耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌（Deinococcus radiodurans）的recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS(DE3)中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E. coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D. radiodurans的recA基因在E. coli细胞中的表达能够完全补偿recA缺损E. coli辐射抗性能力。     

recA基因的克隆及其在λ溶原菌中的生物学功能

通过一对自行设计的引物,以E. coli染色体DNA为模板进行PCR扩增,获得完整的recA基因.将PCR产物和pUC18在体外进行连接后,并分别转入E. coli DH5α和E.coli k12(λ+),筛选出含重组质粒(pUR4)的转化子.在诱导条件和非诱导条件下,分别测定recA基因在不同转化子中的生物学功能.结果表明recA基因在溶原菌中表现出明显的生理功能,能使λ原噬菌体从溶原状态进入裂解循环.这种重组质粒将在进一步研究λ原噬菌体的诱导机理以及受紫外辐射损伤细胞的修复作用等方面成为有效的工具.     

recA基因的克隆及其在λ溶原菌中的生物学功能

通过一对自行设计的引物,以E．coli染色体DNA为模板进行PCR扩增,获得完整的recA基因。将PCR产物和pUCl8在体外进行连接后,并分别转入E.coli DH5α和E.coli k12(λ^ ),筛选出含重组质粒(pUR4)的转化子。在诱导条件和非诱导条件下,分别测定recA基因在不同转化子中的生物学功能。结果表明recA基因在溶原菌中表现出明显的生理功能,能使九原噬菌体从溶原状态进入裂解循环。这种重组质粒将在进一步研究九原噬菌体的诱导机理以及受紫外辐射损伤细胞的修复作用等方面成为有效的工具。     

RP4质粒及其带动的硫杆菌重组质粒psDt125在氧化硫硫杆菌中的接合转移

本实验选择了一种大肠杆菌(E.coli)和氧化硫硫杆菌(Thiobacillusthiooxidans)均能生长的接合培养基。以大肠杆菌E.coli C600(RP4)为供体,以氧化硫硫杆菌T.t-3为受体,通过接合,直接将RP4质粒转移到氧化硫硫杆菌中,其Km~r,Tc~r基因得到表达,但Ap~r基因不表达。再将RP4质粒反向接合转移到E.coliHB101上,其三个抗性基因均表达。并且利用RP4质粒的带动将硫杆菌重组质粒psDt125直接转入氧化硫硫杆菌,其Cm~r基因表达。从而为氧化硫硫杆菌的基因工程改造提供了一条简便可行的遗传信息转移途径。     

铁蛋白DrfE对耐辐射异常球菌抗氧化酶活性的影响

为研究耐辐射异常球菌(Deinococcus radiodurans)中drf E编码的铁蛋白Drf E的功能,通过基因回补试验构建drf E基因的回补菌株(pdrf E∷Δ),对野生型WT、突变株Δdrf E及回补菌株pdrf E∷Δ进行不同浓度H_2O_2和Na Cl胁迫,分别测定野生型WT、突变株Δdrf E和回补菌株pdrf E∷Δ体内Fe2+含量及抗氧化酶类活性。结果显示,drf E基因缺失导致菌株对氧化和盐胁迫敏感,该基因的回补能够恢复菌株对氧化和盐胁迫的抗性。drf E基因缺失导致耐辐射异常球菌体内Fe2+浓度由232μmol/L增加至293μmol/L;80 mmol/L H_2O_2处理30 min后,突变株Δdrf E的CAT活性下降32.74%,SOD下降41.3%。以上结果表明Drf E蛋白可能参与耐辐射异常球菌铁代谢途径,并且在细胞抗氧化体系中发挥重要作用。     

表达载体pHsh对大肠杆菌热休克系统中负调控机制的影响

pHsh是一种由σ32识别和启动外源基因表达的新型高效的大肠杆菌表达载体。正常E.coli细胞在热激诱导条件下,σ32的浓度在5 min内到达高峰,随后被3个负调控蛋白Dnak、DnaJ、GrpE结合导致失活或降解,整个热休克反应持续约12min。在携带有外源基因的高拷贝pHsh 的E.coli细胞中,外源基因却能持续高效表达4～10 h,这一现象表明了此时细胞中的σ32比没有携带质粒的细胞内σ32的浓度要高。σ32浓度的增高有可能是由于3个负调控蛋白Dnak、DnaJ、GrpE在细胞内的含量比正常情况下降低的结果。为了验证这一推测,从E.coli中克隆了Dnak、DnaJ、GrpE的编码基因,表达并初步纯化了其重组蛋白以作分子标记,采用双向电泳技术,分析携带质粒（pHsh+）和不携带质粒的E.coli(pHsh-)细胞在热休克后胞内蛋白质组的差异。该项实验通过与检索到的标准的E.coli蛋白质组图谱进行比较鉴别出的两个蛋白Dnak、GrpE,并通过对比目标点的大小和深浅发现pHsh+中的Dnak均少于pHsh─中的目标蛋白,所得结果与上述假设一致。     

recA基因通过同源重组途径参与由整合质粒发动的大肠杆菌染色体复制

我们在前文中报道由整合的F'质粒所发动的大肠杆菌染色体的复制依赖于recA基因。本文报道有关recA、recB、recC以及lexA等在染色体复制中的作用,实验结果说明,recA基因通过同源重组途径而不是通过SOS途径参与复制,而且recA基因和Chi热点无关。实验结果还说明,RecBC酶的依赖于ATP的双链DNA外切核酸酶活性和recA基因的作用无关。     

