exhibits wild-type Rev function in vivo when exogenously introduced into mammalian cells, confirming that the purification strategy employed retains the biological activity of the proteins. To complement the in vitro reconstitution experiments, we have generated human lymphoid cell lines that express biologically active H6Rev in a tightly regulated, inducible fashion. These cell lines can serve as a source for the isolation of Rev-host factor complexes formed in the mammalian cell. Controlled expression of H6Rev is achieved using a tetracycline-modulated promoter. By varying the concentration of tetracycline in the medium, the activity of this promoter, and hence the level of H6Rev expression, can be modulated over a wide range of concentration.

The entomopoxviruses (EPVs) of eastern spruce budworm (Choristoneura fumiferana, Cf), two-year cycle spruce budworm (C. biennis, Cb) and the Indian red army worm (Amsacta moorei, Am) are the focus of this work. The replication of the A. moorei entomopoxvirus was inhibited by bromodeoxyuridine, whereas the baculovirus of Autographa californica was insensitive to this drug. This result was a biochemical indication that entomopoxviruses contained a kinase that phosphorylated this nucleoside analog and thus viral DNA synthesis was inhibited. TK genes from the three different insect poxviruses, Cb, Cf and Am, were identified, cloned and sequenced. AmEPV TK gene was expressed in Escherichia coli mutants lacking the enzyme. These bacteria were converted to a phenotype that could incorporate radioactive thymidine into their chromosomal DNA. These results provided an impetus for the design of a recombinant entomopoxvirus expression system. / A recombination vector, designated RecVecTK, containing AmEPV TK gene and its flanking sequences, a late gene promoter, and a multiple cloning site, was constructed. $ beta$-Galactosidase reporter gene was cloned into a unique restriction site, and the resulting RecVecLacZ was tested in a transient expression assay. The results indicated that the late promoter used was sufficient to drive expression of $ beta$-galactosidase, whose activity peaked two days after the transfection of the RecVecLacZ into AmEPV-infected cells. / RecVecLacZ and RecVecLuc, a similar vector containing bacterial luciferase as a reporter gene, were transfected into AmEPV-infected cells in an attempt to introduce the reporter genes into the viral genome by a process of homologous recombination. Various infection and transfection parameters were tested but a recombinant virus has not yet been isolated. (Abstract shortened by UMI.)

Abelson murine leukemia virus can induce oligoclonal pre-B and T-cell lymphomas in mice. Expression of the v-abl oncogene in target cells does not appear to be sufficient for tumor formation and additional genetic events are thought to be required. The potential role of the Moloney helper virus as an insertional mutagen was investigated in Abelson lymphomas. A cellular region was found occupied by the helper virus in 16% of Abelson pre-B lymphomas. This new common proviral integration site, designated Ahi-1, was cloned and mapped to the murine chromosome 10. In contrast to pre-B lymphomas, the presence of competent helper virus was not required for efficient induction of Abelson thymomas and Ahi-1 did not appear to be implicated in these tumors. These data suggest a distinct role for the helper virus in Abelson pre-B and T-cell lymphomas and distinct biological requirements for induction of these lymphomas by the v-abl oncogene.

Activation of cell-surface receptors by extracellular signals can generate distinct biological responses. Many of these signals are coordinated through docking proteins, including those in the Gab family (Gab1, Gab2 and Gab3). Following activation of receptor tyrosine kinases (RTKs), cytokine or antigen receptors, docking proteins are recruited to the receptor complex and become phosphorylated on tyrosine residues, providing binding sites for multiple proteins involved in signal transduction. In this manner, they act to diversify and localize signals downstream from receptors by virtue of their ability to assemble multiprotein complexes. / The recruitment of docking proteins to RTKs depends on the ability of the protein to interact directly or indirectly with the receptor. In chapter II, I established that Gab1 and Gab2 can be recruited to RTKs indirectly, through constitutive association of Gab1 or Gab2 with the C-terminal SH3 domain of the adapter protein Grb2. This requires two highly conserved Grb2 binding sites in Gab proteins. One site corresponds to a canonical SH3 domain binding motif, whereas the second contains an atypical PXXXRXXKP motif that I also identified in the unrelated Grb2-binding protein, Slp-76. / In contrast to the other Gab proteins, Gab1 can also interact in a Grb2-independent manner with the Met/Hepatocyte growth factor receptor. In chapter IV, I established that this interaction requires the structural integrity of the Met receptor, phosphorylation of tyrosine 1349 in the Met C-terminus, and a 13 amino acid Met binding motif (MBM) in Gab1. Instead of the expected interaction of a phosphotyrosine-binding domain in Gab1 with a phosphotyrosine-containing motif in the Met receptor, I propose that the activated kinase domain of Met and the negative charge of phosphotyrosine 1349 engage the Gab1 MBM as an extended peptide ligand. / In response to Met receptor stimulation, Gab1 overexpression promotes an invasive morphogenic program in epithelial cells. In contrast, I have shown in chapter III that Gab2 overexpression fails to induce this response. Mutation of the MBM in Gab1 abolishes the ability of Gab1 to promote morphogenesis, whereas its insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote morphogenesis. This indicates that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis. Overall, these studies have identified both common and unique mechanisms through which receptor tyrosine kinases can recruit Gab docking proteins, and have established that Gab1 and Gab2 do not share redundant biological functions in epithelial cells.

The objective of this thesis is to investigate the proteins interacting with specific DNA sequences, termed origins of DNA replication in vivo. Two previously described origin binding proteins, OBA/Ku and CBP/14-3-3 were analyzed. Previously, Ku was shown to bind to A3/4, a 36-bp origin sequence, in vitro, and 14-3-3 isoforms were identified as cruciform binding proteins (CBP) which interact with cruciform structures present in mammalian replication origins. / Here, the in vivo association of Ku and 14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey (CV-1) replication origins ors8 and ors12, by formaldehyde cross-linking, followed by chromatin immunoprecipitation (Chip) and quantitative PCR analysis. The involvement of 14-3-3 in mammalian DNA replication was also analyzed by studying the effect of anti-14-3-3beta, epsilon, gamma, and zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. / Ku and 14-3-3beta, epsilon, gamma, zeta and sigma isoforms were found to be associated with mammalian origins of DNA replication and their association was the highest in cells synchronized at the G1/S phase of the cell cycle. In addition, Anti-14-3-3epsilon, gamma and zeta antibodies inhibited p186 replication by approximately 30--80%. / The Ku80 mutant (xrs-5) and deficient (Ku80-/- MEFs) cell lines were also tested for their ability to replicate p186, in vitro. Whole cell (WCE) and cytoplasmic cell extracts from the xrs-5 cells replicated p186 with the same efficiency as are wild-type (wt) CHO K1 cells. In contrast, xrs-5 nuclear extracts did not possess any detectable replication activity, while the Ku80 -/- WCE had a decrease of ~70% in their ability to support p186 replication, by comparison to the wt Ku80-/- extracts. Furthermore, in vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% by comparison to CHO K1 cells. / The in vivo association of Ku with the Chinese hamster DHFR oribeta or the mouse Adenosine deaminase (ADA) origins of DNA replication was examined in both the Ku80 mutant (xrs-5) and deficient (Ku80-/-) cell lines, and in their respective wild-type counterparts. Anti-Ku antibodies failed to immunoprecipitate a detectable amount of Ku from the either xrs-5 or Ku80-/- cells in the origin-containing-sequence, in contrast to the wild type cells, wherein Ku was found to be associated with the oribeta and ADA origins, respectively. / The data implicate Ku antigen in DNA replication and suggest the existence of another protein in rodent cells that is able to substitute for Ku function. They also indicate a novel function for Ku and the 14-3-3 isoforms beta, epsilon, gamma, zeta and sigma, as origin-binding-proteins in vivo, which provides a better understanding of the chromosomal association and DNA binding sequences of mammalian initiator proteins with origins of replication in their natural chromosomal environment.

Preliminary data suggests that the lipogenic factor Acylation Stimulating Protein (ASP), stimulates the activity of calcium-dependent phospholipase A2 (cPLA2) by increasing intracellular calcium levels [Ca2+]i and by activating extracellular-signal-regulated kinase 1/2 (ERK 1/2). The arachidonic acid (AA) generated by cPLA2 action appears to function as a second messenger in ASP signaling. / ASP also blocks TG breakdown. The calcium-independent PLA2 (iPLA2) zeta has recently been identified as a novel TG-lipase in 3T3-L1 cells. Bromoenol lactone (BEL), a non-reversible iPLA2 inhibitor, has been shown to specifically inhibit the TG-lipase activity of this enzyme. Preliminary data demonstrates that BEL stimulates basal TG synthesis, likely by inhibiting TG breakdown. The effects of BEL in combination with ASP are non-additive, suggesting they act through the same pathway. Furthermore, ASP appears to inhibit 3H-AA release into the media in a concentration-dependent manner. We propose that ASP inhibits an iPLA2 isoform with TG-lipase activity, an effect that can be mimicked by BEL.

Metal particles and ions from hip prostheses have the potential to stimulate macrophages to release various bone-resorbing mediators, leading to periprosthetic loosening and osteolysis around implants. Previous reports have suggested that the imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) activity may contribute to prosthetic loosening. However, the mechanism controlling these enzymes in the periprosthetic environment is unknown. The aim of the present project was to characterize the effect of Co2+ and Cr3+ ions on the expression of genes encoding MMP-1, one of the principal proteinases capable of degrading native fibrillar collagens in the extracellular matrix (ECM), its inhibitor TIMP-1, and TNF-alpha, a cytokine that plays a central role in the induction of implant osteolysis. Human U937 macrophages were incubated with Co2+ and Cr3+ ions. The level of mRNAs was determined by reverse transcription-polymerase chain reaction (RT-PCR). Our results show that both Co2+ and Cr3+ ions induce the expression of MMP-1, TIMP-1, and TNF-alpha in macrophages cultured in suspension. Tyrosine kinase inhibitors (genistein and herbimycin A) partially or completely block induction of MMP-1, TIMP-1, and TNF-alpha by ions. However, Co2+ and Cr3+ ions had no effect on the expression of MMP-1 and TIMP-1 in macrophages cultured on the PC-polymer, suggesting that the attachment of U937 macrophages to the PC-polymer surfaces may modify their gene expression. In fact, MMP-1 and TIMP-1 seem to be constitutively up regulated in this condition. Together, these findings indicate that activation of MMP-1, TIMP-1, and TNF-alpha by Co2+ and Cr3+ ions is regulated by tyrosine kinases and suggest that MMP-1 and TIMP-1 may contribute to the periprosthetic weakening, implant loosening, and osteolysis around implants.