1887

Abstract

A hybrid plasmid. pDB2, was constructed by ligating a 3.24 kb RI/dIII fragment of the chromosome into pBR322. This was used to transform a. mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb RI/dIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-129-6-1889
1983-06-01
2021-05-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/129/6/mic-129-6-1889.html?itemId=/content/journal/micro/10.1099/00221287-129-6-1889&mimeType=html&fmt=ahah

References

  1. Ashworth J. M., Kornberg H. L., Nothmann D. L. 1965; Location of the structural gene for citrate synthase on the chromosome of Escherichia coli K12. Journal of Molecular Biology 11:654–657
    [Google Scholar]
  2. Bloxham D. P., Parmelee D. C., Kumar S., Wade R. D., Ericsson L. H., Neurath H., Walsh K. A., Titani K. 1981; Primary structure of porcine heart citrate synthase. Proceedings of the National Academy of Sciences of the United Stales of America 78:5381–5385
    [Google Scholar]
  3. Bloxham D. P., Parmelee D. C., Kumar S., Walsh K. A., Titani K. 1982; Complete amino acid sequence of porcine heart citrate synthase. Biochemistry 21:2028–2036
    [Google Scholar]
  4. Bolivar F., Backman K. 1979; Plasmids of Escherichia coli as cloning vectors. Methods in Enzymology 68:245–280
    [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of Molecular Biology 41:459–472
    [Google Scholar]
  6. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  7. Clarke L., Carbon J. 1976; A colony bank containing synthetic ColEl hybrid plasmids representative of the entire E. coli genome. Cell 9:91–99
    [Google Scholar]
  8. Davis R. W., Botstein D., Roth J. R. 1980 Advanced Bacterial Genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  9. Flechtner V. R., Hanson V. R. 1969; Coarse and fine control of citrate synthase from Bacillus subtilis. Biochimica et biophysica acta 184:252–262
    [Google Scholar]
  10. Gray C. T., Wimpenny J. W. T., Mossman M. R. 1966; Effects of aerobiosis, anaerobiosis and nutrition on the formation of Krebs cycle enzymes in Escherichia coli. Biochimica et biophysica acta 117:33–41
    [Google Scholar]
  11. Guest J. R. 1981; Hybrid plasmids containing the citrate synthase gene (gitA) of Escherichia coli K12. Journal of General Microbiology 124:17–23
    [Google Scholar]
  12. Kado C. I., Liu S. T. 1981; Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145:1365–1373
    [Google Scholar]
  13. Krebs H. A., Lowenstein J. M. 1960; The tricarboxylic acid cycle. In Metabolic Pathways pp. 129–203 Edited by Greenberg D. M. New York: Academic Press;
    [Google Scholar]
  14. Macrina F. L., Kopecko D. J., Jones K. R., Ayers D. J., Mccowen S. M. 1978; A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1:417–420
    [Google Scholar]
  15. Remington S., Wiegand G., Huber R. 1982; Crystallographic refinement and atomic models of two different forms of citrate synthase at 2µ7 and 1µ7 Å resolution. Journal of Molecular Biology 158:111–152
    [Google Scholar]
  16. Spencer M. E., Guest J. R. 1982; Molecular cloning of four tricarboxylic acid genes of Escherichia coli. Journal of Bacteriology 151:542–552
    [Google Scholar]
  17. Srere P. A. 1969; Citrate synthase. Methods in Enzymology 13:3–11
    [Google Scholar]
  18. Tong E. K., Duckworth H. 1975; The quarternary structure of citrate synthase from Escherichia coli K12. Biochemistry 14:235–241
    [Google Scholar]
  19. Vogel H., Bonner D. M. 1956; A convenient growth medium for E. coliand some other micro-organisms. Microbial Genetics Bulletin 13:43–44
    [Google Scholar]
  20. Weber K., Osborn M. 1969; The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244:4406–4412
    [Google Scholar]
  21. Weitzman P. D. J. 1969; Citrate synthase from Escherichia coli. Methods in Enzymology 13:22–26
    [Google Scholar]
  22. Weitzman P. D. J. 1981; Unity and diversity in some bacterial citric acid-cycle enzymes. Advances in Microbial Physiology 22:185–244
    [Google Scholar]
  23. Weitzman P. D. J., Danson M. J. 1976; Citrate synthase. Current Topics in Cell Regulation 10:161–204
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-129-6-1889
Loading
/content/journal/micro/10.1099/00221287-129-6-1889
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error