Positive Selection Vector Research Articles

Cloning and Expression in Escherichia coli of the Polysaccharide Depolymerase Associated with Bacteriophage-Infected Erwinia amylovora

The bacteriophage-encoded polysaccharide depolymerase produced in Erwinia amylovora has been cloned and expressed in Escherichia coli. The bacteriophage ERA103 genome was observed to consist of five EcoRI fragments, labeled as follows: A, 7.5 kilobases (kb); B, 5.0 kb; C, 2.7 kb; D, 2.1 kb; and E, 1.8 kb. A restriction map for ERA103 was also prepared. Each of the fragments were cloned into the positive-selection vector pOP203(A(2)) and pBR322.

Positive-selection vectors utilizing lethality of the EcoRI endonuclease

The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restricion sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacI Q host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene.

Use of the lysis gene of bacteriophage φX174 for the construction of a positive selection vector

DNA fragments generated by a variety of restriction enzymes can easily be cloned m the small (3.2-kb) positive-selection vector pUH84, which contains the modified lysis gene of bacteriophage φX174 under transcriptional control of the lac promoter. Plasmid pUH84 does not yield transformants after introduction into Escherichia coli unless the lysis gene is inactivated by insertion of foreign DNA into one of the unique PstI, SalI, AccI, HincII, BamHI, or EcoRI sites. This highly efficient positive selection of recombinants requires neither the use of a distinct host strain nor a special induction of the lysis function. Transcription of fragments cloned into pUH84 may be effectively regulated by the lac promoter provided the host cells are cotransformed with the newly constructed plasmid pUH7 which carries the I Q allele of the lac repressor gene.

Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli.

A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed our earlier results [Valerie, K., Henderson, E. E. & de Riel, J. K. (1984) Nucleic Acids Res. 12, 8085-8096] that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe.

Molecular cloning of a pIP501 derivative yields a model replicon for the study of streptococcal conjugation.

We have previously constructed a derivative of the broad host range streptococcal plasmid pIP501, a conjugative plasmid designated pVA797, that confers chloramphenicol resistance and contains a unique EcoRI site in a non-essential region of the plasmid molecule. pVA797 (30.7 kb) when cloned in toto as an EcoRI fragment into the positive selection vector pOP203(A2+) gave a recombinant, pVA904 (37.7 kb), which was able to replicate in Escherichia coli and in streptococcal species. It can be phenotypically monitored in either genus by specific drug resistance markers (chloramphenicol resistance in streptococci, tetracycline resistance in E. coli). pVA904 segregates into E. coli minicells where it specifies the production of at least 13 polypeptides. Many of the polypeptides are missing in minicells containing a transfer-defective, deletion derivative of pVA904. pVA904 is an ideal model replicon for the study of streptococcal conjugation because it is a shuttle plasmid thus enabling manipulation using procedures established for E. coli. Specifically, it should be possible to define the genetic basis of streptococcal conjugation by coupling mutagenesis protocols and minicell protein analyses in E. coli with evaluation of transfer function in streptococci.

Cloning and expression in Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae.

Bacteroides nodosus is the primary causative agent of ovine foot rot. Virulent isolates of this bacterium contain fimbriae which appear to play a major role in both infectivity and protective immunity. This paper presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the fimbriae of B. nodosus. Total DNA was isolated from B. nodosus VCS 1001 (serogroup A), digested with HindIII, and inserted into the positive-selection vector pTR262. Recombinant E. coli clones were screened directly with anti-fimbrial antiserum by using a colony immunoassay. Several positive colonies were identified, each of which contained the same 5.5-kilobase HindIII insert. The prototype has been designated pBA101. Some clones also contained additional flanking sequences from the B. nodosus genome. Western transfer analyses verified that the positive clones were producing the B. nodosus fimbrial structural subunit, molecular weight ca. 17,500. The level of expression of the antigen in E. coli was comparable to that in B. nodosus itself and was unaffected by the insertion site or orientation of the cloned fragment, indicating that synthesis was being directed from an internal promoter. Restriction mapping and deletion analyses localized the fimbrial subunit gene to the vicinity of a PvuII site near the central region of the original HindIII insert. The expressed antigen was located in the membrane-cell wall fraction and may be exposed on the surface of the recombinant E. coli cells.

Positive selection vectors: a small plasmid vector useful for the direct selection of Sau3A-generated overlapping DNA fragments

A positive selection plasmid vector (pLA7), containing a unique BclI site, and its use in the facile selection of a library of overlapping DNA fragments, generated by a partial digest with Sau3A, is described. Selection depends on the 5-fluorouracil + 5′AMP resistance of upp− ush− [pLA7ush:: cloned DNA] cells, whereas upp−ush− [pLA7ush +] cells are sensitive under the same conditions.