A yeast t-SNARE involved in endocytosis.

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

Efficient transcription of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase obtained by a new procedure [corrected].

The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion with ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66.

Genetic study of the CD36 gene in a French diabetic population.

OBJECTIVES: CD36 is a multifunctional membrane receptor widely expressed in different tissues which binds and internalizes oxidized low-density lipoprotein. In rodents, CD36 gene variations modulate glucose homeostasis and contribute to metabolic syndrome associated with type 2 diabetes but the effects in human are unknown. METHODS: We screened the entire coding sequence of the CD36 gene in 272 individuals and we genotyped both rare and frequent variants in 454 T2D subjects and 221 controls. RESULTS: We detected five mutations, P191P and N247S were only found each in one family and did not segregate with diabetes, the three others (A/C-178 in the promoter, A/G-10 in intron 3 and (GGGTTGAGA) insertion in intron 13) being equally frequent in diabetic subjects and in controls. However, adiponectin levels, a marker for insulin sensitivity, were significantly associated with the -178 A/C promoter variant allele (p=0.003, p corrected for multiple testing=0.036), possibly reflecting association with insulin-resistance in the French population. CONCLUSION: Thus, the -178 A/C SNP promoter mutation in the CD36 gene represents a putative genetic marker for insulin-resistance in the French population, although it does not appear to contribute to the genetic risk for T2D.

Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between the rapidly dividing tachyzoite and the slowly replicating bradyzoite in intermediate hosts such as humans and domestic animals. We have recently identified a bradyzoite cDNA encoding a putative phosphatidylinositol (PtdIns) synthase using a subtractive library [Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., and Tomavo, S. (1999) Mol. Biochem. Parasitol. 99, 223-235]. Here, we report the cloning of another cDNA encoding PtdIns synthase that is exclusively expressed in the tachyzoite stage. The two transcripts are encoded by two different genes, which are stage-specifically regulated. The deduced amino-acid sequence (258 amino acids with a calculated total molecular mass of 27.8 kDa) of the tachyzoite-specific cDNA shares a significant degree of identity (between 26.5 and 30.1%) to the PtdIns synthases from human, rat, Arabidopsis thaliana and yeast. Interestingly, the putative protein encompasses an N-terminal extension that is approximately 40 amino-acids longer than that of PtdIns synthases from other organisms. Functional complementation realized by tetrad analysis of segregants of a Saccharomyces cerevisiae PtdIns synthase-deficient mutant (PIS1/pis1:kanMX4) showed that only the T. gondii putative PtdIns synthase truncated at its N-terminal extension is able to restore the viability of the cells. We demonstrate that this protein expressed in yeast transformants is functionally active in the membrane preparation and requires manganese and magnesium ions for activity. To our knowledge, this is the first report on the molecular cloning and functional analysis of a gene encoding a PtdIns synthase in protozoan parasites.

Expression of the 69K movement protein of turnip yellow mosaic virus in insect cells.

The nonstructural 69-kilodalton (K) protein of turnip yellow mosaic virus is necessary for systemic spread of the virus within the plant. To examine the behavior of the 69K protein in vivo, antibodies were raised against the carboxy-terminal region of this protein. The full-length 69K protein was also expressed in insect cells using a recombinant baculovirus. Studies on the posttranslational modifications of the 69K protein in insect cells revealed that the protein is phosphorylated but not glycosylated. Further experiments of subcellular fractionation and indirect immunolocalization in insect cells showed that the 69K protein is localized in the cytoplasm and/or in the plasma membrane.

Disruption of six novel genes from the left arm of chromosome XV of Saccharomyces cerevisiae and basic phenotypic analysis of the generated mutants.

Six open reading frames (ORFs) of unknown function from the left arm of Saccharomyces cerevisiae chromosome XV were deleted in two genetic backgrounds by disruption cassettes with long flanking homology (LFH) (Wach, 1996), within the frame of the research project EUROFAN. The LFH disruption cassettes, obtained by PCR, were made by introducing the kanMX4 marker module between two fragments homologous to the promoter and terminator regions of a given ORF. Transformants resistant to geneticin (G418) were selected. The LFH disruption cassettes were cloned in a bacterial vector. Each cognate gene was also cloned in a centromeric plasmid. Correct deletion of each gene was verified by four different PCR reactions. Sporulation and tetrad analysis of heterozygous deletants revealed that ORF YOL102c is essential. The non-growing haploid spores gave rise to microcolonies. Basic phenotypic analyses were performed on haploid deletants of both mating types of the five non-essential ORFs, YOL018c, YOL098c, YOL101c, YOL104c and YOL105c. Plate growth tests on different media at 15 degrees C, 30 degrees C or 37 degrees C did not reveal any significant differences between parental and mutant cells. Mating and sporulation efficiencies were not affected in any of the viable disruptants as compared to wild-type cells.

Gene expression from viral RNA genomes.

This review is centered on the major strategies used by plant RNA viruses to produce the proteins required for virus multiplication. The strategies at the level of transcription presented here are synthesis of mRNA or subgenomic RNAs from viral RNA templates, and 'cap-snatching'. At the level of translation, several strategies have been evolved by viruses at the steps of initiation, elongation and termination. At the initiation step, the classical scanning mode is the most frequent strategy employed by viruses; however in a vast number of cases, leaky scanning of the initiation complex allows expression of more than one protein from the same RNA sequence. During elongation, frameshift allows the formation of two proteins differing in their carboxy terminus. At the termination step, suppression of termination produces a protein with an elongated carboxy terminus. The last strategy that will be described is co- and/or post-translational cleavage of a polyprotein precursor by virally encoded proteinases. Most (+)-stranded RNA viruses utilize a combination of various strategies.

Uracil-induced down-regulation of the yeast uracil permease.

In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.

In vitro transcripts of turnip yellow mosaic virus encompassing a long 3' extension or produced from a full-length cDNA clone harbouring a 2 kb-long PCR-amplified segment are infectious.

Two types of full-length cDNA clones have been constructed corresponding to the entire genome of turnip yellow mosaic virus (TYMV), from which infectious transcripts devoid of 5' non-viral extensions can be synthesized in vitro. The first type of transcript (tTYFL7) harbours 75 non-viral nucleotides at its 3' end, whereas the second type (tTYFL84) possesses only 2 non-viral nucleotides at its 3' end. The 2 kilobase-long 3' region of tTYFL84 derives from amplification by the polymerase chain reaction of the corresponding TYMV cDNA. Both tTYFL7 and tTYFL84 are infectious in rapeseed protoplasts and plants. tTYFL7 is far less infectious than wild-type TYMV RNA and somewhat less infectious than tTYFL84. The possible effects of the 3' extraviral sequences of tTYFL7 and the heterogeneity observed in the infectivity of other transcripts prepared as was tTYFL84 are discussed.