Cloning and sequence analysis of Mucor circinelloides glyceraldehyde-3-phosphate dehydrogenase gene

A genomic library of Mucor circinelloides ATCC 1216b has been constructed in Lambda Fix II vector. The library has an average insert site of 10 kb and covers the genome 12 times. The M. circinelloides gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The promoter region, containing a consensus TATA and CAAT box and a 298 nucleotid long termination region were also determined.     

Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the zygomycetes fungus Rhizomucor miehei

Rhizomucor miehei is important from a biotechnological aspect in consequence of its content of aspartic proteinase, which has high milk-clotting activity. A genomic library of R. miehei NRRL 5901 has been constructed in a phage (Lambda Fix II) vector. The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by polymerase chain reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 336 amino acids interrupted by 5 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3-phosphate dehydrogenase proteins from yeast and filamentous fungi. The promoter region, containing a consensus TATA box, and 246-bp downstream from the putative stop codon were also determined. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.     

球毛壳菌甘油醛-3-磷酸脱氢酶基因克隆及特性分析

用粗糙脉孢菌(Neurospora crassa,XP_327967)和菜豆炭疽病菌(Colletotrichum lindemuthianu,P35143)的甘油醛_3_磷酸脱氢酶基因(Glyceraldehyde 3_phosphatedehydrogenase,GAPDH)氨基酸序列对球毛壳菌(Chaetomium globosum)菌丝ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌GAPDH全长cDNA序列。该序列长1240bp,开放阅读框1014bp,编码337个氨基酸组成的多肽,蛋白分子量为36.1kD。用PCR方法克隆了该基因的DNA序列,序列长为1556bp,由2个内含子和3个外显子组成。BlastP同源性分析表明该基因与鹅掌柄孢壳(Podosporaanserine)同源性最高为95%;与米曲霉(Aspergillusoryzae)同源性最低为87%。GAPDH酵母转化子生物功能分析表明转化子对Na2CO3和高温有高的耐受性,证明GAPDH为抗胁迫基因。该基因的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY522719,AY593253,AAS01412)。     

Differential expression and sequence analysis of the maize glyceraldehyde-3-phosphate dehydrogenase gene family.

Two cDNA clones for maize cytosolic glyceraldehyde-3-phosphate dehydrogenase are described. One is about 97% similar in coding capacity to a previously published clone [Brinkmann et al. (1987). J. Mol. Evol. 26, 320-328], while the other shows only 88% similarity. Evidence points toward the three cDNAs being the products of three genes, to be called Gpc1, Gpc2, and Gpc3. When the least similar clone, corresponding to Gpc3, was used to analyze RNA gel blots, anaerobic treatment for 6 hours induced RNA accumulation in the shoots 15.6-fold, while a 1-hour shift from 28 degrees C to 40 degrees C increased accumulation 5.1-fold. Roots had a higher basal level of expression, leading to a 6.0-fold anaerobic induction, and a 2.4-fold heat stress induction. RNA gel blot analysis using the clone corresponding to Gpc2 showed decreased RNA accumulation within 6 hours of anaerobiosis, while analysis with the previously published clone, corresponding to Gpc1, showed a decrease within 24 hours. Neither Gpc1 nor Gpc2 showed heat stress induction, while some other known anaerobic genes did. Through the use of hybrid selection, in vitro translation, and immune precipitation, the relative expression of the three genes is shown. The role of the observed changes in gene expression is discussed in relation to stress physiology.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

SEFA-PCR法克隆灵芝鲨烯合酶基因启动子及其序列分析

鲨烯合酶是灵芝三萜生物合成的关键酶,灵芝鲨烯合酶基因的表达和活性的高低决定了灵芝中三萜含量的高低。根据已经获得的灵芝鲨烯合酶全长cDNA序列设计一对专一引物,通过PCR扩增得到了灵芝鲨烯合酶基因的基因组全长,序列长1984bp,含有3个内含子。根据其基因组序列设计引物,采用SEFA-PCR的方法,以总DNA为模板,克隆了灵芝鲨烯合酶基因的启动子序列,长1042bp。序列分析发现灵芝鲨烯合酶基因启动子中没有明显的TATA和CAAT框,但是含有CCAAT-bindingfactor、GATA-1、GC-box、TFⅡD等重要的转录因子的结合位点,以及在人和酿酒酵母鲨烯合酶基因启动子中发现的甾醇调节相关的顺式调控元件。     

The primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae.

The complete nucleotide sequence of the coding, as well as the flanking noncoding regions, of a yeast glyceraldehyde-3-phosphate dehydrogenase gene was determined. Both the 5' and 3' noncoding sequences are extremely AT-rich and regions of partial dyad symmetry are present immediately adjacent to the 5' and 3' ends of the translated portion of the gene. The sequence AAUAAA is present in the 3' noncoding region of this gene and is a part of an extensive region of dyad symmetry which is structurally related to the 3'-terminal portion of both procaryotic mRNAs, as well as some eukaryotic mRNAs. The coding region of this gene does not contain intervening sequences. Establishment of the primary structure of this glyceraldehyde-3-phosphate dehydrogenase gene provides a basis for further studies involving in vitro mutation of the gene and subsequent analysis of gene expression in vivo.     

Structural analysis of glyceraldehyde-3-phosphate dehydrogenase functional diversity

Multifunctional proteins provide a new mechanism to expand exponentially cell information and capability beyond that indicated by conventional gene analyses. As such, examination of their structure–function relationships provides a means to define the mechanisms through which cells accomplish critical yet disparate activities required for cell viability and survival. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be considered the quintessential multidimensional protein which exhibits a variety of functions unrelated to its classical role in energy production. This review discusses new insights into the structure–function mechanisms through which defined GAPDH amino acid domains are utilized for its diverse activities, the importance of its post-translational modification, and, intriguingly, the logic inherent in the presence or the absence of specific signaling domains.     

Cloning and sequencing of the putative glyceraldehyde-3-phosphate dehydrogenase gene from Cephalosporium acremonium and its application to heterologous gene expression

A candidate for Cephalosporium acremonium glyceraldehyde-3-phosphate dehydrogenase gene (GAP) was cloned using the corresponding Saccharomyces cerevisiae gene as a probe. It encoded peptides showing marked similarity with those from S. cerevisiae and Aspergillus nidulans. A fused gene, GAP-HPT, consisting of the putative GAP promoter and the open reading frame of the bacterial hygromycin B phosphotransferase gene (HPT) was constructed, and it was successfully used to transform C. acremonium to a hygromycin B-resistant phenotype.