Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product

Summary Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Summary The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr^- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.     

Cloning and expression of Bacillus subtilis spore genes

Summary Two spore genes, spoOB and spoIIG have been cloned from the B. subtilis genome library, constructed by ligating Sau3A partially digested DNA to the dephosphorylated pHV33 plasmid vector at its BamH1 site.An hybrid plasmid pGsOB2, carrying a 1.7 Kb insert of B. subtilis DNA amplifiable in E. coli was cloned. This recombinant plasmid was capable of transforming the appropriate B. subtilis Rec⁺ and Rec^- recipients to Spo⁺ at very high efficiency. The pGsOB2 was further subcloned and four hybrid plasmids, pGsOB8, pGsOB9, pGsOB10 and pGsOB11 were selected and their restriction enzyme maps established. The four subcloned hybrid plasmids retained their entire transforming activity in both Rec⁺ and Rec^- recipients although two of them carry the insert in an inverse orientation, indicating thus, that the spoOB gene in these plasmids is being transcribed by the B. subtilis RNA polymerase using an internal promotor of the cloned DNA fragment. The adjacent genes spoIVF and pheA, mapped respectively to the right and left of the spoOB locus, that normally show 90% cotransformation, are absent on the cloned DNA fragments. The cloned hybrid plasmids have been expressed in E. coli minicells and it was shown that the spoOB locus encoded a polypeptide of 24 K.We have also cloned the spoIIG gene in two hybrid plasmids, pGsIIG24 and pGsIIG26, carrying respectively inserts of 2 and 3 Kb. From the transforming activity and the endonuclease cleavage maps it was shown that these two hybrid plasmids do not carry the entire spoIIG locus. The use of these plasmids for further cloning of this gene is discussed.     

Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli

Summary The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoIHpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 by downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.     

脂肪储存小滴蛋白5重组腺病毒的制备

目的：利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5（LSDP5）基因的重组腺病毒。方法：从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果：LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论：成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。     

Cloning of rpsO, the gene for ribosomal protein S15 of Escherichia coli

Summary The gene for Escherichia coli ribosomal protein S15 (rpsO) was cloned on the vector pBR322 from F-prime JCH55 DNA. The recombinant plasmid was transformed to Serratia marcescens cells and it was proved that E. coli S15 was synthesized and incorporated into ribosome particles in S. marcescens cells. A DNA fragment containing rpsO was also inserted into the vector pRF3, which changes its copy number depending on the growth temperature in a temperature-sensitive polA host. By use of this recombinant plasmid it was shown that the relative synthesis rate of S15 increased about twice even when the copy number of the plasmid increased more than twenty-fold.     

Insertion of a heterologous gene construct into a non-functional ORF of the Streptococcus thermophilus chromosome

An integrative vector was constructed for inserting heterologous genes within a non-functional open reading frame (ORF) on the chromosome of Streptococcus thermophilus. The vector, pINTRS, contained a temperature sensitive origin of replication and an erythromycin resistance gene for initial selection in S. thermophilus. The region of the vector containing unique cloning sites, for insertion of recombinant genes, was flanked by homologous DNA sequences corresponding to a pseudogene in S. thermophilus to facilitate chromosomal integration. The gene encoding green fluorescent protein, regulated by a plasmid borne hsp promoter of S. thermophilus, was cloned into pINTRS to demonstrate proper functioning of the vector. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Cloning and analysis of the inulinase gene from Kluyveromyces cicerisporus CBS4857

A highly expressed inulinase gene, KcINU1 was cloned and sequenced from Kluyveromyces cicerisporus CBS4857 a strain which secrets high levels of inulinase into the growth medium. The result of DNA sequencing showed that KcINU1 contained a 1665 bp ORF, coding for a 555 amino acid protein, in which a 23 amino acid signal peptide was included. The sequence has the GenBank Accession no. AF 178979. The analysis of conserved domain in the ORF indicated there was a consensus sequence about 470 amino acids long. The 0.7 Kb promoter and 0.9 Kb terminator were also cloned and sequenced.     

Characterization of fall armyworm mitochondrial DNA (Lepidoptera: Noctuidae)

Mitochondrial DNA from the fall armyworm, Spodoptera frugiperda (J.E. Smith), was cloned and characterized using restriction enzyme mapping. Genome size is approximately 16.3 kilobase (Kb), consistent with that of most animals. Three fragments, 8.1 Kb, 6.2 Kb, and 2.0 Kb, were produced by digestion with restriction enzyme Xbal and cloned into a pUC19 vector. Twenty-nine restriction enzymes were used to generate a detailed physical restriction enzyme map of the three cloned fragments. These data represent the first detailed characterization of a lepidopteran mitochondrial genome. © 1992 Wiley-Liss, Inc.     

