苏云金芽孢杆菌以色列亚种130kd杀蚊蛋白基因的...

The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe. The crystal protein containing the component of 130kd toxic protein was purified. The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay. The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E. coli TG1. From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization. The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3. For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation. It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis. The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.     

Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein.

An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy. One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain. The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10). After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein. From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS-polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd.     

Proteolytic processing of the Ada protein that repairs DNA O6-methylguanine residues in E. coli

In extracts of E. coli treated with an adapting regime of MNNG, the induced 39kd Ada protein having O6-MeG-DNA methyltransferase activity is processed to a 19kd active domain corresponding to the C-terminal half of the intact protein. This proteolytic processing has been followed on Western immunoblots using antisera raised against the 19kd fragment. Initial processing at 25 degrees C or 37 degrees C mainly generates a fragment of mol. wt. 24kd which then undergoes a slower second cleavage to generate the 19kd active domain. Preceding this second cleavage site is a sequence of amino acids Thr- -Gly-Met-Thr- -Lys that also occurs at another site in the N-terminal half of the 39kd methyltransferase. It is proposed that this sequence is a recognition site for proteolytic activity. On the basis of cleavage of the Ada protein at either one or both of these sites, fragments may be generated of mol. wt. 24kd and 19kd containing the active site for O6-methylguanine and O4-methylthymine repair, and 15kd and 20kd, containing the active site for methylphosphotriester repair. These observations explain previous reports by others on the existence in cell extracts of multiple methyltransferase activities of different sizes recognizing O-methyl lesions in DNA. The cellular protease involved is resistant to a wide range of protease inhibitors.     

Genomic distribution of repeated and single copy DNA sequences in Penicillium funiculosum

Study of the reassociation kinetics of Penicillium funiculosum DNA (fragment size > 20 kb) revealed an interspersion of repeated and single-copy DNA sequences. The fragment length of interspersed single-copy DNA was estimated to be more than 9 kb from a curve relating the fraction of DNA binding to hydroxyapatite as a function of DNA fragment length. The length of interspersed short and long repeated nucleotide sequences was determined to be 0.55 kb and > 20 kb, respectively, by agarose gel electrophoresis. The genomic organization of P. funiculosum is thus significantly different from that in other fungi.     

圈卷产色链霉菌尼可霉素生物合成基因：—sanJ的克隆及功能研究

以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌cosmid基因文库中筛选到１个大约７．５kb的ＤＮＡ片段,交ＤＮＡ片段克隆到载体pBluescripM13-的KpnⅠ位点,得到了重组质粒pNL2200.对pNL2200中外源ＤＮＡ片段进行了一系列的亚克隆及部分核苷酸序列分析。结果表明,２．３kb的SalⅠ－ＢaｍＨⅠＤＮＡ片段中含有１个完整的开放阅读框,起始密码子为２７１位的ＧＴＧ,终止密码子为１９５４位的ＴＧＡ,该基因的大小为１６８６bp,编码１个大小为５６１个氨基酸的蛋白质产物。利用blastx程序的蛋白质数据库中进行同源比较,结果揭示此基因产物与腺苷酸形成酶超家族的连接酶有４４％的一致性,此外,该基因的破坏导致圈卷产色链霉菌尼可霉素生物合成能力的丧失,证明它是尼可霉素生物合成所必需的,命名为其为sanＪ。     

紫云英根瘤菌共同结瘤基因nodA和nodBC的核苷酸序列

以~(32)p标记的苜蓿根瘤菌(Rhizobium meliloti)2.3kb nod DNA作探针,从紫云英根瘤菌(Rhizobium huakuii即R. astragali)159基因文库中分离到一株能与探针DNA呈阳性反应的克隆pRaN109。同源DNA-DNA杂交及DNA序列分析表明:pRaN109DNA的9kb EcoRI片段上携带了nodD_1BC基因,pRaN109 NDA的18kb EcoRI片段上携带了nodD_2A基因。共同结瘤基因nodA与nodBC两者相距6.7kb。在nodA基因和nodBC基因的上游都存在有结瘤盒(nod box)。与来自不同种属的菌株所报告的结果相比较,紫云英根瘤菌159中的共同结瘤基因有着明显不同的组合。     

Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as l -histidine amino-transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligo-nucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb Pvu II fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in nikkomycin production.     

Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.     

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Cloning and characterization of the mvrC gene of Escherichia coli K-12 which confers resistance against methyl viologen toxicity.

A new gene mvrC conferring resistance to methyl viologen, a powerful superoxide radical propagator, was cloned on 13.5 kilo base (kb) EcoRI DNA fragment. It gave resistance against methyl viologen to even a wild-type strain with gene dosage dependence. From the physical maps obtained by restriction enzyme digestions, it was predicted to locate at 580 kbp (12.3 min) on the physical map of E.coli. This was confirmed by the Southern hybridization of lambda phages covering this region with mvrC probe. The DNA sequence of mvrC gene was determined and its deduced protein encoding a 12 kd hydrophobic protein was confirmed by maxicell labeling of MvrC protein.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning of the DNA BglII fragments of bacteriophage T4 in the vector plasmid pSCC31

An attempt has been made to clone six BglII fragments of T4 DNA in the range of 3.3-8.1 kb in the vector plasmid pSCC31 containing a single BglII site within the gene for endonuclease EcoRI and pL promoter of phage lambda. DNA fragments were extracted from the corresponding bands of agarose gel. The following BglII fragments were cloned: the 3.3 kb fragment No. 9 containing a portion of gene 20, the gene 21 and a portion of gene 22; the 4.2 kb fragment No. 8.1 with genes 17, 18, 19 and a portion of gene 20; the 5.2 kb fragment No. 7.1 with genes 25-29 and a portion of gene 48. In the case of the fragment No. 7.1, the recombinant plasmids pRL705 and pRL707 with different orientation of phage DNA fragment were obtained. An attempt to clone the fragments No. 8.2 (4.2 kb), No. 7.2 (5.45 kb) and No. 6 (8.1 kb) was unsuccessful and this probably indicates the presence of the genes, whose products are deleterious to the growth of bacterial cell.     

Structure of the Abelson murine leukemia virus genome and the homologous cellular gene: studies with cloned viral DNA

Circular double-stranded DNA produced after infection of mouse cells with Abelson murine leukemia virus (A-MuLV) was isolated and cloned in the phage vector Charon 21A. The resulting clones of the A-MuLV genome show homology to the ends of Moloney MuLV and to a 3.5 kb central region containing sequences unique to Abelson virus. A 2.3 kb restriction fragment containing only A-MuLV-specific sequences was subcloned in the plasmid vector pBR322 and used as a probe for the cellular gene that had been acquired by the virus. DNA from all inbred mouse lines examined contains an identical region of homology spread out over 11 to 20 kb. The cellular gene contains intervening sequences which are lacking in the viral genome. Rat, Chinese hamster, rabbit, chicken and human DNA also show homology to the viral probe.     

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

The xcp genes are required for protein secretion by Pseudomonas aeruginosa. They are involved in the second step of the process, i.e. the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion. The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd. Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo. Subcloning and complementation experiments showed that both proteins are required for secretion. The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae. These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae.     

Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5)

Previous work from our laboratory has shown that cosmid clone pFVl00, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFVl00 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb Xhol-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAOl (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.