Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

LEU2 gene homolog in Kluyveromyces lactis.

A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis. The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosomal protein L7.     

Possible gene interchange between plasmid and chromosome in yeast

Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.     

Cloning and sequencing of the PHO80 gene and CEN15 of Saccharomyces cerevisiae

The PHO80 gene, which is one of the regulatory genes exerting negative control in the pho system of Saccharomyces cerevisiae, was cloned. The 1.8 kb DNA fragment carrying the PHO80 gene was sequenced and one open reading frame large enough to encode 293 amino acids was found in the sequence. Northern blot analysis of poly(A)+-RNA isolated from cells grown under repressed and derepressed conditions revealed that (i) the size of the PHO80 message was around 1.4 kb, (ii) the expression of the PHO80 gene was not affected by the presence or absence of inorganic phosphate in the medium, and (iii) the expression of the PHO80 gene was not affected by pho2, pho4, pho81, or by pho80 itself. A centromere sequence was found downstream of the PHO80 coding region.     

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.     

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.     

The SPL1 tRNA splicing gene of Candida maltosa and Candida albicans

The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.     

The 5-aminoimidazole ribonucleotide-carboxylase structural gene of the methylotrophic yeast Pichia methanolica: Cloning,sequencing and homology analysis

The ADE1 gene of the yeast Pichia methanolica encodes phosphoribosyl-5-aminoimidazole-carboxylase (AIRC, EC 4.1.1.21), which is involved in purine biosynthesis. The gene was cloned by complementation of an ade2 mutation in Saccharomyces cerevisiae and a 3077 nucleotide DNA fragment was sequenced. The sequence possessed a single open reading frame, corresponding to a 543 amino acid sequence. The sequence of this putative protein has been compared to the proteins of homologous genes from S. cerevisiae, Schizosaccharomyces pombe, Escherichia coli, chicken and man. The analysis revealed remarkable homology between yeast AIRCs, while for other proteins homology was limited to defined regions.     

甘蔗UGPase DNA及其5’侧翼序列的克隆与序列分析

以甘蔗（FN95-1702）为材料,首次克隆得到甘蔗UGPaseDNA片段,测序结果表明该基因全长约5-3kb,包含21个外显子,20个内含子。开放读码框（ORF）共编码476个氨基酸。通过接头连接PCR方法克隆得到约0．8kb的该基因5’侧翼序列,并对得到的序列进行基础启动子区及调控元件分析,预测在该序列上存在TAT...     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216-1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene "mreA".     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

枯草芽孢杆菌BJ3-2赖氨酸脱羧酶基因yaaO的克隆与序列分析

根据GenBank中的枯草芽孢杆菌（Bacillus subtilis） 168菌株假定的赖氨酸脱羧酶基因（LDC）序列设计同源引物,提取枯草芽孢杆菌BJ3-2基因组DNA进行PCR扩增,并克隆至pGEM-T载体后测序.序列分析结果表明：Bacillus subtilis BJ3-2 yaaO基因开放阅读框（ORE）长为1 443bp,获得基因登录号为KJ561349,BLAST比对与已报道枯草芽孢杆菌的yaaO基因核苷酸序列及氨基酸序列同源性均高达90％以上,对赖氨酸脱羧酶氨基酸序列构建系统进化树分析,发现Bacillus subtilis BJ3-2的LDC与B.subtilis JS亲缘关系最近,SWISS-MODEL预测该蛋白的3D结构与鸟氨酸/赖氨酸/精氨酸脱羧酶蛋白相似性为39.22％,属于典型的Ⅲ型磷酸吡哆醛（PLP）依赖型鸟氨酸/赖氨酸/精氨酸脱羧酶家族成员.yaaO基因序列分析及氨基酸保守结构域的分析,为有效控制水豆豉产品中尸胺含量过高的研究提供了理论基础.     

南方红豆杉GGPP合酶基因的克隆与生物信息学分析

根据GeneBank中公布的GGPPS基因保守区设计特异引物,克隆南方红豆杉GGPPS基因的DNA和cDNA序列,并通过生物信息学方法对其核苷酸序列和蛋白质结构进行分析预测。结果表明,GGPPS基因DNA和cDNA序列都为1 182 bp,DNA序列中无内含子,cDNA序列为一个编码393个氨基酸残基的开放式阅读框;GGPPS的理论相对分子质量为42 590,等电点为5.68。核苷酸同源性分析显示,它与Taxus canadensis(AF081514)同源性为98.8%;推导出的氨基酸序列与Taxus canadensis(AAD16018)同源性达99.0%。利用Protparam、SignalP、ProtScale、SOPMA、Swiss-Modeling、Scan Prosite和MEGA4.0等生物信息学工具分别对其理化性质、信号肽、疏水性、亲水性、二级结构、三级结构和进化树进行分析,为其功能研究和生物合成紫杉醇研究提供分子基础。     

