Showing papers by "Daniel M. Brown published in 1997"

The Mechanism of Mutation Induction by a Hydrogen Bond Ambivalent, Bicyclic N4-oxy-2′-deoxycytidine in Escherichia coli

Abstract: The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.

The synthesis of a tricyclic pyrrolopyrimidine related toN 6-hydroxyadenine

Abstract: The synthesis of 9-methylpyrrolo[4,3,2-de]pyrimido [4,5-c]dihydrooxazepine 34, a tricyclic pyrrolo[2,3-d]pyrimidine analogue of the mutagenic purine N6-hydroxyadenine, and several novel pyrrolo[2,3-d]pyrimidines is described. The presence of the third ring constrains the amino substituent of 34 to an anti orientation and is expected to improve dramatically the base-pairing characteristics of the analogue with both cytosine and thymine when present in DNA. An intramolecular cyclisation reaction of 5-(aminooxyethyl)-4-chloro-7-methyl-2-methylsulfonyl-7H -pyrrolo[2,3-d]pyrimidine 30 gave 33, which was converted into the target molecule 34 via the displacement of the methylsulfonyl group with hydrazine followed by oxidation of the hydrazino group with mercuric oxide. An analogous cyclisation with 5-(aminooxyethyl)-4-chloro-7-methyl-7H-pyrrolo [2,3-d]pyrimidine 31 was less effective, whilst the corresponding 2-amino derivative 32 failed to cyclise.

Properties of oligonucleotides containing the transition bases p and k

Abstract: The properties of the degenerate nucleosides dP and dK, in templates and primers were determined. dP was copied as either pyrimidine, dK as either purine. In primers, an equimolar mixture of the two nucleosides functioned as a universal base equivalent in both sequencing and the polymerase chain reactions. Cloning of DNA containing dP or dK produced transition mutations in vivo.

Nucleoside analogues based on indol-1-yl, pyrrol-1-yl and related base analogues

Abstract: Nucleoside analogues as defined in claims 1 and 11, which are characterised by replacing a natural base by a group M where M is where each of X 1 , X 2 and X 3 are C or N; ```each of R 6 and R 7 is the same or different and each is H, NO 2 , CO, COR 8 , OR 8 , CN, O, CON(R 8 ) 2 , COOR 8 , SO 2 R 8 , SO 3 R 8 , SR 8 , NHCHO, (CH 2 ) n N(R 8 ) 2 , halogen, or a reporter moiety; ```each of R 8 and R 9 is H or hydrocarbyl or a reporter moiety; ```and n is 0-4 are provided. These analogues are substrates for polymerase and terminal transferase enzymes, and thus can be used to extend polynucleotide chains and be incorporated therein. Nucleic acids containing a residue of such an analogue can be detected by using an antibody which binds to M.

Analogues de nucleosides

Abstract: On decrit des analogues de nucleosides contenant l'analogue P de base degenere, ainsi que leurs derives, lesquels comportent des fractions de marqueurs comprenant, de preference, des fractions de signal. Ces analogues de nucleosides sont utiles pour marquer l'ADN ou l'ARN ou pour etre incorpores dans des oligonucleotides.