解淀粉芽胞杆菌启动基因的克隆及其对地衣杆菌α-淀粉酶基因的调控

用大肠杆菌启动基因探针质粒pHE5克隆了解淀粉芽胞杆菌的启动基因。供体菌DNA的HindⅢ片段与pHE5重组后转化大肠杆菌,获得一批带启动基因的抗四环素转化子,其中四株的抗性达到200μg/mL,从这四株高抗性转化子中提取质粒,并对其中的一个重组质粒pAE23的插入片段进行限制性图谱分析和缺失研究,获得了一个缺失了部份片段,四环素抗性仍达到200μg/mL的衍生质粒pAED23,经酶切分析证明其上的启动基因位于0.8kb的EcoR Ⅰ—HindⅢ片段上。点杂交分析证实该片段来自解淀粉芽胞杆菌的DNA。将地衣杆菌的α-淀粉酶基因亚克隆至pAED23启动基因下游的HindⅢ位点上能增强该基因在大肠杆菌中的表达。     

大肠杆菌启动基因的重组和表达——Ⅱ.不同启动基因对抗四环素基因表达的影响

质粒pHE5为pBR322的EcoRI—HindⅢ片段由噬菌体T4基因37的EcoRI-HindⅢ片段取代、构建而成,抗Tc基因的启动基因被破坏。在抗Tc结构基因前插入新的启动基因,可以使抗Tc功能恢复,以此可以筛选各种启动基因。我们用pHE5构建了含大肠杆菌启动基因的重组质粒800株,对它们抗四环素程度的分布作了测定,制作了重组质粒pTP210、pTP213的限制性酶切图谱,并且就应用这二个质粒作为真核基因表达运载体的可能性进行了初步讨论。     

分子无性繁殖载体的组建——Ⅰ.乳糖操纵子的重组和表达

用包含乳糖操纵子的λplac 5 EcoR1 DNA 片段与质粒pBR 322重组,获得一个重组质粒pMG4。几种限制性内切酶的分析结果表明,pMG4上乳糖操纵子(lac)和抗氨基苄青霉素基因的转录方向一致;Iac 插入片段λ区的Hind Ⅲ切点和pBR 322抗四环素基因上的Hind Ⅲ切点相邻近。用pMG4 Hind Ⅲ片段转化大肠杆菌C_(600),筛选Ap~r Tc~s lac~ 转化体,得到另一个lac 重组质粒pMG401,在β-半乳糖苷酶结构基因近羧基端有一个EcoR1切点,插入外源基因可以置于lac 控制下而实现表达。同时,我们还测定了包含pMG4、pMG401或λplac 5不同细胞的β-半乳糖苷酶活力,对lac 的表达作了分析。     

分子无性繁殖载体的组建——Ⅰ.乳糖操纵子的重组和表达

用包含乳糖操纵子的λplac 5 EcoR1 DNA片段与质粒pBR 322重组,获得一个重组质粒pMG4。几种限制性内切酶的分析结果表明,pMG4上乳糖操纵子(lac)和抗氨基苄青霉素基因的转录方向一致;Iac插入片段λ区的Hind Ⅲ切点和pBR 322抗四环素基因上的Hind Ⅲ切点相邻近。用pMG4 Hind Ⅲ片段转化大肠杆菌C_(600),筛选Ap~rTc~s lac~ 转化体,得到另一个lac重组质粒pMG401,在β-半乳糖苷酶结构基因近羧基端有一个EcoR1切点,插入外源基因可以置于lac控制下而实现表达。同时,我们还测定了包含pMG4、pMG401或λplac 5不同细胞的β-半乳糖苷酶活力,对lac的表达作了分析。     

棒杆菌启动子片段的酶谱分析及定位

用大肠杆菌启动子探测质粒pSDS I (Ap^r,Tc^s)从钝齿棒杆菌(Corynebacterium crenatum)6282染色体的HindⅢ酶切片段中,克隆到两个具有启动功能的DNA片段,分别将两个重组质粒命名为pSDB5和pSDB21。含有这两个质粒的菌株均可以在含300μg/ml Tc的平板上生长。通过酶切分析,pSDB5的插入片段为1.6kb,pSDB21的插入片段为3.4kb,并分别作出了它们的限制性酶切图谱。对pSDB21利用其EcoR Ⅰ和BglⅡ位点,通过亚克隆删除了与启动功能无关片段,构建成pSDB210和pSDB211,从而使启动子定位于约0.1kb的BglⅡ/HindⅢ外源片段上。分子杂交实验证明所得到的这两个具有启动功能的DNA片段确实来源于钝齿棒杆菌6282的染色体DNA。     

