(9) Reverse transcriptase inhibitors (ex: 3' azido 3' deoxythymidine
) (10) and natural products (ex.tclomestatin and sulfoquinovosyldiacyl glycerol) (11).
Thymidylate synthase (TS) is a 36-kDa dimeric protein that catalyzes methylation of deoxyuridine monophosphate (BUMP) to deoxythymidine
monophosphate (dTMP), using the reduced folate 5,10-methylenetetrahydrofolate as the methyl donor (53, 54).
The reaction mixture contained 50 mmol KCl; 10 mmol Tris HCl, pH 8.5; 3 mmol Mg[Cl.sub.2]; 0.1% Triton X-100 (Nacalai Tesque, Kyoto, Japan); 2% formamide; 10 pmol of primer pairs; 200 [micro]mol each of deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine
triphosphate; and 2.5 units recombinant Taq polymerase; the mixture then was overlaid with 25 [micro]L mineral oil.
Therefore, T vectors possessing two 3' overhanging deoxythymidines
have been created using different approaches, either by tailing a single deoxythymidine
to the 3' end of linear and blunt ended plasmids (2) or by cutting a modified plasmid with selected restriction enzymes such as Xcm I, to generate linear plasmids with single overhang deoxythymidine
at both 3' ends (3-8).
The amplification solution consisted of the following: 1 [micro]mol/L of each consensus primer; 200 [micro]mol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate; and 140 [micro]mol/L of deoxythymidine
The hydrolyzed samples were then injected into the HPLC and the 5-methyldeoxycytidine and deoxythymidine fractions were collected.
Radioactivity of methyldeoxycytidine and deoxythymidine was measured more specifically after DNA hydrolysis and HPLC analysis (Fig.
Methyldeoxycytidine and deoxythymidine were well separated.