A voltage-gated chloride channel in the yeast Saccharomyces cerevisiae.

We report the sequencing and identification on chromosome X of Saccharomyces cerevisiae of an open reading frame whose product, designated yClC-1, displays significant structural similarity to a voltage-gated Cl- channel family. This putative protein contains 13 hydrophobic domains very similar to transmembrane domains exhibited by known members of this family. Some amino acids in the domains and at the loops between them are well conserved among all members. This is the first voltage-gated Cl- channel described in the yeast S. cerevisiae. The identification of yClC-1 will facilitate the functional analysis of Cl- channels in general, and should also assist in the identification of other ClC genes in higher eukaryotes.

The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence.

The complete nucleotide sequence of the 3877-bp segment spanning the 3' region of intron-6 to the 5' region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exon gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40-55-million-year-old Alu-Se subclass.

Sequence of rat lipoprotein lipase-encoding cDNA.

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.

Using the fluorogenic 5' nuclease assay for high-throughput detection of (CA)n repeats in radiation hybrid mapping.

Here, the power of the 5' nuclease assay to detect PCR products containing (CA)n repeats was compared with that of the classical electrophoretic analysis. This assay, which relies on the use of a unique (CA)10 energy transfer-labeled probe and the 5' nuclease activity of Taq DNA polymerase, was used to construct a dog radiation hybrid map consisting of microsatellite markers. Data from over 7000 PCRs were analyzed in parallel by the fluorogenic assay and the conventional ethidium bromide-stained, agarose gel-based assay. We show that the fluorogenic assay provides a sensitive, reliable and specific method for detecting (CA)n amplimers. Moreover, as no processing is required after the PCR, the risk of carryover contamination and the time required for sample analysis are greatly reduced. All radiation hyrid (RH) assays can be performed using a single PCR protocol, and a standard analysis method has been developed that enables numerically automated data processing. On the whole, using this strategy greatly enhanced the rapidity, throughput and accuracy of the RH mapping of microsatellite markers.

Comparison of the cDNA and amino acid sequences of lipoprotein lipase in eight species.

By aligning nucleotide and amino acid sequences of lipoprotein lipase in eight species (man, pig, cow, sheep, mouse, rat, guinea-pig and chicken), we found that the main domains (catalytic, N-glycosylation and putative heparin binding sites) are well conserved. The longest identical amino acid chain was encoded by a sequence between the end of exon 2 and the beginning of exon 3, emphasizing the importance of this region which encodes the beta 5-loop of the active site, among other domains. Exon 10 is entirely untranslated in the seven mammals studied here and contains species-characteristic deletions, insertions or elements rich in A or A + T. In chicken, the beginning of exon 10 is translated. These eight previously unreported alignments could be a useful tool for further studies on LPL function.

Construction of a cosmid contig and of an EcoRI restriction map of yeast chromosome X.

We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.

[Structural characteristics of the human lipoprotein lipase gene]. / Données structurales sur le gène de la lipoprotéine lipase (LPL) humaine.

Lipoprotein lipase plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. In relation with this study, the fundamental results concerning the structure of human LPL gene are first summarized. Sequencing of this gene enabled us to characterize an Alu sequence. Interest of these repetitive sequences is exposed.

[The importance of the canine model in medical genetics]. / Intérêt du modèle canin pour la génétique médicale.

Dog domestication dates back to as early as 100,000 years ago, or 10,000 years depending upon the data used, and nowadays more than 350 breeds are duly registered in the different kennel clubs around the world. Due to intensive selection in the course of breeding, dog presently comes in any shape, size, color one can imagine, in addition to displaying a wide panel of characters, capacities and behaviours. As a consequence of excessive breeding, numerous breeds are plagued by a large variety of genetic diseases, many of them resembling those observed in human. All this makes dog an attractive model to track down genes and alleles responsible for those phenotypic behavioural or pathological traits, provided a genome map with polymorphic markers, and genes is available.

Can ribozymes be used to regulate procaryote gene expression?

The in vivo activity of ribozymes designed against mRNA coding for E. coli beta-galactosidase was tested both in intramolecular and in intermolecular conditions. When recombinant M13 phage DNA carrying on the same molecule the information for both the ribozyme and the target was transfected into bacterial cells, ribozyme activity was observed. Conversely, a ribozyme coded by a recombinant M13 vector, but targeted against an mRNA transcribed from the F episome including the remaining part of the beta-galactosidase gene, was inefficient.

Detection of B-tropic endogenous type-C virus in viral isolates originating from induced BALB/K-3T3 cells.

Isoelectric focusing (IEF) analysis of class I endogenous type-C virus induced by iododeoxyuridine treatment of BALB/K-3T3 cells revealed, in addition to the major variant of the p30 polypeptide, which has an isoelectric point of 6.1 (pI 6.1 isop30), a minor isop30 with a pI of 5.6. This value was also found for a prototype BALB/c B-tropic endogenous virus isolate. The pI 5.6 isop30 of the N-tropic isolate was amplified by long-term virus replication in B-type mouse cells, and comparative IEF and XC-assay data suggest that it may correspond to a B-tropic subpopulation which has not yet been detected in vitro in mouse cells of embryonic origin.

