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International audience; Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

International audience; Little is known about the major histocompatibility complex (MHC) class 1 in squirrel monkeys ( Saimiri sciureus). We cloned, sequenced and characterized two alleles and the cDNA of the coding region of MHC class 1 in these New World monkeys. Phylogenetic analyses showed that these sequences are related to HLA class 1 genes ( HLA-A and HLA-G). The structure and organization of one of the two identified clones was similar to that of a class 1 MHC gene ( HLA-A2). All the exon/intron splice acceptor/donor sites are conserved and their locations correspond to the HLA-A2 gene. The sequences of the newly described cDNAs reveal that they code for the characteristic class 1 MHC proteins, with all the features thought necessary for cell surface expression. Typical sequences for the leader peptide, alpha(1), alpha(2), alpha(3), transmembrane and cytoplasmic domains were found.

The BRCA2 tumor suppressor has been implicated in the maintenance of genomic integrity through a function in cellular responses to DNA damage. The BRCA2 protein directly associates with Rad51, that is essential for repair of double-strand breaks (DSBs) by homologous recombination (HR). In this report, we study the BRCA2-defective Chinese hamster cell mutant V-C8 for its ability to perform homology-directed repair (HDR) between repeated sequences. V-C8 cells were recently shown to be defective in Rad51 foci formation in response to DNA damage. Strikingly, we find that these BRCA2 mutant cells exhibit a strong stimulation of HDR activity compared to the V79 parental cells, which harbor a wild-type BRCA2. Furthermore, molecular characterization of the HDR products shows that loss of BRCA2 in V-C8 cells leads to significant reduction in Rad51-dependent gene conversion but strong enhancement of Rad51-independent single-strand annealing (SSA) events frequency. These data imply that, when HDR by conservative gene conversion is impaired, DSBs usually repaired by this pathway are instead resolved by other non-conservative HDR subpathways. Therefore, high chromosomal instability in BRCA2-deficient cells presumably results from enhancement of error-prone repair mechanisms, such as SSA.

International audience; Primates have long been used as models to study the basic mechanisms of human disease. The squirrel monkey, Saimiri sciureus,is a New World monkey that can be satisfactorily bred in captivity and infected with various human pathogens. The basic immunological parameters of squirrel monkeys, including immunoglobulin subclasses and markers for lymphocyte subsets, have already been characterized. However, immunological reagents and assays specific for the detection and quantification of squirrel monkey cytokines are required to elucidate the immunological and physiopathological changes that occur during experimental infection of these animals with pathogens. We therefore cloned, sequenced, and characterized the cDNAs encoding various squirrel monkey Th1 and Th2 cytokines, including interleukin (IL)-1 beta, IL-2, IL-5, IL-6, IL-10, tumor necrosis factor alpha, and gamma interferon. We found 91.4%-98.1% homology between the nucleotide sequence of the squirrel monkey cytokine genes and published sequences of equivalent human and non-human primate genes. The aligned sequences of cytokines for Saimiriand several New and Old World primates and human are shown, and a phylogenetic analysis of published sequences of selected cytokines in other species and those of non-human primates is given. In addition, we adapted a previously described quantitative, reverse transcriptase, competitive polymerase chain reaction technique to measure the mRNA expression of those cytokines in squirrel monkeys. The primer and competitor plasmids that allowed quantification are described. We showed that the assay is sensitive and reproducible.

International audience; The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.

Background An important issue concerning the evolution of duplicated genes is to understand why paralogous genes are retained in a genome even though the most likely fate for a redundant duplicated gene is nonfunctionalization and thereby its elimination. Here we study a complex superfamily generated by gene duplications, the snail related genes that play key roles during animal development. We investigate the evolutionary history of these genes by genomic, phylogenetic, and expression data studies. Results We systematically retrieved the full complement of snail related genes in several sequenced genomes. Through phylogenetic analysis, we found that the snail superfamily is composed of three ancestral families, snail, scratchA and scratchB. Analyses of the organization of the encoded proteins point out specific molecular signatures, indicative of functional specificities for Snail, ScratchA and ScratchB proteins. We also report the presence of two snail genes in the annelid Platynereis dumerilii, which have distinct expression patterns in the developing mesoderm, nervous system, and foregut. The combined expression of these two genes is identical to that of two independently duplicated snail genes in another annelid, Capitella spI, but different aspects of the expression patterns are differentially shared among paralogs of Platynereis and Capitella. Conclusion Our study indicates that the snail and scratchB families have expanded through multiple independent gene duplications in the different bilaterian lineages, and highlights potential functional diversifications of Snail and ScratchB proteins following duplications, as, in several instances, paralogous proteins in a given species show different domain organizations. Comparisons of the expression pattern domains of the two Platynereis and Capitella snail paralogs provide evidence for independent subfunctionalization events which have occurred in these two species. We propose that the snail related genes may be especially prone to subfunctionalization, and this would explain why the snail superfamily underwent so many independent duplications leading to maintenance of functional paralogs.