Teresa Ruiz

Cloning and sequence analysis of the invertase geneINV1 from the yeastPichia anomala

A genomic library from the yeastPichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant ofSaccharomyces cerevisiae. The cloned gene,INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also withKluyveromyces marxianus inulinase, a yeast β-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5′ and 3′ non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine‐lacking medium, indicating that it encodes a product able to complement the β‐isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC‐rich palindrome motif identified in the 5′ region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3′‐terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1–PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ294714. Copyright

Cloning and Characterization of a Candida albicans Gene Homologous to Fructose-1,6-Bisphosphatase Genes

By sequencing of the DNA adjacent to the Candida albicans SEC61 gene, an open reading frame encoding a polypeptide of 331 amino acids was found. The predicted protein showed a strong homology with the fructose-1,6-bisphosphatase [FbPase] from other organisms, and conserved regions included the catalytic motif found in all known FbPases. Although the cloned gene did not complement the growth failure of a Saccharomyces cerevisiae fbp1 mutant in media with gluconeogenic carbon sources, it was transcribed in the transformants in a fashion that indicates a partial repression by glucose. A similar control on the transcription of this gene and on FbPase activity was found in wild-type C. albicans, where the cloned gene (CaFBP1) was shown to be localized in a single chromosomal locus in the genome.

The sequence of a 15 769 bp segment of Pichia anomala identifies the SEC61 and FBP1 genes and five new open reading frames

We have determined the sequence of a 15 769 bp DNA segment of Pichia anomala. The sequence contains seven complete open reading frames (ORFs) longer than 100 amino acids and a putative tRNA gene. Two of the ORFs code for the well‐characterized genes SEC61 (which codes for the core subunit of the ER translocation complex) and FBP1 (encoding fructose‐1,6‐bisphosphatase). A gene coding for a protein similar to S. cerevisiae YDL054c was found between the two genes. These three genes show a different organization (intermingled triples) in three yeast species: Saccharomyces cerevisiae, Candida albicans and P. anomala. Two out of the four remaining ORFs show weak homology with different proteins from other species and the other two show non‐significant similarity with previously sequenced genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ306295. Copyright

Effect of anticalmodulin drugs on the action of yeast α factor pheromone

Saccharomyces cerevisiae α factor pheromone arrest growth of cells of the a mating type (MAT a) at the G1 phase of the cell cycle. When treatment of MAT a cells with α factor was carried out in the presence of anticalmodulin drugs, trifluoperazine or chlorpromazine, the extent of cell growth arrest induced by α factor was reduced or even became undetectable. These results lend support to the hypothesis that calmodulin plays a role as mediator in the action of α factor on MAT a cells.

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3.

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane‐spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine‐repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane. Copyright

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.