Cloning,characterization and heterologous expression of the Blakeslea trispora gene encoding orotidine-5'-monophosphate decarboxylase

The pyrG gene of the fungus Blakeslea trispora, encoding orotidine-5'-monophosphate decarboxylase (OMPD) enzyme, was cloned by heterologous hybridization of a genomic library with the Mucor circinelloides pyrG gene. The deduced amino acid sequence of the B. trispora pyrG gene is highly similar to the OMPD from other organisms. Hybridization analyses revealed that the only copy of this gene present in the genome of B. trispora is constitutively expressed. Heterologous complementation of a mutant of M. circinelloides deficient in OMPD activity with the B. trispora pyrG gene and promoter sequence confirmed the function of this gene. This functional complementation demonstrates that heterologous expression in M. circinelloides might be used to investigate the function of genes of B. trispora.     

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.     

Cloning and sequence analysis of a glyceraldehyde-3-phosphate dehydrogenase gene from Ganoderma lucidum

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.     

Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA.

A cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene (AAF) induced rat hepatocellular carcinoma was screened by synthetic DNA probes deduced from a partial amino acid sequence of glutathione S-transferase P subunit that had been isolated from the tumor by two-dimensional gel electrophoresis. One of the four clones analyzed contained an mRNA region encoding the total amino acid sequence of this enzyme subunit and the complete 3'-noncoding region. The nucleotide sequence indicates that this enzyme subunit has 209 amino acids (calculated Mr=23,307) distinct from other glutathione S-transferase subunits such as Ya and Yc. Comparison of the amino acid sequences between these proteins indicates that glutathione S-transferase P subunit gene has been evolved from the ancestral gene at an earlier stage than the separation of Ya and Yc and that there are at least three domains having a considerable homology with each other in these enzymes. The very large increase of this mRNA in chemically induced hepatocellular carcinoma suggests a characteristic derepression of this gene during hepatocarcinogenesis.     

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.     

Isolation and sequencing of a genomic clone encoding aspartic proteinase of Rhizopus niveus.

A gene encoding Rhizopus niveus aspartic proteinase was isolated from an R. niveus genomic library by using oligonucleotides probes corresponding to its partial amino acid sequence, and its nucleotide sequence was determined. By comparing its deduced amino acid sequence with the amino acid sequence of rhizopuspepsin (5, 26), we concluded that the R. niveus aspartic proteinase gene has an intron within its coding region and that it has a preproenzyme sequence of 66 amino acids upstream of the mature enzyme of 323 amino acids.     

Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis.

The gene for beta-isopropylmalate dehydrogenase (EC 1.1.1.85) of Spirulina platensis (leuB) was cloned from a lambda EMBL3 genomic library by heterologous hybridization using the Nostoc UCD 7801 leuB gene as a probe. The sequence of the entire leuB coding region was determined as well as 645 bp of 5' flanking region and 956 bp of 3' flanking region. DNA sequencing revealed an open reading frame of 1065 nucleotides capable of encoding a polypeptide of 355 amino acids. Homologies between the amino acid sequence deduced from the nucleotide sequence of the S. platensis leuB gene and the amino acid sequences published for corresponding proteins either from bacteria or yeasts are 45% or more. Northern hybridization analysis indicated that the S. platensis leuB gene is transcribed as a single monocistronic RNA of approximately 1200 bases.     

Molecular cloning and nucleotide sequence of the streptavidin gene.

Using synthetic oligonucleotides as probes we have cloned the streptavidin gene from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal peptide region and the 3' and 5' flanking regions of the gene. The deduced amino acid sequence shows several interrupted blocks of homology with the amino acid sequence of chicken egg-white avidin. Analysis of the secondary structure suggests a high content of beta-structure in both proteins and considerable overall structural similarity between them.     

Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase.

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.     

Molecular cloning of LMO41, a new human LIM domain gene

We have identified a new human LIM domain gene by isolating an autoantigenic cDNA clone from a human breast tumor cDNA library. The predicted amino acid sequence of the cDNA clone's 495 bp open reading frame contains two tandem LIM domain motifs, and within the LIM domain region there is 62% identity with the analogous region of the LIM-only gene LMO1. The homology to LMO1 is restricted to the 360 bp region encoding the tandemly repeated LIM domains, the rest of the open reading frame as well as the extensive, GC-rich 5' untranslated region, and 3' region of the 2 kb cDNA sequence are unrelated to any known genes. This gene has been designated LMO4.     

