Chromosome XV Research Articles

Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.

Sequencing and Analysis of 51 kb on the Right Arm of Chromosome XV from Saccharomyces cerevisiae Reveals 30 Open Reading Frames

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.

Analysis of a 26 kb region on the left arm of yeast chromosome XV.

In the framework of the EC programme for sequencing yeast chromosome XV, we have determined the nucleotide sequence of a 26 kb region. Subsequent analysis revealed 13 non-overlapping open reading frames, three of which correspond to known yeast genes. A pair of tRNA genes associated with remnant Ty elements were localized in this region. From structural parameters and/or similarity searches with entries in the current data libraries, a preliminary functional assessment of several of the putative novel gene products can be made. The gene density in this region amounts to one gene in 2 kb. Protein coding regions occupy 61% of the total DNA sequence. Within the intergenic regions, potential regulatory elements can be predicted. The data obtained here may serve as a basis for a more detailed biochemical analysis of the novel genes. The complete nucleotide sequence of the 26 kb segment as depicted in Figure 1 has been deposited at the EBI data library under Accession Number X91067.

Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae.

We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product.

DNA sequence analysis of a 13 kbp fragment of the left arm of yeast chromosome XV containing seven new open reading frames.

The sequence of a 13 kbp fragment located in the vicinity of the left telomere of chromosome XV (cosmid pEOA179) has been determined. Seven new open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB629, AOA342, AOC231, AOE555, AOE236, AOA236 and AOE1045). Three of them show no identity with proteins deposited in the data banks. ORF AOB629 (629 amino acids) has some similarity with previously described ferric reductases from Saccharomyces cerevisiae and Schizosaccharomyces pombe. ORF AOA342 encodes a polypeptide reminiscent of dihydroflavonol-4-reductases from a number of plant species. AOE236 displays a high level of identity when compared with peroxisomal membrane proteins previously cloned from the methylotrophic yeast Candida boidinii. Finally, AOE1045 encodes a large protein (1045 residues) with some identity with a hypothetical 147 kDa protein identified during the sequencing of Caenorhabditis elegans chromosome 3.

Sequence analysis of a 44 kb DNA fragment of yeast chromosome XV including the Tyl-H3 retrotransposon, the suf1(+) frameshift suppressor gene for tRNA-Gly, the yeast transfer RNA-Thr-1a and a delta element.

We have sequenced on both strands a 44,019 bp fragment located on the left arm of Saccharomyces cerevisiae chromosome XV. The sequenced segment contains 22 open reading frames (ORFs) of at least 100 amino acids long, one of which probably contains an intron. Six of the 22 ORFs correspond to known proteins: the multicopy suppressor of Snf1 protein 1, the two Tyl-H3 transposon proteins TyA and TyB, the myo-inositol transporter 2, the transcription factor protein Ino4 and the 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase. Of the 16 remaining ORFs, two show highest homologies with the yeast serine/threonine protein kinase Ste20 and the human tryptophanyl-tRNA synthetase. Eight ORFs show slight similarities with protein sequences described in data banks. DNA sequence comparison reveals also the presence of three known sequences: the Tyl-H3 transposable element, the yeast suf1(+) frameshift suppressor gene for tRNA-Gly and the yeast transfer RNA-Thr-1a. A fourth DNA sequence shows striking identities with the yeast delta elements.

Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

XV. Yeast sequencing reports. A 29·425 kb segment on the left arm of yeast chromosome XV contains more than twice as many unknown as known open reading frames

The nucleotide sequence of a 29.425 kb fragment localized on the left arm of chromosome XV from Saccharomyces cerevisiae has been determined. The sequence contains 13 open reading frames (ORFs) of which four encode the known genes ADH1, COQ3, MSH2 and RCF4. Predictions are made concerning the functions of the unknown ORFs. Some of the ORFs contain sequences similar to expressed sequence tags (EST) found in the database made available by TIGR. In particular, the highly expressed ADH1 gene is represented in this database by no less than 20 EST sequences. Two ARS sequences and a putative functional GCN4 motif have also been detected. One ORF (O0953) containing nine putative transmembrane segments is similar to a hypothetical membrane protein of Arabidopsis thaliana. Characteristic features of the other ORFs include ATP/GTP binding sites, a fungal Zn(2)-Cys(6) binuclear centre, an endoplasmic reticulum targeting sequence, a beta-transducin repeat signature and in two instances, good similarity to the prokaryotic lipoprotein signal peptide motif.

