Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

M. J. Orbach, W. P. Schneider, C. Yanofsky

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.

Original languageEnglish (US)
Pages (from-to)2211-2213
Number of pages3
JournalMolecular and cellular biology
Volume8
Issue number5
DOIs
StatePublished - May 1988
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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