氧化硫硫杆菌启动子功能片段在大肠杆菌中的克隆和表达

用DNA体外重组技术,以氧化硫硫杆菌(T.thiooxidans)染色体DNA的EcoPIHindⅢ片段取代pBR322的相应片段,构建成一个重组质粒pSDR12。转化大肠杆菌C600受体菌株后,表现了较强的抗四环素能力。表明了一个专性自养细菌的启动子功能片段在异养细菌中表达。     

酵母启动基因在大肠杆菌中的克隆和表达*

利用启动基因克隆载体pHE5,研究了不同酵母的启动基因在大肠杆菌巾的功能作用。酵母染色体DNA的HindI和Ec0R I片段与pHE5重组,获得一批启动基因重组体,并作了不同启动基因重组体的限制性图谱分折和抗四环索水平的测定。对这启动基因用于组建表达质粒的意义进行了讨论。     

大肠杆菌启动基因的重组和表达 Ⅰ.不同启动基因对β-半乳糖苷酶结构基因表达的影响

质粒pKL6带有β-半乳糖苷酶结构基因(lacZ),启动基因突变失活,lacZ不能表达。在lacZ前插入外源DNA片段,观察lacZ的表达,可以研究启动基因的功能作用。我们用大肠杆菌染色体DNA的HindⅢ片段与pKL6重组,获得一批重组体,对这些重组体的β-半乳糖苷酶的活力水平和限制性图谱作了分析,对它们用作表达载体的可能性进行了讨论。     

体外建成带有λ噬菌体DNA片段的重组质粒

将大肠杆菌抗四环素及抗氨苄青霉素的质粒pBR322的DNA、大肠杆菌λ噬菌体DNA在体外经限制性核酸内切酶Hind Ⅲ切割,并用T4DNA连接酶连接后,以大肠杆菌K12HB101(recA~-r(?)m(?))为受体进行转化,采用插入钝化(insertional inactivation)方法选择重组质粒。利用环丝氨酸浓缩Ap~rTc~3转化体。在648株Ap~r转化体中选出120株无性繁殖系为Ap~rTc~s。随机挑出3株含有重组质粒的无性繁殖系进行大肠杆菌素El(colicin El)免疫特性测定及显微镜观察,证明为大肠杆菌素El敏感,并为典型的大肠杆菌形态。随机挑出一株含有重组质粒pAG-5的无性繁殖系,采用Zasloff等酸酚法进行重组质粒DNA提取,用Hind Ⅲ消化重组质粒DNA后进行琼脂糖凝胶电泳分析,观察到重组质粒pAG-5除pBR322质粒DNA带外含有λ-Hind Ⅲ的第5 DNA带。用重组质粒DNA再次对HB101(recA~-r(?)m(?))及K12 802(Su_(11)~+m_K~+)受体进行转化,转化体在含氨苄青霉素培养基平皿上生长,但在含四环素培养基平皿上不生长,再次证明为Ap~rTc~5.每微克重组质粒DNA Ap~r转化体为4.0—4.6×10~3。     

在枯草杆菌中有启动子功能的噬菌体T5 DNA片段的克隆

用启动子探针质粒pTG 402为载体,用鸟枪射击法克隆了噬菌体T5 DNA的片段。克隆中的2％含有具启动子功能的插入片段,其中pTG 402-20含有启动功能最强的插入片段。DNA-DNA分子杂交实验结果表明,pTG 402-20的插入片段能与T5 DNA Hind Ⅲ酶切的G和B片段杂交。这个插入片段的大小约为0.84kb。     

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.     

解淀粉芽孢杆菌中温α-淀粉酶基因的克隆、表达与酶学性质分析

以中温α-淀粉酶生产菌株Bacillus amyloliquefaciens M23基因组DNA为模板。PCR扩增得到了2．0kb α-淀粉酶基因全长序列。该基因由上游启动子220bp,结构基因1544bp和终止序列320bp构成。将无信号肽的α-淀粉酶结构基因amyQ,克隆入表达载体pET28a,转化E．coli BL21（DE3）,经诱导,测定α-淀粉酶活性。结果表明：α-淀粉酶基因amyQ获得了活性表达,酶活力为2．297U／mL,SDS-PAGE电泳结果显示出分子量约为58kDa特异性蛋白质条带。酶学性质分析表明,重组α-淀粉酶的最适反应温度为60℃,最适反应pH为6．5,在60℃保温15min保持85％以上活性,超过15min,酶迅速失活,在pH5．5～10．0环境下稳定。水解产物分析表明：淀粉水解终产物主要为麦芽寡糖和糊精和少量葡萄糖。     