Cloning and characterization of GPD2, a second gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino-terminal extension of unknown function. GPD2 is located on chromosome VII and cross-hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild-type cells. Analysis of gene transcription by a CAT-reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on nonfermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8-17-fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4 M NaCl.

SSU71, encoding the largest subunit of TFIIF, is located on the right arm of chromosome VII in Saccharomyces cerevisiae.

SSU71 (TFG1) is an essential nuclear gene encoding the largest subunit of the yeast general transcription factor TFIIF. The SSU71 gene was physically mapped to the right arm of chromosome VII, physically linked to QCR9, by hybridization of the cloned gene to CHEF and lambda clone grid blots. This assignment was confirmed by genetic mapping. A search of the nucleotide sequence databases revealed that SSU71 is immediately adjacent to the TYS1 gene, which encodes tRNA(Tyr) synthetase. TYS1 was reported previously to lie on chromosome XV based on sequence overlap with the adjacent UBR1 gene. The mapping data reported here established that TYS1 and UBR1 do not lie on chromosome XV; rather the SSU71-TYS1-UBR1 gene cluster lies on the right arm of chromosome VII, physically linked to QCR9 and genetically linked to ade3 and ser2.

Sequence analysis of the right end of chromosome XV in Saccharomyces cerevisiae: An insight into the structural and functional significance of sub‐telomeric repeat sequences

Approximately 3.9 kb of DNA, centromere proximal to the previously sequenced Y' element at the right end of chromosome XV in Saccharomyces cerevisiae strain YP1, has been sequenced. A number of the known sub-telomeric repeat sequences were identified, including Y', core X and STRs A, B. C and D. Several of these repeat elements contain potentially functional sequences. In addition, two other members of repeated gene families were identified. The first of these shows 61% and 60% DNA sequence identity to Enolases 1 and 2 respectively. The Enolase-like sequence appears to be species specific, with three copies being found in all strains of S. cerevisiae studied. The location of the three copies is the same for all strains. The second repeated sequence has homology with known open reading frames on chromosomes III, V and XI. There are five or six copies of this sequence in all S. cerevisiae and S. paradoxus strains studied and three in S. bayanus strains. The analysis of this region and comparison to sub-telomeric regions on other chromosomes gives some indication as to the potential functional and structural significance of sub-telomeric repeat sequences. In addition, these findings are consistent with the idea that sub-telomeric regions may be targets for unusual recombination events.

Sequence of a 10.27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene.

A 10,270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size.

Cloning and characterization of the essential Saccharomyces cerevisiae RFC4 gene encoding the 37-kDa subunit of replication factor C.

The RFC4 gene encoding the 37-kDa subunit of Saccharomyces cerevisiae replication factor C (RF-C) has been cloned and sequenced. The RFC4 gene encodes a 323-amino acid polypeptide with a molecular weight of 36,126. The deduced amino acid sequence of the RFC4 gene shows high sequence similarity to the other two small subunits of yeast RF-C and the three small subunits of human RF-C (35-60% identity, 55-78% similarity). The similarity is greatest with the 40-kDa subunit of human RF-C (also called Activator 1). Despite the presence of a MluI cell cycle box in the regulatory region of the RFC4 gene, the steady state levels of its mRNA do not change significantly during the yeast mitotic cell cycle. The RFC4 gene is essential for yeast viability and is located on the left arm of chromosome XV, between the SUF1 and ADH1 genes. The essential role of this as well as the other small subunits of RF-C indicates a unique function for each of these subunits, despite their apparent structural redundancy. Rfc4p was overexpressed in Escherichia coli. Despite the presence of consensus ATPase motifs in the primary amino acid sequence, no discernible biochemical function could be ascribed to the purified protein. However, Rfc4p formed a tight complex with the product of the RFC3 gene which encodes the ATPase of RF-C.

