A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes

Lambda derivatives are described that can be used for cloning DNA fragments of about 20 kilobase pairs (kb) generated by restriction enzymes EcoRi, HindIII, BamHI, MboI and BglII. Recombinants can be selected by their Spi- phenotype and their propagation is facilitated by the presence of a chi site.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

Development of cloning vehicles from the Streptomyces plasmid pFJ103

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies     

Purification and characterization of the N gene product of bacteriophage lambda

The N protein (pN) specified by bacteriophage λ is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from λtrp transducing phage bearing N

An improved cosmid vector for the cloning of equine herpesvirus DNA

We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid- cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.     

A new isoschizomer, BnaI, of the BamHI restriction endonuclease

By assaying the yield of phage SPO1 we have identified a new restriction-modification activity in the Bacillus natto B3364 strain. A class II restriction endonuclease, BnaI, isolated from the crude extract of B3364 cells was shown to be a true isoschizomer of the BamHI endonuclease. The Mr, stability and optimal conditions required for DNA digestion were determined for BnaI. Although both enzymes show the same specificity, BnaI and BamHI differ from each other in all the properties specified above.     

A Fluorometric Assay for DNA Cleavage Reactions Characterized with BamHI Restriction Endonuclease

Fluorescently labeled oligonucleotides and DNA fragments have promise in nucleic acid research with applications that include DNA hybridization, automated DNA sequencing, fluorescence anisotropy, and resonance energy transfer studies. Past concerns with fluorescent-labeled DNA arose from interactions between fluorophores and DNA that result in quenched fluorescence. This quenching phenomenon is most problematic in fluorescence resonance energy transfer studies because quenching of the donor fluorescence could result from either resonance energy transfer or nontransfer effects. In the present study, relief of nontransfer quenching of a 14-mer fluorescein 5-isothiocyanate (FITC)-labeled oligonucleotide containing the BamHI restriction site was characterized with both steady-state and time-resolved fluorescence techniques. The FITC-labeled single strand was best fit by a triexponential decay with lifetimes of 0.5, 2.7, and 4.2 ns. The 4.2-ns component was found to contribute more than 80% of the total steady-state intensity. Upon annealing with an unmodified complementary strand, the contribution from the 4.2-ns component was significantly decreased, resulting in twofold quenching of total fluorescence. We reasoned that this quenching phenomenon should be a reversible process and could be employed to study strand separation processes in molecular biology. Hence, cleavage of the fluorescently labeled substrate was examined using DNase I and BamHI restriction endonuclease. Our results show that the quenched fluorescence is totally recovered upon cleavage (compared to that of the single strand). The extent of cleavage measured by fluorescence was confirmed by nondenaturing polyacrylamide gel electrophoresis analysis. We believe this fluorescence "dequenching" technique may be used to quantify the kinetics of other DNA strand separation and cleavage processes in molecular biology.     

Bacteriophage lambda cloning vehicles for studies of genetic recombination

A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination. These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted. (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right. (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites. As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA. Restriction enzyme maps of the vectors have been determined. Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared.     

Isolation of cDNAs encoding GTP cyclohydrolase II from arabidopsis thaliana

A GTP cyclohydrolase II-encoding gene from Arabidopsis thaliana was isolated through functional complementation of a mutant of Escherichia coli, BSV18, deficient in this protein. The derived amino-acid sequence constitutes a polypeptide of 27 kDa and shows 37–58% identity with previously published sequences of Escherichia coli, Bacillus subtilis, Photobacterium leiognathi and P. phosphoreum.     

谷氨酸棒杆菌/大肠杆菌穿梭型启动子探测载体构建

从质粒pXZ10145和pUC19出发,构建了一个谷氨酸棒杆菌/大肠杆菌穿梭载体pAK6。pAK6的大小为5684bp,带有卡那霉素和氨苄青霉素抗性选择标记,以及多克隆位点。在pAK6基础上,构建了以氯霉素乙酰转移酶为报告基因的启动子探测载体pAKC6,pAKC6的大小为6474bp。采用鸟枪法,将经Sau3AI消化的谷氨酸棒杆菌基因组片段连入pAKC6;根据谷氨酸棒杆菌对氯霉素的抗性,从中分离出两个具有启动子功能的插入片段。通过测定报告基因氯霉素乙酰转移酶的活性,对两个启动子片段在谷氨酸棒杆菌中的强度进行了初步的判断;测序后,用启动子预测软件对其结构进行了预测,证实了启动子序列的存在。     

In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1

A technique is presented by which chromosomal mutations may be efficiently transferred onto chimeric multicopy plasmids in vivo. The technique employs the transduction of plasmids using bacteriophage P1 as vector. The utility of this method was demonstrated by cloning a chromosomal ompR mutation of Escherichia coli K-12. The high-frequency transduction of the chimeric plasmid appeared to be dependent on its integration into the chromosome by homologous recombination. The results also suggest that the plasmid was transduced as part of the chromosome and resolved from its integrated state in the recipient cell, resulting in a high yield of mutant plasmid segregants.     

大肠杆菌重组工程

源于噬菌体的大肠杆菌同源重组系统不需要限制性内切酶和DNA连接酶就可以进行DNA克隆和亚克隆,还能快速地改造质粒、细菌人工染色体及细菌基因组染色体,是基因工程技术的一大突破,被称为重组基因工程或重组工程。该技术操作简单,效率较高,可望为功能基因组学研究提供一个有力的工具。     

A eukaryotic expression vector for the study of nuclear localization signals

We describe a new vector designed to produce β-galactosidase fusion proteins which can be used to assess subcellular localization of target peptide fragments or proteins in eukaryotic cells. The vector was constructed in such a way as to produce the peptide of interest in fusion via a short linker of proline residues to the N terminus of the reporter protein. Efficiency of the transport machinery is optimized using this particular protein fusion construction. This vector has potential uses for readily testing putative nuclear localization sequences and identifying their crucial amino-acid residues.     

Paucity of the Sau3AI recognition sequence (GATC) in the genome of Methanococcus voltae

Summary High molecular weight genomic DNA isolated from the archaebacterium Methanococcus voltae by alkaline-SDS lysis was not effectively digested with the restriction enzyme Sau3AI, which recognizes the base sequence GATC. Mc. voltae DNA was also resistant to digestion by MboI and BamHI which recognize sites containing the same GATC sequence. Examination of a Mc. voltae genomic library prepared in Escherichia coli JM83 with a pUC vector revealed that the 5–10 kb inserts were still resistant to Sau3AI digestion, indicating a likely lack of the GATC sequence in Mc. voltae DNA.     

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.