Soybean seed lectin, Le1, is specifically located in seeds of soybean, Glycine max, (L.) Merr., due to its promoter. Gene homologues of Le1 were previously identified as possibly located in other parts of soybean. We cloned two novel promoters from these genes, and show that they drive reporter gene expression in transgenic Arabidopsis. A total of 1.3kb was isolated from each of the Le2 and Le3 5' promoter regions and fused with the GUS reporter gene. A previously cloned Le1 5' promoter was used as a control and the constructs were introduced into Arabidospis. GUS expression in transformed plants reveals that GUS driven by Le3 is found predominantly in vegetative tissues whereas GUS driven by Le2 show low expression in all tissues examined. The expression patterns resulting from the three different lectin promoters are distinct and consistent with regulatory motifs computationally identified in the sequences. / Chez le soja (Glycine max), le promoteur du gene lectine Le1 dirige l'expression spécifique dans les graines. Des homologues de Le1 existent dans le genome du soja et sont exprimées ailleurs dans la plante. Nous avons isolé deux promoteurs de ces homologues de lectine, et décrivons le patron d'expression qu'ils dirigent. Un total de 1.3 kilobase des regions 5' des promoteurs, en amont du gène, a été isolé pour chacune des copies Le2 et Le3, et fusionné avec le gène rapporteur GUS. Le promoteur de Le1 étant déjà connu, il sert de controle. L'Arabidopsis transformée avec ces constructions, montre que le promoteur de Le3 dirige l'expression dans les tissues végétatifs, tandis que le promoteur de Le2 procure un niveau minimal d'expression dans tous les tissus examinés. De plus, des analyses bioinformatiques identifient des motifs spécifiques dans les sequences de promoteurs qui confirment les patrons d'expression que nous avons démontrés.

The alternative splicing of the prosaposin gene results in the inclusion or exclusion of a 9 bp insertion of exon 8, which is located in the saposin B domain of this gene. Thus, exon 8 codes for three amino acid residues (Gin-Asp-Gln), which may potentially be present or absent in human prosaposin. Recently, it was shown that the alternative splicing of the prosaposin gene may be tissue specific and that a possible function of the three amino acid insertion could be to alter the binding specificity of saposin B towards different glycosphingolipids. We have recently cloned the murine SGP-1 gene and found that it also contains the 9 bp exon 8. In the present study we have used reverse transcription-polymerase chain reaction (RT-PCR) to study the distribution of the alternatively spliced mRNAs in several tissues. We have also used Northern Blot analysis to confirm the expression and stability of these transcripts, and light microscope immunocytochemistry to examine whether or not alternative splicing affects the translation of the mRNA transcripts into mature proteins. (Abstract shortened by UMI.)

Chromogranin A (CgA) is an acidic glycoprotein that is specifically expressed in neuroendocrine tissue. It is the best characterized member of the granin family, which includes proteins proposed to be involved in the regulation of peptide hormone and neurotransmitter secretion in the regulated secretors pathway by helping granule formation, targeting peptide hormones and neurotransmitters to the granules, affecting peptide hormone processing and controlling secretion through a feedback mechanism. / Although elaborate in vitro studies have been conducted on CgA, its precise neuroendocrine function and the complete elements that control its neuroendocrine specific expression remain unclear. This project applied transgenic technology to study CgA regulation and function in vivo. For the study of the regulation of CgA gene expression, four constructs, containing one of two promoter portions of the CgA gene (the 184bp proximal promoter portion and the 6kb 5'-flanking region) in two reporter systems (beta-galactosidase and green fluorescent protein systems), were constructed. (Abstract shortened by UMI.)

Obesity and obesity related diseases are increasing at an alarming rate. Elucidating the regulation of three anabolic hormones and their receptors in adipose tissue is an important step to understanding adipose tissue metabolism. The aims of this thesis are to (1) investigate the direct regulation of visfatin and C5L2 in vitro using a 3T3-L1 preadipocyte and adipocyte model with hormone treatments related to insulin resistance and metabolism and (2) investigate the regulation of multiple genes related to insulin resistance and ASP function in ex vivo human adipose tissue from SC vs OM depots in lean and morbidly obese subjects using a microarray technique. We observe a direct regulation of visfatin mRNA in 3T3-L1 preadipocyte and / or adipocytes by insulin, TNFa, progesterone, testosterone, oleate, palmitate, and the TZD treatment rosiglitizone. Further, in human ex vivo adipose tissue we observe a preferential regulation of insulin signaling genes in OM adipose tissue of morbidly obese subjects with relativly higher insulin and glucose (IRO). In contrast, changes in adipose tissue related to ASP-C5L2 metabolism are primarily in SC adipose tissue. From the studies presented in this thesis we have determined that C5L2 is present in human SC and OM adipose tissue, preadipocytes and adipocytes, and in all mouse adipose depots. In the absence of C5L2, ASP does not stimulate TGS. Endogenous C5L2 is downregulated by TNFa and upregulated by a TZD treatment in vitro, and C5L2 is upregulated ex vivo in a population of morbidly obese subjects with high ASP and high TG. Further, in this same morbidly obese population a general trend for the upregulation of anti-inflammatory genes / Le taux d'obésité et des maladies liées à celle-ci augmentent à une vitesse inquiétante. Aussi, la connaissance du mécanisme de régulation de certaines hormones anaboliques et de leurs récepteurs dans le tissu adipeux constitue une étape importante dans la compréhension du métabolisme lipidique. Cette thèse a pour but (i) d'étudier l'impact du traitement hormonal associé à la résistance à l'insuline et au métabolisme sur la régulation directe, in vitro, de la visfatine et du C5L2 en utilisant des modèles de 3T3-L1 préadipocytes et des adipocytes, et (ii) d'explorer, en utilisant des techniques de micro puces à ARN, la régulation de plusieurs gènes impliqués dans la résistance à l'insuline et dans la fonction de l'ASP, ex vivo, dans des tissus adipeux humains provenant de dépôts sous-cutanés (SC) versus omental (OM) extraits de sujets minces et de sujets atteints d'obésité morbide. Nos travaux ont démontré que le traitement à l'insuline, le TNFa, la progestérone, l'oléate, le palmitate et le traitement au TZD rosiglitizone régulent directement l'expression des ARNm de la visfatine dans les 3T3-L1 préadipocytes et/ou adipocytes. De plus, dans les tissus adipeux humains ex vivo, nous avons noté une régulation préférentielle des gènes impliqués dans la signalisation par l'insuline, surtout dans le tissu adipeu omental de sujets atteints d'obésité morbide et ayant des taux d'insuline et de glucose relativement plus élevés. Par contre, les changements touchant la signalisation ASP-C5L2 se trouvent dans le tissu adipeux sous-cutané. Cette étude nous a permis de déterminer la présence du C5L2 dans les tissus adipeux humains

Is a nuclear phosphoprotein synthesized as a 57 kDa protein that is primarily found in the proximity of the nuclear envelope. It is expressed in growth-arrested cells, such as quiescent and senescent human fibroblasts but not in their replicating counterparts (Wang, 1985a), and in terminally differentiated cells (Muggleton-Harris et al., 1989). Hence, statin is a useful marker for nonproliferation and suppressed growth. On the other hand, transformed cells are statin negative. / A WI-38 cDNA library was screened for potential statin clones using the antistatin antibody S44. Clone S44-I was obtained and sequence analysis demonstrated that it shows 99.9% homology to the human C-terminal peroxisomal targeting signal (hPTS1). Northern blot analysis revealed that it is expressed not only in quiescent and senescent, but also in replicating WI-38 as well as in transformed cells. These results suggest that the expression of S44-I is not cell-cycle-arrested-specific. / In vitro translation of S44-I using 35S labeled L-methionine demonstrated that it encodes for a 71 kDa protein which is consistent with the size of the open reading frame (ORF). Moreover, S44-I was transiently transfected and Western blot analysis revealed that S44-I encodes for the expected 71 kDa protein in addition to an 80 kDa polypeptide. Mutation of potential initiation methionines of S44-I for possible alternate initiation of translation mechanism did not eliminate any of the two proteins. / In conclusion, these findings are not consistent with previous studies that have analyzed statin at the protein level. This implies that S44-I does not encode for statin but for the hPTS1 or a related member which may possess a shared antigenic epitope recognizable by the anti-statin antibody.

An important aspect of the regulation of eukaryotic gene expression is the modulation of translation rates, which is most often controlled at the level of initiation. The interaction of proteins of the translational machinery with the mRNA cis-acting elements, governs mRNA translatability. Although both the 5' cap structure and 3' poly(A) tail act independently to stimulate translation, together they synergistically enhance translation. Therefore, an in-depth study of translation initiation would require examining the role of regulatory proteins acting on the 5' and 3' untranslated region (UTR) of the mRNA, and their influence on translation rates. A study of proteins interacting with the mRNA 5' UTR, reveals that translational homeostasis is induced by eIF4E through control of the 4E-BP1 and p70 S6 kinase activities. In an eIF4E tetracycline inducible system, overexpression of eIF4E leads to dephosphorylation of 4E-BP1 and p70 S6 kinase (but not Akt), with the extent of dephosphorylation proportional to the expression level of eIF4E. Therefore, a negative feedback loop is engendered by eIF4E expression that targets a downstream component of PI 3-kinase. The mechanisms of translational control imparted via the 3' UTR of the mRNA was studied by cloning a novel PABP interacting protein-2 (Paip2). Paip2 inhibited translation both in vivo and in vitro, in a dose dependent manner. The dual mechanism by which Paip2 inhibits the stimulatory role of PABP on translation is through (i) preventing the interaction of PABP with the poly(A) and disrupting the repeating structure of the poly(A) ribonucleoprotein structure and (ii) through direct competition with the translational co-activator Paip1 for PABP binding. BIACore data indicates that Paip2 binds PABP using a two site simple fit model. Consistent with this, two Paip2 molecules may be associated with one PABP molecule in vivo . Thus, the functional significance of interaction of Paip2 with PABP acting at the 3' UTR is a

The proprotein convertases (PCs) are involved in the activation of a wide variety of precursors via limited proteolysis at either single or paired basic residues, a crucial regulatory process in both normal and disease states. Seven members of this family were identified including furin, PC I, PC2, PC4, PACE4, PC5 and PC7. All of which exhibit a signal peptide, a prosegment, a catalytic domain, a P-domain and a specific C-terminal segment. The present work concentrated on the characterization of two structural elements, namely the prosegment and the P-domain, which are critical for enzymatic function and cellular trafficking. We examined the biosynthesis, functional activity and cellular localization of two PACE4 isoforms generated by differential splicing, the full length PACE4-A and the C-terminally truncated PACE4-CS that lacks 11 amino acids at the end of its chaperone-like P-domain. Cellular expression demonstrated that PACE4-A codes for a functional secretable enzyme capable of cleaving pro7B2 into 7132. However, PACE4-CS is not secreted and remains in the endoplasmic reticulum as an inactive zymogen form, therefore emphasizing the importance of the integrity of the P-domain. The prosegment is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. We purified recombinant prosegments of furin (pFurin) and PC7 (pPC7) from bacteria. The inhibitory potencies and selectivities of the prosegments were tested in vitro and ex vivo. The pFurin and pPC7 are potent inhibitors (IC50 low nM) of their parent enzymes. Whereas pPC7 is more selective than pFurin. Most of the inhibitory potency seems to reside at the C-terminal region immediately preceding the primary cleavage site of the prosegment with the P1 Arg playing a critical role. Furthermore, overexpression of prosegments in cell lines efficiently blocked precursor processing of the neurotrophins NGF and BDNF. For the first time our results showed that PC prosegments expressed ex