Cloning and disruption of CKB2, the gene encoding the 32-kDa regulatory beta'-subunit of Saccharomyces cerevisiae casein kinase II.

Casein kinase II of Saccharomyces cerevisiae is composed of two distinct catalytic subunits, alpha and alpha', and two distinct regulatory subunits, beta and beta' (Padmanabha, R. and Glover, C. V. C. (1987) J. Biol. Chem. 262, 1829-1835; Bidwai, A. P., Reed, J. C., and Glover, C. V. C. (1994) Arch. Biochem. Biophys. 309, 348-355). We report here the cloning, sequencing, and disruption of the CKB2 gene encoding the beta'-subunit. The deduced amino acid sequence of Ckb2 displays only 40-45% identity to other beta-subunit sequences reported to date, allowing a better definition of conserved features of this protein. Most notable is the conservation of a cysteine-containing sequence, CPX3C-X22-CPXC, which may constitute a novel metal-binding motif. The degree of sequence divergence of Ckb2 is comparable to that of the Drosophila Stellate protein, a testis-specific protein of unknown function, suggesting that the latter may function as a second beta-subunit in Drosophila. CKB2 is located on the right arm of chromosome XV between the HIR2 and WHI2 loci and has not been previously identified genetically. Haploid and homozygous diploid cells harboring a ckb2 null allele are viable, demonstrating that the beta'-subunit does not have an essential function distinct from that of beta. Strains lacking a functional CKB2 gene appear to grow normally on both fermentable and non-fermentable carbon sources, mate and sporulate normally, and display normal resistance to nitrogen starvation and heat shock. However, haploid strains harboring disruptions of both the beta' gene and either of the catalytic subunit genes exhibit a synthetic phenotype consisting of slow growth and flocculation in rich glucose medium. The occurrence of this synthetic phenotype implies that the beta'-subunit interacts physically and/or functionally with both the alpha- and alpha'-subunits in vivo.

The gene LEO1 on yeast chromosome XV encodes a non-essential, extremely hydrophilic protein

A 5.6 kbp segment of DNA from Saccharomyces cerevisiae chromosome XV has been isolated and sequenced. Genetic and nucleotide sequence analyses revealed that this region is closely linked to the ADE2 marker on chromosome XV and densely packed with genetic information. We show the gene organization of the entire region and report the nucleotide sequence of the gene, LEO1, which occurs in single copy in the haploid genome. The deduced amino acid sequence specifies an extremely hydrophilic protein with pronounced domain structure (molecular mass 53.9 kDa). The gene is constitutively expressed at a low level and is non-essential, as indicated by the absence of a phenotype from gene disruption mutants.

A pleiotropic phospholipid biosynthetic regulatory mutation in Saccharomyces cerevisiae is allelic to sin3 (sdi1, ume4, rpd1).

Three mutants were identified in a genetic screen using an INO1-lacZ fusion to detect altered INO1 regulation in Saccharomyces cerevisiae. These strains harbor mutations that render the cell unable to fully repress expression of INO1, the structural gene for inositol-1-phosphate synthase. The Cpe-(constitutive phospholipid gene expression) phenotype associated with these mutations segregated 2:2, indicating that it was the result of a single gene mutation. The mutations were shown to be recessive and allelic. A strain carrying the tightest of the three alleles was examined in detail and was found to express the set of co-regulated phospholipid structural genes (INO1, CHO1, CHO2 and OP13) constitutively. The Cpe- mutants also exhibited a pleiotropic defect in sporulation. The mutations were mapped to the right arm of chromosome XV, close to the centromere, where it was discovered that they were allelic to the previously identified regulatory mutation sin3 (sdi1, ume4, rpd1, gam2). A sin3 null mutation failed to complement the mutation conferring the Cpe- phenotype. A mutant harboring a sin3 null allele exhibited the same altered INO1 expression pattern observed in strains carrying the Cpe- mutations isolated in this study.

Cloning and nucleotide sequence of a gene conferring ability to grow at a low temperature on Saccharomyces cerevisiae tryptophan auxotrophs

A gene conferring on yeast YNN140 the ability to grow at a low temperature was cloned and designated LTG3 ( low temperature growth gene). Disruption of the LTG3 gene resulted in no growth at a low temperature. It was shown that the effect of the presence of the gene in multicopy was dependent on the strain. DNA sequencing analysis of the LTG3 gene revealed an open reading frame which can encode 592 amino acids. The deduced amino acid sequence of the protein was homologous with that of some amino acid permeases. Since the addition of a large amount of l-tryptophan to YPAD medium enabled the YNN140 strain to grow at a low temperature, it is possible that a limiting step for YNN140 to grow at a low temperature is to take up tryptophan from the medium. A part of the nucleotide sequence of the cloned DNA containing the LTG3 gene coincided with that of the SUP3 gene, indicating that the LTG3 gene exists on chromosome XV.

Isolation and characterization of a thiamin pyrophosphokinase gene, THI80, from Saccharomyces cerevisiae

The thi80 mutant of Saccharomyces cerevisiae (Nishimura, H., Kawasaki, Y., Nosaka, K., Kaneko, Y., and Iwashima, A. (1991) J. Bacteriol. 173, 2716-2719) shows markedly reduced activity of thiamin pyrophosphokinase (TPK; EC 2.7.6.2). We have isolated a DNA fragment carrying the THI80 gene from a yeast genomic library by its ability to complement constitutive synthesis of the thiamin-repressible acid phosphatase, encoded by the PHO3 gene, of thi80 mutant cells. On the other hand, the thi80 locus was found to be located 3.3 centimorgans proximal to the smp3 locus on the right arm of chromosome XV by genetic mapping analysis, and one more fragment bearing the THI80 gene trailing SMP3 gene was obtained by the plasmid eviction method. The nucleotide sequence of the overlapped region between the two isolated DNAs contained an open reading frame of 957 base pairs, encoding a 319-amino acid polypeptide with a calculated molecular weight of 36,616. When the intact THI80 open reading frame was expressed as a fusion protein carrying three vector-encoded amino acids at its N terminus in Escherichia coli lacking TPK, marked TPK activity was detected in the procaryotic cells, proving that the THI80 gene of S. cerevisiae encodes a structural gene of TPK. A gene disruption experiment demonstrated that the THI80 gene was essential for growth, and therefore, revealed that TPK is the only enzyme capable of synthesizing thiamin pyrophosphate in yeast. Studies of Northern blot analysis and the enzyme assay demonstrated that the THI80 gene expression is regulated mainly at the mRNA level by the intracellular thiamin pyrophosphate and requires the positive regulatory factors encoded by THI2 and THI3 genes. However, unlike thiamin-repressible acid phosphatase and the enzymes involved in thiamin synthesis of S. cerevisiae, TPK was found to be expressed constitutively at a low level and incompletely repressed by exogenous thiamin.

Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene.

The isolation and characterization of MGM1, an yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2. Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.

Genetic mapping of 1,3-beta-glucanase-encoding genes in Saccharomyces cerevisiae.

The map position of three 1,3-beta-glucanase-encoding genes in S. cerevisiae has been determined following conventional meiotic and mitotic mapping combined with recombinant DNA techniques. EXG1, EXG2 and SSG1 were localized to chromosomes XII, IV and XV, respectively, by hybridizing the cloned genes to Southern blots of chromosomes separated by pulsed-field gel electrophoresis, in conjunction with the rad52-1-dependent chromosome-loss mapping technique. Meiotic tetrad analyses further localized the EXG1 gene 6.1 centimorgans centromere-proximal to CDC25 on the right arm of chromosome XII. EXG2 was positioned between LYS4 and GCN2 on the right arm of chromosome IV, at distances of 6.2 centimorgans from LYS4 and 4.9 centimorgans from GCN2. Finally, the SSG1 locus mapped on the right arm of chromosome XV, about 8.2 centimorgans to the centromere-proximal side of HIS3.