Deletion Mutants Of Simian Virus Research Articles

Viable deletion mutants of simian virus 40: selective isolation by means of a restriction endonuclease from Hemophilus parainfluenzae.

Resistance of simian virus 40 (SV40) DNA to cleavage by Hemophilus parainfluenzae II (HpaII) restriction endonuclease has been used as a positive, in vitro selection for mutants lacking the one HpaII endonuclease-cleavage site of wild-type SV40 DNA. Each of 10 viable mutants isolated by this procedure multiplies significantly more slowly than wild-type virus and contains a small deletion (80 to 190 base pairs in size) of the region of the genome that includes the HpaII endonuclease-recognition sequence. These well-defined mutants, having a selective disadvantage for growth, would not have been readily obtained by conventional methods used to screen for viral mutants. Therefore, in certain circumstances, restriction endonucleases are effective reagents for the selection of new classes of mutants. Because these small deletions can be visualized in heteroduplexes, these mutants provide internal markers for mapping other alterations or features of the simian virus 40 genome.

Defective simian virus 40 genomes: Isolation and growth of individual clones

Eleven defective deletion mutants of simian virus 40 (SV40) have been isolated. The methodology developed for this purpose involves the use of temperature-sensitive mutants as helpers. Included are procedures for screening for the presence of mutants, plaque-purifying, propagating, and assaying the titer of stocks of these mutants, and assigning mutants to complementation groups. These same procedures may be adaptable to enable isolation of deletion mutants of other animal viruses as well.

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40 † † Abbreviations used: SV40, simian virus 40; DNA form I, closed circular SV40 DNA; DNA form II, open circular SV40 DNA; endo R ·. Hind, Hpa, Eco, restriction endonucleases from H. influenzae strain d, H. parainfluenzae and Escherichia coli, respectively; dl, deletion mutant; ts, temperature-sensitive mutants. . For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.