Completed DNA sequences and organization of repressor-binding sites in the operators of phage lambda.
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Biomedical subjects
Publications and source records attributed to Z Humayun.
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DNA bearing lambda operator sequences was methylated by dimethyl sulfate (DMS) in the presence or absence of lambda repressor. Under the experimental conditions, DMS methylates only the purine residues. The presence of lambda repressor affects only the methylation of certain G residues in the operators. Repressor blocks the methylation of certain G's and enhances the methylation of other G's. Since the reactive ring-nitrogen of G lies in the major groove of double-stranded DNA, and the reactive ring-nitrogen of A lies in the minor groove, the above results imply that the repressor makes contacts in the major groove of the helix. The repressor effect on G-methylation is sharply confined to the three 17 base pair units within each lambda operator previously proposed as the repressor-binding sites.
The nucleotide sequence of 57 base pairs near the end of the cI gene in phage lambda is presented. This sequence was determined by direct sequencing techniques and includes the codons for 11 carboxyterminal aminoacids of the cI product, the lambda repressor. The sequence reveals that the cI gene, which has recently been shown to have a unique initiation region, is terminated by a UGA codon. A GUG triplet, which could act as a translation start signal for the rex gene occurs 8 base pairs beyond the cI termination codon. This GUG triplet is preceded by a sequence that could serve as a strong ribosome binding site for the rex message.
The sequences in lambda DNA in and around six sites cut by Hph, a restriction enzyme isolated from Haemophilus parahaemolyticus, are compared. The enzyme produces a staggered cut around an AT or TA base pair, but the sequences immediately surroinding the cleavage sites bear no obvious relation to one another. Eight (in some cases nine) base pairs to one side of each cleavage site is the common sequence TCACC AGTGG. Two lines of evidence indicate that these bases constitute part or all of the Hph recognition site. First, mutations in this sequence prevent Hph cutting. Second, dimethylsulfate-mediated methylation of Gs and As in this site prevent cutting, whereas methylation of purines in the region between this sequence and the cleavage sites has no such effect. There is discernible 2-fold rotational symmetry neither in the common sequence nor around the cleavage sites.