The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae

We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library. The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A)+ RNA and by complementation of the mutant phenotype with subclones. The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an Mr of 61310. The nucleotide sequence of the gene as well as the 5' and 3' flanking regions are presented. The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor. The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.     

紫杆柽柳与其它物种脂质转运蛋白基因编码蛋白的同源性比较

根据从柽柳cDNA文库克隆获得的脂质转运蛋白(LTP)的部分序列,用RACE技术克隆出其全长cDNA序列.基因的5'非翻译区96bp,3'非翻译区222bp,开放阅读框285bp,编码94个氨基酸,预计蛋白的分子量为9.9 kD,等电点为8.02.此基因有8个位置保守的Cys残基及26个氨基酸的信号肽,为典型的植物脂质转运蛋白基因.其基因序列数据库(GenBank)登录号为AY574218(基因)和AAS79106(蛋白).     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

柽柳(Tamarix androssowii)Tadir基因的克隆及分析

在柽柳cDNA文库测序中获得了Tadir基因的全长cDNA序列,去除PolyA后,该基因全长724bp。其中5′非翻译区26bp,3′非翻译区143bp,开放阅读框(ORF)长555bp,编码184个氨基酸。基因编码蛋白的分子量为19.69kD,理论等电点为6.96。疏水性分析表明,蛋白的前41个氨基酸为亲水性的。该基因的Genbank登录号为DQ462418(基因),ABE73781(蛋白)。实时荧光定量PCR分析结果表明,0.4mol·L-1NaCl和NaHCO3胁迫后该基因表达量发生变化,其可能与柽柳的耐盐性有关。     

Identification of genomic DNA coding for chicken type II procollagen

A segment of the type II procollagen gene has been isolated by screening a lambda Charon 4A library containing fragments of chicken genomic DNA. The specific clone, LgCOL(II), was selected by hybridization using overlapping inserts from two cDNA clones which are specific for a cartilage procollagen (Vuorio, E., Sandell, L., Kravis, D., Sheffield, V. C., Vuorio, T., Dorfman, A., and Upholt, W. B. (1982) Nucleic Acids Res. 10, 1175-1192). DNA sequence analysis of LgCOL(II) in the COOH-telopeptide region of the protein, shows conclusively that this DNA corresponds to the chicken type II procollagen gene. Hybridization of cDNA probes to restriction fragment gel blots together with DNA sequence analysis have established the orientation and position of the procollagen gene within the lambda Charon 4A vector and indicate that LgCOL(II) contains approximately 6 kilobase pairs of the type II procollagen gene plus additional DNA flanking the 3' end of the gene. DNA sequence analysis shows directly that LgCOL(II) contains DNA sequences identical with those in the cDNA clones. The portion of the gene from amino acid 578 of the triple helical region to the COOH-terminal end of the protein (approximately 700 amino acids) is contained within the clone, corresponding to approximately 50% of the amino acid coding sequence of the gene. This region of the chicken alpha 1 (type II) procollagen gene is encoded within a shorter segment of the chicken genome than is the corresponding region of the alpha 2(type I) procollagen gene.     

特异种质烟草HZNH的Fe-SOD基因的克隆与表达

超氧化物歧化酶(superoxidedismutase,SOD)是一种广泛存在于动物、植物、微生物体内的金属酶,按其结合的金属性离子可分为Fe SOD、Mn SOD和CuZn SOD三种,它们通过催化超氧阴离子自由基O·-2发生歧化反应,达到清除O·-2的效果,具有防御氧毒性、增强机体抗辐射损伤能力、防衰老,治疗某些肿瘤、炎症、自身免疫疾病等功效,在农业、医药、食品、化工等产业中的应用前景广阔,因此广受国内外科研工作者的关注和重视[1].而试图通过转SOD基因技术来培育高抗逆农作物新品种和基因克隆与表达技术来实现SOD的大规模发酵生产,已成为国内外SOD…