Nucleotide sequence adjacent to polyadenylic acid in globin messenger RNA

Proudfoot, Nick J.; Brownlee, George G.

doi:10.1016/0014-5793(74)80108-0

Cited by 30 publications

(7 citation statements)

References 13 publications

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“…This base has been reported to be a guanine residue in both poliovirus and EMC RNAs (11,18). We confirmed these results by determining that, for both RNAs, dCTP alone labeled oligo(dT) in the presence of enzyme (10,19). Hence Fig.…”

Section: Resultssupporting

confidence: 76%

3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors.

Zimmern¹,

Kaesberg²

1978

Proc. Natl. Acad. Sci. U.S.A.

165

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We have adapted the chain-termination method for determining the nucleotide sequence of DNA of Sanger, Nicklen, and Coulson [(1977) Proc. NatL Acad. Sci. USA 74,[5463][5464][5465][5466][5467] for use with reverse transcriptase (RNA-directed DNA nucleotidyltransferase) on RNA templates. With this method and using a primer (the octanucleotide pdT7rC) directed at the 3'-terminal poly(A), we have determined a sequence of 166 residues in the genomic RNA of the picornavirus encephalomyocarditis virus.Techniques for the primary structural analysis of large RNA molecules have improved steadily (1-6), though not as rapidly as in DNA structural analysis after the introduction of restriction enzymes, recombinant DNA technology, and sequence determination on gels. Indeed, two of the recent techniques for determining the nucleotide sequence in RNA simply involve prior conversion into DNA (4-6), and all are ultimately derived from the new DNA technology. One highly successful method for determining the nucleotide sequence in DNA is the "plus and minus" technique of Sanger and Coulson (7), which has been adapted for use on RNA templates by Brownlee and Cartwright (1). Sanger et al. (8) have recently described a second generation rapid method for determining the nucleotide sequence of DNA, relying, like the plus and minus method, on enzymatic synthesis of a complementary strand, and improving on it in speed, accuracy, sensitivity, convenience, and range. The method involves incorporation of chain-terminating deoxynucleoside triphosphate analogues [such as the 2',3'-dideoxy analogues (ddNTPs)] of each base in turn into the reaction mixture, and hence into the growing chain, thus generating four nested sets of fragments each terminated opposite one of the four bases in the template.Since ddNTPs are competitive inhibitors of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) (9, 10), it seemed to us that this technique could also be adapted for use in determining the nucleotide sequence of RNA. We have found that a straightforward adaptation in the spirit of Brownlee and Cartwright yields a powerful technique with which we have been able to determine the nucleotide sequence adjacent to the 3'-terminal poly(adenylic acid) tract in the picornavirus encephalomyocarditis virus (EMC virus). The only major modification necessary was to lower the ddNTP concentration considerably, since, as others have noted (9), reverse transcriptase is much more sensitive to these inhibitors than is Escherichia coli DNA polymerase I. The method offers many of the advantages of its parent DNA technique and promises to be especially useful in sequence analysis of large RNA molecules. A special advantage of direct methods such as this for studying pathogens of higher organisms is that they avoid whatever risks may be associated with techniques involving cloning of complementary DNA in E. coli (6).We chose the poly(A)-adjacent sequences of picornavirus genomic RNAs, particularly that of EMC virus, for development of the RNA dideoxy ...

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Section: Resultssupporting

confidence: 76%

3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors.

Zimmern¹,

Kaesberg²

1978

Proc. Natl. Acad. Sci. U.S.A.

165

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“…Proudfoot and Brownlee have identified a unique nucleotide sequence (AUUGC) adjacent to the poly(A) sequence of rabbit globin mRNA, by sequence analysis of short cDNA transcripts obtained by incubating rabbit globin mRNA with E. coli DNA polymerase I (40).…”

mentioning

confidence: 99%

Nucleotide Sequences of Human Globin Messenger Rna *

Forget

Marotta

Weissman

et al. 1974

Annals of the New York Academy of Sciences

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“…(i) Using DNA polymerase I (E. coli). Rabbit globin mRNA [purified from rabbit reticulocytes as previously described (2)] was copied into 32P-labelled cDNA using subtilisin-treated DNA polymerase I (Boehringer Mannheim Corp., West Germany) in the presence of Mn2+ (8,12) with oligo (dT)10 (P.L. Biochemicals Inc., Wis., U.S.A.) as primer.…”

Section: Materials and Methods (A) Transcription Methodologymentioning

confidence: 99%

“…Proudfoot and Brownlee (1974) (7) used a somewhat different approach to sequence the 3' terminal region of rabbit p-globin mRNA. They obtained short 32P-labelled cDNA using the reverse transcriptase activity of DNA polymerase I (E. coli) (8,9) and directly sequenced this using various DNA sequencing procedures (endonuclease IV digestion (10)(11)(12) and partial exonuclease digestion (13,14) in particular). This approach has been successfully applied to other mRNAs (15-17) as well as to both human (18) and rabbit globin mRNAs (12), so that substantial parts of the 3' non-coding regions of several different eukaryotic mRNAs are now sequenced.…”

Section: Introductionmentioning

confidence: 99%