Paulo Guerreiro

Expression of YAP4 in Saccharomyces cerevisiae under osmotic stress

YAP4, a member of the yeast activator protein ( YAP ) gene family, is induced in response to osmotic shock in the yeast Saccharomyces cerevisiae. The null mutant displays mild and moderate growth sensitivity at 0.4 M and 0.8 M NaCl respectively, a fact that led us to analyse YAP4 mRNA levels in the hog1 (high osmolarity glycerol) mutant. The data obtained show a complete abolition of YAP4 gene expression in this mutant, placing YAP4 under the HOG response pathway. YAP4 overexpression not only suppresses the osmosensitivity phenotype of the yap4 mutant but also relieves that of the hog1 mutant. Induction, under the conditions tested so far, requires the presence of the transcription factor Msn2p, but not of Msn4p, as YAP4 mRNA levels are depleted by at least 75% in the msn2 mutant. This result was further substantiated by the fact that full YAP4 induction requires the two more proximal stress response elements. Furthermore we find that GCY1, encoding a putative glycerol dehydrogenase, GPP2, encoding a NAD-dependent glycerol-3-phosphate phosphatase, and DCS2, a homologue to a decapping enzyme, have decreased mRNA levels in the yap4 -deleted strain. Our data point to a possible, as yet not entirely understood, role of the YAP4 in osmotic stress response.

The Tetrahymena chaperonin subunit CCTη gene is coexpressed with CCTγ gene during cilia biogenesis and cell sexual reproduction

We report here the cloning and the characterization of the T. pyriformis CCTη gene (TpCCTη) and also a partial sequence of the corresponding T. thermophila gene (TtCCTη). The TpCCTη gene encodes a protein sharing a 60.3% identity with the mouse CCTη. We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction. These genes have similar patterns of expression to those of the previously identified TpCCTγ gene. Indeed, the Tetrahymena CCTη and CCTγ genes are upregulated at 60–120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place. Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.

Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein

SummaryThe genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.

The macronuclear polyubiquitin gene of the ciliate Tetrahymena pyriformis

The presence of ubiquitin in ciliates was first demonstrated in Tetrahymena pyriformis. One clone--pTU2--presents two incomplete open reading frames and the putative polyubiquitin genes have been shown to be highly similar to those of other organisms. To further analyze the organization of this multigene family, several fragments of macronuclear DNA were cloned. We report here the isolation and characterization of one genomic clone (pTU20) that encodes a polyubiquitin gene (TU20) with five tandem repeats and presenting only one extra triplet CAA (Gln) upstream from the TGA. The promoter region of TU20 also presents a consensus heat shock element. The specific detection of RNA species with a synthetic oligonucleotide probe reveals that it corresponds to the 1.8 kb mRNA species whose expression is increased by temperature stress.

Analysis of a 17·9 kb Region from Saccharomyces cerevisiae Chromosome VII Reveals the Presence of Eight Open Reading Frames, Including BRF1 (TFIIIB70) and GCN5 Genes

We report the nucleotide sequence of a 17 898 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5′ portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat‐shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins, YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases. This sequence has been submitted to the EMBL data library under Accession Number Y07703.

Clusters of 5S rRNAs in the intergenic region of ubiquitin genes in Tetrahymena pyriformis.

Here, we report the molecular analysis of two independent 5S rRNA clusters found in the intergenic region of two ubiquitin genomic clones isolated from Tetrahymena pyriformis. Each cluster contains two 120-bp-long coding regions organized in tandem with 142/145-bp-long spacers.

Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII

A 9·9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the β‐subunit of the succinyl‐CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII. The sequence is in the EMBL data library under Accession Numbers Z73024, Z73025, Z73026, Z73028 and Z73029.©1997 John Wiley & Sons, Ltd.

Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short‐flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non‐essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the β‐subunit of the succinyl‐CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild‐type gene was cloned in pRS416. Copyright