A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

用蛋白质工程的方法改良枯草杆菌蛋白酶E(Subtilisin E)的抗氧化性

以含有蛋白酶E基因(aprE)的单链M13mp18-aprE DNA为模板,合成的寡核苷酸5′-3′为诱变引物,用缺口双链法对aprE进行Met-222-Ala点突变。经菌落印迹杂交筛选,选出阳性噬斑。用SaⅡ酶解M13mp18-aprE得到aprE,将它和pPZW103重组,转化中性、碱性蛋白酶缺失宿主菌DB104。经含卡那霉素和脱脂奶粉板筛选和比较aprE限制性内切酶NcoⅠ和SacⅡ水解电泳图谱分析,完成构建一个分泌抗氧化的枯草杆菌蛋白酶E的工程菌PW8888。     

A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI

An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed. The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence. Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.     

New versatile cloning and sequencing vectors based on bacteriophage M13

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.     

Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA.

Double-stranded cDNA has been synthesized from RNA of a severe strain of potato spindle tuber viroid using a synthetic oligodeoxyribonucleotide as a primer. Upon cloning in bacteriophage M13mp9, two recombinant phages were selected to construct a pBR322-derived plasmid containing a complete viroid DNA copy. Elucidation of the nucleotide sequence revealed four differences with the previously established sequence of another PSTV strain, three of which were base exchanges and one a deletion.     

The making of strand-specific M13 probes

A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (?) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, 24, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.     

Bvui: a site-specific endonuclease from Bacillus vulgatis

A site-specific endodeoxyribonuclease was partially purified from an extract of osmotically shocked spheroplasts of a Bacillus vulgatis strain; the enzyme has been designated BvuI and its activity was characterized. The locations of BvuI-generated cleavages on bacteriophage lambda and M13 derivatives mp7, mp8 and mp9, SV40 and PBR322 DNA molecules were determined. BvuI was shown to recognize the DNA sequence decreases 5'-G-Pu-G-C-Py-C-3' 3'-C-Py-C-G-Pu-G-5' increases and cleaves it at the positions indicated by arrows. Two identical BvuI recognition sites separated by fourteen nucleotide pairs were shown to occur within the tetracycline resistance gene of PBR322; BvuI should be useful for molecular cloning in that plasmid vector.     

A versatile primer for DNA sequencing in the M13mp2 cloning system

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.     

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.     

A direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13.

A new cloning strategy is described which utilizes direct selection of recombinants for shotgun sequencing in the filamentous bacteriophage M13. Direct selection is accomplished by insertional inactivation of the M13 gene X protein, a powerful inhibitor of phage-specific DNA synthesis when overproduced. An extra copy of gene X was inserted into the intergenic region of M13 and placed under the control of the bacteriophage T7 gene 10 promoter and RBS. Random fragments are cloned into the EcoRV cloning site of the new gene X cistron and recombinants are selected in an E. coli male strain producing T7 RNA polymerase. Cloning efficiencies obtained with M13-100 or phosphatase-treated M13mp19 vector are comparable. The direct selection capability of M13-100 was demonstrated to have the following advantages: (a) consistently achieved high ratios of recombinants to religated vector in the libraries, averaging about 500:1 (0.2% background), and (b) the elimination of the need for phosphatase treatment of the vector to reduce background. The direct selection strategy significantly improves the efficiency of shotgun library construction in M13, and should therefore facilitate the cloning aspects of large scale sequencing projects.     

Development of a genetically modified bacteriophage for use in tracing sources of pollution

Bacteriophage are frequently used as biotracers to identify the source of water pollutants. Genetic manipulation of bacteriophage M13mp18 has been used to enhance this technique by creating a library in which each recombinant bacteriophage genome contains a unique identification sequence. Techniques that identify a recombinant bacteriophage by the presence of the identification sequence, including polymerase chain reaction, restriction site polymorphism and plaque hybridization, have been developed. Recombinant bacteriophage can be used to test a large number of suspected sources simultaneously. The identification sequence also eliminates confusion with natural bacteriophage present in water samples. The performance of the modified bacteriophage and the techniques were assessed in simulated field trials on a restricted site carried out under a consent for environmental release of a genetically modified organism. The techniques were also field tested at sites in northwest England using wild-type M13 bacteriophage.     

Modification of the bacteriophage vector M13mp2: introduction of new restriction sites for cloning

The construction of two new derivatives of the bacteriophage cloning vector M13mp2 is described. One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site. These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment of Escherichia coli beta-galactosidase within the phage. The new restriction sites do not disrupt the blue color detection system of M13mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.     

Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites

Twenty-nine early promoters from bacteriophage T4 and 14 early promoters from bacteriophage T6 were isolated using vector M13HDL17, a promoterless derivative of M13mp8 carrying a linker sequence, the bacteriophage lambda-terminator tR1, and the lacZ' gene including part of its ribosome-binding site. The consensus sequence for the T4 promoters is: (sequence; see text). Ribosome-binding sites of T4 share the sequence: 5'...g.GGAga..aA.ATGAa.a...3' The consensus sequence of the T4 early promoter regions is significantly different in sequence and length from that of Escherichia coli promoters. Only one of the promoters detected with vector M13HDL17 resembled a typical bacterial promoter. The high information content raises the possibility that additional proteins recognize and contact nucleotides within the promoter region. All T4 early promoters also carry DNA sequences that could support DNA curving, a structural feature that might contribute to promoter recognition.