Showing papers by "Phillip A. Sharp published in 1972"
TL;DR: Asymmetric RNA was synthesized in vitro from SV40 component I DNA using Escherichia coli DNA-dependent RNA polymerase and hybridized to RNA extracted at different stages of lytic infection and to RNA from transformed cells.
169 citations
TL;DR: 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.
Abstract: 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.
140 citations
TL;DR: A population of double-stranded replicative form of DNA molecules from bacteriophage ϕX-174 carrying a deletion of about 9% of the wild-type DNA has been discovered in a sample cultivated under conditions where the phage lysozyme gene is nonessential.
Abstract: A population of double-stranded replicative form of DNA molecules from bacteriophage ϕX-174 carrying a deletion of about 9% of the wild-type DNA has been discovered in a sample cultivated under conditions where the phage lysozyme gene is nonessential. The structures of deleted monomers, dimers, and trimers were studied by the electron microscope heteroduplex method. The dimers and trimers are head-to-tail repeats of the deleted monomers. Some interesting examples of the dynamical phenomenon of branch migration in vitro have been observed in heteroduplexes of deleted dimer and trimer strands with undeleted monomer viral strands from the wild-type phage.
33 citations
TL;DR: Direct physical evidence confirming the Campbell model for the structure of prophage λ was obtained by observing the structure in the electron microscope of a heteroduplex between one strand of an episome, F450(λ ++ ), bearing a λ prophages and the complementary strand of λ b 5 DNA.
12 citations