Cloning and applications of the two/three-base restriction endonuclease R·CviJI from IL-3A virus-infected chlorella

Piotr M. Skowron, Neela Swaminathan, Karolyn McMaster, David George, James L. Van Etten, David A. Mead

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

The gene (cviJIR) encoding the two/three-base R·CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R·CviJI required overexpression of the gene encoding the M·CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R·CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced 'star' activity (R·CviJI cleaves at RGCY, while R·CviJI* also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R·CviJI. The very high frequency of DNA cleavage by R·CviJI* was exploited in the development of a quasi-random shotgun library method. R·CviJI*-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.

Original languageEnglish (US)
Pages (from-to)37-41
Number of pages5
JournalGene
Volume157
Issue number1-2
DOIs
StatePublished - May 19 1995

Keywords

  • 'star' activity
  • ATP stimulation
  • Algae
  • DNA methyltransferase
  • phycoDNAviruses
  • recombinant DNA
  • restriction-modification

ASJC Scopus subject areas

  • Genetics

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