Detection and expression of a gene encoding a new bacteriorhodopsin from an extreme halophile strain HT (JCM 9743) which does not possess bacteriorhodopsin activity

Membrane vesicles prepared from an extreme halophile strain, HT (JCM 9743), showed no bacteriorhodopsin activity. However, a DNA fragment, amplified by polymerase chain reaction (PCR), appeared to encode the C to G helices of a bacteriorhodopsin(bR)-like protein. With the PCR product as a probe, the gene coding for a novel bacteriorhodopsin was cloned from the genomic DNA of the strain HT. The open reading frame of the gene was ligated with the promoter region of the bop gene of Halobacterium salinarum bR, and expressed in a bR-deficient host strain, L33, using the plasmid vector pXLNov-R. The purplish membrane fraction purified from cells of a transformant exhibited a cyclic photoreaction characteristic of bacteriorhodopsin. Received: August 12, 1997 / Accepted: October 20, 1997     

Expression of cDNA fragment encoding sperm membrane peptide inE. coli

A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the P _R -P _L promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. TheE. coli cells transformed with pRSD-220 were propagated at 30 °C, then incubated at 42 °C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.     

The cya gene region of Erwinia chrysanthemi B374: organisation and gene products

Summary A DNA fragment containing the cya gene region of Erwinia chrysanthemi, B374 was cloned in vivo and transferred into cells of E. coli using a plasmid pULB113 derived from RP4 followed by subcloning in vitro into the vector pBR322. The cya gene encodes a 95 kDal protein that complemented E. coli cya mutants. Apparently, cya genes truncated at the 3 end could still produce proteins complementing cya-defective strains, thus showing that adenylate cyclase truncated at its carboxy-terminal end could synthesise cAMP. A protein of unknown function (40 kDal) is encoded by a gene that is transcribed divergently from the control region of the adenylate cyclase gene.     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

Isolation,cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis

Vegetative insecticidal protein (Vip) is a newly discovered family of toxin protein isolated from Bacillus thuringiensis (Bt). An 88.5-kDa Vip3Aa protein was secreted by a local strain of the bacterium during the vegetative growth phase. The full length of the coding region ‘2.3 kbp’ of the vip3Aa gene was isolated from plasmid DNA, cloned in pGEM-T vector and finally cloned in pQE-30 expression vector. Nucleotide sequence revealed 98% homology with that of the previously isolated genes. Expression of the vip3Aa in Escherichia coli was carried out and the expressed protein was detected in the concentrated supernatant, not in the pellet. This indicated that vip3Aa is secreted into the culture medium. Expressed protein was purified, blotted, and assayed against the cotton leaf worm Spodoptera littoralis. The LC₅₀ was found to be 142.4 µ/mL while the LC₅₀ was 90 ppm for the wild strain. These results suggest the use of either the isolated Bt strains or the expressed vip3Aa in an integrated pest management program against lepidopteran insect pests.     

Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

Efficient transformation of Bacillus thuringiensis and B. cereus via electroporation: Transformation of acrystalliferous strains with a cloned delta-endotoxin gene

Summary Electroporation was used as a method to transform intact cells of Bacillus thuringiensis and B. cereus. With our optimized method a range of plasmid vectors could be transformed into strains of B. thuringiensis at frequencies of up to 10⁷ transformants/g DNA. This high frequency allows cloning experiments to be bone directly in B. thuringiensis. A bifunctional vector capable of replicating in Escherichia coli and in Bacillus spp. was constructed. The kurhd1 protoxin gene was cloned into this shuttle vector to produce plasmid pXI93, then transformed into B. thuringiensis HDl cryB and B. cereus 569K. The cloned protoxin gene was expressed in sporulating cultures of both strain HD1 cryB (pXI93) and 569K (pXI93), producing crystal protein active in biotests against larvae of Heliothis virescens. This demonstrates the usefulness of the electroporation method for the introduction of cloned toxin genes, in either their native or modified form, into a variety of host strains.     

食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达

目的:构建能够稳定表达萤火虫荧光素酶报告基因(luc)的乳酸乳球菌(Lactococcus lactis, L.lactis)食品级表达系统,以便后续研究对目的基因进行示踪。方法:从pGL4.10质粒中PCR扩增萤火虫荧光素酶报告基因,测序,克隆至载体pNZ8149,构建pNZ8149-luc表达质粒;电击转化宿主乳酸乳球菌NZ3900,采用乳糖筛选法获得重组的乳酸乳球菌,Nisin诱导,采用微孔板发光检测仪检测荧光素酶的存在,Western Blot检测目标蛋白luc的表达。结果:PCR扩增的荧光素酶报告基因成功克隆至pNZ8149质粒,并电击转化宿主乳酸乳球菌NZ3900,得到乳酸乳球菌表达系统NZ3900/pNZ8149-luc。Nisin诱导后,检测到荧光素酶随诱导时间的延长活性逐渐增强,时间超过24 h之后荧光素酶活性逐渐下降。Western Blot检测到目标蛋白luc在胞内表达。结论:成功构建了p NZ8149-luc表达载体,并能够在乳酸乳球菌体内稳定表达。