Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    That the two cohesive ends represent complementary sequences was established by quantitative studies of the nucleotides incorporated by the action of DNA polymerase I. Wu and Kaiser (1968) reported that the reaction reaches a plateau after incorporation of: 13 dGMP; 13 dCMP; 7 dAMP; 7 dTMP. Although the total number of nucleotides incorporated in this reaction has since been shown to be 12 at each end rather than 20, the equality between dGMP and dCMP and between dAMP and dTMP makes the point that the two cohesive ends probably are complementary to one another. (An alternative explanation would be that each cohesive end contains sequences that are self-complementary.) If the two cohesive ends have the same length and are complementary, the first nucleotide added to one 3' end must be the same as the 5' terminal nucleotide present at the other end of the molecule. By using polynu cleotide kinase to attach 32 P-phosphoryl residues to the 5' termini, and then hydrolyzing the DNA to 5' mononucleotides, Wu and Kaiser ( 1967) were able to show that the label enters (only) equal amounts of AMP and GMP. The use of half molecules showed that the left end terminates in 5' G and the right end terminates in 5' A. Wu and Kaiser (1968) then showed that when each nucleotide is tested alone (that is, in the absence of the other three nucleotides) for its ability to be incorporated into \ DNA, a single dAMP or 4 dGMP residues can be added; dCMP and dTMP are not added. This suggests that one dAMP can be added to the 3' left end and four dGMP residues to the 3' right end, consistent with the 5' terminal bases at the respective opposite ends. This approach has been extended into an analysis of the complete sequence of bases added at the right end. Wu and Kaiser found that when dCTP is added as well as dGTP, a total of 3 dCMP residues and 7 dGMP residues could be incorporated. Nearest neighbor analysis showed that all the dCMP residues were adjacent to GMP residues, suggesting that the initial run of dGMP bases is succeeded by an interspersion of dCMP and dGMP. Using these techniques, Wu (1970) and Wu and Taylor (1971) obtained the complete sequence shown in Figure 4.6. The sequence added in vitro at the right 3' end must (of course) be complementary to the protruding 5' terminal single strand at the right end and must be identical to the protruding 5' terminal single strand at the left end; this is the basis on which the cohesive sequences shown in the figure were calculated. The total length of each cohesive end is 12 bases (for review see Yarmolinsky, 1971). Formation of Prophage When sensitive bacteria are infected with lambda, those cells in which the phage enters the lytic cycle ultimately are lysed to yield the next genera-