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At least 109 records · Page 6Linked to original sources
PubMed1983

Genetic recombination of homologous plasmids catalyzed by cell-free extracts of Saccharomyces cerevisiae.

We have developed an in vitro system utilizing yeast cell-free extracts to catalyze recombination events between homologous plasmids containing different mutant alleles of the Tet or ARG4 genes. The reaction increased the frequency of Tcr or Arg+ transformants (recombinants) from 2 X 10(-6) to 1-3 X 10(-3). Linearizing one substrate between the two tet mutations stimulated the reaction 2 to 4 fold. The reaction required rATP, Mg++, NAD, and DTT. The rad52-1 mutation decreased the reaction between linear and circular substrates 5 to 6 fold but had little effect with circular substrates. The structures of Tcr plasmids was analyzed by restriction endonuclease mapping and was consistent with a recombination reaction involving crossing-over and gene conversion. Recombination products were also observed directly by subjecting reaction mixtures to electrophoretic analysis. These results indicate that recombination events were catalyzed by the yeast extract.

Cell-Free System↗
PubMedDate not supplied

RecA-DNA helical filaments in genetic recombination.

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.

Adenosine Triphosphate↗
PubMed1991

Genetic recombination in a chromosomal translocation t(2;8)(p11;q24) of a Burkitt's lymphoma cell line, KOBK101.

We analyzed a chromosomal translocation, t(2;8)(p11;q24), in a Burkitt's lymphoma cell line, KOBK101. The translocation reciprocally occurred between a site about 150 bp upstream from the J5 segment in the Ig kappa-encoding gene on chromosome 2 and the A-rich end of an Alu repetitive element located far downstream from the c-myc gene on chromosome 8. Short segments of both parental chromosomes were deleted at the rearrangement site. A sequence related to the heptamer recognition signal for the V-J recombination of Ig genes and a topoisomerase I-recognition sequence were detected at the breakpoints. The V-J recombination occurred on both chromosome 2 and the translocated chromosome 2p- at the J3 and J4 segments, respectively. The J region on the translocated chromosomes was mutated, as compared with that on the untranslocated chromosome, while the Alu element and its upstream sequence were conserved. These results suggest the following aspects to the chromosomal translocation of this cell line. A V-J recombination seems to have occurred at the proximal end of the J4 segment first, and then the translocation took place in the region between the J4 and J5 segments. The translocation may have been mediated by the functions of topoisomerase I and the Alu repetitive sequence located at the breakpoint, although the possibility cannot be ruled out that the recombination machinery for Ig gene rearrangements functioned irregularly.

Alleles↗
PubMed1999

Production of an immunoenzymatic tracer combining a scFv and the acetylcholinesterase of Bungarus fasciatus by genetic recombination.

We constructed a plasmid containing a chimeric gene composed of the gene encoding acetylcholinesterase (AChE) from Bungarus fasciatus venom and a gene encoding a single chain antibody fragment (scFv) directed against one of the two subunits of a presynaptic neurotoxin from rattlesnake. Large quantities of the fusion protein were produced in the culture medium of transfected COS cells. Fusion to AChE did not affect the ability of the scFv to recognise its antigen. Similarly, the AChE activity was not impaired in the fusion. The fusion protein was purified from the culture medium in a single step by affinity chromatography. The immunoconjugate obtained consisted of a soluble monomeric form of AChE fused to scFv. It was monovalent and had a molecular weight of 94 kDa. The properties of this scFv-AChE fusion show that the simple, reproducible preparation of various recombinant monovalent immunoenzymatic tracers with low molecular weight is possible. In addition, in the construct presented, the scFv domain can be easily changed to another one taking advantage of the SfiI-NotI restriction sites surrounding this domain.

Acetylcholinesterase↗
PubMed2000

Initiation of genetic recombination and recombination-dependent replication.

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.

DNA Damage↗