The nucleotide sequence of the cloned DNA, 1,478 bp in length coding for glutathione synthetase (GSH-II) of E. coli B has been determined. Amino acid and nucleotide sequence analyses have assigned the open reading frame for GSH-II, starting with the ATG near its 5' terminus. The molecular weight calculated from the predicted amino acid sequence is 35,559 daltons, being in good agreement with that of a GSH-II subunit estimated by the SDS-PAGE method. Several signal sequences conserved in the promoter regions of E. coli were found in the non-coding regions of the gsh-II gene. They include the Shine-Dalgarno sequence, the Pribnow box and the sequence conserved in the "-35 region" with a preferable spacing from each other for an efficient transcription. Downstream from the termination codon, the inverted repeat sequences were present, followed by 6 successive T's. These structural features found in the non-coding regions have suggested to be involved in regulatory functions for the gsh-II gene expression.

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In order to promote shotgun DNAsequencing,1>2) the method for constructing Ml3 clone libraries originally developed by Deininger1* was modified, including the

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