Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis

We have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructose-2,6-bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol-grown cells, in contrast to the constitutive expression of PFK in Saccharomyces cerevisiae. We show here that phosphofructokinase of the yeast K. lactis is composed of two non-identical types of sub-units, encoded by the genes KIPFK1 and KIPFK2. We have cloned and sequenced both genes. KIPFK1 and KIPFK2 encode the α- and the β-PFK subunits with deduced molecular weights of 109.336 Da and 104.074Da, respectively. Sequence analysis indicates that the genes evolved from a double duplication event. Null mutants in either of the genes lack detectable PFK activity in vitro and the respective subunits cannot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow on glucose. However. Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the pentose phosphate pathway and respiration play a substantial role in glucose utilization by K. lactis. The K. lactis PFK genes can be expressed independently in S. cerevisiae and each of them complements the glucose-negative phenotype of pfk1 pfk2 double deletion mutants in this yeast. Expression of both K. lactis PFK genes simultaneously in S. cerevisiae pfk double deletion mutants complements for PFK activity. However, expression of a combination of PFK genes from K. lactis and S. cerevisiae does not lead to the production of a functional enzyme.     

白番红花CrCBF1基因的克隆及功能分析

该研究选取新疆地区耐寒植物白番红花为研究材料,采用RT-PCR方法,从白番红花中克隆得到白番红花CBF1的全长cDNA序列,命名为CrCBF1(GenBank登录号MF681787)。结果表明,CrCBF1基因完整开放阅读框ORF长642bp,编码213个氨基酸,分子量为23.8kD,理论等电点为4.99,具有CBF家族典型的AP2保守结构域;亚细胞定位分析显示,CrCBF1基因编码的蛋白定位于细胞核;酵母自激活分析显示,CrCBF1转录因子具有转录激活活性;酵母功能验证分析显示,过表达CrCBF1基因可以明显提高酵母的抗寒性。     

Cloning,expression and genomic structure of a novel human GNB2L1 gene,which encodes a receptor of activated protein kinase C (RACK)*

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.     

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.     

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

Summary A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn²⁺, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.     

High level expression of isocitrate lyase gene of n-alkane-utilizing yeast Candida tropicalis in Sacchromyces cerevisiae

The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.Abbreviations ICL isocitrate lyase - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物. 以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象，检测酿酒酵母双链RNA介导的基因沉默效应. 以pESC-LEU为骨架，构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499. 用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调. 结果表明，双链RNA介导的基因沉默技术，能够用作降低酿酒酵母某一特定基因表达水平的工具. 并有助于理解芽殖酵母的RNA干扰现象.     

Isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from the edible basidiomycetous mushroom, <Emphasis Type="Italic">Lentinula edodes</Emphasis>, and its expression in the course of fruit-body formation

We isolated and characterized the genomic and complementary DNAs encoding a chitin synthase from an edible basidiomycetous mushroom, Lentinula edodes. The gene (which we designated Lechs1) contains a large open reading frame encoding a polypeptide of 1937 amino acid residues. The open reading frame is interrupted by 14 small introns (49–116 bp). The gene product (LeChs1) consists of a myosin motor-like domain in its N-terminal half and a chitin synthase domain in its C-terminal half, analogous to the class V and VI chitin synthases of other filamentous fungi. Phylogenetic analysis demonstrated that LeChs1 is classified into class VI chitin synthases. Southern blot analysis indicated that Lechs1 is a single-copy gene per haploid genome and that L. edodes has no other highly homologous chitin synthase genes. Northern blot analysis revealed that Lechs1 is expressed throughout the whole stages of fruit-body formation of L. edodes, but its expression level gradually declines in a fruit body-maturation-dependent manner with highest expression in vegetative mycelia and fruit body at the early stage of maturation (immature fruit body). This is the first report on the isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from basidiomycetes.     

The KNS1 gene of Saccharomyces cerevisiae encodes a nonessential protein kinase homologue that is distantly related to members of the CDC28/cdc2 gene family

Summary A novel protein kinase homologue (KNS1) has been identified in Saccharomyces cerevisiae. KNS1 contains an open reading frame of 720 codons. The carboxy-terminal portion of the predicted protein sequence is similar to that of many other protein kinases, exhibiting 36% identity to the cdc2 gene product of Schizosaccharomyces pombe and 34% identity to the CDC28 gene product of S. cerevisiae. Deletion mutations were constructed in the KNS1 gene. kns1 mutants grow at the same rate as wild-type cells using several different carbon sources. They mate at normal efficiencies, and they sporulate successfully. No defects were found in entry into or exit from stationary phase. Thus, the KNS1 gene is not essential for cell growth and a variety of other cellular processes in yeast.     

Translation elongation factor 1-α gene from <Emphasis Type="Italic">Pichia pastoris</Emphasis>: molecular cloning,sequence, and use of its promoter

The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P _TEF1 ) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P _GAP ) by using a bacterial lipase gene as a reporter gene. P _TEF1 demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P _GAP . The sequence of the gene was deposited in GenBank under accession no. EF014948.     

The respiratory complex I in yeast: Isolation of a geneNUO51 coding for the nucleotide-binding subunit of NADH: Ubiquinone oxidoreductase from the obligately aerobic yeastYarrowia lipolytica

We have isolated a geneNUO51 coding for a homologue of the nucleotide-binding subunit of mitochondrial respiratory chain linked NADH:ubiquinone oxidoreductase from the obligately aerobic yeastYarrowia lipolytica. DNA sequencing revealed a 1464 bp open reading frame encoding a protein with predicted molar mass of about 53.7 kDa. The sequence is highly conserved with its counterparts from filamentous fungi and represents the first yeast homologue of the NADH-binding subunit (51 kDa) of the respiratory complex I. In addition, PFGE and Southern hybridization analysis indicate thatNUO51 is a single copy gene in the genome ofY. lipolytica. The expression ofNUO51 by Northern blot analysis was also examined.     

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

Summary The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including -tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.     

Transcriptional analysis of the bglP gene from Streptococcus mutans

Background  

An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II.     

Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.