Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1978 Nov;5(11):4141–4153. doi: 10.1093/nar/5.11.4141

Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome

David Baulcombe 1, Desh Pal S Verma 1
PMCID: PMC342739  PMID: 569292

Abstract

In soybean root nodules, leghaemoglobin (Lb) accounts for 25-30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb invitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 × 10−3 and isolation of the hybridising cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.

Full text

PDF
4141

Images in this article

4146Image
on p.4146
4148Image
on p.4148

Selected References

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

  1. 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]
  2. Bishop J. O., Morton J. G., Rosbash M., Richardson M. Three abundance classes in HeLa cell messenger RNA. Nature. 1974 Jul 19;250(463):199–204. doi: 10.1038/250199a0. [DOI] [PubMed] [Google Scholar]
  3. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  5. Burr B., Burr F. A., Rubenstein I., Simon M. N. Purification and translation of zein messenger RNA from maize endosperm protein bodies. Proc Natl Acad Sci U S A. 1978 Feb;75(2):696–700. doi: 10.1073/pnas.75.2.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cutting J. A., Schulman H. M. The biogenesis of leghemoglobin. The determinant in the Rhizobium-legume symbiosis for leghemoglobin specificity. Biochim Biophys Acta. 1971 Jan 19;229(1):58–62. [PubMed] [Google Scholar]
  7. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  8. Dilworth M. J. The plant as the genetic determinant of leghaemoglobin production in the legume root nodule. Biochim Biophys Acta. 1969 Jul 30;184(2):432–441. doi: 10.1016/0304-4165(69)90047-6. [DOI] [PubMed] [Google Scholar]
  9. Efstratiadis A., Kafatos F. C., Maniatis T. The primary structure of rabbit beta-globin mRNA as determined from cloned DNA. Cell. 1977 Apr;10(4):571–585. doi: 10.1016/0092-8674(77)90090-3. [DOI] [PubMed] [Google Scholar]
  10. Ellfolk N., Sievers G. Correction of the amino acid sequence of soybean leghemoglobin alpha. Acta Chem Scand B. 1974;28(10):1245–1246. doi: 10.3891/acta.chem.scand.28b-1245. [DOI] [PubMed] [Google Scholar]
  11. Harrison P. R., Gilmour R. S., Affara N. A., Conkie D., Paul J. Globin messenger RNA synthesis and processing during haemoglobin induction in Friend cells. II. Evidence for post-transcriptional control in clone 707. Cell Differ. 1974 Jun;3(1):23–30. doi: 10.1016/0045-6039(74)90037-2. [DOI] [PubMed] [Google Scholar]
  12. Humphries S., Windass J., Williamson R. Mouse globin gene expression in erythroid and non-erythroid tissues. Cell. 1976 Feb;7(2):267–277. doi: 10.1016/0092-8674(76)90026-x. [DOI] [PubMed] [Google Scholar]
  13. Hurrell J. G., Leach S. J. The amino acid sequence of soybean leghaemoglobin c2. FEBS Lett. 1977 Aug 1;80(1):23–26. doi: 10.1016/0014-5793(77)80398-0. [DOI] [PubMed] [Google Scholar]
  14. Kacian D. L., Myers J. C. Synthesis of extensive, possibly complete, DNA copies of poliovirus RNA in high yields and at high specific activities. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2191–2195. doi: 10.1073/pnas.73.7.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Larkins B. A., Jones R. A., Tsai C. Y. Isolation and in vitro translation of zein messenger ribonucleic acid. Biochemistry. 1976 Dec 14;15(25):5506–5511. doi: 10.1021/bi00670a014. [DOI] [PubMed] [Google Scholar]
  16. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Marcu K., Dudock B. Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ. Nucleic Acids Res. 1974 Nov;1(11):1385–1397. doi: 10.1093/nar/1.11.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Old J., Clegg J. B., Weatherall D. J., Ottolenghi S., Comi P., Giglioni B., Mitchell J., Tolstoshev P., Williamson R. A direct estimate of the number of human gamma-globin genes. Cell. 1976 May;8(1):13–18. doi: 10.1016/0092-8674(76)90180-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Ragg H., Schröder J., Hahlbrock K. Translation of poly(A)-containing and poly(A)-free messenger RNA for phenylalanine ammonia-lyase, a plant-specific protein, in a reticulocyte lysate. Biochim Biophys Acta. 1977 Jan 20;474(2):226–233. doi: 10.1016/0005-2787(77)90197-6. [DOI] [PubMed] [Google Scholar]
  22. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  24. Scott N. S., Ingle J. The genes for cytoplasmic ribosomal ribonucleic Acid in higher plants. Plant Physiol. 1973 Apr;51(4):677–684. doi: 10.1104/pp.51.4.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sidloi-Lumbroso R., Schulman H. M. Purification and properties of soybean leghemoglobin messenger RNA. Biochim Biophys Acta. 1977 Jun 17;476(4):295–302. doi: 10.1016/0005-2787(77)90294-5. [DOI] [PubMed] [Google Scholar]
  26. Spradling A., Penman S., Campo M. S., Bishop J. O. Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells. Cell. 1974 Sep;3(1):23–30. doi: 10.1016/0092-8674(74)90033-6. [DOI] [PubMed] [Google Scholar]
  27. Sutton W. D. Some features of the DNA of Rhizobium bacteroids and bacteria. Biochim Biophys Acta. 1974 Sep 27;366(1):1–10. doi: 10.1016/0005-2787(74)90312-8. [DOI] [PubMed] [Google Scholar]
  28. Verma D. P., Bal A. K. Intracellular site of synthesis and localization of leghemoglobin in root nodules. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3843–3847. doi: 10.1073/pnas.73.11.3843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Verma D. P., Nash D. T., Schulman H. M. Isolation and in vitro translation of soybean leghaemoglobin mRNA. Nature. 1974 Sep 6;251(5470):74–77. doi: 10.1038/251074a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES