John Davison

Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites.

The construction of a new wide-host-range, restriction-site bank, cosmid-cloning vehicle (pJRD215) is described. The wide-host-range properties and the ability to be transferred by conjugation, extend genetic engineering to those Gram-negative species that cannot be transformed. The vector permits the cloning of genes from Gram-negative bacteria using a complementation screening procedure in a mutant host. This procedure is simplified by the possibility of construction of a cosmid gene bank so that only a few hundred clones need to be screened. Subsequent subcloning of the gene of interest is facilitated by the presence of at least 23 unique cloning sites.

Mechanism of control of DNA replication and incompatibility in ColE1-type plasmids —a review

The related phenomena of ColE1 DNA replication control and plasmid incompatibility are analysed in detail. Replication is negatively regulated in different ways by two gene products, RNA I and gene rop-coded polypeptide, while plasmid incompatibility is specified only by RNA I. Thus, copy-number mutants in the RNA I region usually have altered incompatibility and vice versa. RNA I is transcribed from the opposite strand to RNA II; the processed RNA II transcript serves as the primer for DNA replication. The base-pairing interaction between RNA I and RNA II controls DNA synthesis and incompatibility by preventing primer formation. A model is proposed to explain the phenotype of RNA I mutants in terms of the changes they cause in the secondary structure of RNA I or RNA II.

Vectors with restriction site banks IV. pJRD184, a 3793-bp plasmid vector having 43 unique cloning sites

Abstract An improved restriction site bank vector has been constructed from plasmid pJRD158. The new version is smaller and contains 43 unique restriction sites. It should greatly facilitate cloning versatility by providing unique sites for most commercially available restriction enzymes.

Cloning of Bacteriophage-t5 Dna Fragments in Plasmid Pbr322 and Bacteriophage-lambda Gtwes

Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.

Restriction insensitivity in bacteriophage T5: III. Characterization of EcoRI-sensitive mutants by restriction analysis

Abstract Despite the fact that its DNA carries six EcoRI cleavage sites, bacteriophage T5 is able to grow on an EcoRI restricting host, suggesting that it specifies a restriction protection system. In the hope of identifying this protection system, mutants of T5 have been isolated which are unable to grow on an EcoRI restricting host. Analysis of the DNA of such mutants shows that they have each acquired two new EcoRI sites per molecule as a consequence of a single EcoRI site (ris) mutation located in the terminally repetitious, first step transfer (FST) region of the genome. The EcoRI sites generated by the ris mutations differ from the natural EcoRI sites in that the latter are situated on the second step transfer (SST) DNA, which suggests that the in vivo sensitivity of ris mutants is a consequence of having an EcoRI site on the FST DNA. This is understandable, if the hypothetical restriction protection genes are also located on the FST DNA. While expression of these genes would protect natural sites on the SST DNA, the ris sites would, on the contrary, enter an environment in which the protection, products had not yet been synthesized. Construction of double and triple ris mutants has allowed the ordering of the ris sites and the construction of an EcoRI restriction map of the FST region. In addition, the ris mutants allow estimation of the size of the terminal repetition of T5 DNA as 5.9 × 106 to 6.0 × 106 daltons. Correlation of the physical map of the FST region with the already established genetic map of this region allows orientation of the pre-early genes on the genetic and physical maps, and approximate localization of two amber mutations on the physical map.

Bacteriophage T7 RNA polymerase-controlled specific gene expression in Pseudomonas.

The rifampicin (Rif)-resistant RNA polymerase of phage T7 has proved invaluable for the exclusive over-expression, in Escherichia coli, of genes cloned downstream from the T7 phi 10 promoter [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078]. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible wide host range plasmids. As an example, the Rif-resistant in vivo synthesis and specific radiolabelling of E. coli galactokinase in Pseudomonas ATCC19151, is demonstrated. The incidental observation that 30 min after treatment with Rif, two polypeptides continue to be synthesized in plasmid-free Pseudomonas ATCC19151, indicates that these proteins are produced by very stable mRNA species.

Vectors with restriction-site banks. III. Escherichia coli-Saccharomyces cerevisiae shuttle vectors.

The bank of unique restriction sites present in plasmid pJRD158 has been incorporated into new vectors carrying selective markers and replicons derived from commonly used Escherichia coli-Saccharomyces cerevisiae shuttle vectors pJDB207 and YRp7. The new vectors pMH158 and pJO158 have 21 and 23 unique restriction sites, respectively, and their complete DNA sequences are known.

Cloning and Expression of Trypanosoma-brucei Kinetoplast Dna in Escherichia-coli

The kinetoplast DNA of Trypanosoma brucei is made of two components: mini-circles (1 kb, 90% of total kDNA) and maxi-circles (20 kb, 10%) of total kDNA). These are interlocked to form a network of about 10 000 kb. In order to analyse the components of such a network structure, we have cloned individual mini-circle molecules and two of the three EcoRI maxi-circle fragments in E. coli. Cloned mini-circles are somewhat heterogeneous in size and their restriction patterns are completely different. Despite this heterogeneity all are found to contain a homologous region(s) defined by DNA/DNA hybridization. The maxi-circles probably correspond to the mitochondrial DNA of other organisms and, in contrast to mini-circles, do not show sequence heterogeneity. One of the two cloned maxi-circle EcoRI fragments is able to direct the synthesis of two polypeptides of 10 300 and 13 500 daltons in E. coli mini-cells. Detailed analysis of this phenomenon shows that both structural genes and promoter(s) are located within the cloned maxi-circle fragment.

Restriction Site Bank Vectors for Cloning in Gram-negative Bacteria and Yeast

Publisher Summary This chapter describes the application of the restriction site banks for the construction of Escherichia coli vectors, wide-host-range cosmid vectors (able to grow in most gram-negative bacteria), and E. coli –yeast shuttle vectors. The chapter summarizes the restriction site bank series of vectors that have been constructed as tools for the genetic manipulation of DNA. The advantage of the restriction site bank vectors is that they maximize the choice of restriction enzymes that can be used for cloning and facilitate subsequent subcloning and deletion analysis. Thus, they permit, in a single plasmid, a variety of operations that could otherwise be done only with a combination of several different vector systems. Of the known restriction endonucleases that recognize 6-bp palindromes, 39 have unique recognition sites in E. coli vector pJRD184 and four others, while not unique, can also be used for cloning without disturbing essential vector functions. The example illustrated in the chapter involves the cloning of the genes for vanillate utilization from an uncharacterized pseudomonad ATCC 19151. Vanillate is a key intermediate in the degradation of the lignin molecule, one of the worlds most abundant carbon sources and a potential source of industrial raw material and energy. This system is also a useful choice to demonstrate the generality of the cosmid cloning, conjugal transfer, and complementation method of gene isolation.

A Method for the Generation of Small Predetermined Deletions in Plasmid Dna - Deletion Analysis of the Tetr Region of Vector Pbr322

A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.