Mapping of the three species of polyoma mRNA.

Late in the lytic cycle of simian virus 40 (SV40) there are two distinct classes of viral cytoplasmic RNA that sediment in sucrose gradients at 19 S and 16 S (1, 2). The 19S class includes a low level of 19S early RNA species and a majority of true 149S late RNA species. The early RNA species is complementary to only one of the two strands (the "E" strand), while the late 16S and 19S mRNA species are complementary to the other (the "L" strand) (3, 4). The late RNA species have been mapped by hybridization with SV40 Hind fragments (5, 6). However, by this approach the positioning of the 5' end of 19S late mRNA was uncertain. The topography of polyoma virus mRNA molecules appears to be very similar (7,8). Several investigators have suggested that the map position of the 5' end of early and late mRNA coincides with the site for initiation of DNA synthesis [0.67 map unit (9)] (10, 11), and that this may have some possible biologic significance.Recently a technique has been developed by Thomas et al. (12) [3H]mRNA (about 0.5 ,ug/ml) were incubated for 28 hr in hybridization medium. The final composition of this medium was 65% formamide, 2 X SSC, (0.3 M NaCI, 0.03 M Na citrate), 0.1% sodium dodecyl sulfate, and 0.01 M TES [TES = N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid; Calbiochem, A Grade] pH 7.4. The 100% formamide that was used to prepare hybridization medium was repurified (17).Electron Microscopy. Immediately after hybridization, the nucleic acids were precipitated with ethanol. The pellet was redissolved in 10 mM Tris, 1 mM EDTA, pH 7.5, for electron microscopy analysis, which was performed as described by Westphal et al. (13).Isopycnic Centrifugation in Cs2SO4 was performed in 4.5 ml of a solution of Cs2SO4 (P20 = 1.554 g/cm3) in 10 mM Tris,

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confidence: 99%

Mapping of transcription sites of simian virus 40-specific late 16S and 19S mRNA by electron microscopy.

May¹,

Maizel²,

Salzman³

1977

“…1) gives rise to "giant" RNA molecules up to 3-4 times the size of the viral genome (2)(3)(4)(5). These giant polyoma-specific RNAs are limited to the nucleus (6) where transcription takes place; viral mRNAs in the cytoplasm are one-quarter to one-half the size of the viral DNA (6)(7)(8). Most late nuclear polyoma-specific RNA is transcribed from the L strand of viral DNA (9); all sequences of the L strand are represented in late nuclear transcripts (10,11).…”

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confidence: 99%

Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

Acheson¹

1978

ABSTRACr The bulk of late virusspecific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of polyoma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the Hindu-i fragment of polyoma DNA, which contains 91% of the viral genome. Hybridimolecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in Hind]-i), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.Transcription of polyoma DNA during the late phase of productive infection (for review, see ref. 1) gives rise to "giant" RNA molecules up to 3-4 times the size of the viral genome (2-5). These giant polyoma-specific RNAs are limited to the nucleus (6) where transcription takes place; viral mRNAs in the cytoplasm are one-quarter to one-half the size of the viral DNA (6-8). Most late nuclear polyoma-specific RNA is transcribed from the L strand of viral DNA (9); all sequences of the L strand are represented in late nuclear transcripts (10,11).To try to understand the mechanism by which these giant RNA molecules are synthesized, I have hybridized this RNA with a fragment (HindII-l) of polyoma DNA consisting of 91% of the genome (12,13) (Amersham,25 Ci/mmol) per culture dish in 1 ml of reinforced Eagle's medium. Cells were scraped into cold 10 mM triethanolamine, pH 8.5/10 mM NaCl/1.5 mM MgCl2 and lysed by addition of Nonidet P40 (Shell) to 0.5% final concentration. Nuclei were pelleted and resuspended in the same buffer. RNA was extracted by addition of 1 vol of 10 mM triethanolamine (pH 8.5)/100 mM NaCl/10 mM EDTA/4% triisopropylnaphthylenesulfonate (Serva) (14, 15) and 2 vol of phenol/chloroform/isoamyl alcohol, 50:50:1 (vol/vol). After three successive extractions at room temperature, nucleic acids in the aqueous phase were precipitated three times in succession with ethanol and then incubated at 00 for 20 min with DNase I (RNase-free; Worthington; 10,Mg/ml) in 10 mM Na acetate, pH 5.1/100 mM NaCI/2 mM MnCI2. RNA was extracted with phenol/chloroform in the presence of 1% sodium dodecyl sulfate (NaDodSO4) and ethanol-precipitated two times.Preparative Hybridization of RNA to Polyoma DNA. Polyoma form I DNA was denatured and bound to nitr...

“…By contrast, the polyadenylylated cytoplasmic viral mRNAs are complementary to only one-half of the sequences of each DNA strand (17,18). Therefore, regulation of the final sequence composition of the viral mRNA seems to be accomplished at the post-transcriptional level.…”

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confidence: 98%

“…

ABSTRACTThe (17,18). Therefore, regulation of the final sequence composition of the viral mRNA seems to be accomplished at the post-transcriptional level.

…”

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confidence: 99%

Transcription of a defective polyoma virus genome.

Condit¹,

Cowie²,

Kamen³

et al. 1978

The (17,18). Therefore, regulation of the final sequence composition of the viral mRNA seems to be accomplished at the post-transcriptional level. (19)(20)(21). The 17% of the wild-type DNA sequence that is repeated in D-50 molecules, which we shall refer to as the D-50 "subunit" (see Fig. 1), includes the origin of viral DNA replication at 71 map units (22,23). Previous experiments have suggested that the 5' ends of both early and late viral mRNAs map near the replication origin (17). MATERIALS AND METHODSVirus. Plaque-purified wild-type polyoma virus of the A-2 strain (22) was grown at low multiplicity and checked for the absence of defectives as described (17,19). The D-50 virus has been described previously (19)(20)(21) Purification of Transcription Complexes. Sixty 90-mm dishes of the 3T6 line of mouse fibroblasts, grown to a density of 5 X 106 cells/dish, were infected with either 50 PFU of wild-type polyoma virus per cell as described previously (4) or with an amount of D-50 virus stock that contained 10 PFU. After 30-hr incubation at 370, transcription complex was purified from the infected cells through fraction III, and stored in aliquots in liquid N2 as described previously (4). The total yield of wild-type and defective virus DNA in each of two Abbreviations: SV40, simian virus 40; PFU, plaque-forming units.