Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1

The gene encoding a ferredoxin (Fd) from Haloarcula japonica strain TR-1 was cloned and sequenced. Sequence analysis of the cloned Ha. japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids. The deduced amino acid sequence of Ha. japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea. The Ha. japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. Ha. japonica Fd could then be produced as a fusion with HisTag (6xHis) in Ha. japonica host cells. The absorption and ESR spectra of the Fd/HisTag fusion protein revealed the presence of a [2Fe-2S] cluster which is characteristic of native Ha. japonica Fd.     

Purification and characterization of a ferredoxin from Haloarcula japonica strain TR-1

A ferredoxin (Fd) was purified from the extremely halophilic archaeon, Haloarcula japonica strain TR- 1, to electrophoretic homogeneity. The apparent molecular weight (M _r) of the Fd was estimated to be 24,000 on SDS-polyacrylamide gel electrophoresis. The amino acid composition analysis revealed that the Fd composed of a number of acidic amino acids (uncorrected for amides). The N-terminal amino acid sequence (30 residues) was determined to be: PTVEYLNYEVVDDNGWDMYDDDVFAEASDM. The iron content was 3.42±0.04 mol/mol-Fd on the basis of the apparent M _r value. The absorption and ESR spectra of the Fd showed similarity to those of Fds from plant and Halobacterium halobium. These results led us to conclude that the H. japonica Fd contained a [2Fe-2S] cluster.     

Purification and partial characterization of cell surface glycoprotein from extremely halophilic archaeon Haloarcula japonica strain TR-1

Summary Haloarculajaponica has a glycoprotein on its cell surface. The cell surface glycoprotein of H. japonica was purified and characterized. It had an apparent molecular mass of 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carbohydrate content was about 12% (wt/wt). The polypeptide portion contained a large proportion of acidic amino acids, and the sequence of 18 N-terminal amino acids was determined.     

Cloning and sequencing of ftsZ homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding FtZ was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible ftsZ gene revealed that the structural gene consisted of an open reading frame of 1,182 nucleotides encoding 394 amino acids. The deduced amino acid sequence of the Ha. japonica FtsZ showed high identities with those Halobacterium salinarom, Haloferax volcanii and Haloferax mediterranei FtsZs.     

用PCR突变技术克隆艾滋病病毒蛋白酶基因

作者设计并合成了一对用于PCR技术的突变引物HIV-1 Pr1和HIV-1Pr2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、HindⅢ和TAA序列,便于HIV-1 Pr基因的定向克隆和表达。用HIV-1 Pr1和HIV-1 Pr2作引物,采用PCR方法从HIV-1基因组DNA中扩增出了一个360bp长的DNA片段,用EcoRI和HindⅢ双酶切法将此片段定向克隆入pUC19质粒,将克隆基因插入M13mp18进行DNA序列分析。结果表明,该基因序列的读框完全正确,从而为HIV-1 Pr基因的表达及抑制剂的研究奠定了基础。     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

Cloning and Sequencing of the Gene Encoding 10 kD Zein of Maize

The results of cloning and sequencing of the gene encoding 10 kD zein of maize (Zea mays L. ) with polymerase chain reaction (PCR) technique are here with presented. The genomic DNA template was extracted from sterilized seedlings of maize. Primered with a pair of synthetic 5′and 3′ PCR primers, a 0.57 kb DNA fragment was obtained after 30 PCR amplification cycles. The restriction map of the DNA fragment has been determined. The result indicated that the entire coding sequence of 10 kD zein gene has been cloned. The homologies of the DNA sequence and deduced amino acid sequences between our result and those published abroad are 96% and 90% respectively. As l0 kD zein is rich in sulphur, this gene might be used to improve the quality of crops, especially, the forage legumes by genetic engineering.     

用PCR技术克隆细小病毒H－1部分非结构蛋白基因

应用ＰＣＲ技术定向克隆了细小病毒Ｈ－１的非结构蛋白（ＮＳ）部分基因片段。自行设计并合成了ＰＣＲ引物△Ｐ３和△Ｐ４,在两个引物中分别引入两个突变碱基,使扩增后的ＤＮＡ片段的两端含有限制性核酸内切酶ＨｉｎｄⅢ或ＢａｍＨＩ的酶切位点,经双酿切法把该ＤＮＡ片段重组到ｐＵＣ１１８质粒中。对插入片段的ＤＮＡ序列测定和分析结果证实该片段为Ｈ－１ＮＳ－１基因序列。以此重组质粒为探针,采用分子杂交的方法,分别测定了Ｈ－１及ＭＶＭＤＮＡ在细胞内的复制水平。这一基因的克隆为制备Ｈ－１的质量监测、Ｈ－１及ＭＶＭＮＳ－１蛋白抑瘤作用机理及其在肿瘤细胞及正常组织中的转录表达等研究奠定了基础。     

