地中海拟无枝菌酸菌U32中amrC／amkC基因的序列分析及在大肠杆 …

从力复霉素ＳＶ产生菌－－地中海拟无枝菌酸菌（Ａｍｙｃｏｌａｔｏｐｓｉｓ ｍｅｄｉｔｅｒｒａｎｅｉ）Ｕ３２的硝酸盐同化基因簇的上游克隆了一个２．６ｋｂ的ＥｃｏＲＩ－ＸｈｏＩ ＤＮＡ片段并测定其序列。序列分析表明,该ＤＮＡ片段编码两个完整的开放阅读框架（ＯＲＦ）,ＯＲＦ２的起始密码子ＧＴＧ与ＯＲＦ１的终止密码子ＴＧＡ在ＴＧ处重叠。ＯＲＦ１编码一个含２２４个氨基酸的多肽,它同放线菌中典型的应答调节蛋白包     

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli.

A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1 [alpha beta] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.     

Lysyl-tRNA synthetase of Bacillus stearothermophilus molecular cloning and expression of the gene

The gene of the lysyl-tRNA synthetase of Bacillus stearothermophilus NCA1503 was cloned and sequenced. The gene consists of 1485 bp nucleotides commencing with an ATG start codon and ending with a TAA stop codon, and encodes a polypeptide of 493 amino acids. The recombinant enzymes were expressed in E. coli using an expression plasmid containing the T7 RNA polymerase/promoter.     

Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.

The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL-exon. The alternative ORF encodes ALEX, a very basic (pI 11.8), proline-rich protein of 356 amino acids. Both XLalphas and ALEX are translated from the same mRNA. Like XLalphas, ALEX is expressed in neuroendocrine cells and tightly associated with the cytoplasmic leaflet of the plasma membrane. Remarkably, ALEX binds to the XL-domain of XLalphas. Our results reveal a mechanism of gene usage that is without precedent in mammalian genomes.     

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.     

Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non-overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene. The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.     

斜纹夜蛾核多角体病毒几丁质酶基因上游4.0kb的序列分析

本文报道了斜纹夜蛾核多角体病毒几丁质酶基因(chiA)上游约4.0 kb范围内的序列,它包括了六个读码框(ORF1～6),其长度分别为156 bp、297 bp、540 bp、369 bp、1281 bp和228 bp,可编码的氨基酸长度分别为51、98、179、122、426和75个,分子量分别为6.15?kD、11.46 kD、21.70 kD、14.69 kD、47.59 kD和9.09 kD。在ORF1、ORF2、ORF3起始密码前分别有一个、二个及一个杆状病毒早期启动子基序CAGT;在ORF4、ORF5起始密码前各有一个及二个杆状病毒晚期启动子基序TAAG。在ORF1、ORF4、ORF5终止密码下游有真核生物mRNA转录poly(A)加尾信号。ORF4为AcMNPVORF53、BmNPVORF42、OpMNPVORF56、LdMNPVORF54的同源基因。ORF1、ORF2、ORF6与已知的杆状病毒基因没有同源性,可能为三个新的基因。     

Reactivity of E. coli-derived trans-activating protein of human T lymphotropic virus type III with sera from patients with acquired immune deficiency syndrome

Randomly sheared DNA fragments from HTLV-III proviral DNA were cloned into an E. coli open reading frame (ORF) expression vector. The inserted ORF DNA was expressed in E. coli transformants as a polypeptide fused to the lambda CI protein at the amino terminus and to beta-galactosidase at the carboxyl terminus. The reactivity of the recombinant peptides with antibodies from sera of AIDS patients was determined by the Western blot technique. The coordinates of the DNA inserts of the immunoreactive clones were then determined by DNA sequencing. A clone, ORF 628, was found to contain a short DNA segment located between the sor and env genes (nucleotide positions 5367 to 5597), a region previously thought to be noncoding. Inspection of the DNA sequences of this clone and of other HTLV-III isolates revealed the presence of a small ORF located between nucleotide position 5411 and 5625, capable of encoding a polypeptide of 72 amino acids. The biosynthesis of the polypeptide of ORF 628 initiates from an ATG codon within the HTLV-III insert. The fusion protein of ORF 628 was partially purified by affinity chromatography on CH Sepharose 4B coupled to a beta-galactosidase ligand, and tested against a panel of sera from AIDS patients by Western blot analysis. Approximately 35% of the sera from patients with AIDS or ARC contained antibodies reactive with the peptide. The DNA region spanned by ORF 628 is now thought to be the major functional element of the trans-activator gene, tat.     

A new D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new D-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although alpha-ketoisoleucine was the most favourable of all alpha-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.