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. 1981 Sep 25;9(18):4485–4494. doi: 10.1093/nar/9.18.4485

Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing.

P H Hamlyn, M J Gait, C Milstein
PMCID: PMC327452  PMID: 6170937

Abstract

The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent for comparative studies of mouse k-chain mRNAs because they can be made on impure mRNA preparations. The MOPC 21 light chain mRNA is 943 nucleotides in length excluding the poly(A) region. An unexpected finding was that there are only three bases in the 5' non-coding region and its significance in terms of ribosome binding is discussed; 87 code for the precursor or leader sequence of the protein, 642 for the mature protein and 211 for the 3' non-coding region. The codons for the precursor region allows the previously undetermined amino acid sequence to be predicted. In common with other precursor regions a high proportion of the predicted amino acids are hydrophobic.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baralle F. E., Brownlee G. G. AUG is the only recognisable signal sequence in the 5' non-coding regions of eukaryotic mRNA. Nature. 1978 Jul 6;274(5666):84–87. doi: 10.1038/274084a0. [DOI] [PubMed] [Google Scholar]
  2. Baralle F. Complete nucleotide sequence of the 5' noncoding region of human alpha-and beta-globin mRNA. Cell. 1977 Dec;12(4):1085–1095. doi: 10.1016/0092-8674(77)90171-4. [DOI] [PubMed] [Google Scholar]
  3. Brownlee G. G., Cartwright E. M. Rapid gel sequencing of RNA by primed synthesis with reverse transcriptase. J Mol Biol. 1977 Jul;114(1):93–117. doi: 10.1016/0022-2836(77)90285-6. [DOI] [PubMed] [Google Scholar]
  4. Cheng C. C., Brownlee G. G., Carey N. H., Doel M. T., Gillam S., Smith M. The 3' terminal sequence of chicken ovalbumin messenger RNA and its comparison with other messenger RNA molecules. J Mol Biol. 1976 Nov 15;107(4):527–547. doi: 10.1016/s0022-2836(76)80081-2. [DOI] [PubMed] [Google Scholar]
  5. Dunnick W., Rabbitts T. H., Milstein C. A mouse immunoglobulin heavy chain deletion mutant: isolation of a cDNA clone and sequence analysis of the mRNA. Nucleic Acids Res. 1980 Apr 11;8(7):1475–1484. doi: 10.1093/nar/8.7.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gait M. J., Sheppard R. C. Rapid synthesis of oligodeoxyribonucleotides. III. Effect of added carboxylate ion on the efficiency of internucleotide bond formation. Solid-phase synthesis of the dodecanucleotide, d(pT-A-A-C-T-G-C-T-C-A-C-T). Nucleic Acids Res. 1979 Apr;6(4):1259–1268. doi: 10.1093/nar/6.4.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gait M. J., Singh M., Sheppard R. C., Edge M. D., Greene A. R., Heathcliffe G. R., Atkinson T. C., Newton C. R., Markham A. F. Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates. Nucleic Acids Res. 1980 Mar 11;8(5):1081–1096. doi: 10.1093/nar/8.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hamlyn P. H., Browniee G. G., Cheng C. C., Gait M. J., Milstein C. Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing. Cell. 1978 Nov;15(3):1067–1075. doi: 10.1016/0092-8674(78)90290-8. [DOI] [PubMed] [Google Scholar]
  9. Hamlyn P. H., Gillam S., Smith M., Milstein C. Sequence analysis of the 3' non-coding region of mouse immunoglobulin light chain messenger RNA. Nucleic Acids Res. 1977 Apr;4(4):1123–1134. doi: 10.1093/nar/4.4.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harrison T. M., Brownlee G. G., Milstein C. Studies on polysome-membrane interactions in mouse myeloma cells. Eur J Biochem. 1974 Sep 16;47(3):613–620. doi: 10.1111/j.1432-1033.1974.tb03733.x. [DOI] [PubMed] [Google Scholar]
  11. Hefti E., Bishop D. H., Dubin D. T., Stollar V. 5' nucleotide sequence of sindbis viral RNA. J Virol. 1975 Jan;17(1):149–159. doi: 10.1128/jvi.17.1.149-159.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  13. Lewin B. Alternatives for splicing: recognizing the ends of introns. Cell. 1980 Nov;22(2 Pt 2):324–326. doi: 10.1016/0092-8674(80)90340-2. [DOI] [PubMed] [Google Scholar]
  14. Malek L. T., Eschenfeldt W. H., Munns T. W., Rhoads R. E. Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid. Nucleic Acids Res. 1981 Apr 10;9(7):1657–1673. doi: 10.1093/nar/9.7.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Max E. E., Seidman J. G., Leder P. Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3450–3454. doi: 10.1073/pnas.76.7.3450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Max E. E., Seidman J. G., Miller H., Leder P. Variation in the crossover point of kappa immunoglobulin gene V-J recombination: evidence from a cryptic gene. Cell. 1980 Oct;21(3):793–799. doi: 10.1016/0092-8674(80)90442-0. [DOI] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McReynolds L., O'Malley B. W., Nisbet A. D., Fothergill J. E., Givol D., Fields S., Robertson M., Brownlee G. G. Sequence of chicken ovalbumin mRNA. Nature. 1978 Jun 29;273(5665):723–728. doi: 10.1038/273723a0. [DOI] [PubMed] [Google Scholar]
  19. Milstein C., Brownlee G. G., Cartwright E. M., Jarvis J. M., Proudfoot N. J. Sequence analysis of immunoglobulin light chain messenger RNA. Nature. 1974 Nov 29;252(5482):354–359. doi: 10.1038/252354a0. [DOI] [PubMed] [Google Scholar]
  20. Milstein C., Brownlee G. G., Harrison T. M., Mathews M. B. A possible precursor of immunoglobulin light chains. Nat New Biol. 1972 Sep 27;239(91):117–120. doi: 10.1038/newbio239117a0. [DOI] [PubMed] [Google Scholar]
  21. Noyes B. E., Mevarech M., Stein R., Agarwal K. L. Detection and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1770–1774. doi: 10.1073/pnas.76.4.1770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Proudfoot N. J. Complete 3' noncoding region sequences of rabbit and human beta-globin messenger RNAs. Cell. 1977 Apr;10(4):559–570. doi: 10.1016/0092-8674(77)90089-7. [DOI] [PubMed] [Google Scholar]
  23. Rose J. K. Complete sequences of the ribosome recognition sites in vesicular stomatitis virus mRNAs: recognition by the 40S and 80S complexes. Cell. 1978 Jun;14(2):345–353. doi: 10.1016/0092-8674(78)90120-4. [DOI] [PubMed] [Google Scholar]
  24. Rose S. M., Kuehl W. M., Smith G. P. Cloned MPC 11 myeloma cells express two kappa genes: a gene for a complete light chain and a gene for a constant region polypeptide. Cell. 1977 Oct;12(2):453–462. doi: 10.1016/0092-8674(77)90121-0. [DOI] [PubMed] [Google Scholar]
  25. Sakano H., Hüppi K., Heinrich G., Tonegawa S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature. 1979 Jul 26;280(5720):288–294. doi: 10.1038/280288a0. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Svasti J., Milstein C. The complete amino acid sequence of a mouse kappa light chain. Biochem J. 1972 Jun;128(2):427–444. doi: 10.1042/bj1280427. [DOI] [PMC free article] [PubMed] [Google Scholar]

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