Sequence heterogeneity in closed simian virus 40 deoxyribonucleic acid.
The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002499:Centrifugation, Density Gradient; D004277:DNA, Single-Stranded; D004279:DNA, Viral / Q000032:analysis* / Q000145:classification / Q000302:isolation & purification; D003851:Deoxyribonucleases; D005559:Formamides; D000882:Haplorhini; D007668:Kidney; D008854:Microscopy, Electron; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D013539:Simian virus 40 / Q000032:analysis* / Q000254:growth & development / Q000302:isolation & purification; D014776:Virus Cultivation
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005559:Formamides; D003851:Deoxyribonucleases