PubMed Health⌕ Search

Biomedical subjects

A C Boyd

Publications and source records attributed to A C Boyd.

24 records · Page 2Linked to original sources
PubMed1993

Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.

The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting on pairs of lox sites within directly repeated vector molecules flanking insert DNA. Recombinants are introduced into cells conventionally by transformation or electroporation. In both a model system and the cloning of PCR products, yields approaching those obtainable in cohesive-end cloning were achieved. Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.

Base Sequence↗
PubMed1989

Characterization of the ColE1 mobilization region and its protein products.

A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. The mob region is genetically complex: two mob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis of mob. Four mob genes essential for mobilization by R64drd11 were thus identified. Fragments of mob were subcloned under control of the Ptac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of the mob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essential mob genes are conserved is also presented.

Amino Acid Sequence↗
PubMed1986

Isolation and computer-aided characterization of MmeI, a type II restriction endonuclease from Methylophilus methylotrophus.

A Type II restriction endonuclease, MmeI, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-palindromic recognition sequence 5'-T C C Pu A C (N)20-3', 3'-A G G Py T G (N)18-5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved using two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules.

Chromosome Mapping↗
PubMed1986

Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin.

Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5' end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a lambda dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.

Bacteriocin Plasmids↗
PubMedDate not supplied

Insertion of natural intron 6a-6b into a human cDNA-derived gene therapy vector for cystic fibrosis improves plasmid stability and permits facile RNA/DNA discrimination.

BACKGROUND: The gene therapy vector pCMV-CFTR containing human CFTR cDNA shows high segregational instability during growth in Escherichia coli. METHODS: By host strain screening and optimization of fermentation, satisfactory levels of pCMV-CFTR production were achieved. However, the vector was also vulnerable to structural instability manifested by the appearance during fermentation of a more stable mutant form in which the bacterial insertion sequence IS1 had transposed into exon 7 of plasmidborne CFTR. The instability of pCMV-CFTR is attributable to transcription from an upstream cryptic promoter leading to the production of CFTR peptide fragments known to be toxic when expressed in E. coli. To address this, we inserted the 1.1 kb natural human 6a-6b intron into pCMV-CFTR. RESULTS: The new vector pCMV-CFTR-int6ab is more stable in E. coli than either pCMV-CFTR or the IS1 mutant, grows to high cell density giving higher DNA yields and expresses CFTR appropriately in transfected cells. Thus, the intron has a stabilizing effect comparable to the IS1 insertion yet retains full functionality for gene therapy. We describe a PCR assay using primers directed to sequences flanking the intron that allows differentiation between DNA and mature mRNA. The T936C mutation present only in vector DNA has also been exploited to allow transgene CFTR to be distinguished and its dose-dependent expression to be detected in human cellular backgrounds. CONCLUSIONS: Instability of a plasmid vector for gene therapy has been minimized by rational modification. The introduction of an intron for this purpose offers the additional advantage of providing a discriminatory RT-PCR assay.

Amino Acid Sequence↗