Evelyne Dubois

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.

Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development.

In all organisms, correct development, growth and function depends on the precise and integrated control of the expression of their genes. Often, gene regulation depends upon the cooperative binding of proteins to DNA and upon protein-protein interactions. Eukaryotes have widely exploited combinatorial strategies to create gene regulatory networks. MADS box proteins constitute the perfect example of cellular coordinators. These proteins belong to a large family of transcription factors present in most eukaryotic organisms and are involved in diverse and important biological functions. MADS box proteins are combinatorial transcription factors in that they often derive their regulatory specificity from other DNA binding or accessory factors. This review is aimed at analyzing how MADS box proteins combine with a variety of cofactors to achieve functional diversity.

Methylamine/ammonia uptake systems in Saccharomyces cerevisiae: multiplicity and regulation

SummaryThree lines of evidence show that the uptake of methylamine/ammonia in Saccharomyces cerevisiae is mediated by at least two functionally distinct mechanisms.1.Lineweaver-Burk plots for methylamine uptake show an abrupt transition between apparently linear sections, and both functions are inhibited competitively by ammonia.2.These functions can be lost separately as a result of the two genetically unlinked mutations mep-1 and mep-2. Double (mep-1, mep-2) mutants grow very slowly in medium containing low concentrations of ammonia as sole nitrogen source, while both single mutants are little affected. Resistance to methylamine is linked to the mep-1 mutation which abolishes the high capacity metylamine uptake function.3.both components of methylamine/ammonia uptake are subject to nitrogen catabolite repression. This control is relieved in a gdhCR mutant as well as in a gdhA−mutant. In a mutant with thermosensitive glutamine synthetase (glnts)grown on ammonia at 29°C, repression is lost for the high affinity component only, which indicates that glutamine is a necessary effector for the repression of only one of the components of the methylamine/ammonia transport mechanisms. This differential effect of the glntsmutation further supports the existence of the functionally distinct transport mechanisms. The npr-1 mutation seems to affect a pleiotrophic element involved either in the regulation or as a component of at least four uptake systems: the general aminoacid permease, the proline permease, the ureidosuccinic acid permease, and the high capacity methylamine/ammonia uptake function, all of which are strongly depressed in ammonia grown wild-type cells.Indications for a third ammonia uptake system were also obtained.

TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis.

Ty and Ty-mediated gene expression observed in haploid cells of Saccharomyces cerevisiae depends on several determinants, some of which are required for the expression of haploid-specific genes. We report here the cloning and molecular analysis of TEC1. TEC1 encodes a 486-amino-acid protein that is a trans-acting factor required for full Ty1 expression and Ty1-mediated gene activation. However, mutation or deletion of the TEC1 gene had little effect on total Ty2 transcript levels. Our analysis provides clear evidence that TEC1 is not involved in mating or sporulation processes. Unlike most of the proteins involved in Ty and adjacent gene expression, the product of TEC1 has no known cellular function. Although there was no mating-type effect on TEC1 expression, our results indicate that the TEC1 and the a/alpha diploid controls on Ty1 expression are probably not cumulative.

Characterization of two genes, ARGRI and ARGRIII required for specific regulation of arginine metabolism in yeast

SummaryThree unlinked genes ARGRI, ARGRII and ARGRIII are necessary to control the synthesis of the arginine anabolic and catabolic genes. The three genes have been cloned and sequenced and we report here the results for the ARGRI and ARGRIII genes. They encode proteins of 177 and 355 amino acids, respectively. The ARGRIII protein has a very acidic carboxy-terminus (17 aspartate residues). From a comparison of the sequences, the ARGRI and ARGRIII gene products do not show the common characteristics of other DNA binding proteins (nuclear localization and putative DNA binding site) in contrast to the ARGRII regulatory protein. The transcription of both genes is not affected by the presence of arginine in the growth medium.

Genetic evidence for a role for MCM1 in the regulation of arginine metabolism in Saccharomyces cerevisiae.

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We have shown that MCM1 is part of the ARGR regulatory complex, by in vitro binding experiments, at the ARGR5,6 promoter. The participation of MCM1 in the regulation of arginine metabolism is confirmed by the behavior of an mcm1-gcn4 mutant, which is affected in the repression of arginine anabolic genes. In this mcm1 mutant, synthesis of the catabolic enzymes is rather constitutive, but this derepression requires the integrity of the ARGR system and of the target sequences of these proteins in the CAR1 promoter. Our in vitro binding experiments confirm the presence of MCM1 in the protein complex interacting with the promoters of the catabolic CAR1 and CAR2 genes. This is the first in vivo transcription role ascribed to MCM1 other than its role in the transcription of cell-type-specific genes.

Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast

In Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cells response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82Δ or kcs1Δ cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR‐regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine‐responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1‐dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS‐box proteins against degradation.

Ammonia assimilation in Saccharomyces cerevisiae as mediated by the two glutamate dehydrogenases

SummaryMutants lacking NADP-linked glutamate dehydrogenase (NADP-GDH) activity have been isolated by several procedures.Complementation tests in diploids as well as tetrad analysis show that they map within a short chromosome segment, the gdhA locus, which is allelic to the ure1 locus described previously.That the gdhA locus is a structural gene for NADP-GDH is supported by two kinds of evidence. First, intracistronic complementation, as well as negative complementation were observed between some of the gdhA- mutants. This is in agreement with the multimeric structure of the NADP-GDH in Saccharomyces cerevisiae shown by Venard and Fourcade (1972). Secondly, some of the mutants at the gdhA locus have a NADP-GDH with modified properties, including: five-fold higher Km for 2-oxoglutarate, hundred-fold higher Km for NH4+, loss of inhibition by excess of substrate (2-oxoglutarate), and lower thermostability.Mutants with derepressed NAD-GDH activity have been isolated from gdhA- strains on the basis of their faster growth on ammonia as sole nitrogen source. They define the gdhCR locus, which is allelic to ure2 and usu described previously. This is a strong indication that residual growth of the gdhA- mutants on ammonia as sole nitrogen source is due to the NAD-GDH activity.

Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation.

The product of the LYS14 gene of Saccharomyces cerevisiae activates the transcription of at least four genes involved in lysine biosynthesis. Physiological and genetic studies indicate that this activation is dependent on the inducer alpha-aminoadipate semialdehyde, an intermediate of the pathway. The gene LYS14 was sequenced and, from its nucleotide sequence, predicted to encode a 790-amino-acid protein carrying a cysteine-rich DNA-binding motif of the Zn(II)2Cys6 type in its N-terminal portion. Deletion of this N-terminal portion including the cysteine-rich domain resulted in the loss of LYS14 function. To test the function of Lys14 as a transcriptional activator, this protein without its DNA-binding motif was fused to the DNA-binding domain of the Escherichia coli LexA protein. The resulting LexA-Lys14 hybrid protein was capable of activating transcription from a promoter containing a lexA operator, thus confirming the transcriptional activation function of Lys14. Furthermore, evidence that this function, which is dependent on the presence of alpha-aminoadipate semialdehyde, is antagonized by lysine was obtained. Such findings suggest that activation by alpha-aminoadipate semialdehyde and the apparent repression by lysine are related mechanisms. Lysine possibly acts by limiting the supply of the coinducer, alpha-aminoadipate semialdehyde.

Participation of transcriptional and post-transcriptional regulatory mechanisms in the control of arginine metabolism in yeast.

SummaryIn yeast, as in other organisms, amino acid biosynthetic pathways share a common regulatory control. The manifestation of this control is that derepression of the enzymes belonging to several amino acid biosynthetic pathways follows amino acid starvation or tRNA discharging.The arginine anabolic and catabolic pathways are, in addition, regulated specifically by arginine in opposite ways by common regulators. We have measured the mRNA levels for four genes subject to the general amino acid control: HIS4, ARG3, ARG4 and CPAII and compared them to the corresponding enzyme levels. Similarly we have measured the mRNA levels for two genes subject to the arginine specific regulation: ARG3 and CAR1, the former gene belongs to the arginine anabolic pathway and the latter to the arginine catabolic one.HIS4, ARG4 and CPAII enzyme and messenger amounts are perfectly coordinated in all the conditions of general repression or derepression tested. However, arginine does not reduce the level of the ARG3 mRNA enough to explain the reduction of ornithine carbamoyltransferase activity nor does it increase the level of the CAR1 mRNA enough to explain the extent of induction of arginase. Coordination of enzyme and ARG3 mRNA is achieved only when the specific control is eliminated.The half-lives of the ARG3 and CAR1 messengers are enhanced in mutants leading to constitutive expression of ornithine carbamoyltransferase and arginase.These data suggest that the control that coordinates the synthesis of all the amino acids in the yeast cell operates at the level of transcription while the arginine specific regulatory mechanism seems to operate at a post-transcriptional level.