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Plasmid proviral molecular clones of the LAI isolate of HIV-1 and the ROD isolate of HIV-2 were originally prepared in moderate copy number plasmids derived from pBR322, which contains the colE1 origin of replication. In these plasmid vectors, the HIV sequences are stable to continuous passage in bacteria. However, when the colE1 origin was replaced by the mutant origin from pUC18, pGEM, or pBluescript plasmids, which replicates to much higher copy numbers in bacteria, then deletions of HIV sequences occurred even in RecA defective strains. Deletions occurred in two different media, at room temperature and 37 degrees C, and with or without plasmid amplification in the presence of chloramphenicol. These results raise a cautionary note when cloning immunodeficiency viral sequences into plasmid vectors containing a high copy number origin of replication. |
