Nucleotide sequences of 5' termini of adenovirus 2 early transforming region Ela and Elb messenger ribonucleic acids

Hashimoto, Shuichi; Wold, William S.M.; Brackmann, Karl H.; Green, Maurice

doi:10.1021/bi00526a019

Cited by 10 publications

(14 citation statements)

References 48 publications

(53 reference statements)

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“…In previous studies, we have purified mRNA specific to each Ad2 early gene region and characterized the 5'-termini of the purified mRNA. We observed heterogeneous 5'-termini in EIA, E1B, and E4, and we were able to identify "major" termini from each region which we sequenced and mapped on the Ad2 DNA genome (13)(14)(15). Similar results were reported by Baker and Ziff (16).…”

Section: Introductionsupporting

confidence: 82%

“…Ad2 DNA and restriction fragments Ad2 DNA was isolated and EcoRI restriction fragments were prepared as described (22). In vitro labeling of 5'-termini of poly(A ) RNA The 5'-terminal m7Gppp in poly(A ) RNA from uninfected or Ad2 early infected cells was removed by periodate oxidation, 1-elimination, and phosphatase treatment (15). To remove m7Gppp efficiently from poly(A ) RNA, these treatments were repeated twice.…”

Section: Cell Fractionation and Isolation Of Poly(a ) Rnamentioning

confidence: 99%

“…To remove m7Gppp efficiently from poly(A ) RNA, these treatments were repeated twice. The mRNA molecules with m7Gppp removed were labeled at the new 5'-terminal Nm residues with [y-32P]ATP (1000 Ci/ mmol) and polynucleotide kinase (15).…”

Section: Cell Fractionation and Isolation Of Poly(a ) Rnamentioning

confidence: 99%

“…The 5'-terminal nucleotides of hybridization purified, Ad2 early region mRNA were identified by nuclease P1 digestion of 5'-32P-labeled RNA and two dimensional thin layer chromatography (2D-TLC) as described (15). Purified mRNA was digested in 10 pl of 20 mM sodium acetate, pH 5.3, containing 50 pg of carrier tRNA, with 2 pg of nuclease P1 (Boehringer Mannheim) at 370 for 2h, mixed with pNm markers (pA m,pm Am,pCmpUm,pGm, 0.2 A260 units each, rnm P-L Biochemicals), and applied to cellulose TLC plates (10 x 10 cm, EM Laboratories, Inc.).…”

Section: Nucleic Acids Researchmentioning

confidence: 99%

“…Purified mRNA was digested in 10 pl of 20 mM sodium acetate, pH 5.3, containing 50 pg of carrier tRNA, with 2 pg of nuclease P1 (Boehringer Mannheim) at 370 for 2h, mixed with pNm markers (pA m,pm Am,pCmpUm,pGm, 0.2 A260 units each, rnm P-L Biochemicals), and applied to cellulose TLC plates (10 x 10 cm, EM Laboratories, Inc.). 2D-TLC was performed with isobutyric acid/0.5 N NH40H (5:3) in the first dimension and isopropyl alcohol/concentrated HCl/H20 (70:15:15) in the second dimension (15 (14). 32pAGp was prepared by digestion of 5'-32P-labeled RNA with RNase TI and purification by 2D-homochromatography (24).…”

Section: Nucleic Acids Researchmentioning

confidence: 99%

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Unusual heterogeneity of the 5′-termini of human adenovirus type 2 early region E2 mRNA

Hashimoto

Green

1984

Nucl Acids Res

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The 5'-terminal structures of human adenovirus type 2 (Ad2) early region 2 (E2) mRNA were investigated. The E2 transcription unit has several interesting properties, including the presence of a TATA-like box that matches the consensus sequence poorly, delayed transcription during early stages of infection, and a switch in promoter recognition late after infection. E2-specific RNA, 5'-labeled in vitro to high specific activity was analyzed. Purified E2 mRNA was digested with RNase A or RNase T1 and the resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Remarkably, as many as sixteen 5'-terminal RNase A oligonucleotides were identified and their sequences were deduced. The most common 5'-termini in the RNase A digest were p(m6)AmCp, p(m6)AmA(m)Cp, pGmA(m)Cp, and p(m6)AmG(m)Cp. Two RNase A oligonucleotides originated from the E4 promoter region, consistent with electron microscopic observations. The sequence encoding these potential initiation sites covered about 90 nucleotides. Eleven of the sequences of the 5'-terminal RNase A oligonucleotides were aligned with the Ad2 DNA sequence in the Ad2 E2 promoter region. If the heterogeneous termini in the E2 promoter region were generated by a process of transcription initiation, their existence cannot be explained by stuttering of RNA polymerase II. This suggests that the transcription of Ad early region 2 has features which differ from those of other Ad2 early gene transcription units. Perhaps this is due to the absence of a conventional TATA box which is believed to position the initiation site. Alternatively, it is conceivable that the E2 promoter represents an alternate class of RNA polymerase II promoters containing different signals with different requirements for activation and/or that an E1A gene product modifies transcription initiation.

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Section: Introductionsupporting

confidence: 82%

Section: Cell Fractionation and Isolation Of Poly(a ) Rnamentioning

confidence: 99%

Section: Cell Fractionation and Isolation Of Poly(a ) Rnamentioning

confidence: 99%

Section: Nucleic Acids Researchmentioning

confidence: 99%