低温菌启动子探针质粒的构建

为了在宿主菌Acinetobacter sp.DWC6中构建低温菌蛋白表达载体,以pBR322质粒为基础,去除质粒上β-内酰胺酶基因的启动子片段,取而代之为来源于质粒pJRD215的卡那霉素抗性基因片段,并在pBR322中插入Acinetobacter菌属特异性ori的DNA片段,构建了能在Acinetobacter sp.DWC6和E.coli中正常复制的启动子探针质粒pBAP1。通过在质粒pBAP1中的β-内酰胺酶基因上游随机导入Acinetobacter sp.DWC6基因组片段,通过检测宿主细胞的氨苄青霉素抗性和β-内酰胺酶活性,来筛选强启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。     

大肠杆菌中整合的F′质粒带动细菌染色体复制需要recA基因

大肠杆菌的dnaA46突变能被F′质粒整合抑制。整合抑制的菌林(Sin菌株)在通过转导引入了recA56突变后又变得不能在40℃中生长。标记转移、吖啶橙敏感性,F′质粒消除和mini-染色体质粒转化等实验说明,Sin菌株中F′质粒始终处于整合状态,并且在40℃中细菌染色体的复制由整合状态的F′质粒所带动。比较了Sin recA~ 和Sin recA~-菌株在不同温度中的DNA、蛋白质的生物合成情况。实验结果说明recA基因在DNA复制过程中起作用。前人的工作证明了recA基因在DNA重组和DHA损伤应急修复(SOS)过程中是一个关键的基因。本文的工作为recA基因的功能提供了新的认识。     

带有特定染色体片段的mini-F质粒所发动的染色体复制对于recA基因的依赖性

构建了一些带有大肠杆菌的特定染色体片段的mini-F质粒,使它们通过同源重组整合在dnaA46细菌的染色体的预定位置上,然后测定整合抑制菌株(sin)在40℃中染色体复制对recA基因的依赖性。实验结果说明,Sin菌株对recA基因的依赖性决定在质粒的整合位置。整合在oriC近旁的Sin菌株不依赖于recA基因;整合在oriC和terC中间的只在丰富培养基上是依赖的;整合在rerC附近的在不丰富的培养基上也依赖于recA基因。     

Expression of recA in Deinococcus radiodurans.

Deinococcus (formerly Micrococcus) radiodurans is remarkable for its extraordinary resistance to ionizing and UV irradiation and many other agents that damage DNA. This organism can repair > 100 double-strand breaks per chromosome induced by ionizing radiation without lethality or mutagenesis. We have previously observed that expression of D. radiodurans recA in Escherichia coli appears lethal. We now find that the RecA protein of D. radiodurans is ot detectable in D. radiodurans except in the setting of DNA damage and that termination of its synthesis is associated with the onset of deinococcal growth. The synthesis of Shigella flexneri RecA (protein sequence identical to that of E. coli RecA) in recA-defective D. radiodurans is described. Despite a large accumulation of the S. flexneri RecA in D. radiodurans, there is no complementation of any D. radiodurans recA phenotype, including DNA damage sensitivity, inhibition of natural transformation, or inability to support a plasmid that requires RecA for replication. To ensure that the cloned S. flexneri recA gene was not inactivated, it was rescued from D. radiodurans and was shown to function normally in E. coli. We conclude that neither D. radiodurans nor S. flexneri RecA is functional in the other species, nor are the kinetics of induction and suppression similar to each other, indicating a difference between these two proteins in their modes of action.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Functional properties of Borrelia burgdorferi recA

Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 microJ/cm(2). The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli. Furthermore, E. coli lambda phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi. The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.     

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.     

Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO.

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.     

Molecular cloning and expression in Escherichia coli of the recA gene of Legionella pneumophila

Interspecific complementation of an Escherichia coli recA mutant with a Legionella pneumophila genomic library was used to identify a recombinant plasmid encoding the L. pneumophila recA gene. Recombinant E. coli strains harbouring the L. pneumophila recA gene were isolated by replica-plating bacterial colonies on medium containing methyl methanesulphonate (MMS). MMS-resistant clones were identified as encoding the L. pneumophila recA analogue by their ability to protect E. coli HB101 from UV exposure and promote homologous recombination. Subcloning of selected restriction fragments and Tn5 mutagenesis localized the recA gene to a 1.7 kb Bg/II-EcoRI fragment. Analysis of minicell preparations harbouring a 1.9 kb EcoRI fragment containing the recA coding segment revealed a single 37.5 kDa protein. Insertional inactivation of the cloned recA gene by Tn5 resulted in the disappearance of the 37.5 kDa protein, concomitant with the loss of RecA function. The L. pneumophila recA gene product did not promote induction of a lambda lysogen; instead, the presence of the heterologous recA gene caused a significant reduction in spontaneous and mitomycin-C-induced prophage induction in recA+ and recA E. coli backgrounds. Despite the lack of significant genetic homology between the L. pneumophila recA gene and the E. coli counterpart, the L. pneumophila RecA protein was nearly identical to that of E. coli in molecular mass, and the two proteins showed antigenic cross-reactivity. Western blot analysis of UV-treated L. pneumophila revealed a significant increase in RecA antigen in irradiated versus control cells, suggesting that the L. pneumophila recA gene is regulated in a manner similar to that of E. coli recA.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

The recA gene of Chlamydia trachomatis: Cloning, sequence, and characterization in Escherichia coli

Abstract The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.     

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.