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.     

Genome organization of the linear cytoplasmic element pPE1B from Pichia etchellsii.

The linear cytoplasmic element pPE1B from Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) was totally sequenced. It consists of 12835 bp and has a remarkable high A+T content of 77.3%. The termini of pPE1B were found to consist of inversely orientated identical nucleotide repetitions 161 base pairs long, to which proteins are probably covalently linked at the 5' ends. Ten putative genes (open reading frames, ORFs) were identified, covering 96.5% of the total sequence. The predicted polypeptides correspond to proteins encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis and pSKL of Saccharomyces kluyveri. ORF1, existing on both latter elements, is lacking on pPE1B. An upstream conserved sequence motif (UCS) is located at the expected distance from the start codon of each of the 10 ORFs. As the arbitrarily chosen UCS6 was able to drive expression of a reporter gene in the heterologous pGKL-encoded killer system of K. lactis, extranuclear promoter function is probable. The almost congruent genome organization of pPE1B and other autonomous linear yeast plasmids sequenced so far, i.e. pGKL2 and pSKL, suggests a common, presumably viral, ancestor.     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

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.     

Translational regulator RpL10p/Grc5p interacts physically and functionally with Sed1p,a dynamic component of the yeast cell surface

Biogenesis of an active ribosome complement and a dynamic cell surface complement are two major determinants of cellular growth. In yeast, the 60S ribosomal subunit protein RpL10p/Grc5p functions during successive stages in ribosome biogenesis, specifically rRNA processing, nucle(ol)ar preribosomal subunit assembly, nucleo-cytoplasmic transport and cytoplasmic maturation of ribosomes. Here, we report that a two-hybrid screen identified yeast genes SED1, ACS2 and PLB3 as encoding proteins physically interacting with both ribosomal RpL10p/Grc5p and its human homologue hRpL10p/QMp. SED1 encodes a differentially expressed cell wall protein which is proposed to be first transiently secreted to the plasma membrane as a GPI (glycosylated derivative of phosphoinositol)-anchored form and to be then transferred to the glucan layer of the cell wall. Ectopic expression of SED1 rescues both the aberrant growth phenotype and the translation defect of grc5-1(ts) temperature-sensitive cells. Furthermore, we report that Sed1p associates with translating ribosomes suggesting a novel, cytoplasmic role for Sed1p. ACS2 encodes one of the two yeast acetyl-CoA synthases and represents a key enzyme in one of several metabolic routes to produce acetyl-CoA, which in turn is indispensable for lipid biosynthesis. PLB3 encodes a phospholipase, which is active in the breakdown of membrane lipids. Our results support the view that Grc5p/RpL10p links ribosome function to membrane turnover and cell surface biogenesis.     

DNA Sequencing and Analysis of 130 kb from Yeast Chromosome XV

We have determined the nucleotide sequence of 129 524 bases of yeast (Saccharomyces cerevisiae) chromosome XV. Sequence analysis revealed the presence of 59 non-overlapping open reading frames (ORFs) of length >300 bp, three tRNA genes, four delta elements and one Ty-element. Among the 21 previously known yeast genes (36% of all ORFs in this fragment) were nucleoporin (NUP1), ras protein (RAS1), RNA polymerase III (RPC1) and elongation factor 2 (EF2). Further, 31 ORFs (53% of the total) were found to be homologous to known protein or DNA sequences, or sequence patterns. For seven ORFs (11% of the total) no homology was found. Among the most interesting protein identifications in this DNA fragment are an inositol polyphosphatase, the second gene of this type found in yeast (homologous to the human OCRL gene involved in Lowe's syndrome), a new ADP ribosylation factor of the arf6 subfamily, the first protein containing three C2 domains, and an ORF similar to a Bacillus subtilis cell-cycle related protein. For each ORF detailed sequence analysis was carried out, with a full consideration of its biological function and pointing out key regions of interest for further functional analysis. The sequence has been submitted to the EMBL data library under Accession Number X94335.© 1997 John Wiley & Sons, Ltd.     

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.     

