大肠杆菌glgC基因的克隆及原核表达

以大肠杆菌BL21染色体DNA为模板,根据glgC基因的全序列设计了1对引物,在优化的PCR反应条件下扩增出了glgC基因片段,测序结果显示该片段大小为1296 bp,编码432个氨基酸残基。将该基因克隆到原核表达载体pET-28a-c( )中,重组载体pET-glgC转化至大肠杆菌BL21(DE3),经IPTG诱导后,SDS-PAGE电泳鉴定,得到了与理论推算的glgC基因表达产物分子质量(约53 kD)相符的特异蛋白条带。     

大肠杆菌嘌呤核苷磷酸化酶基因的克隆与真核表达载体的构建

根据Ｇｅｎｂａｎｋ中大肠杆菌嘌呤核苷磷酸化酶（ＰＮＰ）基因的核苷酸序列,设计并合成了一对引物,以大肠杆菌基因组ＤＮＡ为模板,进行ＰＣＲ扩增,并将扩增产物定向连接到克隆、测序及真核表达载体ＰＣＤＮＡ３中,进行酶切鉴定、测序及序列分析。结果表明ＰＣＲ扩增出７４１ｂｐ大小的片段,通过酶切和序列分析证明含完整的ＰＮＰ基因序列且基因插入方向正确,此序列与文献报道的ＰＮＰ基因的同源性为９９．７％。说明克隆的ＰＮＰ基因与文献报道的基本一致,ｐｃＤＮＡ３－ＰＮＰ的构建成功为今后用其进行基因转染来研究ＰＮＰ／Ｍｅｐ－ｄＲ自杀基因系统在肿瘤基因治疗中的应用打下了基础。     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

Cloning and structure of the hem A gene of Escherichia coli K-12

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor 1(RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA [Li et al., J Bacteriol. 171 (1989b) 2547-2552].     

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.     

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.     

Cloning of a DNA fragment encoding the 5'-terminus of the botulinum type E toxin gene from Clostridium butyricum strain BL6340

Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using lambda gt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.     

Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

禽白血病病毒p19基因末端片段在大肠杆菌中的表达

根据禽白血病病毒（ALV）p19基因末端序列合成一条82bp的双链DNA片段,将其克隆到表达质粒pGE-MEX-1中,序列分析结果与设计的相符。重组表达质粒转化的大肠杆菌BL21（ＤＥ３）经ＩＰＴＧ诱导后产生３４kD融合表达产物,与理论值相符;Western-blot分析表明该表达产物能与兔抗ＡＬＶ血清发生反应。     

抗冻蛋白结构基因片段的克隆

为了研究和开发利用抗冻蛋白或多肽,木文通过聚合酶链式反应（ＰＣＲ）合成了抗冻蛋白１１０ｂｐ的结构基因片段,然后将该基因片段克隆到大肠杆菌质粒载体Ｐ（Ｂｌｕｅｓｃｒｉｐｔ）Ⅱｋｓ＋／－上,获得了重组质粒,经酶切证明,获得的重组质粒中含有抗冻蛋白结构基因片段,以供该基因在大肠杆菌或酵母中表达抗冻蛋白。     

Construction of derivatives of the diphtheria toxin gene and their expression in Escherichia coli cells

A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested.     

禽白血病病毒p19基因末端片段在大肠杆菌中的表达

根据禽白血病病毒（ALV）p19基因末端序列合成一条82 bp的双链DNA片段,将其克隆到表达质粒pGEMEX-1中,序列分析结果与设计的相符。重组表达质粒转化的大肠杆菌BL21（DE3）经IPTG诱导后产生34kD融合表达产物,与理论值相符;Western-blot分析表明该表达产物能与兔抗ALV血清发生反应。     

Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli.

A 3.4-kilobase EcoRI restriction endonuclease fragment has been cloned from the facultatively photoheterotrophic bacterium Rhodobacter sphaeroides and shown to contain the structural gene (prkA) for phosphoribulokinase (PRK) activity. The PRK activity was characterized in Escherichia coli, and the product of the reaction was identified. The prkA gene was localized to a 1,565-base-pair EcoRI-PstI restriction endonuclease fragment and gave rise to a 33-kilodalton polypeptide both in vivo and in vitro. The gene product produced in E. coli was shown to be identical to the gene product produced in R. sphaeroides. The amino acid sequence for the amino-terminal region deduced from the DNA sequence confirmed that derived for partially purified PRK derived from both E. coli and R. sphaeroides. In addition, the 3.4-kilobase EcoRI restriction endonuclease fragment coded for a 37-kilodalton polypeptide of unknown function, and preliminary evidence indicates that this DNA fragment is linked to genes coding for other activities significant in photosynthetic carbon assimilation. The genetic organization and proposed operon structure of this DNA fragment are discussed.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY

A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis SecY homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.     

Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh.

By using a DNA fragment of Escherichia coli ffh as a probe, the Bacillus subtilis ffh gene was cloned. The complete nucleotide sequence of the cloned DNA revealed that it contained three open reading frames (ORFs). Their order in the region, given by the gene product, was suggested to be ORF1-Ffh-S16, according to their similarity to the gene products of E. coli, although ORF1 exhibited no significant identity with any other known proteins. The orf1 and ffh genes are organized into an operon. Genetic mapping of the ffh locus showed that the B. subtilis ffh gene is located near the pyr locus on the chromosome. The gene product of B. subtilis ffh shared 53.9 and 32.6% amino acid identity with E. coli Ffh and the canine 54-kDa subunit of signal recognition particle, respectively. Although there was low amino acid identity with the 54-kDa subunit of mammalian signal recognition particle, three GTP-binding motifs in the NH2-terminal half and amphipathic helical cores in the COOH-terminus were conserved. The depletion of ffh in B. subtilis led to growth arrest and drastic morphological changes. Furthermore, the translocation of beta-lactamase and alpha-amylase under the depleted condition was also defective.