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.     

Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors

pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors. In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli. pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers. These vectors could be introduced into S. suis, E. coli, Salmonella typhimurium, S. pneumoniae, and S. equi ssp. equi by electrotransformation. The recA gene was cloned from S. suis and sequenced, and this information was used in the construction of a recA mutant of S. suis. Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S. suis and E. coli recA mutants compared with the parental strains. These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S. suis and E. coli. Moreover, cloning of the functional S. suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S. suis but not in E. coli. These results showed that pSET vectors are useful tools for cloning and analyzing S. suis genes in S. suis strains directly.     

EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis.

For shotgun cloning into M13 vectors, a double-stranded cassette of synthetic oligonucleotides containing a SmaI site within the two halves of an EcoK site, has been introduced into the vector M13mp8. Cloning of blunt end DNA into the SmaI site destroys the EcoK site, and recombinants are therefore preferentially selected on transfection into a K strain of E.coli. For deletion mutagenesis using synthetic oligonucleotides, an M13 vector with four copies of the EcoK cassette has been made to facilitate the joining of lacZ or a Factor Xa cleavage site to any protein reading frame.     

Isolation and characterization of naturally occurring hairpin structures in single-stranded DNA of coliphage M13

With precise conditions of digestion with single-strand-specific nucleases, namely, endonuclease S1 of $\text{Aspergillus oryzae}$ and exonuclease I of $\text{Escherichia coli}$, nuclease-resistant DNA cores can be obtained reproducibly from single-stranded M13 DNA. The DNA cores are composed almost exclusively of two sizes (60 and 44 nucleotides long). These have high (G + C)-contents relative to that of intact M13 DNA, and arise from restricted regions of the M13 genome. The resistance of these fragments to single-strand-specific nucleases and their nondenaturability strongly suggest the presence of double-stranded segments in these core pieces. That the core pieces are only partially double-stranded is shown by their lack of complete base complementarity and their pattern of elution from hydroxyapatite.     

Construction of new shuttle plasmid vectors for Escherichia coli-Bacteroides transgeneric cloning

Abstract An Escherichia coli-Bacteroides shuttle vehicle (pKBF367-1) was constructed by combining the pBR322 derivative pKC7 (5.9 kb) with [1] a 4.6 kb cryptic plasmid from Bacteroides fragilis ; and [2] the 4.2 kb Eco RI-B fragment of the B. fragilis plasmid pBFTM10. This latter component allowed selection of clindamycin-resistant transconjugants upon helper plasmid-mediated transfer to a recipient strain of Bacteroides distasonis . To improve the potential of pKBF367-1 (14.7 kb) as cloning vector, successive deletions generated derivatives of 12.8, 10.5 and 9.3 kb, which were still able to replicate in B. distasonis 419. These bifunctional vectors were successfully employed to introduce transposon Tn 501 (Hg^r) into B. distasonis 419, but expression of mercury resistance was not observed. This plasmid vehicles series may be useful for cloning Bacteroides genes in E. coli and studying their expression in a heterologous Bacteroides strain.     

M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates

M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.     

Construction of first-generation lactococcal integrative cloning vectors

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (ery ^r) gene obtained from L. lactis IL1837. Integration of the ery ^r gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nis ^r) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nis ^r fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for >200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (Nis ^r ) transformant suggested that the integrated nis ^r gene may be amplifying within the host chromosome. Correspondence to: S. K. Harlander     

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.     

A new primer for sequencing DNA fragments cloned in M13mp vectors

Tridecadeoxyribonucleotide d(CCAGGGTTTTCCC) was prepared by solid phase crown-ether-catalyzed phosphotriester method and proposed a new sequencing primer. This primer expands capacities of the Sanger dideoxy-chain-terminating method to solve various sequencing problems as compared to well-known universal primers. Criteria of the choice of an oligonucleotide primer without computer analysis of nucleotide sequence are described.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

Fractionation of membrane vesicles from coliphage M13-infected Escherichia coli.

Membrane vesicles were prepared by osmotic lysis of spheroplasts from M13-infected Escherichia coli. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (reduced NAD: oxidoreductase, EC 1.6.99.3) and Mg2+-Ca2+-activated adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3), which are normally localized to the inner surface of the cytoplasmic membrane, were 50% acceesible to their polar substrates in these vesicles. The major coat protein of coliphage M13 is also bound to the cytoplasmic membrane (prior to phage assembly) but with its antigenic sites exposed to the exterior of the cell. Antibody to M13 coat protein was used to fractionate membrane vesicles. Neither agglutinated nor unagglutinated vesicles had altered NADH oxidase and adenosine triphosphatase specific activities. This is inconsistent with such vesicles being a mixture of correctly oriented and completely inverted membrane sacs and suggests that NADH oxidase, adenosine triphosphatase, M13 coat protein, or all three proteins rearrange during vesicle preparation.     

Rapid purification of bacterial plasmids and coliphage M13 RF without CsCl centrifugation

A simple and rapid procedure for the purification of plasmids from Escherichia coli K12 has been developed. Bacterial cells are subjected to the boiling procedure [D. S. Holmes, and M. Quigley Anal. Biochem.114, 193–197 (1981)] followed by removal of contaminating RNA by chromatography on Sepharose 2B and of genomic DNA by acid-phenol extraction. Plasmids are recovered with good yield. They can be restricted and ligated and will transform host cells. A simple modification of the procedure allows it to be used for the isolation of coliphage M13 RF DNA.