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, sequencing and characterization of the alpha-aminoadipate reductase gene (LYS2) from Saccharomycopsis fibuligera

The gene putatively encoding alpha-aminoadipate reductase (AAR) was isolated successfully by degenerate PCR and chromosome walking, based on cassette PCR methods, from the dimorphous yeast Saccharomycopsis fibuligera PD70 and was named SfLYS2. Sequence analysis revealed that it contained a putative open reading frame (ORF) of 4161 bp and encoded a polypeptide of 1386 amino acids. The deduced translation product shared an identity of 53% and 51% to the Lys2p homologues of Candida albicans and Saccharomyces cerevisiae, respectively. An atypical TATA box and a GCN4-box element were found in the 5'-upstream region. Genomic Southern hybridization suggested the presence of a single locus of SfLYS2 in the S. fibuligera genome. Expression of the ORF of SfLYS2 in a lys2(-) strain of S. cerevisiae could functionally complement the lysine mutant of the S. cerevisiae strain. S. fibuligera could use lysine as the sole nitrogen source but its growth was inhibited on the alpha-aminoadipate (AA) medium. Approximately 90% of the mutants of S. cerevisiae resistant to AA are lysine auxotrophs; in contrast all the mutants of S. fibuligera resistant to AA recovered in this work were not lysine auxotrophs.     

Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74·1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Number of the HIS3 gene is U69170. © 1998 John Wiley & Sons, Ltd.     

Establishment of a simple system to analyse the molecular interaction in the agglutination of Saccharomyces cerevisiae

Saccharomyces cerevisiae a-agglutinin, which is involved in mating and covalently anchoring to the cell wall, consists of two components, Aga1p and Aga2p, whose syntheses are individually regulated. To facilitate the analysis of the protein-protein interaction on agglutination between a- and alpha-agglutinins, the construction of a yeast strain (MATa) with the functional protein prepared by genetic fusion of Aga1p- and Aga2p-encoding genes and by the expression system using the UPR-ICL promoter derived from the n-alkane-assimilating yeast, Candida tropicalis, which is functional under the condition of lower glucose concentration was tried and the agglutination ability of the constructed strain was evaluated with a yeast strain (MATa) which expressed AGalpha1 encoding alpha-agglutinin under the control of the same promoter. The genes were integrated into the yeast chromosomes. Cell agglutination between both (MATa) strains was observed microscopically when these two strains were mix-cultured to a glucose-decreased concentration. The agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed Aga1p-Aga2p fusion protein was enoughly functional for the interaction with the Agalpha1 protein, and that this phenomenon occurred dependent on glucose concentration, but independent of the peptide pheromones secreted by the cells of the opposite mating types. Using this system, the role of two disulphide linkages between Aga1p and Aga2p on the binding activity between Aga2p and Aga1p was first evaluated. Under the treatment by the SH-compound (dithiothreitol), in which Agalpha2p is easily released into the medium from the intact cell surface, the Aga1p and Aga2p fusion protein was a good tool to make clear the role of the disulphide linkages. As a result, the linkages had a significant effect on not only the assembly but also the binding activity. The novel and simple system described here may further facilitate the study of molecular interaction in agglutination.     

DNA sequence of the beta-isopropylmalate dehydrogenase gene and phylogenetic analysis of the yeast Saccharomyces exiguus Yp74L-3

The beta-isopropylmalate dehydrogenase (LEU2) gene from a homothallic wild-type yeast, Saccharomyces exiguus Yp74L-3, was analyzed to estimate the phylogenetic position of this strain in yeasts. The beta-isopropylmalate dehydrogenase gene of Yp74L-3 was first isolated as a clone complementing the leu2 mutation of Saccharomyces cerevisiae, and then confirmed to complement the haploid leu2 mutant derived from strain Yp74L-3 through genetic transformation. The nucleotide sequence of the cloned DNA revealed an open reading frame (ORF) encoding the beta-isopropylmalate dehydrogenase composed of 365 amino acids. The beta-isopropylmalate dehydrogenase coding sequence from the Yp74L-3 strain displayed 76.7% similarity to that of S. cerevisiae. Candidates for a UAS and a TATA-box in the 5'-upstream region and for a poly-A attachment site in the 3'-downstream region were found. A phylogenetic tree constructed from the nucleotide sequences of the beta-isopropylmalate dehydrogenase coding regions revealed that Yp74L-3 is located between S. cerevisiae and the Kluyveromyces yeasts. The LEU2 gene cloned from Yp74L-3 will serve as an effective genetic marker for constructing the transformation system in S. exiguus Yp74L-3.