Topography of polyoma virus-specific giant nuclear RNA molecules containing poly(A) sequences.

The distribution of viral RNA sequences with respect to poly(A) tracts has been determined for giant polyoma virus-specific nuclear RNA molecules. Polyadenylated nuclear RNA sedimenting faster than 30 S was isolated from polyoma virus-infected cells and cleaved into fragments of various sizes by partial alkaline hydrolysis. Fragments containing poly(A) were separated from nonpolyadenylated RNA. The viral sequence composition of polyadenylated or nonpolyadenylated fragments, and of total polyadenylated giant RNA, was determined by hybridization to ³²P-labeled separated strands of specific restriction endonuclease fragments of polyoma virus DNA. The results of this analysis showed that in all polyadenylated giant RNA molecules transcribed from the L DNA strand the portion adjacent to the poly(A) segment hybridizes to the 3′-terminal half of the late region of the viral genome. The next portion of these chains hybridizes to the remainder of the late region, the more distal part hybridizes to the early region, and the portions furthest from the poly(A) contain sequences complementary to the L DNA strand of both early and late regions. This finding shows that polyadenylation of Py giant nuclear RNA is non-random and further suggests that the poly(A)-linked RNA sequence(s) maps within the same region of the viral genome as the poly(A)-linked sequence found in mature cytoplasmic mRNA.     

Adenovirus transcription: III. Mapping of viral RNA sequences in cells productively infected by adenovirus type 5

Separated strands of restriction endonuclease Eco R1 and Hpa 1 fragments of ³²P-labeled Adenovirus 5 DNA have been used in saturation hybridization experiments with cytoplasmic RNA extracted from human cells infected with Adenovirus type 5. The results of such experiments have allowed the construction of maps of the regions of the viral genome complementary to both “early” and “late” Adenovirus 5 RNA. At early times during the lytic cycle, about 25% of the viral genome is expressed as mRNA. Adenovirus 5 DNA sequences complementary to “early” mRNA comprise four discrete regions, two on each strand, of the viral genome: except in one instance, these correspond to the regions of the Adenovirus 2 genome complementary to “early” Adenovirus 2 mRNA. In the exceptional case, “early” Adenovirus 5 mRNA appears to contain not only sequences corresponding, at least in position, to those expressed in Adenovirus 2-infected cells, but also some additional, adjacent sequences, reminiscent of the situation in human cells infected with the Adenovirus 2/Simian virus 40 hybrid viruses, Ad2⁺ND₁ and Ad2⁺ND₃ (Flint et al., 1975a).All, or almost all, the information encoded by the Adenovirus 5 genome is expressed as mRNA during the lytic cycle and most of the exclusively “late” mRNA is complementary to the r strand of Adenovirus 5 DNA. The separated strands of fragments of ³²P-labeled Adenovirus 2 DNA generated by the restriction endonuclease Bam Hl have been used to improve the resolution of maps of both Adenovirus 2 and Adenovirus 5 “late” mRNA, which are described here.     

Human cytomegalovirus DNA: restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs

Complete physical maps for human cytomegalovirus (Davis strain) DNA were constructed from fragments produced by restriction endonucleases HindIII, XbaI, and EcoRI. The results showed that the human cytomegalovirus genome has a structural organization similar to that of the herpes simplex virus genome: a long (L) segment, comprising 82% of the genome, joined to a short (S) segment, comprising the remaining 18% of the genome. Permutations of the S and L segments produce four different molecular arrangements which are represented in equimolar proportions in virion DNA preparations. Heterogeneity in the size of the DNA was detected within fragments which map at one terminus of the S segment and also in fragments which map at one terminus of the L segment. The regions of the genome from which RNA synthesized at various times after infection originates was determined. Immediate-early RNA (RNA synthesized in the presence of cycloheximide) was restricted to a few regions on the genome but hybridized in abundance to a region which mapped between 0.686 and 0.733 units (within the L segment). Early RNA (synthesized in the absence of viral DNA synthesis) hybridized to most areas of the genome, but in particular abundance to a small region which mapped between 0 and 0.046 units (also within the L segment). RNA which accumulated during the late phase of infection was not characterized by particular abundances to any region of the genome.