An efficient synthetic primer for the M13 cloning dideoxy sequencing system

The deoxytetradecamer d(AAAACGACGGCCAG) has been shown to be an excellent universal primer for sequence determination of DNA cloned into the bacteriophage M13 mp7, mp8, and mp9 series. This new primer offers several advantages over others currently available and it has been used to define the cloning of Hinf I fragments of bacteriophage S13 DNA into the Eco RI site of M13 mp7, utilizing the homologous complementary base pairing of the two restriction sites. Of the four possible sequence derivatives of the Hinf I GANTC recognition site, only those corresponding to GAATC and GATTC have been found at cloning sites in chimeras.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

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.     

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.     

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.     

A new site-specific endonuclease, ScaI, from Streptomyces caespitosus.

A new site-specific endonuclease has been isolated from Streptomyces caespitosus and named ScaI. Based on analysis of sequences around the restriction sites in pBR322 and pBR325, the recognition sequence of ScaI endonuclease was deduced to be a new hexanucleotide 5'-AGTACT-3'. The cleavage site was determined by comparing the ScaI-cleaved product of a primer-extended M13mp18-SCA DNA, which contains an AGTACT sequence, with dideoxy chain terminator ladders of the same DNA. ScaI was found to cleave the recognition sequence between the internal T and A, leaving flush ends to the cleaved fragments.     

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.     

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.     

Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.     

Preparation and characterization of a viral DNA molecule containing a site-specific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens

The synthetic oligonucleotide heptamer 5'-ATCCGTC-3' was reacted in vitro with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene and the resulting product isolated by reverse-phase high-performance liquid chromatography (HPLC). This purified oligonucleotide, which was shown by chemical and enzymatic analysis to be a heptamer containing a single N-(deoxyguanin-8-yl)-2-aminofluorene adduct, was then used to situate the putatively mutagenic aminofluorene lesion within the genome of M13 mp9 by ligating it into a complementary single-stranded region located at a specific site in the negative strand of the duplex M13 mp9 DNA molecule. The presence of the adduct at the anticipated location was confirmed by taking advantage of the facts that AF adducts inhibit many restriction enzymes when located in or near their restriction sites and that the AF moiety should be contained within the HincII recognition sequence on M13 mp9 DNA. Upon attempted cleavage of the M13 DNA containing the site-specific AF adduct with HincII, we find that the large majority of the DNA remained circular, demonstrating the incorporation of the AF adduct in high yield into the DNA molecule at this location. This system should prove useful in vivo for the study of mutagenesis by chemical carcinogens and in vitro to study the interaction of purified DNA metabolizing proteins with a template containing a site-specific lesion.     

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.     

Human methylated DNA-binding protein. Determinants of a pBR322 recognition site

Methylated DNA-binding protein (MDBP) from human placenta has a high affinity for a site in pBR322 (pB site 1) when that site is methylated at its CpG dinucleotides. Dimethyl sulfate interference analysis and experiments with ligands prepared by oligonucleotide-directed mutagenesis indicate that 15 contiguous base pairs, 14 of which exhibit hyphenated dyad symmetry, influence MDBP binding to pB site 1. These 14 base pairs, 5'-RTMGYCAMGG(M/T)GAY-3' (M, 5-methylcytosine), suffice for recognition by MDBP as demonstrated with a double-stranded, MpG-containing oligonucleotide used as a free ligand or cloned into M13mp19 and subsequently methylated. Seven single-site mutations at different positions of this 14-base pair region largely eliminated binding, and several others increased binding up to 2-fold when compared to the nonmutant, triply methylated sequence. However, MDBP recognizes a site in hemimethylated M13mp19 replicative form DNA, which was homology to pB site 1 at only 10 of 14 base pairs, and all four of these different base pairs are equivalent to transversions. Based upon the above data, a mixed oligonucleotide probe was constructed that contains variants of pB site 1 which should be recognized by MDBP. This 14-base probe hybridizes under stringent conditions to a number of discrete fragments in restriction digests of human DNA. this suggests that there are multiple pB site 1-related sequences in human DNA that might, when methylated, bind MDBP in vivo.     

A vector for directional cloning and expression of polymerase chain reaction products in Escherichia coli

This paper describes the construction of a modified vector for the cloning and expression of protein-encoding genes in Escherichia coli. The vector, pfXblue, is derived from the system originally developed by Nagai and Th?gerson [Nature 309 (1984) 810-812], but contains a modified multiple cloning site (MCS) from M13mp18 to allow directional insertion of foreign coding sequences. The MCS is located within the M13mp18 lacZ' gene and thus allows blue/white screening of colonies for inserts. The inserted gene is expressed as a fusion protein, which, when cleaved by the coagulation factor Xa protease, yields the mature product. This vector was successfully used for the production of a mitochondrial [2Fe-2S]ferredoxin using polymerase chain reaction products generated from a chick kidney cDNA library.     

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.     

Complementary-addressed elimination of E1a sequence of simian adenovirus oncogene SA7 from circular single-stranded DNA of recombinant phage M13

The G fragment of the simian adenovirus SA7 oncogene corresponding to E1a region was cloned into M13mp8 and M13mp9 phages. Single-stranded DNAs of the recombinant phages thus obtained (mp8G and mp9G) partially digested with DNAse II were used to synthesize polyalkylating derivatives capable of specific hybridisation and subsequent alkylation of complementary G sequences of corresponding phage DNAs. After incubation of complementary alkylated DNA in the presence of lysine, the preselected region (G fragment) was specifically eliminated without damaging vector sequences. The method of complementary-addressed cleavage proved to be useful for precise analysis of reactions of polyalkylating derivatives within complementary complexes.     

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.     

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.     

A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments

The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.