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Genetics, Vol 125, 261-273, Copyright © 1990
INVESTIGATIONS |
Physical Analysis of Spontaneous and Mutagen-Induced Mutants of Escherichia coli K-12 Expressing DNA Exonuclease VIII Activity
S. K. Mahajan, C. C. Chu, D. K. Willis, A. Templin and A. J. Clark
Present address: Molecular Biology and Agriculture Division, Bhabha Atomic Research Center, Bombay 400 085, India.
We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VIII (ExoVIII). One mutation, sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and the trg gene. Western blot analysis shows that the protein produced is larger than wild type ExoVIII. The nucleotide sequence shows that a translational gene fusion has occurred. The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call sfcA. Analysis of the protein sequence encoded by sfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme. We discuss the possibility that sfcA is identical to maeA which encodes NAD-linked malic enzyme from Escherichia coli. Restriction nuclease analysis of a second deletion, sbcA81, by Southern blot technique indicates that about 105 kb of DNA have been deleted and a transcriptional gene fusion has occurred between recE and the regulatory region of an E. coli chromosomal gene. We also examined eight other sbc mutations that result in ExoVIII production. Five have no effect on restriction nucleotide fragment sizes detected by complementarity to {lambda} rev as probe. These are presumed point mutations. Three seem to produce additional restriction nucleotide fragments complementary to {lambda} rev. The possible nature of these sbc mutations is discussed.
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