Biomedical subjects

M Chasles

Publications and source records attributed to M Chasles.

9 recordsLinked to original sources

PubMed2006

Two Hrd1p homologues in the yeast Yarrowia lipolytica which act in different pathways.

The endoplasmic reticulum associated degradation (ERAD) is a process widespread in eukaryotes that enable cells to get rid of unfolded or unassembled polypeptides which jam the endoplasmic reticulum compartment. In order to improve understanding of the initial steps of the secretory pathway and their relationship, we focused on components of the ERAD ubiquitylation machinery in the yeast Yarrowia lipolytica. Two Hrd1p homologues, Hrd1p and Hrh1p, were identified in Y. lipolytica. A study of the fate of the heterologous CPY* reporter protein showed that YlHrd1p is involved in the elimination of this misfolded polypeptide, while YlHrh1p is not. Moreover, the different phenotypic pattern displayed by Deltahrd1 and Deltahrh1 cells suggests that the two putative E3 enzymes function in separate ways. Our results bring some evidence of a coupling between the ERAD pathway and the co-translational translocation process and show that studies in Y. lipolytica can give new insights into events that take place in the ER.

Amino Acid Sequence↗

PubMed2001

Only centromeres can supply the partition system required for ARS function in the yeast Yarrowia lipolytica.

Autonomously replicating sequences (ARSs) in the yeast Yarrowia lipolytica require two components: an origin of replication (ORI) and centromere (CEN) DNA, both of which are necessary for extrachromosomal maintenance. To investigate this cooperation in more detail, we performed a screen for genomic sequences able to confer high frequency of transformation to a plasmid-borne ORI. Our results confirm a cooperation between ORI and CEN sequences to form an ARS, since all sequences identified in this screen displayed features of centromeric DNA and included the previously characterized CEN1-1, CEN3-1 and CEN5-1 fragments. Two new centromeric DNAs were identified as they are unique, map to different chromosomes (II and IV) and induce chromosome breakage after genomic integration. A third sequence, which is adjacent to, but distinct from the previously characterized CEN1-1 region was isolated from chromosome I. Although these CEN sequences do not share significant sequence similarities, they display a complex pattern of short repeats, including conserved blocks of 9 to 14 bp and regions of dyad symmetry. Consistent with their A+T-richness and strong negative roll angle, Y. lipolytica CEN-derived sequences, but not ORIs, were capable of binding isolated Drosophila nuclear scaffolds. However, a Drosophila scaffold attachment region that functions as an ARS in other yeasts was unable to confer autonomous replication to an ORI-containing plasmid. Deletion analysis of CEN1-1 showed that the sequences responsible for the induction of chromosome breakage could be eliminated without compromising extrachromosomal maintenance. We propose that, while Y. lipolytica CEN DNA is essential for plasmid maintenance, this function can be supplied by several sub-fragments which, together, form the active chromosomal centromere. This complex organization of Y. lipolytica centromeres is reminiscent of the regional structures described in the yeast Schizosaccharomyces pombe or in multicellular eukaryotes.

Base Sequence↗

PubMed1999

Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica.

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

Centromere↗

PubMed1997

An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica.

Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P. Fournier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Gaillardin, Proc. Natl. Acad. Sci. USA 90:4912-4916, 1993; and P. Fournier, L. Guyaneux, M. Chasles, and C. Gaillardin, Yeast 7:25-36, 1991). We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.

Base Sequence↗

PubMed1993

Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica.

Two sequences (ARS18 and ARS68) displaying autonomous replication activity were previously cloned in the yeast Yarrowia lipolytica. The smallest fragment (1-1.3 kb) required for extrachromosomal replication of a plasmid is significantly larger in Y. lipolytica than in Saccharomyces cerevisiae. Neither autonomously replicating sequence (ARS) is homologous with known ARS or centromere (CEN) consensus sequences. They share short regions of sequence similarity with each other. These ARS fragments also contain Y. lipolytica centromeres: (i) integration of marker genes at the ARS loci results in a CEN-linked segregation of the markers, (ii) an ARS on a plasmid largely maintains sister chromatid attachment in meiosis I, and (iii) integration of these sequences at the LEU2 locus leads to chromosome breakage. Deletions performed on ARS18 show that CEN and ARS functions can be physically separated, but both are needed to establish a replicating plasmid.

Base Sequence↗

PubMed1992

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica.

The mating type gene MATA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.

Chromosome Deletion↗

PubMed1991

Scarcity of ars sequences isolated in a morphogenesis mutant of the yeast Yarrowia lipolytica.

Previous attempts is isolate autonomously replicating sequences (ars) from the dimorphic yeast Yarrowia lipolytica have been unsuccessful. We isolated a Fil- mutant unable to produce hyphae and growing only in a yeast form to facilitate ars isolation. This mutant was transformed with a Y. lipolytica DNA bank and several unstable clones were obtained. Extrachromosomal plasmids were evidenced in yeast, recovered in Escherichia coli and characterized by restriction mapping. They were able to retransform Fil- and Fil+ yeast strains at high frequency and transformants displayed a slightly unstable phenotype. The detailed analysis of the plasmids showed that only two different ars sequences had been isolated, each of them corresponding to a unique sequence in the Y. lipolytica genome. We concluded that functional ars sequences that can be cloned on plasmids are rare in this yeast.

Base Sequence↗

PubMed1991

Use of ars18 based vectors to increase protein production in Yarrowia lipolytica.

The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991). Vectors containing ars18 have been used to increase homologous and heterologous protein production. Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin. A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.

Animals↗

PubMed1990

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Amino Acid Sequence↗