Home LiteratureArticle Details
PMID: 6207484 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.

Nucleic acids research ·Vol. 12 ·No. 18 ·1984-09-25 ·Pages 7057-70

Krieg PA, Melton DA

Abstract

We describe a method for the synthesis of microgram quantities of eucaryotic messenger RNAs. Injection into the cytoplasm of frog oocytes and addition to wheat germ extracts show that these synthetic RNAs function efficiently as messenger RNAs. We confirm that a 5' cap on the mRNA is essential for translation in injected oocytes and show that most of the 3' flanking region, including the poly A tail, can be deleted without the abolition of protein synthesis. The method of mRNA synthesis involves in vitro transcription of cDNAs which have been cloned into SP6 vectors (described in the accompanying paper). This method enables one to produce large amounts of mRNA and consequently protein from any cDNA clone.

MeSH Terms
Animals Cloning, Molecular DNA/metabolism Female Globins/genetics Interferons/genetics Oocytes/metabolism Plants/metabolism Protein Biosynthesis RNA, Messenger/genetics Salmonella Phages/genetics Salmonella typhimurium/genetics Transcription, Genetic Triticum/metabolism Xenopus
Chemicals
RNA, Messenger Globins DNA Interferons
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Krieg P A
Melton D A
References (31)
31 references, click to expand
  1. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  2. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells.
    Nature. 1971 Sep 17;233(5316):177-82 PMID: 4939175
  3. Message stability in injected frog oocytes: long life of mammalian alpha and beta globin messages.
    J Mol Biol. 1973 Nov 5;80(3):539-51 PMID: 4796973
  4. Role of the polyadenylate segment in the translation of globin messenger RNA in Xenopus oocytes.
    Proc Natl Acad Sci U S A. 1974 Aug;71(8):3143-6 PMID: 4528715
  5. The degradation of ribonucleic acids injected into Xenopus laevis oocytes.
    Cell. 1974 Jul;2(3):189-96 PMID: 4370441
  6. Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system.
    Proc Natl Acad Sci U S A. 1975 May;72(5):1922-6 PMID: 168582
  7. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.
    Eur J Biochem. 1975 Aug 15;56(2):335-41 PMID: 1175627
  8. Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.
    J Biol Chem. 1975 Dec 25;250(24):9322-9 PMID: 1194286
  9. Readenylation of polyadenylate-free globin messenger RNA restores its stability in vivo.
    Eur J Biochem. 1975 Nov 15;59(2):589-92 PMID: 1107033
  10. Processing of RNA.
    Annu Rev Biochem. 1976;45:605-29 PMID: 786158
  11. Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal.
    J Embryol Exp Morphol. 1976 Dec;36(3):523-40 PMID: 1010977
  12. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells.
    Cell. 1977 Jan;10(1):67-78 PMID: 837445
  13. Synthesis of human interferon by Xenopus laevis oocytes: two structural genes for interferons in human cells.
    Proc Natl Acad Sci U S A. 1977 Aug;74(8):3287-91 PMID: 269391
  14. Purification of mRNA guanylyltransferase from vaccinia virions.
    J Biol Chem. 1978 Jun 25;253(12):4481-9 PMID: 659428
  15. Requirement for 7-methylguanosine in translation of globin mRNA in vivo.
    Nucleic Acids Res. 1978 Sep;5(9):3237-47 PMID: 212716
  16. The 3' noncoding region of beta-globin mRNA is not essential for in vitro translation.
    Nucleic Acids Res. 1979 Jan;6(1):153-66 PMID: 424286
  17. Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure.
    Nature. 1979 Jun 21;279(5715):692-6 PMID: 450118
  18. Export of proteins from oocytes of Xenopus laevis.
    Cell. 1979 Jul;17(3):517-26 PMID: 476830
  19. Transcription units for mRNA production in eukaryotic cells and their DNA viruses.
    Prog Nucleic Acid Res Mol Biol. 1979;22:327-53 PMID: 523634
  20. Human leukocyte and fibroblast interferons are structurally related.
    Nature. 1980 Jun 19;285(5766):547-9 PMID: 6157095
  21. The nucleotide sequence of the major beta-globin mRNA from Xenopus laevis.
    Nucleic Acids Res. 1980 Sep 25;8(18):4247-58 PMID: 7433108
  22. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.
    Proc Natl Acad Sci U S A. 1980 Oct;77(10):5794-8 PMID: 6777776
  23. The mechanism for increased protein synthesis during Xenopus oocyte maturation.
    Dev Biol. 1982 Jan;89(1):159-67 PMID: 7033020
  24. Differential capacity for translation and lack of competition between mRNAs that segregate to free and membrane-bound polysomes.
    Cell. 1981 Nov;27(1 Pt 2):183-91 PMID: 7198941
  25. Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme.
    J Biol Chem. 1982 May 25;257(10):5772-8 PMID: 7040372
  26. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei.
    Cell. 1983 Mar;32(3):681-94 PMID: 6550524
  27. The complete nucleotide sequence of the major adult beta globin gene of Xenopus laevis.
    J Biol Chem. 1983 Jul 25;258(14):8521-3 PMID: 6305990
  28. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems.
    Cell. 1983 Oct;34(3):759-66 PMID: 6313206
  29. Formation of the 3' end of histone mRNA by post-transcriptional processing.
    Nature. 1984 Mar 8-14;308(5955):203-6 PMID: 6700722
  30. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.
    Cell. 1984 Apr;36(4):993-1005 PMID: 6323033
  31. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.
    Cell. 1984 Aug;38(1):317-31 PMID: 6088074
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/12.18.7057
Published
1984-09-25
Pages
7057-70
Language
English
Region
England
NLM ID
0411011
PMCID
PMC320142
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/12.18.7057 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2