The enzymatic activity of the protein tyrosine phosphatase, CD45 has been demonstrated to play an absolutely required role in the regulation of Src family protein tyrosine kinases in T lymphocytes. CD45 function during early events of antigen receptor signaling has been well established, however, the role of CD45 during later stages of T cell activation is only beginning to emerge. Transient tyrosine phosphorylation of CD45 has previously been demonstrated. We show this phosphorylation can be sustained through treatment of a T cell hybridoma cell line with the PTPase inhibitor, pervanadate. Tyrosine phosphorylation of CD45 is significantly increased in the presence of an activated form of Lck. Tyrosine phosphorylated CD45 is correlated with the association of a subset of signaling molecules containing SH2 domains. These molecules include p56Lck, p59Fyn, rasGAP, Grb2 and Csk. Although CD45 becomes abundantly tyrosine phosphorylated upon pervanadate treatment, no other SH2-containing molecules that we had tested demonstrated association with CD45. The interaction between CD45 and Grb2 in particular was found to be dependent upon CD45 tyrosine phosphorylation and mediated through the SH2 domain of Grb2. Interestingly, both Grb2 and a close homolog, Grap are able to bind CD45 in in vitro binding assays. Grap does not, however, associate with CD45 in vivo as was observed for Grb2. / CD45 contains two Grb2 consensus binding sequences. Deletion of both results in a greatly diminished capacity for CD45- Grb2 association. However, interaction between the two is not completely abolished and appears to result from an unconventional peptide sequence as observed by 2-dimensional phosphopeptide mapping studies. In the process of mapping the phosphotyrosine sequences required for CD45-Grb2 interaction, many tyrosine-phosphorylated peptides have been identified and assigned to tyrosine residues in CD45.

The expression of different myosin heavy chain (MHC) isoforms is under the control of several factors, including the state of innervation and thyroid hormone levels. Muscle regeneration following injection of venom from the snake Notechis scutatus scutatus into rat soleus was used to study the role of innervation and excess thyroid hormone in influencing MHC gene expression. MHC protein and mRNA content was determined in muscles regenerating in the presence/absence of innervation or in the hyperthyroid state. / In summary, MHC production appears to be primarily controlled at the level of transcription or mRNA stability; however, translational or post-translational control may play a role for IIA MHC protein production. The results also suggest that satellite cells are not preprogrammed to express a particular MHC isoform, but rather are capable of expressing either fast or slow MHC. Subsequent expression of MHC isoforms is modified by factors such as innervation and thyroid hormone.

The eukaryotic mRNA 5$ sp prime$ cap structure m$ sp7$GpppX (where X is any nucleotide) is of crucial importance in translation initiation and protects messenger RNAs (mRNAs) against exonucleolytic degradation. L-A and L-BC are double-stranded RNA viruses which are persistently maintained in the cytoplasm of many Saccharomyces cerevisiae strains. Unexpectedly, their coat proteins exhibited a strong cap-binding activity. Unlike any other cap-binding protein, the coat protein (gag) of the L-A virus was shown to attach covalently to the cap structure of mRNAs, resulting in the cleavage of the cap structure and in the capture of m$ sp7$Gp. The linkage was determined to be a phosphoroimidazole bond between the $ alpha$ phosphate of the cap structure and a nitrogen ($N sp1$ or $N sp3$) in the gag His154 imidazole side-chain. Mutation of His154 abrogated the ability of gag to covalently link to the cap structure, without affecting cap recognition, viral particle formation from an L-A cDNA clone in vivo, or specific binding and replication of (+) single-stranded RNA in vitro. However, genetic analysis demonstrated that His154 was essential for the expression of viral proteins, possibly because efficient translation of the viral mRNA requires capping of the initially ppG-terminated viral transcripts by transfer of the m$ sp7$Gp captured by gag.