嗜热硫杆菌启动子的克隆及定位

用DNA体外重组技术,将嗜热硫扦菌(Thiobacillus sp．)染色体DNA的Hind III片段插人启动子探测质粒pSDSI(Apr、Tc2)的Hind III位点,在四环素平板上获得一批转化子。四环素抗性能力测定表明,有12个转化子可以在含240／μg／ml四环素的平板上生长,有2个可以在360μg／ml的四环素平板上生长,对这二个抗性表达能力较高、分子量较小的质粒pSDH7和pSDH11作了限制性酶切图谱分析,并利用PstI位点,通过亚克隆将pSDH11插人片段上的启动子定位在0．25kb片段内。分子杂交实验证明克隆的启动子活性片段确实来源于嗜热硫杆菌染色体DNA。     

Cloning of a probe for a previously undescribed enterobacterial tetracycline resistance gene

An 8.35 kb BamHI fragment was cloned from the plasmid R714a. It encoded resistances to chloramphenicol, streptomycin, spectinomycin and tetracycline. Tetracycline resistance was determined by a locus without homology to the known enterobacterial gene classes, TetA-TetE. Further subcloning of the fragment located an unusual tetracycline resistance gene on a 4.7 kb BamHI-BglII fragment. A physical-genetic map of this fragment indicated that the gene was bisected by a PstI site. Deletion analysis and insertion mutagenesis were used to define a suitable probe. An intragenic PstI-AvaI fragment of 1.2 kb was identified, and used as a non-radioactive probe, being specific for this previously undescribed enterobacterial Tet gene.     

Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. II. Increased level of gene expression resulting from IS1 element integration

The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.     

Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon

We have sub-cloned from the Eco RI-H fragments of the IncFII plasmid R100 a 260-bp EcoRI fragment, using the promoter-cloning vehicle, pBRH4, (The Inc FII plasmid codes for the mer operon, and pBRH4 expresses tetracycline resistance only when the deleted tet promoter has been replaced by another sequence that can serve as a promotor). With the 260-bp fragment inserted, the derivative plasmid, pFB4, directs the expression of tetracycline resistance only if there is a second plasmid in the strain that carries the merR-positive regulatory element. Under these conditions, the level of tetracycline resistance is directly proportional to the concentration of Hg2+ present in the medium. The 260-bp fragment also allows low-level constitutive expression of tet resistance when transactivated with merR mutants that have a "micro-constitutive" phenotype. The 260-bp mer promoter fragment contains a single HincII site; there is also but one HincII site in the EcoRI-H fragment of R100 from which the promoter fragment was derived. Restriction analysis of purified Eco RI-H DNA shows that the single HincII site is at 550 bp from the "right"terminus of the IS1b element, which is also present in the EcoRI-H fragment. Because of its biological activity and its location within the "H" fragment, this promoter is very likely a promoter for the structural genes of the operon.     

Analysis of a Bacteroides conjugative transposon using a novel "targeted capture" model system

Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such as CTn341, are able to mobilize unlinked genetic elements such as plasmids and mobilizable transposons in a tetracycline-inducible manner. To study the molecular properties of these unique elements, a vector was designed to capture CTn's for analysis in heterologous hosts. This plasmid, pFD670, consisted of the low-copy vector pWSK29, the RK2 oriT, an ermF gene, and a tetQ gene fragment containing the N-terminus and promoter. The vector was transferred into Bacteroides recipients containing CTn341 where it integrated into the tetQ gene by homologous recombination. This integrated construct then was transferred back into an Escherichia coli host where it replicated as a plasmid, pFD699, about 56 kb in size. Further analysis showed that pFD699 could be transferred into Bacteroides hosts where it displayed the same tetracycline-inducible properties as the native CTn341. The captured element appeared to utilize a circular intermediate in both transfer and transposition, and integration into the chromosome seemed to be random. Hybridization studies with a range of Bacteroides CTn's encoding tetracycline resistance revealed a great deal of homology between most of the CTn's but there was much variation seen in the restriction patterns of these elements, suggesting great diversity among this group.     

Identification of the tetracycline resistance promoter and repressor in transposon Tn10.

The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region. Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance. These various sequences conferred altered levels of tetracycline resistance. Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene.