Document 3318
 DOCN  M94A3318
 TI    LTR-directed homologous recombination of HIV-1 proviral clone in recA(-)
       bacteria.
 DT    9412
 AU    Yamada K; Nakano T; Okamoto T; Dept. Mol. Genetics, Nagoya City Univ.
       Med. School, Aichi, Japan.
 SO    Int Conf AIDS. 1994 Aug 7-12;10(1):103 (abstract no. PA0029). Unique
       Identifier : AIDSLINE ICA10/94369257
 AB    OBJECTIVE: In this study we have attempted to clarify the genetic
       mechanism of frequent homologous recombination (HR) during cloning of a
       full length HIV-1 plasmid in recA(-) host bacteria. METHODS: A plasmid
       containing the full length HIV-1 sequence, pNL432, was transfected into
       various recA(-) bacterial strains. The frequency of the appearance of HR
       was measured in bacterial colonies when transfected with a closed
       circular or linealized plasmid DNA. RESULTS: When pNL432 DNA was
       digested at a unique site within the HIV sequence, we frequently
       observed generation of mutant truncated clones lacking the entire
       proviral sequence except one copy of the LTR (HR). The frequency of HR
       was compared with different DNA forms in various recA(-) hosts.
       Interestingly, those bacterial strains previously characterized with its
       low-rate recombination showed higher rate of HR when the linealized
       plasmid was transfected. The nucleotide sequences at the junction point
       will be presented at the meeting. DISCUSSION AND CONCLUSIONS: From the
       above observations the following possibilities were entertained: (1) HR
       might occur due to an unknown mechanism other than the recA system or,
       (2) recA(-) phenotype in the tested bacteria might be leaky and it
       became detectable by using the linealized plasmid. Therefore, caution
       should be taken when plasmid constructions are attempted using a
       full-length virus HIV-1 clone. It is noteworthy to consider this
       possibility when genetic engineering using a retroviral full length
       clone is attempted. Moreover, this system might be useful for the study
       of molecular mechanism of HR.
 DE    Bacteria/GENETICS  Cloning, Molecular  Genes, Bacterial  Genetic
       Engineering  *HIV Long Terminal Repeat  HIV-1/*GENETICS  Phenotype
       Plasmids/GENETICS  Proviruses/*GENETICS  *Recombination, Genetic
       Transfection  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
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