Strand Orientation of Simian Virus 40 Transcription in Productively Infected Cells

Small amounts of fractionated, denatured, 32P-labeled DNA /Ci/ml), as well as fresh phosphate-free Eagle's medium, were added 24-30 hr after infection. Virus was prepared (6) from cells and medium 7-8 days later. SV40 DNA I was isolated from purified virus after it was treated with 1% sodium dodecyl sulfate for 30 min at 500 and centrifuged isopycnically in CsCl in the presence of ethidium bromide (200,ug/ml) (7,3). Labeled DNA preparations had specific activities from 0.7 to 2.6 X 101 cpm/,ug. The SV40 [32P]DNA I was mechanically sheared (50,000 psi) in a Ribi Cell Fractionator to a molecular size of about 3.1 X 105 daltons (4).

Section: Discussionsupporting

confidence: 81%

Patterns of Simian Virus 40 DNA Transcription after Acute Infection of Permissive and Nonpermissive Cells

Khoury

Byrne

Martin

1972

“…One class is present at both early and late times after infection and is complementary to about 30-35% of the E, or (-), strand of viral DNA. The second class, which becomes detectable only after SV40 DNA synthesis has begun, is com-plementary to about 65-70%o of the sequences of the L, or strand (1)(2)(3).…”

Section: Introductionmentioning

confidence: 99%

Transcription of Simian Virus 40. III. Mapping of “Early” and “Late” Species of RNA

Sambrook

Sugden

Keller

et al. 1973

To determine the orientation of transcription of the E and L strands of DNA from simian virus 40 (SV40), we used linear DNA prepared by cleavage of superhelical viral DNA by endonuclease RARE from Escherichia coli as a primer, template for DNA polymerase. The resulting molecules, which were labeled only at the 3' end of each DNA strand, were then cleaved with Hemophilus parainfluenzae endonuclease Hpa I. The ensuing four DNA fragments, whose locations on the viral genome are known, were separated by electrophoresis, denatured, and hybridized to asymmetric SV40 complementary RNA. From the pattern of hybridization of the fragments containing the labeled 3' ends, we conclude that transcription of SV40 proceeds in a clockwise direction on the L strand and in a counterclockwise direction on the E strand as drawn on the conventional SV40 map.To map the "early" and "late" regions of the viral genome, we extracted RNA from lytically infected cells and hybridized it to the separated strands of the four fragments of 32P-labeled SV40 DNA. Early after infection, RNA complementary to part of the E strand of the contiguous fragments A and C was detected. Late polysomal RNA was complementary to part of the L strand sequences of fragments A and C and to the total L strand sequences of fragments B and D. During infection of permissive cells by simian virus 40 (SV40), two distinct classes of virus-specific, stable RNA are-synthesized. One class is present at both early and late times after infection and is complementary to about 30-35% of the E, or (-), strand of viral DNA. The second class, which becomes detectable only after SV40 DNA synthesis has begun, is complementary to about 65-70%o of the sequences of the L, orTo analyze the pattern of viral transcription in lytically infected cells in more detail, we have used specific fragments of viral DNA. The restricting endonuclease R * R1, isolated from Escherichia coli (4), cleaves SV40 DNA at a unique site (5-7) by making single-strand scissions that are four bases apart (8), thereby producing cohesive ends (9). Hemophilus parainfluenzae (10) also contains restriction activities (10) that cleave SV40 DNA (11,12 on the SV40 genome is known (11, 13). We have used the fragments to analyze the orientation of RNA synthesis relative to the R * R1 cleavage site on SV40 DNA and to identify the regions of the SV40 genome that generate the "early" and "late" classes of viral RNA. MATERIALS AND METHODSEnzymes. Endonuclease R -R1 was prepared from E. coli strain RY-13 as described elsewhere (8). Endonculease Hpa I was prepared from H. parainfluenzae as described previously (13). DNA polymerase from avian myeloblastosis virus (kindly provided by Drs. Dorothy and Joseph Beard, Duke University), was purified as outlined previously (15).Preparation of 32P-labeled SV40 DNA. 82P-Labeled SV40 DNA (specific activity 5 X 105 cpm/Mug) was prepared as described previously (13).Isolation of Specific Fragments of SV40 DNA. Ten micrograms of 82P-labeled SV40 component I DNA was converted to linear...

“…The homology distribution of SV40 cRNA.-I between heavy and light strands of Ad2+ND1 is shown in Table 1B. Only the light strand has significant homology with this RNA, and the very small amount of hybridization exhibited by the heavy strand most likely arises from a small amount of symmetrical transcription during in vitro synthesis of the cRNA (9). This result confirms that the two DNA strands are well separated and, in addition, serves to identify the SV40 moiety present in the light strand of Ad2+ND, as a portion of thestrand of SV 40 DNA (early RNA template) (13 tidine (8).…”

mentioning

confidence: 99%

“…The entire genome is transcribed after viral DNA replication has begun., so that the RNA synthesized late in. infection conlists of both early and late sequences (early-plus-late RNA) (7)(8)(9). Although the strands of SV40 cannot be sel)arated with synthetic polyribonucleotides (10), Westphal (11) demonstrated that su)erhelical SV40 DNA (Form I) is transcribed asymmetrically (from one strand) by E. coli RNA polymerase in vitro.…”

mentioning

confidence: 99%

Evidence for a Transcription-Control Region of Simian Virus 40 in the Adenovirus 2—Simian Virus 40 Hybrid, Ad2 ⁺ ND ₁

Patch

Lewis

Levine

1972

The complementary DNA strands of the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2+ND,, were separated by isopycnic banding in a CsCl density gradient in the presence of synthetic polyribonucleotides. Separated strands were used in DNA-RNA hybridization reactions with RNA from cells productively infected by Ad2 or SV40, and with complementary SV40 RNA transcribed asymmetrically in vitro. About five times as much Ad2 RNA hybridized to the SV40 cRNA-I hybridized with only one strand (the strand) which was selcarated from the unhybridized strand (the "-+" strand) by hydroxyapatite chromatography.Using seIzarated strands, Khoury et al. have shown that about one-third of the -strand of SV40 DNA is transcribed before viral )-AN synithesis begins, while none of the + strand is transcribed during the early phase of infection. On the other hand, the RNA transcribed in the late phase of infection is homologous with one-third of the -strand and two-thirds of the + strand. Thus, the early DNA template occul)ies about one-third of the -strand and the late D)NA template occupies about two-thirds of the + strand.The nondefective Ad2-SV40 hybrid virus Ad2+NDj (14) contains about 10