Physical and functional analyses of the syrA and syrB genes involved in syringomycin production by Pseudomonas syringae pv. syringae.

The syrA and syrB genes involved in syringomycin production in Pseudomonas syringae pv. syringae B301D were identified from an EcoRI-pLAFR3 cosmid library and then physically and functionally analyzed in relation to plant pathogenicity. Homologous recombination of the genes required for syringomycin production from cosmids pGX183 (syrA) and pGX56 (syrB), respectively, introduced into nontoxigenic (Tox-) Tn5 mutants W4S2545 and W4S770 resulted in the concomitant restoration of toxin production and full virulence. The disease indices of the Tox+ strains obtained by recombination of the cloned, homologous DNA into the corresponding Tn5 mutant were essentially equivalent to that of strain B301D-R and significantly higher than those of W4S2545 and W4S770. A 12-kilobase (kb) EcoRI fragment from pGX183 was subcloned (i.e., pGX15) and found to contain the sequences necessary for syringomycin production. A map of pGX15 prepared by a combination of restriction endonuclease digestions and Tn5 mutagenesis showed that the syrA sequence was 2.3 to 2.8 kb. Marker exchange of syrA::Tn5 from pGX15 into B301D-R yielded nonpathogenic phenotypes, indicating that syrA is a regulatory gene since it is necessary for both syringomycin production and pathogenicity. The 4.9-kb EcoRI fragment from pGX56 was subcloned (i.e., pGX4) and shown to carry the syrB sequence which was 2.4 to 3.3 kb. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of protein extracts from B301D-R associated five proteins, ranging from approximately 130,000 to approximately 470,000 in molecular weight, with syringomycin production. The syrA and syrB genes were required for the formation of proteins SR4 (approximately 350,000) and SR5 (approximately 130,000), which are believed to be components of the syringomycin synthetase complex.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Nucleotide sequence of the ipaBCD structural genes of Shigella dysenteriae

A 9 kb EcoRI and two PstI fragments from the virulence plasmid of Shigella dysenteriae CG097 were shown to contain all ipa genes by probing with Shigella flexneri ipaB, -C, -D and -A gene probes. The DNA sequences of S. dysenteriae ipaBC genes were very similar to those of S. flexneri M90T and S. flexneri YSH6000, but ipaD differed by 22 codons from that of S. flexneri. The differences in ipaD may account for the different in vitro host specificities shown by S. dysenteriae and S. flexneri. The nucleotide composition of ipa genes revealed an unusually large number of codons that are rarely used in Escherichia coli chromosomal genes, indicating a different origin.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

麦迪霉素产生菌酮基还原酶基因的研究

将麦迪霉素产生菌基因文库中与放线紫红索酮基还原酶基因actⅢ有同源性的4·0kb DNA片段克隆到质粒载体pWHM3中,构成重组质粒pCB4。将质粒pCB4转入酮基还原酶基因缺陷菌株——加利利链霉菌ATCC3167l中,得到转化子。转化子发酵产物经TLC和HPLC分析证明是阿克拉菌酮,与加利利链霉菌原株ATCC31133的产物相同,说明麦迪霉素产生菌酮基还原酶基因互补了加利利链霉菌ATCC31671中缺陷的酮基还原酶基因,使其恢复了产生阿克拉菌酮的能力。4．Okb DNA片段插入方向相反的重组质粒pCBR4在加利利链霉菌ATCC31671中发酵产物经TLC分析证明也是阿克拉菌酮,这说明4．0kbDNA片段中麦迪霉素产生菌酮基还原酶基因具有自身的启动子。对4．0kb DNA片段进行了限制酶酶切分析,建立了其酶切图谱。以actⅢ基因为探针,经分子杂交以及亚克隆和DNA转化实验,将麦迪霉索产生菌酮基还原酶基因定位于BssH Ⅱ—BamH Ⅰ 1．3kb DNA片段上。对1．3kb DNA片段核苷酸序列分析结果表明：此1．3kbDNA片段中含有一个独立的ORF,起始密码ATG,终止密码TAG,含783bp;在起始密码上游有GGAGG5个核苷酸SD序列;此ORF编码260个氨基酸,与actⅢ基因编码的261个氨基酸相似性为77．4%,相同性为66．7％,对麦迪霉素产生苗酮基还原酶基因的可能作用进行了讨论。     

Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene

Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.     

Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli

Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184. Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively. These two genes showed no homology. The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain. Expression of these genes in E. coli was found not to depend on their orientation in the cloning vector. Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected.     

C型产气荚膜梭菌β1毒素基因表达

利用PCR技术,从C型产气荚膜梭菌染色体DNA中扩增出β1毒素基因,然后用限制性核酸内切酶BamHI和EcoRI对其进行双酶切处理,回收0．95kb的β1毒素基因片段,最后将其定向克隆在事先经同样内切酶处理的载体pET-28c中相应位点上,转化至受体菌B121(DE3)qh。经BamHI和Eco RI双酶切鉴定和核苷酸序列测定证实,构建的重组质粒pETXBl含有B毒素基因,并且具有正确的基因序列和阅读框架。重组菌株BI21(DE3)(pETXB1)经IPIG诱导后,其表达产物经ELISA检测和SDS-PAGE分析,结果表明重组菌株可以高效表达β1毒素蛋白,该蛋白占菌体总蛋白相对含量的12．24％。     

Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.