人骨保护素成熟肽段基因的克隆和序列测定

人骨保护素（OPG）具有抑制破骨细胞分化、抑制成熟破骨细胞的活性并诱导其凋亡的功能,具有广泛的研究和应用前景。采用RT－PCR的方法从重组骨形成蛋白2（BMP－2）刺激的人骨肉瘤细胞系MG63细胞中克隆编码人OPG的成熟肽段基因,并将其克隆到pUC18中,序列分析结果表明,所克隆的人OPG／OCIF成熟肽段基因与献报道的完全一致。为进一步基因表达及功能研究奠定了基础。     

Cloning and sequencing analysis of Trp1 gene of Flammulina velutipes

The genomic TRP1 gene from basidiomycete Flammulina velutipes was cloned by complementation of yeast Saccharomyces cerevisiae trp1 mutation. Sequencing analysis revealed that the TRP1 gene encoded a single protein consisting of three catalytic functional domains; glutamine amidotransferase, indole-3-glycerol phosphate synthase ) and N-(5'-phosphoribosyl) anthranilate isomerase, in order of NH2-glutamine amidotransferase-indole-3-glycerol phosphate synthase N-(5'-phosphoribosyl) anthranilate isomerase-COOH. The coding sequence of the TRP1 gene was interrupted by a single intron of 48 bases, the position and flanking sequences of which were highly homologous to those of basidiomycete Phanerochaete chrysosporium trpC.     

AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA Acetyl-CoA + AMP + PP_i). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37°C. Using the N-terminal amino acid sequence an open reading frame — coding for a 74 kDa protein — was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.     

Cloning and Sequencing of phbB and phbC in Alcaligenes eutrophus H16 and Construction of Their Plant Expression Vectors

NADPH-dependent acetoacetyl-CoA reductase gene (phbB) and poly-β-hydroxybu-tyrate (PHB) synthase gene (phbC) for biosynthesis of PHB were amplified and cloned from chromosomal DNA of Alcaligenes eutrophus H16 using PCR. The restriction maps and sequencing results show that both phbB and phbC have been cloned. It was found that the two genes cloned were highly homologous in DNA sequences to those being reported abroad. By DNA processing, the authors have constructed three tuber-specific plant expression vectors: pPSAGB (containing phbB), pBIBGC (containing phbC) and pPSAGCB (containing both phbB and phbC).     

Presence of an additional PstI fragment in intron 1 of the chicken growth hormone-encoding gene

A previously unreported 196-bp PstI fragment was found in intron 1 of the gene encoding chicken growth hormone (cGH) when a PCR assay for an MspI restriction fragment length polymorphism was established. A pair of PCR primers was designed according to the published cGH sequence and used to amplify a fragment which contained two MspI sites, one polymorphic and another non-polymorphic. However, amplification of genomic DNA from two strains of meat-type chickens and three strains of White Leghorn chickens yielded a PCR product which was about 200 bp larger than expected. The fragment from one of the meat-type chickens was subcloned into the vector pCR-Script SK⁺, and sequenced. It revealed the presence of an extra fragment of 196 bp which was flanked by the PstI sites and occurred at nt + 308 of the previously reported cGH sequence.     

Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin)

Abstract The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fim A, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.     

Cloning and sequencing of a novel metallothionein gene in Mytilus galloprovincialis Lam

Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 5' promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http://www.unizh.ch/~mtpage/classif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.     

人粒细胞集落刺激因子cDNA的克隆、序列鉴定及初步表达

利用多聚酶链反应技术,从人膀胱癌细胞系5637细胞中快速扩增并克隆了人粒细胞集落刺激因子cDNA,序列分析证明,该cDNA包含人粒细胞集落刺激因子的全部编码基因,全长612bp,编码30个氨基酸的信号肽和174个氨基酸的成熟蛋白。其中第43位codon出现一个碱基的突变(CAC→TAC)导至第43位氨基酸的改变(组氨酸→酪氨酸)。经逆转录病毒导入SP2/0细胞并初步表达。结果表明:该基因产物具有G-CSF活性。