PubMed Health⌕ Search

Biomedical subjects

A Vassarotti

Publications and source records attributed to A Vassarotti.

16 recordsLinked to original sources
PubMed1994

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Base Composition↗
PubMed1987

Sequences distal to the mitochondrial targeting sequences are necessary for the maturation of the F1-ATPase beta-subunit precursor in mitochondria.

The beta-subunit of the mitochondrial F1-ATPase is synthesized as a precursor in the cytoplasm which is delivered through two bilayers bounding the mitochondria prior to its assembly with other proteins into a functional complex. In order to determine the role of the amino-terminal 50 residues of the precursor on its localization, maturation, and assembly, a set of deletions within this region of the ATP2 gene encoding the beta-subunit has been analyzed. These studies reveal that deletions between residue 10 of the F1 beta-presequence and residue 36 can still direct in vivo mitochondrial import and assembly of the mutant subunit into a functional complex. Deletions within ATP2 which contain less than the first 10 residues of the precursor are not imported. Thus, the extreme amino terminus (about half of the transient presequence) of the F1 beta-subunit can direct its mitochondrial import. The wild-type F1 beta-subunit precursor is matured by the matrix-located metalloprotease at Lys19-Gln20; however, small in-frame deletions up to 17 residues distal to this site fail to be matured either in vitro or in vivo. This nonmatured F1 beta-subunit is also assembled into a functional enzyme and supports growth of its host on a nonfermentable carbon source. These data indicate that maturation of the F1 beta-subunit precursor is dependent on a protein sequence located distal to the proteolytic maturation site which is distinct from the mitochondrial targeting sequence.

Amino Acid Sequence↗
PubMed1987

Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import.

Intracellular delivery of the mitochondrial F1-ATPase beta-subunit precursor from the cytoplasm into the matrix of mitochondria is prevented by deletion of its mitochondrial import signal, a basic amphipathic alpha-helix at its amino terminus. Using a complementation assay, we have selected spontaneous mutations which restore the correct in vivo localization of the protein containing the import signal deletion. Analysis of these mutations revealed that different functional surrogate mitochondrial targeting signals formed within a narrow region of the extreme amino terminus of the import signal deleted beta-subunit. These modifications specifically replace different acidic residues with neutral or basic residues to generate a less acidic amphipathic helix within a region of the protein which is accessible for interaction with the membrane surface. The observations of this study confirm the requirement for amphipathicity as part of the mitochondrial import signal and suggest how mitochondrial targeting signals may have evolved within the extreme amino terminus of mitochondrial proteins.

Amino Acid Sequence↗
PubMed1986

The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal.

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.

Amino Acid Sequence↗
PubMed1985

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor.

The yeast nuclear gene ATP2 encodes a F1-ATPase beta-subunit protein of 509 amino acids with a predicted mass of 54,575 daltons. In contrast to the ATPase beta-subunit proteins determined previously from Escherichia coli and various plant sources, the yeast mitochondrial precursor peptide contains a unique cysteine residue within its immediate amino terminus. Expression of an in-frame deletion in ATP2 between residues 28 and 34 to eliminate this single cysteine residue located near the processing site of the matrix protease does not prevent the in vivo delivery of the subunit to mitochondria or its assembly into a functional ATPase complex. Thus, the import F1 beta-subunit into mitochondria does not require a covalent modification of the type utilized for the secretion of the major lipoprotein from E. coli. In addition, analysis of the level of the major F1-ATPase subunits in mitochondria prepared from an atp2- disruption mutant demonstrates that the in vivo import of these catalytic subunits is not dependent on each other. These data and additional studies, therefore, suggest that the determinants for mitochondrial delivery reside within the amino terminus of the individual precursors.

Amino Acid Sequence↗
PubMed1985

Isolation of the fructose-1,6-bisphosphatase gene of the yeast Schizosaccharomyces pombe. Evidence for transcriptional regulation.

