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R Stucka

Publications and source records attributed to R Stucka.

35 records · Page 2Linked to original sources
PubMed1992

Molecular cloning of CIF1, a yeast gene necessary for growth on glucose.

The cif1 mutation of Saccharomyces cerevisiae (Navon et al., Biochemistry 18, 4487-4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of function and located it in a 2.75 kb SphI-BstEII fragment situated at ca. 18 kb centromere distal of LYS2 and ca. 80 kb centromere proximal of TYR1 on chromosome II. Southern analysis demonstrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1.8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increase. cAMP concentration increased in response to galactose or 2,4-dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1 background did not restore growth on glucose, suggesting that the absence of cAMP signal is not the primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.

Adenosine Triphosphate↗
PubMed1992

Molecular analysis of yeast chromosome II between CMD1 and LYS2: the excision repair gene RAD16 located in this region belongs to a novel group of double-finger proteins.

We have analysed a region some 30 kb centromere distal from PHO5 on the right arm of yeast chromosome II and determined the nucleotide sequence of a 8.95 kb DNA segment from this region. By this analysis we were able to derive the precise location and the transcriptional orientation of CMD1, ALG1, SSN6 and LYS2. An open reading frame of 2370 bp was localized between SSN6 and LYS2, which has recently been identified (Schild et al., 1991) to be the RAD16 gene. The putative gene product, 790 amino acids in length, reveals several interesting features. It contains a nuclear target signature and shares several blocks of similarity with the yeast recombinational repair protein RAD54 and the nuclear factor SNF2 (SWI2), which is required for the transcriptional activation of a number of yeast genes. The similarity blocks in these three proteins are reminiscent of those found in the helicase superfamily. Furthermore, RAD16 contains a novel 'double-finger' motif, which has been encountered in a variety of proteins from different organisms that are suggested to interact with DNA and are involved in diverse functions including site-specific recombination, DNA repair, and transcriptional regulation. The putative gene product of RAD16 then is the first example of a protein in which the novel double-finger motif is found to be combined with a potential DNA helicase framework.

Adenosine Triphosphatases↗
PubMed1992

DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls.

A mutant screen has been designed to isolate mutants in Saccharomyces cerevisiae deficient in spore wall dityrosine. As shown by electron microscopy, most of the mutant spores lacked only the outermost, dityrosine-rich layer of the spore wall. Mutant dit101, however, was additionally lacking the chitosan layer of the spore wall. Chemical measurements showed that this mutant does not synthesize chitosan during sporulation. The mutant spores were viable but sensitive to lytic enzymes (glusulase or zymolyase). Unlike most of the dit-mutants, dit101 did show a distinctive phenotype in vegetative cells: they grew normally but contained very little chitin and were therefore resistant to the toxic chitin-binding dye, Calcofluor White. The cells showed barely detectable staining of the walls with Calcofluor White or primulin. The decrease in the amount of chitin in vegetative cells and the absence of chitosan in spores suggested that the mutant dit101 could be defective in a chitin synthase. Indeed, a genomic yeast clone harboring the gene, CSD2, sharing significant sequence similarity with yeast chitin synthases I and II (C. E. Bulawa (1992), Mol. Cell. Biol. 12, 1764-1776), complemented our mutant and was shown to correspond to the chromosomal locus of dit101. Thus, the mutations dit101 and csd2 (and probably also call; M. H. Valdivieso et al., (1991), J. Cell Biol. 114, 101-109) were shown to be allelic. The gene was mapped to chromosome II and was located about 3 kb distal of GAL1. Using this DNA clone, a transcript of about 3500-4000 nucleotides was detected. Comparing RNA isolated from vegetative cells and from sporulating cells at different times throughout the sporulation process, no significant differences in DIT101 transcript levels could be detected indicating absence of sporulation-specific transcriptional regulation. However, the amount of DIT101 transcript changed significantly at different stages of the mitotic cell cycle, peaking after septum formation, but before cytokinesis. As most of the chitin synthesis of vegetative cells occurs at this stage of the cell division cycle, chitin synthesis mediated by DIT101 could be primarily regulated at the level of transcription in vegetatively growing cells.

Cell Cycle↗
PubMed1991

The use of a synthetic tRNA gene as a novel approach to study in vivo transcription and chromatin structure in yeast.

