Yeast sequencing reports. Cloning and DNA sequence of a Kluyveromyces lactis ERD1 homologue

The ERD1 gene product is required for the correct localization of soluble proteins that normally reside in the endoplasmic reticulum (ER). Cell lacking ERD1 secrete resident ER proteins and, in addition, exhibit defects in the processing of glycoproteins. Here, the molecular characterization of the Kluyveromyces lactis ERD1 homologue is described. A comparison of the predicted sequences of the Saccharomyces cerevisiae and K. lactis Erd1 proteins indicates that they are about 30% identical and 50% similar in sequence. Despite low sequence identity, these proteins are predicted to be conserved structurally. Furthermore, the K. lactis protein can functionally complement an S. cerevisiae mutant containing a deletion of the entire ERD1 gene, indicating these two proteins are functional homologues. The GenBank data library Accession Number for the DNA sequence reported in this paper is UO4714.     

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7·1 kilobase pairs‐long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria‐associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria‐associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide. Copyright © 1999 John Wiley & Sons, Ltd.     

Cloning and characterization of WMSU1, a Williopsis saturnus var. mrakii gene encoding a new yeast SUN protein involved in the cell wall structure

SUN proteins of Saccharomyces cerevisiae have been defined on the basis of high homologies in their C-terminal domain. Recently, two of these four proteins were shown to be involved in cell wall morphogenesis (Mouassite et al., 2000a). In the present study, we have isolated WMSU1 (Accession No. AF418983), a new SUN-related gene, from W. saturnus var. mrakii MUCL 41968. Sequencing of the gene revealed an open reading frame coding for 402 amino acids. The predicted amino acid sequence of WMSU1 is closely related to the S. cerevisiae SUN proteins and to other yeast proteins involved in cell wall metabolism. WMSU1 is proposed to encode a cell wall protein since its predicted product contains a signal sequence, a Kex2p cleavage site and a serine/threonine-rich N-terminal domain. Southern blot analysis of the W. saturnus var. mrakii MUCL 41968 genome using the highly conserved domain of WMSU1 as a probe suggested that the isolated gene belongs to a multigenic family. Expression of WMSU1 in E. coli led to a 45 kDa protein, which appeared to be toxic to this host. Scanning electron microscopy analysis of a recombinant S. cerevisiae producing Wmsu1p showed that this strain exhibited an altered cell wall, thus pointing to a probable role of this protein in the cell wall structure.     

Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.     

Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ～ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.     

II. Yeast sequencing reports. The nucleotide sequence of TTP1, a gene encoding a predicted type II membrane protein

The DNA sequence of a 2967 bp fragment located near the centromere of chromosome II, between the CEN2 and FUR4 genes, was determined. The segment contains a new open reading frame of 1794 bp. The product encoded by the gene, designated TTP1, is a predicted type II membrane protein of 597 amino acid residues with a short cytoplasmic NH₂-terminus, a membrane-spanning region and a large COOH-terminal region containing three potential N-glycosylation sites. Gene disruption indicated that TTP1 is not essential for cell growth. The sequence has been deposited in the GenBank data library under Accession Number U05211.     

XI. Yeast sequencing reports. Analysis of an 11·7 kb DNA fragment of chromosome XI reveals a new tRNA gene and four new open reading frames including a leucine zipper protein and a homologue to the yeast mitochondrial regulator ABF2

We report the nucleotide sequence of an 11·7 kb fragment from the left arm of Saccharomyces cerevisiae chromosome XI. Analysis reveals a new tRNA for valine and four unknown open reading frames among which YKL245 shows homology with a yeast mitochondrial regulatory protein and YKL244, YKL246 and YKL247 are unknown.     

XIV. Yeast sequencing reports. Sequence analysis of a 30 kb DNA segment from yeast chromosome XIV carrying a ribosomal protein gene cluster,the genes encoding a plasma membrane protein and a subunit of replication factor C,and a novel putative serine/threonine protein kinase gene

We have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV of Saccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp. NO422 and NO425 correspond to the split ribosomal protein genes encoding S16A and rp28, respectively, NO450 displays a striking similarity with serine/threonine protein kinase genes, in particular with STE20, and therefore may encode a novel member of this protein family. NO453 is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known. NO530 encodes the plasma membrane protein Mid1p and NO533 corresponds to the gene coding for a 40 kDa subunit of replication factor C. The remaining ORFs show weak or no homology with proteins in the data bases. The sequence has been submitted to the EMBL data library under Accession Number U23084.     

II. Yeast sequencing reports. The sequence of 12·5 kb from the right arm of chromosome II predicts a new N-terminal sequence for the IRA1 protein and reveals two new genes,one of which is a DEAD-box helicase

We have determined the complete nucleotide sequence of a 12·5 kb segment from the right arm of chromosome II carried by the cosmid α20. The sequence encodes the 5′ end of the IRA1 gene. Two complete new open reading frames and the 3′ non-coding region of the SUP1 (SUP45) gene. A comparison of our sequence with the data bank reveals a 154 amino acid extension at the N-terminus of Ira1p compared to the previously predicted sequence. According to the 11th edition of the Saccharomyces cerevisiae genetic map, our sequence should encode the MAK5 gene, which is necessary for the maintenance of dsRNA killer plasmids. One of the two new open reading frames, YBR1119, is predicted to encode an RNA helicase, thus YBR1119 may correspond to the MAK5 gene. The sequence has been deposited in the EMBL data library under Accession Number X78937.     

XIV. Yeast sequencing reports. A 21·7 kb DNA segment on the left arm of yeast chromosome XIV carries WHI3, GCR2, SPX18, SPX19, an homologue to the heat shock gene SSB1 and 8 new open reading frames of unknown function

We report the amino acid sequence of 13 open reading frames (ORF > 299 bp) located on a 21·7 kb DNA segment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Five open reading frames had been entirely or partially sequenced previously: WHI3, GCR2, SPX19, SPX18 and a heat shock gene similar to SSB1. The products of 8 other ORFs are new putative proteins among which N1394 is probably a membrane protein. N1346 contains a leucine zipper pattern and the corresponding ORF presents an HAP (global regulator of respiratory genes) upstream activating sequence in the promoting region. N1386 shares homologies with the DNA structure-specific recognition protein family SSRPs and the corresponding ORF is preceded by an MCB (MluI cell cycle box) upstream activating factor. The sequence has been deposited in the EMBL data library under Accession Number X78898.     

X. Yeast sequencing reports. The sequence of a 36 kb segment on the left arm of yeast chromosome X identifies 24 open reading frames including NUC1, PRP21 (SPP91), CDC6, CRY2, the gene for S24, a homologue to the aconitase gene ACO1 and two homologues to chromosome III genes

A 36 kb fragment from the left arm of chromosome X, located at about 50 kb from the telomere, was sequenced and analysed. The segment contains a new putative ARS, a new tRNA for threonine, remnants of a solo delta and 24 open reading frames (ORFs) numbered from J0310 to J0355. Six of them, NUC1, PRP21 (also called SPP91), CDC6, CRY2, the gene encoding the ribosomal protein S24 and the gene coding for a hypothetical protein of 599 amino acids, have been sequenced previously. Three ORFs show high homology to the yeast gene ACO1 encoding mitochondrial aconitase and to the chromosome III genes YCR34W and YCR37C of unknown function. Three other ORFs show lower but significant homology: a first one to UNP, a gene related to the tre-2 oncogene from mouse and to the gene coding for the yeast deubiquitinating enzyme DOA2; a second one to SLY41, a suppressor of the functional loss of YPT1 and a third one to the gene encoding the proline utilization activator PUT3. The complete nucleotide sequence of 36 016 bp was submitted to the EMBL database (accession number X77688).     

XI. Yeast sequencing reports. The complete sequencing of a 24·6 kb segment of yeast chromosome XI identified the known loci URA1, SAC1 and TRP3, and revealed 6 new open reading frames including homologues to the threonine dehydratases,membrane transporters,hydantoinases and the phospholipase A2-activating protein

We report the entire sequence of a 26·4 kb segment of chromosome XI of Saccharomyces cerevisiae. Identification of the known loci URA1, TRP3 and SAC1 revealed a translocation compared to the genetic map. Additionally, six unknown open reading frames have been identified. One of them is similar to catabolic threonine dehydratases. Another one contains characteristic features of membrane transporters. A third one is homologous in half of its length to the prokaryotic hydantoinase HyuA and in the other half to hydatoinase HyuB. A fourth one is homologous to the mammalian phospholipase A₂-activating protein. A fifth one, finally, is homologous to the hypothetical open reading frame YCR007C of chromosome III. The sequence has been deposited in the EMBL data library under Accession Number X75951.