A possible yeast homolog of human active-gene-repairing helicase ERCC6+.

We report here the sequencing and identification on the chromosome X of S. cerevisiae of an open reading frame, designated GTA1085, encoding a protein 1085 amino acids in size that displays significant homology to a of helicase subfamily. The highest similarity score is with ERCC6, a human putative helicase involved in the repair of active genes, with 53.3% identity over a stretch of 589 amino acids. This putative protein contains all seven consecutive domains conserved among DNA and RNA helicases. Thus, it apparently constitutes a novel member of this subfamily and might be involved, like ERCC6, in the preferential repair of active genes in yeast.

Analysis of a 42.5 kb DNA sequence of chromosome X reveals three tRNA genes and 14 new open reading frames including a gene most probably belonging to the family of ubiquitin-protein ligases.

We have sequenced a 42,500 bp stretch located on chromosome X of Saccharomyces cerevisiae between the genes MET3 and CDC8. This stretch contains 24 open reading frames (ORFs) of at least 100 amino acids. Ten of these correspond to previously published sequences, whereas of the 14 remaining ORFs, only one, GTD892, has significant similarity to proteins from yeast or other organisms. It may belong to the family of ubiquitin-protein ligases and be involved in the ubiquitin-dependent proteolytic pathway. In addition, three tRNA genes were recognized, two of which had not been hitherto localized.

[Isoelectric variants of the principal polypeptide (p 30) of different strains of murine oncornaviruses]. / Répartition des variants iso-électriques du polypeptide principal (p 30) dans différentes souches d'oncornavirus murins

The major murine oncornavirus polypeptide p 30 (30,000 daltons), which bears the conventional group-specific (gs) determinants, can be separated by isoelectric focusing into four classes, with isoelectric points (pI) of 5.5, 5.9, 6.7 and 7.1. All the Moloney stocks tested have a pI 5.9 p 30, while p 30's with three to four different pI values are found to coexist in other stocks, with the pI 6.7 form predominating in Gross virus stocks.

The isoelectric point of the p30 polypeptide as a marker of mouse endogenous viruses.

The isoelectric point (PI) of the p30 polypeptide of members of the three known classes of mouse C-type endogenous viruses was determined both by column and by thin-layer gel isoelectric focusing. Each class was found to be characterized by a particular variant of p30 (isop30), with pI values of 6.1 for class I (ecotropic), 5.7 for class II (xenotropic), and 5.5 for class III (NZB, NIH, ATS124, also xenotropic). The 6.1-isop30 was found as a minor component of rat-grown NZB virus and of a number of laboratory strains of mouse C-type viruses.

Search for endogenous C-type viruses in cultures of non leukemic human cells.

Cultures of cells derived from non-leukemic human tissues were submitted to treatments known to induce endogenous C-type viruses of a number of animal species. Virus expression was evaluated by reverse transcriptase (RT) assays in the growth medium. Of 20 cultures treated or untreated with bromodeoxyuridine, 18 were totally negative in RT assays. Of the 2 positive cultures, one was a human-mouse hybrid in which the induction of RT activity coincided with the expression of murine antigens. The other culture, a diploid strain which exhibited borderline enzymatic activity, was subjected to more detailed analysis after treatment with several chemical inducers and/or irradiation, but none of these procedures gave clearly positive results. The apparent lack of an endogenous virus synthesis in these human cells is discussed.

Revised nucleotide sequence of the COR region of yeast Saccharomyces cerevisiae chromosome X.

The COR region, a gene cluster located on chromosome X of Saccharomyces cerevisiae and including genes CYC1, UTR1, UTR3, OSM1, tRNA(Gly) and RAD7, was sequenced within the framework of the European Union genome systematic sequencing project. It was compared with previously published sequences to be found in GenBank under the acronym YSCCORA. While some of the discrepancies observed can be readily ascribed to polymorphism, others most probably result from sequencing errors. A revised version of the sequence of the COR cluster is given.

Construction and optimization of a dog whole-genome radiation hybrid panel.

A dog whole-genome radiation hybrid (WGRH) panel including 126 clones was constructed by fusing dog fibroblasts irradiated at 5000 rads with thymidine kinase-deficient hamster cells. The average retention frequency of the panel designated as RHDF5000 is 21%, and its resolution power is estimated at 600 kb. The data provided by typing 400 markers were used to estimate linkage power changes subsequent to panel reduction. These changes were analyzed by recomputing typing data from five reduced panels. From these simulations, the parameters allowing investigation of the evolution of the linkage power in the course of panel reduction were determined. Guidelines for constructing a WGRH panel are proposed.

Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae.

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374-376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33.4%, 26.7%, 23.4% and 29.2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68.4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle.

Analysis of a 62 kb DNA sequence of chromosome X reveals 36 open reading frames and a gene cluster with a counterpart on chromosome XI.

We have sequenced a 61.989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X. This stretch contains 36 open reading frames (ORFs) of at least 100 codons. Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1. The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S. cerevisiae serine/threonine-specific protein kinase. Schizosaccharomyces pombe Act2 protein, S. cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively. Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence. In addition, three tRNA genes have been recognized. Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI.