LTR-directed homologous recombination of full-length HIV-1 provirus clone inrecA(?) bacteria

Abstract

During molecular cloning of full-length retroviral plasmid clones occurrence of homologous recombination (HR) between LTR regions is frequently observed. In order to evaluate appropriate host bacterial strains for cloning such HR-prone plasmids, we utilized a linearized template plasmid containing a full-length HIV-1 proviral sequence. The plasmid was linearized within the viral sequence so that plasmid transformed bacteria would grow only when the plasmid was circularized by HR. Using this genetic system for detecting HR, we evaluated the frequency of HR in various recA(-) bacterial strains which are commercially available and in some recA-null strains in which recA-defective phenotype was constructed by P1 transduction. We found that HR occurred even in recA-null strains although in lesser frequencies. The nucleotide sequence analysis at the junction of recombination revealed no loss, insertion or duplication of DNA sequence. It is suggested that recombination machinery other than the RecA system is involved.

Construction and Characterization of a Full-Length Infectious Molecular Clone from the HIV Type 1 Subtype Thai-B Isolated in Henan Province, China

“…Many laboratories have experienced genetic instability of such clones, including frequent homologous recombination (HR) at the site of the homologous region, even in various recA(-) host bacteria strains. 14 We also experienced a similar phenomenon with various plasmids and bacterial strains in the process of constructing pCNHN24. Either the high-copy plasmid, pUC18, or the medium-copy plasmid, pBR322, was used to construct our infectious molecular clones, but large fragment rearrangements and deletions occurred, and frequent HR was observed until we finally adopted the very low-copy plasmid, pLG338 (copy number 1-2).…”

Targeted rearrangement of a chromosomal repeat sequence by transfection of a homologous DNA sequence using purified integrase

“…A highly efficient LTR-directed homologous recombination has also been observed in an HIV-1 proviral clone. 26,27 Bischerour et al 28 suggest that Vpr stimulates HIV-1 integrase-mediated homologous strand transfer of miniviral DNA. Furthermore, Delviks and Pathak 29 have described a murine leukemia virus-based retroviral vector with a high-frequency deletion of direct repeats that can excise a drug resistance gene during reverse transcription.…”

Shuttle System Allowing Simplified Cloning of Expression Cassettes into Advanced Generation Lentiviral Vectors

“…In addition, during the cloning of full-length viral plasmid clones, homologous recombination between long terminal repeat (LTR) regions is frequently observed (3,4) even in recA , recB , recJ , and sbcC mutant bacterial strains and often results in the loss of the entire viral sequence except a single copy of an LTR (4). …”

“…Only for the very last cloning step, the insertion of complete expression cassettes between the viral LTRs of pLentiShuttle, is it recommended to use special (usually expensive) commercially available recombination-deficient E. coli strains, such as Max Efficiency Stbl2, Stbl3, or ElectroMax Stbl4 (all from Invitrogen). Other commonly used recombinations impeding E. coli strains, such as HB101 (Takara, Otsu, Japan), are less efficient in preventing recombinations of lentiviral transfer vectors during cloning (4). …”