Rinji Akada

TheSaccharomyces cerevisiae genes (CMP1 andCMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B

SummarySaccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a λgt11 expression library using125I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 andCMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62999 Da) and CMP2 protein (68496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 213 (calcineurin). The products of theCMP1 andCMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62000 and 64000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.

Initiation of meiosis and sporulation in Saccharomyces cerevisiae requires a novel protein kinase homologue

SummarySME1 was cloned due to its high copy number effect: it enabled MATα/MATα diploid cells to undergo meiosis and sporulation in a vegetative medium. Disruption of SME1 resulted in a recessive Spo− phenotype. These results suggest that SME1 is a positive regulator for meiosis. DNA sequencing analysis revealed an open reading frame of 645 amino acids. An amino terminal peptide of ca 400 amino acids in the deduced protein was similar to known protein kinases. Transcription of SME1 was regulated negatively by nitrogen and glucose and positively by MATα/MATα and IME1, another positive regulator gene of meiosis. By complementation analysis, SME1 was found to be identical to IME2, which had been shown to be important in meiosis. These results suggest that IME1 product stimulates meiosis by activating transcription of SME1 (IME2) and that protein phosphorylation is required for initiation of meiosis.

Screening and identification of yeast sequences that cause growth inhibition when overexpressed

Abstract To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter. In all, 240 000 yeast transformants were screened for lethality on galactose medium. From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained. Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII. Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition. The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule. Overexpression of the BNI1ΔN and BOI1ΔN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation. Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively. The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA.

Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides.

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

Positive and negative elements upstream of the meiosis-specific glucoamylase gene in Saccbaromyces cerevisiae

SummaryThe SGA1 gene encoding glucoamylase is specifically expressed late in meiotic development of the yeast Saccharomyces cerevisiae. We found that accumulation of both enzyme activity and transcripts was regulated negatively by both nutritional signals and a haploid-specific negative regulator gene of meiosis, RME1, and positively by the inducer genes for meiosis, IME1 and IME2. To study the role of sequences upstream of the SGA1 gene in its expression and regulation, we generated internal deletions in the 5′ non-coding region of the gene and chimeric genes with portions of the upstream sequence inserted into a reporter gene. By analyzing the expression of these genes, we have identified both a 19 by upstream activation sequence (UAS) and a 49 by negatively regulating element (NRE). The UAS activated transcription with no requirement for heterozygosity at the mating-type locus, but this activation was still under negative control by nutrients. The NRE showed no UAS-like activity but conferred IME2-dependent (or meiosis-specific) expression on a heterologous promoter. These results suggest that meiosis-specific expression of the SGA1 gene is established by a regulatory hierarchy including positive and negative factors, the actions of which are mediated through the two separate upstream regulatory elements, UAS and NRE, respectively. Also, that two independently acting cascades exist for the regulation of SGA1 expression: one transduces both the mating-type and nutritional signals and includes the IME2 product, which acts to relieve the repression through NRE ; and another transduces only the nutritional signal independently of the above pathway and inhibits positive factors acting on UAS.

Genomic organization of multiple genes coding for rhodotorucine A, a lipopeptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Rhodotorucine A, a lipopeptide mating pheromone, is secreted from mating type A cells of Rhodosporidium toruloides and induces sexual differentiation of the opposite mating type a cells. Genome of A-type cells contains three homologous genes (RHA1, RHA2, and RHA3) encoding rhodotorucine A. Genomic Southern blot analysis using RHA1 DNA as a probe showed that RHA1 strongly hybridize with A-type genomic DNA but weakly with a-type, suggesting that the sequences of RHA genes were dissimilar in the opposite a-type genome. The range of dissimilar regions in a-type genome was searched using RHA-flanking DNA segments as probes. The result suggests that a-type genome lacks sequences coding for rhodotorucine A and its 5′ upstream but contains its 3′ non-coding sequences. The absence of mating pheromone genes in the opposite mating type genome suggests that the expression of mating-type-specific genes in R. toruloides is not controlled trans-criptionally, as shown in the yeast Saccharomyces cerevisiae.

Involvement of Ca2+/calmodulin in sexual differentiation induced by mating pheromone rhodotorucine A in Rhodosporidium toruloides

Summary: Rhodotorucine A is a mating pheromone produced by mating type A cells of Rhodosporidium toruloides which induces mating tube formation in mating type a cells. Rhodotorucine A induced conspicuous changes in Ca2+ fluxes in the target cells which consisted of a very rapid, transient Ca2+ influx immediately after pheromone addition and a gradual Ca2+ accumulation during mating tube elongation. The magnitude of the pheromone-induced changes of Ca2+ fluxes was correlated with the efficiency with which the pheromone elicits biological responses in mating type a cells. Compared to vegetative growth, the response to the pheromone was highly sensitive to trifluoperazine, an inhibitor of calmodulin. Trifluoperazine inhibited exclusively the emergence and the elongation of mating tubes but not the signalling reaction.

Mating types and mating-inducing factors (gamones) in the ciliate Euplotes patella syngen 2

Relationships between mating type genes and mating-inducing factors (gamones) were investigated in the ciliate Euplotes patella syngen 2. Ten mating types were distinguished, and genetic data indicated that the ten mating types were determined by four codominant alleles in possible combinations of two of them. There were six heterozygous types ( mt1 / mt2 , mt3 / mt4 , etc.) and four homozygous types ( mt1 / mt1 , mt2 / mt2 , etc.). Conjugation-conditioned fluid (CCF) obtained from a mixture of cells of homozygous types could induce homotypic pair formation in cells of all mating types except for a particular type. Genetic data of cell-CCF combination experiments suggest that each mating type allele controls the production of a specific gamone which induces pair formation in cells which do not produce the same gamone. Gamones and their hypothetical receptors are discussed.

Cloning of a gene coding for rhodotorucine A, a farnesyl peptide mating pheromone of rhodosporidium toruloides

A putative structural gene (RHA). coding for rhodotorucine A, a farnesyl peptide mating pheromone in cells of mating type A of Rhodosporidium toruloides, has been cloned using synthetic oligonucleotides corresponding to the amino acid sequence of rhodotorucine A. The RHA gene contains sequences for four tandem copies of the rhodotorucine A peptide, each followed by a spacer peptide of three or four amino acids. The spacer peptides are hypothesized to contain signals for proteolytic processing and farnesylation. The results of Southern blot analysis revealed that the RHA gene is specifically contained in the genome of A-type cells, and not in that of complementary a-type cells. The existence of an additional homologous gene in the genome of A-type cells is also suggested.

Relationships between cell cycle and conjugation in the ciliate Euplotes patella syngen 2

Summary Analyses of the relationships between the cell cycle and the early conjugation process were extended to Euplotes patella syngen 2. This species had longer cell cycle and S phase than the other ciliates so far analyzed for these problems. Stages of macronuclear S phase were arbitrarily subdivided into three stages by means of the cytologic marker, replication bands, which migrated from the tips of the C-shape macronucleus to the middle along with the progress of S phase. Cells in each stage were examined for the mating capability. This species enter conjugation during not only macronuclear Gl phase but also S phase except for the terminal stage. Moreoever, early conjugants with oral primordia for cell division and/or swelled micronuclei which showed vegetative mitotic prophase were observed occasionally. However, cells in terminal S phase and G2-D phase did not conjugate. These results suggested that in the long cell cycle species E. patella syngen 2 the mating capability was associated with the cell division but not with the stages of the cell cycle, such as G1 and S phase.