Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jul 5;91(14):6614–6618. doi: 10.1073/pnas.91.14.6614

A yeast system for expression of human cystathionine beta-synthase: structural and functional conservation of the human and yeast genes.

W D Kruger 1, D R Cox 1
PMCID: PMC44253  PMID: 8022826

Abstract

Human cystathionine beta-synthase (CBS; EC 4.2.1.22) deficiency results in a recessive genetic disorder whose clinical and biochemical manifestations vary greatly among affected individuals. In an effort to identify and analyze mutations in the human CBS gene, we have developed a yeast expression system for human CBS. We have cloned and sequenced a human cDNA that codes for CBS and have expressed the human CBS protein in yeast cells lacking endogenous CBS. The human enzyme produced in yeast is functional both in vitro and in vivo. We have also cloned and sequenced the yeast gene, CYS4, that codes for CBS. The predicted human and yeast CBS proteins are 38% identical and 72% similar to each other, as well as sharing significant similarity with bacterial cysteine synthase. These results demonstrate the evolutionary conservation of CBS and establish the utility of a yeast expression system for studying human CBS.

Full text

PDF
6617

Images in this article

6617Image
on p.6617
6618Image
on p.6618

Selected References

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

  1. Avramopoulos D., Cox T., Kraus J. P., Chakravarti A., Antonarakis S. E. Linkage mapping of the cystathionine beta-synthase (CBS) gene on human chromosome 21 using a DNA polymorphism in the 3' untranslated region. Hum Genet. 1993 Jan;90(5):566–568. doi: 10.1007/BF00217460. [DOI] [PubMed] [Google Scholar]
  2. Bittles A. H., Carson N. A. Homocystinuria: studies on cystathionine beta-synthase, S-adenosylmethionine synthetase and cystathionase activities in skin fibroblasts. J Inherit Metab Dis. 1981;4(1):3–6. doi: 10.1007/BF02263573. [DOI] [PubMed] [Google Scholar]
  3. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  4. Boers G. H., Smals A. G., Trijbels F. J., Fowler B., Bakkeren J. A., Schoonderwaldt H. C., Kleijer W. J., Kloppenborg P. W. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med. 1985 Sep 19;313(12):709–715. doi: 10.1056/NEJM198509193131201. [DOI] [PubMed] [Google Scholar]
  5. Brattstrom L. E., Hardebo J. E., Hultberg B. L. Moderate homocysteinemia--a possible risk factor for arteriosclerotic cerebrovascular disease. Stroke. 1984 Nov-Dec;15(6):1012–1016. doi: 10.1161/01.str.15.6.1012. [DOI] [PubMed] [Google Scholar]
  6. Byrne C. R., Monroe R. S., Ward K. A., Kredich N. M. DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH. J Bacteriol. 1988 Jul;170(7):3150–3157. doi: 10.1128/jb.170.7.3150-3157.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cherest H., Thomas D., Surdin-Kerjan Y. Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment. J Bacteriol. 1993 Sep;175(17):5366–5374. doi: 10.1128/jb.175.17.5366-5374.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clarke R., Daly L., Robinson K., Naughten E., Cahalane S., Fowler B., Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med. 1991 Apr 25;324(17):1149–1155. doi: 10.1056/NEJM199104253241701. [DOI] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Fowler B., Kraus J., Packman S., Rosenberg L. E. Homocystinuria. Evidence for three distinct classes of cystathionine beta-synthase mutants in cultured fibroblasts. J Clin Invest. 1978 Mar;61(3):645–653. doi: 10.1172/JCI108976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fridovich-Keil J. L., Jinks-Robertson S. A yeast expression system for human galactose-1-phosphate uridylyltransferase. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):398–402. doi: 10.1073/pnas.90.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hemraj F., Griffiths R. Enzyme studies in cystathione-beta-synthase deficiency: a possible effect of elevated intracellular levels of homocystine in kinetic studies. J Inherit Metab Dis. 1978;1(4):171–174. doi: 10.1007/BF01805590. [DOI] [PubMed] [Google Scholar]
