Abstract
The effects of substituents at position 5 in the pyrimidine ring of a variety of phage DNAs upon EcoRI endonuclease and methylase activities have been examined. The replacement of cytidine in DNA with glucosylated hydroxymethylcytidine confers resistance to cleavage by the EcoRI endonuclease. Substitution of thymidine in DNA by hydroxy-methyluridine(a change in the methyl at position 5 of thymidine for a hydroxymethyl) lowers the maximal velocity of endonucleolytic cleavage 20-fold, but has no detectable effect upon the Km. Substitution of thymidine in DNA by uridine (a change in the methyl at position 5 of thymidine for a hydrogen atom) has no effect upon either the maximal velocity or the Km. The effect of these modifications upon EcoRI methylase activity was markedly different. DNA containing glucosylated hydroxymethylcytidine is methylated as well as normal DNA. DNA containing uridine or hydroxy-methyluridine, in place of thymidine, is much more poorly methylated than normal DNA. These different sensitivities of the EcoRI endonuclease and methylase to modifications in the pyrimidine rings of DNA suggest there are significant differences in the manner by which these enzymes recognize and bind to the canonical EcoRI sequence.
Highlights
The effects of substituents at position 5 in the pyrimidine ring of a variety of phage DNAs upon EcoRI endonuclease and methylase activities have been examined
Analysis of Digestion of DNAs by EcoRI Endonuclease -As a qualitative approach to examine the susceptibility of the various phage DNAs to cleavage by the EcoRI endonuclease, each was incubated with sufficient enzyme to produce a limit digest, and the products were analyzed by electrophoresis through agarose slab gels (Fig. 2)
Restriction endonucleases and methylases which act on distinct nucleotide sequences provide an excellent opportunity to explore DNA-protein interactions, since they produce an quantifiable event
Summary
15575) was grown to an A,,,,,, of 1.0 in media containing, per liter, 10 g of Bacto-tryptone,. 5 g of Difco yeast extract, and 10 g of NaCl supplemented with 0.14% glucose, 10 rnM MgSO,, and 100 pM MnCl,, and infected with PBS2 at a multiplicity of infection After stirring for 90 min at 37” the culture was chilled and the phage purified as described below. B. subtilis 3610 (ATCC 6051) was grown at 37” in media containing, per liter, 10 g of Bacto-tryptone,. Upon reaching an A,,,, of 0.3, the cells were infected with he at a multiplicity of infection of 0.1, and the culture agitated until it was clear, indicating complete lysis Price) was grown at 37” in media containing 80 mM NaCl, 20 mM KCl, 20 rnM NH,Cl, 1 mM MgCl,, 0.2.
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