The fructose-1,6-bisphosphatase structural gene (FBP+) of Schizosaccharomyces pombe has been isolated by genetic complementation of a deficient mutant, which is characterized by the inability to grow on a nonfermentable carbon source such as glycerol. Growth on glycerol-containing medium was restored in a S. pombe fructose-1,6-bisphosphatase-deficient mutant (fbp-16) when it was transformed with a plasmid (pAVO4) carrying FBP+. The transformant displayed a 5-fold increase in enzymatic activity when compared to the parental S. pombe strain. Subcloning of DNA fragments from the 8.5-kilobase (kb) insert of pAVO4 defined a 4-kb DNA fragment which contained the functional FBP+ gene and its regulatory region. When this gene was placed under the control of the lac promoter-operator, functional expression in Escherichia coli was obtained, as deduced by complementation of bacterial fructose-1,6-bisphosphatase mutants. The FBP+ gene encodes a 1.9-kb glucose-repressible transcript whose appearance in S. pombe is correlated with fructose-1,6-bisphosphatase derepression in glycerol-containing medium. We suggest that the regulation of the S. pombe FBP+ gene is exerted at the transcriptional level. The S. pombe FBP+ gene gave rise to a 1.9-kb transcript in Saccharomyces cerevisiae, but not to measurable enzymatic activity.

Ascomycota↗
PubMed1984

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe.

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.

Animals↗
PubMed1984

Independent loci for the structural genes of the yeast mitochondrial alpha and beta ATPase subunits.

In the yeast Schizosaccharomyces pombe, the structural gene mutations A23-13 (alpha-) and B59-1 (beta-) which totally prevent the expression of either the alpha or the beta subunits of the mitochondrial ATPase, were shown by classical genetic mapping studies to be both located on chromosome I but genetically unlinked. It is concluded that the structural genes ATP1 and ATP2 for the alpha and beta subunits of the mitochondrial ATPase are not organized in a cluster. By both meiotic recombination frequency analysis and gene transfer studies, three single nuclear mutations affecting to different extents the electrophoretic mobility of the beta polypeptide were located on the chromosome I very close to the mutation B59-1 (beta-). Two mutations involved a defective ATPase activity and the inability to grow on glycerol (gly). One of these mutants E5-23 (beta") exhibited a beta subunit of slightly reduced electrophoretic mobility. The other mutation F1-10 (beta) was associated with a beta subunit of normal electrophoretic mobility. The plasmid pMa2 (Boutry, M., Vassarotti, A., Ghislain, M., Douglas, M., Goffeau, A. (1984) J. Biol. Chem. 259, 2840-2844) containing the structural gene for the beta subunit complemented the mutants E5-23 (beta") and F1-10 (beta) as well as B59-1 (beta-). These three mutations are therefore likely to affect the beta structural gene itself or a very contiguous gene contained in the 5.4-kilobase genomic insert of pMa2. The mutation F1-10 (beta) was mapped between E5-23 (beta") and B59-1 (beta-) by analysis of the meiotic recombination frequencies. Another mutation F25-28-11 (beta') was responsible for an appreciable decrease of electrophoretic mobility of the beta subunit which, however, did not affect either the ATPase activity or the ability to grow on glycerol (GLY). This mutant transformed by pMa2 was able to express the structural gene for the wild type beta subunit and the resulting transformants synthesized and assembled both the beta and beta' subunits. It is concluded that the mutation F25-28-11 (beta') also affects the structural gene for the beta subunit and does not affect genes controlling the processing machinery.

Ascomycota↗
PubMed1984

Molecular cloning and biosynthetic regulation of cry1 gene of Saccharomyces cerevisiae.

The cryptopleurine resistance gene, cry1, of Saccharomyces cerevisiae has been molecularly cloned using genetic complementation of cryptopleurine sensitivity by the cryptopleurine resistance gene contained in a clone library prepared from DNA of a cryptopleurine resistant strain. Analysis of RNA transcripts indicated that the cry1 gene is the template for a transcript of approximately 900 bases and that the primary transcript contains an intron of approximately 300 bases. In vitro hybrid selection translation experiments indicated that this transcript encodes a protein of molecular weight 17 kilodaltons which on two-dimensional SDS polyacrylamide gels exactly coincides with ribosomal protein rp59. Further analysis showed that when the gene was present on a plasmid of about five copies per cell the amount of messenger RNA was elevated approximately five-fold compared to a cell that had only a single chromosomal copy. The rate of synthesis of ribosomal protein rp59 was not detectably elevated. These data suggest that the cry1 gene is regulated, at least in part, post-transcriptionally.

Alkaloids↗
PubMedDate not supplied

Sequencing the yeast genome: the European effort.

For ethical, practical and economic reasons, scientists have traditionally relied on model organisms for biological research. Although model organisms do not always quite constitute the 'real thing', the significant advantages of their use contribute to making their study a viable alternative. The decision to use a specific model, particularly in large-scale studies such as genome projects, will be governed not only by biological consideration, but also by the prevailing financial and organizational infrastructure and expertise of the research community.

Chromosome Mapping↗