Abstract
Poly(A)-RNA enriched for prothrombin was isolated by specific immunoprecipitation of bovine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RR1 under P3-EK1 conditions. Sixty-three tetracycline-resistant clones were obtained that hybridized to 32P-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a [3H]cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700, 500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.
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- Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Busslinger M., Portmann R., Birnsteil M. L. A regulatory sequence near the 3' end of sea urchin histone genes. Nucleic Acids Res. 1979 Jul 11;6(9):2997–3008. doi: 10.1093/nar/6.9.2997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Butkowski R. J., Elion J., Downing M. R., Mann K. G. Primary structure of human prethrombin 2 and alpha-thrombin. J Biol Chem. 1977 Jul 25;252(14):4942–4957. [PubMed] [Google Scholar]
- Chang A. C., Erlich H. A., Gunsalus R. P., Nunberg J. H., Kaufman R. J., Schimke R. T., Cohen S. N. Initiation of protein synthesis in bacteria at a translational start codon of mamalian cDNA: effects of the preceding nucleotide sequence. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1442–1446. doi: 10.1073/pnas.77.3.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davie E. W., Fujikawa K., Kurachi K., Kisiel W. The role of serine proteases in the blood coagulation cascade. Adv Enzymol Relat Areas Mol Biol. 1979;48:277–318. doi: 10.1002/9780470122938.ch6. [DOI] [PubMed] [Google Scholar]
- Fitch W. M. Is there selection against wobble in codon-anticodon pairing? Science. 1976 Dec 10;194(4270):1173–1174. doi: 10.1126/science.996548. [DOI] [PubMed] [Google Scholar]
- Gough N. M., Adams J. M. Immunoprecipitation of specific polysomes using Staphylococcus aureus: purification of the immunoglobulin k chain messenger RNA from the mouse myeloma MPC11. Biochemistry. 1978 Dec 12;17(25):5560–5566. doi: 10.1021/bi00618a036. [DOI] [PubMed] [Google Scholar]
- Grantham R., Gautier C., Gouy M., Mercier R., Pavé A. Codon catalog usage and the genome hypothesis. Nucleic Acids Res. 1980 Jan 11;8(1):r49–r62. doi: 10.1093/nar/8.1.197-c. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Katz L., Williams P. H., Sato S., Leavitt R. W., Helinski D. R. Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli. Biochemistry. 1977 Apr 19;16(8):1677–1683. doi: 10.1021/bi00627a024. [DOI] [PubMed] [Google Scholar]
- Konkel D. A., Tilghman S. M., Leder P. The sequence of the chromosomal mouse beta-globin major gene: homologies in capping, splicing and poly(A) sites. Cell. 1978 Dec;15(4):1125–1132. doi: 10.1016/0092-8674(78)90040-5. [DOI] [PubMed] [Google Scholar]
- Kronenberg H. M., McDevitt B. E., Majzoub J. A., Nathans J., Sharp P. A., Potts J. T., Jr, Rich A. Cloning and nucleotide sequence of DNA coding for bovine preproparathyroid hormone. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4981–4985. doi: 10.1073/pnas.76.10.4981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
- 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]
- McGillivray R. T., Chung D. W., Davie E. W. Biosynthesis of bovine plasma proteins in a cell-free system. Amino-terminal sequence of preproalbumin. Eur J Biochem. 1979 Aug 1;98(2):477–485. doi: 10.1111/j.1432-1033.1979.tb13209.x. [DOI] [PubMed] [Google Scholar]
- 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]
- NOMENCLATURE of blood clotting factors; four factors, their characterization and international number. J Am Med Assoc. 1959 May 16;170(3):325–328. [PubMed] [Google Scholar]
- Nakanishi S., Inoue A., Kita T., Nakamura M., Chang A. C., Cohen S. N., Numa S. Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature. 1979 Mar 29;278(5703):423–427. doi: 10.1038/278423a0. [DOI] [PubMed] [Google Scholar]
- Norgard M. V., Keem K., Monahan J. J. Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA. Gene. 1978 Jul;3(4):279–292. doi: 10.1016/0378-1119(78)90038-0. [DOI] [PubMed] [Google Scholar]
- Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
- Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
- Reddy V. B., Ghosh P. K., Lebowitz P., Piatak M., Weissman S. M. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. J Virol. 1979 Apr;30(1):279–296. doi: 10.1128/jvi.30.1.279-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reddy V. B., Ghosh P. K., Lebowitz P., Weissman S. M. Gaps and duplicated sequences in the leaders of SV40 16S RNA. Nucleic Acids Res. 1978 Nov;5(11):4195–4213. doi: 10.1093/nar/5.11.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
- Schutz G., Kieval S., Groner B., Sippel A. E., Kurtz D., Feigelson P. Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody. Nucleic Acids Res. 1977 Jan;4(1):71–84. doi: 10.1093/nar/4.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith M., Leung D. W., Gillam S., Astell C. R., Montgomery D. L., Hall B. D. Sequence of the gene for iso-1-cytochrome c in Saccharomyces cerevisiae. Cell. 1979 Apr;16(4):753–761. doi: 10.1016/0092-8674(79)90091-6. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Staden R. Further procedures for sequence analysis by computer. Nucleic Acids Res. 1978 Mar;5(3):1013–1016. doi: 10.1093/nar/5.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Staden R. Sequence data handling by computer. Nucleic Acids Res. 1977 Nov;4(11):4037–4051. doi: 10.1093/nar/4.11.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stein J. P., Catterall J. F., Woo S. L., Means A. R., O'Malley B. W. Molecular cloning of ovomucoid gene sequences from partially purified ovomucoid messenger RNA. Biochemistry. 1978 Dec 26;17(26):5763–5772. doi: 10.1021/bi00619a025. [DOI] [PubMed] [Google Scholar]
- Sutcliffe J. G. pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 1978 Aug;5(8):2721–2728. doi: 10.1093/nar/5.8.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]