To monitor in vivo transcription and chromatin structure of yeast tRNA genes, we constructed a synthetic tRNA gene that can be used as a reporter. Constructs in which this synthetic tRNA gene is combined with different flanking regions can be integrated into the genome as single copies. The artificial tRNA gene is tagged by the insertion of an intron-like sequence that cannot be spliced out from the precursor and transcripts can thus be identified and quantitated. By several criteria, the artificial tRNA gene behaves like a resident tRNA gene. By measuring the accessibility towards DNaseI in chromatin, we found that the artificial tRNA gene exhibits the same characteristic pattern as resident tRNA genes. Three DNaseI-sensitive sites across the transcribed part of the gene and the immediate flanking regions reflect the formation of the stable transcription complex; positioned nucleosomes are observed in the upstream flanking region. We are confident that the system we have established will prove useful for studying regulatory aspects of tRNA gene expression as well as aspects of pre-tRNA processing and splicing.

Animals↗
PubMed1991

DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains.

A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

Amino Acid Sequence↗
PubMed1990

An element of symmetry in yeast TATA-box binding protein transcription factor IID. Consequence of an ancestral duplication?

TATA-box binding factor TFIID is one of the key factors in transcriptional activation. Surprisingly, the yeast TFDII protein [(1989) Nature 341, 299-303; (1989) Cell 56, 1173-1181; (1989) Proc. Natl. Acad. Sci USA 86, 7785-7789] reveals only limited homology with other DNA-binding proteins. From computer-assisted searches we infer that yeast TFIID possesses a domain structure in which homologous segments are repeated. The greatest similarity is found between two segments, each 33 amino acids in length, in which the positions of four basic residues are strictly conserved. The high homology is also reflected at the gene level. Implications of this novel type of domain structure for possible interactions in transcriptional activation are discussed.

Amino Acid Sequence↗
PubMed1989

Characterization of the prephenate dehydrogenase-encoding gene, TYR1, from Saccharomyces cerevisiae.

TYR1, the gene from Saccharomyces cerevisiae, which encodes prephenate dehydrogenase, one of the tyrosine biosynthetic enzymes, has been cloned by complementing a yeast tyr1 mutant strain. The DNA fragment containing the gene is part of a 45-kb cosmid clone which represents a region of chromosome II covering the genetically mapped tyr1 locus. The nucleotide sequence of a 3.1-kb region carrying the TYR1 gene and adjacent regions has been determined. The open reading frame contains 441 codons, corresponding to about 52.2 kDa for the encoded protein. The canonical NAD-binding domain is located within the first 45 amino acids of the protein. By primer extension, we show that there is one transcription start point. Presumably, the expression of TYR1 is not under the general GCN4 control. Instead, we find a dependence on the presence or absence of phenylalanine. These data were obtained by analysing CAT activity in constructs containing promoter fragments of TYR1 and the cat reporter gene.

Amino Acid Sequence↗
PubMed1989

Ty4, a novel low-copy number element in Saccharomyces cerevisiae: one copy is located in a cluster of Ty elements and tRNA genes.

We have identified a composite element, Ty4, in S. cerevisiae that is ca 6.3 kb in length and contains two tau sequences as long terminal repeats. According to hybridization analyses, Ty4 occurs in low but varying copy number (one to four copies) in different yeast strains. By several criteria, Ty4 is a novel type of retroelement which is similar but not related to the other Ty elements in yeast. Two cosmid clones from strain C836 (c90 and c476) carrying individual copies of Ty4 were isolated. By restriction analysis and nucleotide sequence we show that c476 derives from the 'transposition right arm hot spot' of chromosome III [1]. The analysis of c476 revealed that an initiator tRNA(Met) gene is present at this locus and that an unusual concentration of different Ty elements has occurred: in addition to the Ty4, a Ty1 and a Ty2 element were detected in this region, confirming its highly polymorphic character.

Base Sequence↗
PubMed1989

A hot-spot for transposition of various Ty elements on chromosome V in Saccharomyces cerevisiae.

Ty4 is a novel transposable element in the yeast, Saccharomyces cerevisiae, which is present in only a few copies in the genome (Stucka et al. 1989). In strain C836 one of the three copies (Ty4-90) is contained in cosmid clone c90, where it resides on chromosome V. Analysis of this region reveals a "hot-spot" of transposition: in addition to Ty4-90, the locus contains a complete Ty3 element and seven singular delta, sigma and tau elements. Three tRNA genes (for His, Lys, and Ile) are located in this region, and these are closely associated with one or the other of the elements, a phenomenon commonly observed in yeast. A comparison of c90 with corresponding regions from other strains shows that the locus is highly polymorphic and that this polymorphism is explicitly associated with Ty transposition and recombination events.

Base Sequence↗
PubMed1988

Analysis of yeast chromosomal regions carrying members of the glutamate tRNA gene family: various transposable elements are associated with them.

We carried out an analysis on the genomic organisation of the tRNA(Glu) family in S. cerevisiae; eight clones were characterized by restriction mapping, hybridization and sequencing. These data taken together with our earlier findings show that the individual tRNA(Glu3) copies are identical only in their structural part but embedded in entirely different genomic environments. All of the tRNA genes identified here are flanked by elements such as Ty, delta, sigma, and tau. In some cases, sequences from different elements form complex patterns indicating a sophisticated history of these chromosomal regions. A novel observation is that Ty and delta in the regions analyzed are exclusively associated with the tRNA genes. The observed patterns imply that the tRNA genes mark regions of multiple transposition and subsequent excision events, but that these have occurred after the individual tRNA gene copies had been fixed in their present locations. Transcription experiments by the use of micro-injection into Xenopus oocytes suggest that the elements flanking the tRNA genes exert a modulating effect on their expression.

Animals↗
PubMed1987

One member of the tRNA(Glu) gene family in yeast codes for a minor GAGtRNA(Glu) species and is associated with several short transposable elements.

During characterization of the whole tRNA-(Glu) family from the yeast, Saccharomyces cerevisiae, we isolated one cosmid clone bearing a tRNA(Glu) gene copy that is deviant from the major tRNA(Glu3) gene members in only five positions. This divergent tRNA-(Glu) is a minor species and is represented by a single gene copy. One of the nucleotide exchanges concerns the anticodon which is modified from T-T-C in the tRNA(Glu3) gene to C-T-C which implies that this tRNA serves the codon triplet G-A-G. Two other minor yeast tRNA species have been reported which appear to be particularly designed for the translation of those codons that have a G in its third (Wobble) position. The low abundance of such minor tRNA species correlates positively to the low occurrence of most of the N-N-G codons in yeast. Furthermore, the GAGtRNA-(Glu) locus represents another case of the general phenomenon in which the majority of the tRNA genes in yeast are associated with one or several transposable elements forming complex patterns. In this particular case, divergent segments of delta and tau are present in the 5' flanking region of the tRNA gene and arranged in a novel configuration. The sequence data lend support to the view that tau is not an evolutionary young element as was earlier anticipated.

Base Sequence↗
PubMed1986

Conserved and non-conserved features among the yeast Ty elements.

We have isolated and characterized a Ty element from a yeast cosmid library which exhibits several unusual features: it is flanked by non-homologous delta elements and directly associated with a singular delta element. A tRNA(Glu3) gene and tRNA(Cys) gene are found in conjunction with this element, located in opposite orientation on either end of it. The sequence information now available for several Ty elements has been used in a detailed comparative analysis to determine conserved features among the Ty elements, preferably between class I elements and a class II element. Highly conserved sequence motifs appear to be located at the borders of particular segments that correspond to the putative protein domains of the Tys. Furthermore, we include a comparison of the best-conserved amino acid homologies for these putative proteins of Ty elements, transposable elements from other organisms and several retroviral proviruses to confirm their close structural resemblance.

Amino Acid Sequence↗
PubMed1985

Different patterns of transposable elements in the vicinity of tRNA genes in yeast: a possible clue to transcriptional modulation.

We have extended the catalogue of yeast tRNA genes that are found associated with repetitive (transposable) elements. We determined the nucleotide sequences of loci containing the genes for a tRNAGln and a tRNASer2 (pY66), a tRNAGlu3 (pY80), and a tRNALys1 (pY109). Our analyses revealed that complex patterns exist in which different types of elements (Ty, delta, sigma, and tau) are involved. We could further demonstrate that in several there are alleles of which one contains a particular element and the other lacks it; such differences are also found when comparing hybridization patterns of DNA from a diploid and a haploid yeast strain. In order to investigate a possible functional role of the elements in conjunction with the tRNA genes, we compared the transcriptional activities of several tRNA genes by microinjection into Xenopus oocyte nuclei. The observed differences in expression may be attributed to the presence or absence of different elements in the vicinity of the tRNA genes.

Alleles↗