Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1)

The yeast Kluyveromyces marxianus strain BKM Y-719 produces an efficient pectin-degrading endopolygalacturonase (EPG) that cleaves the internal alpha-1,4-D-glycosidic linkages to yield oligomers of varying sizes. The EPG1 gene encoding this industrially important EPG was cloned by using the polymerase chain reaction (PCR) technique and degenerate primers to generate a 135 bp DNA fragment with which a genomic library was screened. The cloned fragment contained an open reading frame (ORF) of 1083 bp, encoding a 361 amino acid polypeptide. The predicted amino acid (aa) sequence of EPG showed similarity with polygalacturonases (PGs) of fungi. Analysis of the aa sequence indicated that the first 25 aa constitute a signal sequence and a motif (C218XGGHGXSIGSVG230) that is usually associated with a PG active site. Pulsed-field gel electrophoresis resolved chromosomal bands for K. marxianus BKM Y-719 and using chromoblotting it seems that EPG1 is present as only a single copy in the genome.     

Molecular cloning and sequence analysis of Zygosaccharomyces rouxii ADE2 gene encoding a phosphoribosyl-aminoimidazole carboxylase.

The nucleotide sequence of a 2.8 kb fragment containing the ADE2 gene of the osmotolerant yeast Zygosaccharomyces rouxii has been determined. The gene was cloned from a Z. rouxii genomic DNA library by complementation of the Saccharomyces cerevisae ade2 mutant strain. The sequenced DNA fragment contains a 1710 bp open reading frame predicting a protein of 570 amino acids. The deduced amino acid sequence shares a high degree of homology with Ade2p homologues in five other yeast species.     

Molecular and functional analysis of a MIG1 homologue from the yeast Schwanniomyces occidentalis

A putative glucose repressor MIG1-homologue (SoMIG1) was isolated from the amylolytic yeast Schwanniomyces occidentalis. Degenerate primers were designed from the conserved zinc finger regions of Mig1 and CreA proteins from different organisms. PCR using these primers and S. occidentalis genomic DNA as template yielded a single 128 bp product. This fragment was used as a DNA probe to screen a S. occidentalis genomic library. Analysis of the positive clones led to the isolation by PCR of a DNA fragment, which contained an open reading frame (ORF) that would encode a 458 amino acid polypeptide. The DNA binding and effector domains of this putative protein showed an identity of 71% and 15%, respectively, to those of the Mig1 protein from Saccharomyces cerevisiae. The SoMIG1 gene complemented a mig1 mutant of this yeast, which suggests that in S. occidentalis SoMIG1 is a glucose repressor. The Accession No. is AJ417892.     

Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.     

Rapid DNA purification for Hal gene PCR diagnosis in porcine tissues and extension to other meat species

Two different halothane (Hal) gene polymerase chain reaction (PCR) tests were applied to genomic DNA extracted from porcine blood, semen, muscle and fat tissues by a rapid and simple Chelex-100 based method. One of the PCR procedure is designed from the ryanodine receptor coding sequence to produce a 81 base pair (bp) fragment, while the other is designed from pig intron sequences to produce a 659 bp fragment. Oligonucleotide primers derived from the coding sequence were also used for other meat species. Amplification products obtained from porcine, bovine, ovine, equine and deer genomic DNA were successfully digested with Hha I restriction enzyme to produce the same electrophoretic pattern as in the normal homozygous (NN) pig. No PCR products could be amplified from chicken and turkey DNA.     

Yeast Derlin Dfm1 interacts with Cdc48 and functions in ER homeostasis

Recent studies have identified Derlin-1, a protein that associates with the AAA-ATPase p97 and is implicated in late steps in ER-associated protein degradation (ERAD). Derlin-1 has two Saccharomyces cerevisiae homologues, Der1p and Dfm1p. While Der1p has been studied extensively, little is known about Dfm1p. Accordingly, we investigated the role of Dfm1p in ERAD, ER homeostasis and interactions with the yeast p97 homologue Cdc48p. Dfm1p was not involved in the degradation of a number of Der1-dependent or -independent ERAD substrates, neither was it redundant with either Der1p or Sec61p in ERAD. However, Dfm1p had a role in ER homeostasis, since Dfm1p loss or overexpression could stimulate the unfolded protein response (UPR). Furthermore, Dfm1p interacted both genetically and physically with Cdc48p, the yeast p97 homologue, and this interaction required an eight amino acid sequence found in the C-terminus of Dfm1p that we have termed the SHP box. Our genetic studies are consistent with the lack of a role for Dfm1p in ERAD, but indicate it participates in ER-related Cdc48p actions distinct from retrotranslocation. Finally, sequence analysis indicated that the UPR-related and Cdc48p interaction functions of Dfm1p could be separated, implying this protein probably has numerous actions in the cell. Thus, the interaction between Derlins and p97 is conserved between yeast and mammals, although its function in ERAD is not. Furthermore, Dfm1p interacts with Cdc48p through its SHP boxes, and so defines a new motif for interaction with this widely-employed AAA-ATPase.     

Cloning, sequencing, and expression of H.a.YNR1 and H.a.YNI1, encoding nitrate and nitrite reductases in the yeast Hansenula anomala

A single Hansenula anomala genomic DNA fragment containing the genes H.a.YNR1 (yeast nitrate reductase) and H.a.YNI1 (yeast nitrite reductase) encoding nitrate and nitrite reductase, respectively, was isolated from a lambda EMBL3 genomic DNA library. As probe, a 3.2 kb DNA fragment isolated from a lambda gt11 H. anomala genomic DNA library screened with antiserum anti-NR from H. anomala was used. H. a.YNR1 and H.a.YNI1 genes are separated by 473 bp and encode putative proteins of 870 and 1077 amino acids, respectively, with great similarity to nitrate and nitrite reductases from other organisms. Northern blot analysis revealed that both genes are highly expressed in nitrate, very low in nitrate plus ammonium, and no expression was detected in ammonium or nitrogen-free media. Levels of nitrate reductase and nitrite reductase were very low or undetectable by Western blot analysis in nitrogen-free and ammonium media, whereas both proteins were present in nitrate and ammonium plus nitrate media. The nucleotide sequence Accession No. is AF123281.     

DNA sequence of the mat2,3 region of Schizosaccharomyces kambucha shares high homology with the corresponding sequence from Sz. pombe

To define conserved sequences for mat1 imprinting and silencing of the mat2,3 region of Schizosaccharomyces pombe, we determined the DNA sequence of the cognate region (mat2,3 region) of another fission yeast, Sz. kambucha, a yeast species isolated from Kambucha tea mix. The entire mat2,3 region shows more than 98% identity between the two species. Sequence similarity is even higher (99.3%) for mating-type cassettes; deduced amino acid sequences of three of the four Mat peptides (Pi, Pc and Mi) are identical between the two species, while the fourth (Mc) has a single amino acid polymorphism. Comparison of the sequence motif of the imprint site essential for mat1 switching shows that mat-P of Sz. kambucha has a sequence identical to the conserved motif present in Sz. pombe. However, this sequence motif of nine bases differs by one base for mat-M of Sz. kambucha. The sequence of the K region shows about 98% identity between the two species, with the cenH region showing 98.3% homology. Thus, the arrangement of the mat2,3 region in both yeasts is conserved and shows 1-2% nucleotide sequence variation throughout the region. The DNA sequence of the mat2,3 region from Sz. kambucha has been submitted to GenBank under Accession No. AY271822.     

Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology: Direct Sequencing of the Amplified DNA and Its Introduction into Yeast

A DNA fragment longer than 10 kb can be amplified by the long and accurate polymerase chain reaction (LA-PCR) method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA. © 1997 John Wiley & Sons, Ltd.     

Molecular cloning and sequence analysis of the Zygosaccharomyces rouxiiLEU2 gene encoding a beta-isopropylmalate dehydrogenase.

A DNA fragment carrying the LEU2 gene of osmotolerant yeast Zygosaccharomyces rouxii was isolated. The sequenced DNA fragment (2630 bp) contained two ORFs; one of them (1086 bp long, predicting a protein of 362 amino acids) shared a high degree of similarity with LEU2 genes of other yeast species. The cloned DNA fragment fully complemented the leu2 mutations of Saccharomyces cerevisiae and Z. rouxii.     

Identification of an UDP-Glc:glycoprotein glucosyltransferase in the yeast Yarrowia lipolytica

The UDP-Glc:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that plays a determining part in the mechanism by which unfolded, partially folded or misfolded glycoproteins are retained into the ER. We have identified an UGT in the yeast Yarrowia lipolytica. This protein, of a predicted molecular weight of 165.7 kDa, is encoded by a 5054 bp coding sequence containing a 643 bp intron at position 682-1323. The N-terminal part of the protein displays a signal sequence whereas its C-terminal part carries an ER retrieval signal HDEL. An interruption of the gene that removes the 1075 last nucleotides of its sequence did not lead to any evident phenotype except for a slight increased sensitivity to tunicamycin. YlUGT1 mRNA levels respond to tunicamycin treatment by an induction factor of 2-4, which indicates that the gene product participates in the quality control mechanism in this yeast. Finally, an immunofluorescence study of the protein localization, shows that the protein distribution is different from that of previously studied ER resident proteins. This could indicate that UGT distribution in the secretory pathway is not confined to the ER.     

一种真菌漆酶的克隆与序列分析

比较了几种不同真菌DNA提取方法对血红密孔菌基因组的提取的影响,根据GenBank中真菌漆酶氨基酸保守序列设计的简并引物,以血红密孔菌基因组总DNA为模板,扩增到一条长度为1084bp的DNA片段。通过BLAST比对,其基因序列与密孔菌属同源性最高,为99%,氨基酸同源性为88%,说明扩增的片段确为密孔菌漆酶基因。     

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.