Cloning and analysis of CoEXG1, a secreted 1,3-beta-glucanase of the yeast biocontrol agent Candida oleophila

Lytic enzymes may have a role in the biological control of fungi. The yeast biocontrol agent, Candida oleophila, is an excellent subject to research this matter. In the present study, CoEXG1, which encodes for a secreted 1,3-beta-glucanase, is the first gene to be cloned from C. oleophila. It was isolated from a partial genomic library and analysed. Its open reading frame and putative promoter were expressed in baker's yeast, Saccharomyces cerevisiae. The reading frame, expressed under the inducible GAL1 promoter, caused an increased secretion of beta-glucanase, and the putative promoter region activated the lacZ reporter gene, to which it was fused. Sequencing analysis revealed that CoEXG1 carries the signature pattern of the 5 glycohydrolases family and has a putative secretion leader, as well as a high degree of identity to yeast 1,3-beta-glucanases. The GenBank Accession No. of CoEXG1 is AF393806.     

The PET18 locus of Saccharomyces cerevisiae: a complex locus containing multiple genes

The basis of pleiotropy shown by the pet18 mutants of Saccharomyces cerevisiae (rho-0,KIL-0 and temperature sensitive growth) was examined by cloning the fragment which complements the defect in growth at 37 degrees C of the pet18 mutants. The cloned DNA could complement the defect in the maintenance of the killer plasmid but did not give the cell the ability to maintain mitochondrial DNA. Sequence analysis of the cloned DNA revealed the presence of four open reading frames, at least two of which are necessary for the complementation activity. By using the cloned DNA as a probe, we found that two independent pet18 mutants have a deletion covering the entire sequence contained in the probe. From these results we predict that the traits of the pet18 mutants that concern temperature sensitivity and killer of the pet18 mutants are controlled by a separate gene(s) from that which participates in the maintenance of mitochondrial DNA.     

深海古菌Thermococcus siculi HJ21高温普鲁兰酶基因的克隆及表达

根据NCBI 上公布的普鲁兰酶基因的保守序列设计简并引物,以Thermococcus siculi HJ21 基因组DNA 为模板进行PCR,得到T. siculi HJ21 普鲁兰酶的基因,测序后通过Blast(NCBI)数据库比对和分析表明扩增基因有一个4056bp、编码1351 个氨基酸的开放阅读框,为一新的普鲁兰酶基因。将该基因插入表达载体pET28a,并转化Escherichia coli BL21(DE3),经IPTG 诱导,测定普鲁兰酶比活力。重组转化子的细胞破碎液有高温普鲁兰酶活性,SDS-PAGE 电泳结果显示出分子质量约为150kD 的特异性条带。     

Cloning and sequence analysis of the Candida boidinii ADE2 gene

Candida boidinii ADE2 gene (phosphoribosyl-5-aminoimidazole carboxylase; AIRC, EC 4. 1. 1. 21) has been cloned by homology to the Saccharomyces cerevisiae ADE2 gene. The cloned C. boidinii ADE2 gene complemented the ade2 mutation of S. cerevisiae. Sequence analysis showed a single open reading frame of 1719 nucleotides coding for a polypeptide of 573 residues. Comparison of the deduced amino acid sequence with those of AIRC enzymes from other yeasts showed marked homology among yeast AIRCs.     

枯草芽孢杆菌BJ3-2精氨酸脱羧酶基因speA的克隆与序列分析

根据GenBank中枯草芽孢杆菌168菌株的精氨酸脱羧酶基因speA序列设计特异性引物,提取Bacillus subtilis BJ3-2基因组DNA进行PCR扩增,并克隆至pGEM-T载体后测序。序列分析结果表明,Bacillus subtilis BJ3-2 speA基因ORF长为1 473 bp,可编码490个氨基酸,分子质量为53.53ku,基因登录号为KJ561348,与已报道枯草芽孢杆菌的speA基因核苷酸序列和氨基酸序列同源性均达93%以上,精氨酸脱羧酶氨基酸序列系统进化树分析,发现Bacillus subtilis BJ3-2的ADC与Bacillus subtilis 168亲缘关系最近,属于典型的III型PLP依赖型鸟氨酸/赖氨酸/精氨酸脱羧酶家族成员。speA基因序列分析及其蛋白保守结构域的分析,为有效控制水豆豉产品中生物胺含量的研究提供了理论依据。