  13. Kashiwamata S., Greenberg D. M. Studies on cystathionine synthase of rat liver. Properties of the highly purified enzyme. Biochim Biophys Acta. 1970 Sep 16;212(3):488–500. doi: 10.1016/0005-2744(70)90255-x. [DOI] [PubMed] [Google Scholar]
  14. Kozich V., Kraus J. P. Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency. Hum Mutat. 1992;1(2):113–123. doi: 10.1002/humu.1380010206. [DOI] [PubMed] [Google Scholar]
  15. Kraus J. P., Le K., Swaroop M., Ohura T., Tahara T., Rosenberg L. E., Roper M. D., Kozich V. Human cystathionine beta-synthase cDNA: sequence, alternative splicing and expression in cultured cells. Hum Mol Genet. 1993 Oct;2(10):1633–1638. doi: 10.1093/hmg/2.10.1633. [DOI] [PubMed] [Google Scholar]
  16. Kraus J., Packman S., Fowler B., Rosenberg L. E. Purification and properties of cystathionine beta-synthase from human liver. Evidence for identical subunits. J Biol Chem. 1978 Sep 25;253(18):6523–6528. [PubMed] [Google Scholar]
  17. Lee R. T., Peterson C. L., Calman A. F., Herskowitz I., O'Donnell J. J. Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10887–10891. doi: 10.1073/pnas.89.22.10887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Malinow M. R., Kang S. S., Taylor L. M., Wong P. W., Coull B., Inahara T., Mukerjee D., Sexton G., Upson B. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989 Jun;79(6):1180–1188. doi: 10.1161/01.cir.79.6.1180. [DOI] [PubMed] [Google Scholar]
  19. McGill J. J., Mettler G., Rosenblatt D. S., Scriver C. R. Detection of heterozygotes for recessive alleles. Homocyst(e)inemia: paradigm of pitfalls in phenotypes. Am J Med Genet. 1990 May;36(1):45–52. doi: 10.1002/ajmg.1320360111. [DOI] [PubMed] [Google Scholar]
  20. Mudd S. H., Skovby F., Levy H. L., Pettigrew K. D., Wilcken B., Pyeritz R. E., Andria G., Boers G. H., Bromberg I. L., Cerone R. The natural history of homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet. 1985 Jan;37(1):1–31. [PMC free article] [PubMed] [Google Scholar]
  21. Ono B., Shirahige Y., Nanjoh A., Andou N., Ohue H., Ishino-Arao Y. Cysteine biosynthesis in Saccharomyces cerevisiae: mutation that confers cystathionine beta-synthase deficiency. J Bacteriol. 1988 Dec;170(12):5883–5889. doi: 10.1128/jb.170.12.5883-5889.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peterson C. L., Kruger W., Herskowitz I. A functional interaction between the C-terminal domain of RNA polymerase II and the negative regulator SIN1. Cell. 1991 Mar 22;64(6):1135–1143. doi: 10.1016/0092-8674(91)90268-4. [DOI] [PubMed] [Google Scholar]
  23. Schild D., Brake A. J., Kiefer M. C., Young D., Barr P. J. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2916–2920. doi: 10.1073/pnas.87.8.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Skovby F., Kraus J. P., Rosenberg L. E. Biosynthesis and proteolytic activation of cystathionine beta-synthase in rat liver. J Biol Chem. 1984 Jan 10;259(1):588–593. [PubMed] [Google Scholar]
  26. Swaroop M., Bradley K., Ohura T., Tahara T., Roper M. D., Rosenberg L. E., Kraus J. P. Rat cystathionine beta-synthase. Gene organization and alternative splicing. J Biol Chem. 1992 Jun 5;267(16):11455–11461. [PubMed] [Google Scholar]
  27. Uhlendorf B. W., Conerly E. B., Mudd S. H. Homocystinuria: studies in tissue culture. Pediatr Res. 1973 Jul;7(7):645–658. doi: 10.1203/00006450-197307000-00008. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES