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
. 1983 Jan;80(2):402–406. doi: 10.1073/pnas.80.2.402

Cloned restriction/modification system from Pseudomonas aeruginosa.

T R Gingeras, J E Brooks
PMCID: PMC393385  PMID: 6300841

Abstract

DNA fragments from Pseudomonas aeruginosa carrying the PaeR7 restriction/modification genes have been cloned in the plasmid vector pBR322 and propagated in Escherichia coli. A subclone (pPAORM3.8) has been constructed that contains the complete restriction/modification system on a 3.8-kilobase DNA fragment. Digestion of the pPAORM3.8 plasmid with nuclease BAL-31 has yielded two types of clones. One type contains an active methylase gene but no active endonuclease gene; such clones will modify the DNA but not restrict the growth of incoming phage in vivo. The second type contains an active endonuclease gene but no active methylase gene, as judged both by in vivo tests and by the activity of the cell extracts in vitro. Although extracts of cells containing these plasmids display restriction endonuclease activity, these bacteria are unable to restrict the growth of incoming phage. Furthermore, chromosomal and phage DNA isolated from these host cells are not protected against cleavage by PaeR7 in vitro. The properties of PaeR7 endonuclease and methylase enzymes have also been examined. The PaeR7 restriction endonuclease recognizes and cleaves the sequence C decreased T-C-G-A-G, as does Xho I. However, there exists a canonical Xho I site at 26.5% on the adenovirus 2 genome which is totally refractory to PaeR7 cleavage but is cut by Xho I. Under conditions of low salt, high glycerol, and high enzyme concentrations, a "PaeR7" activity is found that is similar to that observed for EcoRI. Finally, evidence is presented that the PaeR7 methylase modifies the adenine residue within the recognition sequence.

Full text

PDF
404

Images in this article

404Image
on p.404
405Image
on p.405
405Image
on p.405
405Image
on p.405

Selected References

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

  1. Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Brooks J. E., Roberts R. J. Modification profiles of bacterial genomes. Nucleic Acids Res. 1982 Feb 11;10(3):913–934. doi: 10.1093/nar/10.3.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chaconas G., van de Sande J. H. 5'-32P labeling of RNA and DNA restriction fragments. Methods Enzymol. 1980;65(1):75–85. doi: 10.1016/s0076-6879(80)65012-5. [DOI] [PubMed] [Google Scholar]
  6. Clewell D. B., Helinski D. R. Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli. J Bacteriol. 1972 Jun;110(3):1135–1146. doi: 10.1128/jb.110.3.1135-1146.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Forsblom S., Rigler R., Ehrenberg M., Philipson L. Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI. Nucleic Acids Res. 1976 Dec;3(12):3255–3269. doi: 10.1093/nar/3.12.3255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  10. Hinkle N. F., Miller R. V. pMG7-mediated restriction of Pseudomonas aeruginosa phage DNAs is determined by a class II restriction endonuclease. Plasmid. 1979 Jul;2(3):387–393. doi: 10.1016/0147-619x(79)90022-2. [DOI] [PubMed] [Google Scholar]
  11. Humphreys G. O., Willshaw G. A., Smith H. R., Anderson E. S. Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids. Mol Gen Genet. 1976 Apr 23;145(1):101–108. doi: 10.1007/BF00331564. [DOI] [PubMed] [Google Scholar]
  12. Jacoby G. A., Sutton L. Restriction and modification determined by a Pseudomonas R plasmid. Plasmid. 1977 Nov;1(1):115–116. doi: 10.1016/0147-619x(77)90012-9. [DOI] [PubMed] [Google Scholar]
  13. Kosykh V. G., Buryanov Y. I., Bayev A. A. Molecular cloning of EcoRII endonuclease and methylase genes. Mol Gen Genet. 1980;178(3):717–718. doi: 10.1007/BF00337884. [DOI] [PubMed] [Google Scholar]
  14. Mann M. B., Rao R. N., Smith H. O. Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus. Gene. 1978 Apr;3(2):97–112. doi: 10.1016/0378-1119(78)90054-9. [DOI] [PubMed] [Google Scholar]
  15. McClelland M. Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI. Nucleic Acids Res. 1981 Dec 21;9(24):6795–6804. doi: 10.1093/nar/9.24.6795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Modrich P. Structures and mechanisms of DNA restriction and modification enzymes. Q Rev Biophys. 1979 Aug;12(3):315–369. doi: 10.1017/s0033583500005461. [DOI] [PubMed] [Google Scholar]
  17. Pettersson U., Sambrook J. Amount of viral DNA in the genome of cells transformed by adenovirus type 2. J Mol Biol. 1973 Jan;73(1):125–130. doi: 10.1016/0022-2836(73)90164-2. [DOI] [PubMed] [Google Scholar]
  18. Sato S., Shinomiya T. An isochizomer of TaqI from Thermus thermophilus HB8. J Biochem. 1978 Nov;84(5):1319–1321. doi: 10.1093/oxfordjournals.jbchem.a132252. [DOI] [PubMed] [Google Scholar]
  19. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  20. Smith H. O. Nucleotide sequence specificity of restriction endonucleases. Science. 1979 Aug 3;205(4405):455–462. doi: 10.1126/science.377492. [DOI] [PubMed] [Google Scholar]
  21. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  22. Walder R. Y., Hartley J. L., Donelson J. E., Walder J. A. Cloning and expression of the Pst I restriction-modification system in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1503–1507. doi: 10.1073/pnas.78.3.1503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yuan R. Structure and mechanism of multifunctional restriction endonucleases. Annu Rev Biochem. 1981;50:285–319. doi: 10.1146/annurev.bi.50.070181.001441. [DOI] [PubMed] [Google Scholar]
  24. van der Ploeg L. H., Flavell R. A. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980 Apr;19(4):947–958. doi: 10.1016/0092-8674(80)90086-0. [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