Lentivirus envelope sequences and proviral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors.
A promoter-selection vector (pKK232-8) was used to identify sequences with strong Escherichia coli promoter activity positioned near the start of the envelope-encoding genes (env) of two lentiviruses, simian immunodeficiency virus (SIV) and equine infectious anemia virus (EIAV). For EIAV, cloning the cryptic promoter sequences together with downstream sequences encoding the envelope glycoprotein (gp90) in moderate- to high-copy-number (hcn) plasmid vectors, such as pBR322 or pUC, resulted in rearrangements and point mutations in env when propagated in E. coli. To alleviate this problem, low-copy-number (lcn) cloning vectors, pLG338-30 and pLG339-SPORT, were constructed. The plasmids carry resistance markers for ampicillin (ApR) or kanamycin (KmR), the pSC101 origin of replication (ori) from plasmid pLG338 [Stoker et al., Gene 18 (1982) 335-341], and a multiple cloning site (MCS) from plasmids pIBI30 or pSPORT. Full-length env and genomic proviral sequences of EIAV were genetically stable when subcloned into these lcn vectors. Proviral sequences of an SIV clone (pBK28-SIV) that are genetically unstable in the hcn vector pUC18 were also stabilized and remained fully infectious when subcloned into the lcn vector pLG339-SPORT. These lcn vectors appear to be generally useful in stabilizing lentivirus genomic sequences for subcloning, propagation, and manipulation in E. coli, possibly as a result of reducing the level of toxic gene expression from cryptic promoter sequences.
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261.
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Cunningham
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Montelaro
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Rushlow
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015686:Gene Products, env / Q000235:genetics*; D015752:Genes, env; D005822:Genetic Vectors; D016679:Genome, Viral; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics*; D015183:Restriction Mapping; D015302:Simian Immunodeficiency Virus / Q000235:genetics*
D015686:Gene Products, env