Faculty Bibliography

Single-stranded, linear DNA of simian virus 40 (SV40) created by denaturing the endonuclease EcoRI- or Hpa II-generated, linear, double-stranded products from form I DNA of SV40 was analyzed for regions of inverted repeated sequences by visualization with the electron microscope. Six hairpin loops were found at positions 0.11-0.30 (two loops forming a 'rabbit ears' structure), 0.47-0.52, 0.63-0.68, 0.70-0.76, and 0.90-0.96. The nucleotide sequences within all of these inverted repeats may be related since the looped regions can crosshybridize with one another and, thus, the SV40 genome may contain regions of interspersed repeated and unique sequences. The map positions of the 3' and 5' ends of the early and late messenger RNAs, as determined by others, lie within regions of inverted repeated sequences. Previously recorded recombination events that occurred either within the SV40 genome or between SV40 DNA and other genomes have apparently occurred frequently at positions of inverted repeated sequences within the SV40 DNA

Nuclei isolated from HeLa cells 15 hr after infection with Ad2 synthesize an RNA transcript approximately 25 KB long, beginning between 0.2 and 0.3 on the physical map and extending to (or close to) the right end of the genome. The majority (60-92%) of the RNA transcribed from fragments between 0.2 and 1.0 is synthesized as part of this large molecule which represents the major product of late Ad2 RNA synthesis. Smaller transcripts are also formed mainly from the left end of the Ad2 genome.

The smallest size class of mRNA (12S) synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed.

The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.

Poly(A)-containing HeLa cell mRNA prepared from cells labeled with [methyl-(3)H]methionine or [(32)P]phosphate was found to contain a variety of methylated, blocked 5'-terminal structures of two general types: m(7)GpppN(7)-Np and m(7)GpppN(m)-N(m)-Np. In addition, about one-third of the [(3)H]methyl label was present in the N(6)-methyladenosine; this labeled nucleoside was not found in the 3'-terminal one-third of the mRNA chain and thus may also be in the 5' portion of the mRNA.