No abstract

A key to the development of our knowledge about yeast replication origins was the autonomous replicating sequence (ARS) 1 assay; genomic fragments cloned into prokaryotic vectors were found to function as yeast replication origins. ARS plasmids transform yeast at a high frequency, replicate autonomously, and can be maintained in vivo as episomal genetic elements (1). These constructs, however, are lost without selective pressure because of imperfect partition and can integrate into the genome during long term culture. ARS plasmids were invaluable in identifying and defining replication origins in yeast (2, 3). Progress in understanding other eukaryotic DNA replication, particularly in mammalian cells, has been slower (for review, see Refs. 4 and 5).Our studies with small fragments of DNA which can support autonomous replication of a plasmid in mammalian cells (6 -10) encouraged us to look further for putative replicator sequences. We report here the identification and testing of a putative consensus sequence that will aid in identification of initiation sites (origins) of DNA replication in mammalian and higher eukaryotic cells. We used four mammalian autonomously replicating sequences containing ␣-satellite sequence and a reiterative process between pairs of African green monkey and human sequences to minimize derivation of an ␣-satellite consensus. The resultant consensus sequence was 36 bp.

An enantioselective assay has been developed for the determination of the enantiomers of ketorolac and its metabolite p-hydroxyketorolac in plasma and urine. The analytical method utilizes a coupled achiral-chiral HPLC system where the initial separation of ketorolac from P-hydroxyketorolac and matrix interferences was achieved on a CISstationary phase and the enantioselective separations of the two target solutes were accomplished on a human serum albumin-based chiral stationary phase. The two columns were attached in sequence and the assay was carried out without the necessity of column-switching techniques. The method has been validated for use in pharmacokinetic and metabolic studies and represents the initial report of the determination of ketorolac and p-hydroxyketorolac enantiomers in urine. The results of the study indicate that after the administration of racemic ketorolac there was an enantioselective distribution of ketorolac enantiomers in plasma [(R)-ketorolac: (S)-ketorolac = 3.89 2 0.93 (n = 6) and urine (R)-ketorolac: (S)-ketorolac = 1.26 k 0.09 (n = 7)]. The mean ratio of the p-hydroxyketorolac enantiomers was 1.77 2 0.46 (n = 7). Both ketorolac and P-hydroxyketorolac are glucuronized in the acyl carboxyl moiety and the results of this study indicate that this process is not enantiospecific. Ketorolac [(2)-5 benzoyl-2,3-dihydro-~-pyrrolizine-lcarboxylic acid, Ket, Fig. 13 is an analgesic and nonsteroidal antiinnammatory drug (NSAID) which inhibits the arachidonic acid cascade at the cyclooxygenase level. ' It displays nonirritating ophthalmic antiinnammatory activity and it has been used with success in the treatment of postoperative cystoid macular edema, a frequent cause of decreased vision after cataract extraction.Ket is a chiral compound which is marketed as the racemic mixture. Previous animal studies have shown that the (S)-enantiomer is more potent than the (R)-enantiomer of KeL3The pharmacokinetics profiles of the Ket enantiomers has also been studied in humans and (S)-Ket has a higher clearance (CL) and a greater volume of distribution (Vss) than (R)-Ket, while (R)-Ket has a longer elimination half-life.4 The major metabolic route of Ket in humans is acyl glucuronidation which accounts for 28% of the administered dose and a second pathway accounting for 12% of the administered dose is P-hydroxylation, which produces p-OH-Ket.' To our knowledge the enantioselectivity of the urinary excretion of Ket and p-OH-Ket in humans has not been determined.The enantioselective disposition of Ket demonstrated the necessity of using enantioselective analytical methods. Earlier human and animal HPLC studies have been reported for the analysis of Ket in biological fliuds, however, most did not 0 1994 Wiley-Liss, Inc. discriminate between the Ket enantiomers. I X~ An enantioselective assay for (S)-Ket and (R)-Ket in human plasma has been r e p~r t e d .~ This method involved derivatization of Ket with 6)-1-phenylethylamine followed by stereochemical resolution of the diastereomers using reversed-...

DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases alpha and delta) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication.