The Linear Plasmid pDHL1 from Debaryomyces hansenii Encodes a Protein Highly Homologous to the pGKL1-Plasmid DNA Polymerase

Both the linear plasmids, pDHL1 (8·4 kb) and pDHL2 (9·2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15·0 kb) in the same host cell for their replication. A 3·5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3·5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids. The 4·2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis. The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids. Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the α subunit of the K. lactis killer toxin, although killer activity was not known in the DHL system. Analysis of the 5′ non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs. The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids. © John Wiley & Sons, Ltd.     

Nucleotide sequence and chromosomal localization of the gene encoding the old yellow enzyme from Kluyveromyces lactis

A 6·6 kb genomic DNA fragment from the yeast Kluyveromyces lactis was isolated. Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H⁺-ATPase and the other for the amino terminus of an unidentified product. In the complementary strand, a full-length ORF which encodes for a protein homologous to the yeast NADPH-dependent Old Yellow Enzyme was found. The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevisiae. In addition, an internal region showed considerable similarity to the bile acid-inducible polypeptide from Eubacterium sp., to the NADH oxidase from Thermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W₃A₁ and to the estrogen-binding protein from Candida albicans, suggesting a functional or structural relationship between them. Inactivation of the KYE1 (K luyveromyces Y ellow E nzyme) gene by deletion of 0·6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time. Haploid cells carrying the disrupted allele showed one-third of the NADPH oxidase activity, compared to wild-type cells. Southern blotting analysis of digested DNA and chromosomes separated by contour-clamped homogeneous electric field electrophoresis from K. lactis indicated that this is a single-copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1·3 Mb. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L37452).     

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.     

The Genes Encoding the Transcription Factor yTAFII60, the G4p1 Protein and a Putative Glucose Transporter are Contained in a 12·3 kb DNA Fragment on the Left Arm of Saccharomyces cerevisiae Chromosome VII

We report the nucleotide sequence of a DNA fragment of 12 325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAF_II60. The G3075 ORF is 47·8% identical to the hypothetical yeast protein YB88. G3080 shows 36·7% identity to the eel calmodulin. G3085 shows 94·9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46·7% identity with the probable glucose transport protein yBR1625. The DNA sequence has been submitted to the EMBL data library under Accession Number X97644. © 1997 by John Wiley & Sons, Ltd.     

Cloning,sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.     

VII. Yeast sequencing reports. The sequence of an 11·1 kb fragment on the left arm of Saccharomyces cerevisiae chromosome VII reveals six open reading frames including NSP49, KEM1 and four putative new genes

We report the sequence of an 11·1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer than 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins. The sequence has been submitted to the EMBL data library under Accession Number X84705.     

Sequence analysis of a 5·6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).     

II. Yeast sequencing reports. Sequence analysis of a 78·6 kb segment of the left end of Saccharomyces cerevisiae chromosome II

We report the sequence analysis of a 78,601 bp DNA segment on the left arm of chromosome II of Saccharomyces cerevisiae. This 78·6 kb segment spans the region from the start of a subtelomeric Y′ element up to the ILS1 gene. It contains 49 open reading frames (ORFs) with more than 100 amino acids length including 14 internal and five overlapping ORFs. The gene density, excluding the internal ORFs, was calculated as one ORF per 2·2 kb. Eight ORFs (PKC1, TyA, TyB, ATP1, ROX3, RPL17a, PET112 and ILS1) correspond to previously characterized genes. ORF YBL0718 was identified as CDC27; YBL0706 as TEL1. Four other ORFs show strong similarities to already known genes. The gene product of YBL0838 is 60% identical to the ribosomal protein RPL32 from rat, mouse and man. YBL0701 encodes a protein with significant similarity to the initiation factor eIF2 associated p67 glycoprotein from rat. Eight ORFs were disrupted and the resulting yeast strains analysed with respect to their phenotype. The sequence has been deposited in the EMBL Nucleotide Sequence Database under the Accession Number X79489.     

Sequence and analysis of an aldose (xylose) reductase gene from the xylose-fermenting yeast Pachysolen tannophilus

A xylose reductase gene was isolated from the xylose-fermenting yeast Pachysolen tannophilus as a cDNA clone by selecting clones that hybridized specifically to xylose-inducible messenger RNA. Use of the cDNA clone as a probe in Northern hybridizations identified a xylose-inducible mRNA species large enough to encode a 36kDa xylose reductase protein known to be produced by this yeast. A corresponding genomic clone was isolated as a 3kb EcoRI fragment that specifically hybridized to the cDNA clone. The sequence of the cDNA and the largest open reading frame of the genomic clone are identical. The predicted translation product exhibits: (1) significant sequence identity with a previously published N-terminal amino acid sequence from purified P. tannophilus xylose (aldose) reductase protein exhibiting NADH/NADPH-dependent activities (aldose reductase, EC 1.1.1.21); (2) identity with a protein composed of 317 amino acid residues with a calculated molecular mass of 36·2kDa, equivalent to that reported for purified P. tannophilus xylose reductase; and (3) considerable sequence similarity to, and features of, a superfamily of oxidoreductases. This sequence is deposited as GenBank Accession Number U40706.     

Isolation and nucleotide sequence of the gene encoding phosphoenolpyruvate carboxykinase from Kluyveromyces lactis

The KlPCK1 gene encoding phosphoenolpyruvate carboxykinase (PEPCK; ATP-dependent) was cloned from the Kluyveromyces lactis genome using a PCR amplicon from Saccharomyces cerevisiae PCK1 gene as a probe. A DNA fragment of about 4·8 kb containing KlPCK1 complemented PEPCK activity of the mutant of S. cerevisiae defective in PEPCK. The KlPCK1 gene has an open reading frame of 1629 bp (543 amino acids). The KlPCK1 nucleotide sequence and deduced amino acid sequence showed 76% and 84% homologies to those of S. cerevisiae PCK1, respectively. Multiple alignment of ATP-dependent PEPCK genes shows highly conserved regions. The nucleotide sequence of KlPCK1 has been submitted to the DDBJ/GenBank/EMBL data bank with Accession Number U88575. © 1998 John Wiley & Sons, Ltd.     

The KlPMR1 gene of Kluyveromyces lactis encodes for a P‐type Ca2+‐ATPase

A novel P‐type Ca²⁺‐ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102 437 Da. Analysis of the deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P‐type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with YlPmr1p of Yarrowia lipolytica and 52% with the Ca²⁺‐ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca²⁺‐ATPase) of the P‐type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue. The sequence described can be retrieved under EMBL Accession No. AJ001018. Copyright © 1999 John Wiley & Sons, Ltd.     

Molecular Cloning of a Candida albicans Gene (SSB1) Coding for a Protein Related to the Hsp70 Family

We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2·1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans. The sequence data has been deposited in the GenBank data library under the Accession Number X97723. © John Wiley & Sons, Ltd.     

IV. Yeast sequencing reports. GUF1, a gene encoding a novel evolutionarily conserved gtpase in budding yeast

While sequencing a region of chromosome IV adjacent to the checkpoint gene MEC3, we identified a gene we call GUF1 (G TPase of U nknown F unction), which predicts a 586 amino acid GTPase of the elongation factor-type class. The predicted Guf1p protein bears striking sequence similarity to both LepA from Escherichia coli (43% identical) and LK1236·1 from Caenorhabditis elegans (42% identical). Analysis of both a guf1δ deletion and a putative constitutive-activating mutant (GUF1HG) revealed that GUF1 is not essential nor did mutant cells reveal any marked phenotype. The GenBank Accession Number for the GUF1 sequence is U22360.     

Characterization of adenylyl cyclases in cultured human granulosa cells

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.