Daniel Dupret

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

Abstract The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERSs collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

STUDIES TOWARDS THE DESIGN OF A MODIFIED GC BASE PAIR WITH STABILITY SIMILAR TO THAT OF THE AT BASE PAIR

Abstract Modified G ∗ C, ∗ GC or ∗ G ∗ C base pairs have been incorporated at the 3 rd and 8 th positions of a self-complementary decadeoxyoligonucleotide. The influence of these modifications on duplex stabilities has been studied by absorption spectroscopy. It has been found that a few of them have thermal stabilities similar to that of the AT base pair.

Design of modified A*U and G*C base pairs with similar stability. Implication for the DNA sequencing by hybridization

Abstract Modified G*C, or A*U base pairs have been incorporated at the 4 th position of a duplex composed with nine base pairs. The influence of these modifications on duplex stabilities has been studied by absorption spectroscopy. It has been found that G 4Me C has thermal stability similar to that of the A 5Prop U base pair. Thus duplexes involving G 4Me C or A 5Prop U base pairs exhibit the same Tm.