Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.     

Transposable elements in yeasts

With the development of new sequencing technologies in the past decade, yeast genomes have been extensively sequenced and their structures investigated. Transposable elements (TEs) are ubiquitous in eukaryotes and constitute a limited part of yeast genomes. However, due to their ability to move in genomes and generate dispersed repeated sequences, they contribute to modeling yeast genomes and thereby induce plasticity. This review assesses the TE contents of yeast genomes investigated so far. Their diversity and abundance at the inter- and intraspecific levels are presented, and their effects on gene expression and genome stability is considered. Recent results concerning TE-host interactions are also analyzed.     

Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Multiple-mutation reaction: a method for simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene.     

Testing the antagonistic effect of follistatin on BMP family members in ovine granulosa cells

Follistatin was first demonstrated as an activin-binding protein, neutralizing its actions. However, there is emerging evidence that follistatin inhibits the action of other members of the transforming growth factor beta(TGFbeta) / bone morphogenetic protein (BMP) superfamily. Recently, numerous BMP factors have been shown to play important roles in regulating folliculogenesis and ovulation rate in mammals, and such a potential antagonistic role of follistatin is of particular interest in the context of ovarian function. Using a biological test based on progesterone production by ovine primary granulosa cells in culture, we show that follistatin was a strong antagonist of activin A, but not BMP-2 or BMP-4 actions. In contrast, noggin, a known specific BMP antagonist, had no effect on activin A but strongly neutralized BMP-2 and BMP-4 actions. BMP-6 action was only slightly reduced by both follistatin and noggin. Our data led to the conclusion that follistatin would not represent a determinant physiological modulator of the biological effect of BMP factors on granulosa cells.     

BAG-1 p29 protein prevents drug-induced cell death in the presence of EGF and enhances resistance to anoikis in SKOV3 human ovarian cancer cells

BAG-1 is a multi-functional protein that exists in three major isoforms, BAG-1 p50, p46, and p36. A fourth isoform of 29 kDa also exists but its function remains mostly unknown. To further understand the role of this smaller isoform in ovarian cancer cells, the SKOV3 cell line was transfected with a doxycycline-inducible human BAG-1 p29 isoform or control plasmid. Ovexpression of BAG-1 p29 promotes protection from apoptosis in the presence of EGF as shown by decreased cell death measured by XTT assay and caspase-3 activity. Unexpectedly, however, BAG-1 p29 does not associate with the EGF receptor. When BAG-1 p29 transfectants were incubated in hydrogel-coated plates, BAG-1 p29-expressing SKOV3 cells were significantly more resistant to anoikis as compared to controls, and this correlated with decreased activation of caspase-3. The results of this study implicate BAG-1 p29 in the regulation of both the EGF signaling cascade and the apoptotic cascade induced by loss of anchorage.     

Bone morphogenetic protein 5 expression in the rat ovary: biological effects on granulosa cell proliferation and steroidogenesis

Recently, the role of several elements of the bone morphogenetic protein (BMP) family has been studied in the ovary, some of them being crucial for ovarian function. In the present work, we have studied bone morphogenetic protein 5 (BMP5) expression and its biological role in the rat ovary. BMP5 is expressed by rat granulosa cells (GCs) and exerts specific biological effects on proliferation and steroidogenesis of these cells in an autocrine manner. These effects were shown to be associated with an increase in cyclin D2 protein level and a decrease in steroidogenic acute regulatory (StAR) protein expression in GCs in vitro. Ultimately, BMP5 actions were inhibited by follistatin. Overall, these data show that BMP5 is a novel element of the BMP family that might play a fully paracrine role in rodent ovarian folliculogenesis.