Resistance to tetracycline and lincosamide antibiotics was transferred en bloc from a strain of Bacteroidesfragilis (V503) to a plasmidless strain of Bacteroides uniformis (V528) during in vitro filter matings. Resistance transfer was detected at frequencies of 1i-5 to 10-6 drug-resistant progeny per input donor cell and was dependent on cell-to-cell contact of donors and recipients. Transfer was insensitive to DNase and was not mediated by chloroform-or filter-sterilized donor broth cultures. A determinant for resistance to cefoxitin in V503 was not transferred in this system. V503 contained a 3.7 x 106-dalton plasmid (pVA503). Drug-resistant progeny of V503 x V528 matings usually contained pVA503, but up to 20o of the total progeny of such crosses were plasmid free. Filter blot DNA hybridization studies (Southern method) confirmed that pVA503 was not integrated into the host chromosome of the plasmidless progeny. Drug-resistant progeny from V503 x V528 matings (with or without pVA503) conjugally transferred clindamycin resistance and tetracycline resistance to a suitable recipient strain. None of the drug resistance determinants of V503 were affected by treatment with standard plasmid curing regimens, and methods designed to detect very large plasmid molecules failed to suggest the involvement of extrachromosomal DNA in this resistance transfer system. The well-characterized Bacteroides R plasmid, pBF4 (conferring clindamycin resistance), was found to share hybridizing sequences with bulk cellular V503 DNA when examined by filter blot hybridization. Similarly sized sequences were found in drug-resistant progeny recovered from matings. Neither of the two pBF4 derivatives carrying deletions that abolished clindamycin resistance hybridized with V503 DNA.In the last 3 years there have been several reports describing self-transferable resistance (R) plasmids isolated from anaerobic bacteria.Brefort et al. (2)

Analysis of the properties of the DNA polymerase (pol) system as a function of fundamental factors of the assay environment allowed a rather accurate estimation of its dependence on the HeLa cell cycle. For pol a, the temperature and pH optima were 38.10C and 8.0, respectively; for pol (3, these optima were 36.20C and pH 7.4. Pol y showed a pH optimum at 7.7. Optimum activity for both the a and (3 enzymes was observed at 60 mM Tris. The maximal activity at 36.20C and pH 7.4 was associated with resistance to N-ethylmaleimide (MaINEt), whereas that at 38.10C and pH 8.0 was sensitive to MaINEt. Incorporation of [3HjdTTP was maximal after 1 hr of incubation for the former activity and after 4 hr, for the latter.In extracts from cells in early S phase, the pol activity decreased after 1 hr of incubation, was MaINEt-resistant, and was characterized by temperature and pH optima at 36.20C and 7.4, respectively. In extracts of late S-phase cells, the polcatalyzed incorporation of [3H]dTTP continued after 4 hr of incubation, was MalNEt-sensitive, and was characterized by temperature and pH optima at 38.10C and 8.0, respectively. Thus, a pol (3-type activity appeared in early S phase, whereas a pol a-type activity appeared in late S. During the G1, M, and G2 phases, a background level of pol activity was observed that showed intermediate kinetic properties.The information available about the physicochemical properties and regulation of the eukaryotic DNA polymerases a, A, and y (pol a, /3, and y) has been insufficient and not always devoid of contradictions (1-3). Little has been known about either the kinetics or the temperature, pH, and ionic strength optima for these enzymes. In fact, assays for pol a, (, or y are variously carried out through 30-60 min incubations at 370C and pH 7.4-9.2, using 20-40 mM concentrations of the Tris buffer (1-3). Studies of regulation of the pol system as a function of the cell cycle often have given inconsistent results. For instance, whereas one analysis showed a rise in the pol a activity during the S phase (4), another suggested that the rise and fall in pol a activity is independent of DNA synthesis (1).In (6,7). DNA biosynthesis and the lengths of the cell-cycle phases were measured as described (7).Crude Cytoplasmic Pol Extract. This extract was prepared from synchronized or unsynchronized cells by using the procedure described by Pedrali-Noy and Weissbach (8), with minor modifications. All operations were carried out at 40C in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (9). The extracts were always assayed for DNA (10) and protein (11).Purification of Pol a, (3, and y. The procedure followed to obtain partially purified enzyme preparations (approximately 300-, 200-, and 150-fold for pol a, (3, and y, respectively) was essentially that proposed by Lewis et al. (12).Enzyme Assay. The enzyme activity was determined as described (13,14). A crude extract or a purified enzyme sample was added to an equal volume of incubation mixture containing "activat...

To clarify the relationship between the pre-S antigens and other serological markers of hepatitis B virus (HBV) replication, we followed up 27 patients: 21 presented with symptoms of acute hepatitis (two progressed to chronicity) and six suffered from chronic hepatitis. Pre-S1, pre-S2, HBV DNA, IgM antihepatitis core antigen (HBc), hepatitis B e antigen (HBeAg), and anti-HBe were detected in about 200 sera serially collected at different times for at least 6-12 months from the onset of clinical observation. In the early symptomatic phase of acute hepatitis, the pre-S1 and pre-S2 antigens were present in 95% of the cases and correlated well with high levels of alanine-transferase (ALT) and IgM anti-HBc, while HBV DNA was present in the sera of only six (28.6%) patients (P less than 0.0001). This was the first marker to disappear (1 month after the initial stage). All of the HBV DNA-positive patients were also HBeAg positive, whereas no HBeAg-negative subjects were found with serum HBV DNA. In the six chronic patients, pre-S antigens were always present independently of the HBeAg/anti-HBe status; HBV DNA was detected in three of them, even if transiently, and in two of these it reappeared together with pre-S2 epitope. The follow-up data suggest that, in acute hepatitis, the clearance of pre-S antigens can be considered as a prognostic index of clinical resolution and that, in chronic hepatitis, the persistence of pre-S antigens seems to indicate progression of the disease. In particular, pre-S2, in patients in whom it is intermittent, can be considered as an index of reactivation.

The HIV viral burden and RNA expression in a selected group of infected, clinically non-progressor patients were investigated. Five fast-progressor patients and 10 AIDS cases were included as controls. The HIV viral load was investigated by semiquantitative polymerase chain reaction (PCR) in adherent macrophages and in genomic and extragenomic fractions of lymphocytes. HIV DNA was not found in macrophages in the non-progressor subjects, was weakly positive in 2 of 5 fast-progressors and strongly positive in most of the AIDS patients. The number of HIV proviruses found in lymphocytes of the non-progressor subjects varied from 5 to 160 copies/microgram DNA, values ten times lower than those recorded in fast-progressors and AIDS patients. The extragenomic HIV DNA (2 LTR forms) was absent or barely detectable in the lymphocytes from non-progressors and abundant in the other groups. HIV RNA was not found in the lymphocytes of all non-progressors. This may indicate that a latent state of HIV provirus exists in the lymphocytes of these subjects. To investigate this point, cultivation and stimulation with PHA (phytohemoagglutinin) and PMA (phorbol 12-myristate 13-acetate) of lymphocytes from these subjects were attempted but after 6 days HIV RNA (RT-PCR for gag region) was still absent or barely detectable in these patients. There are no other reports of the absence of HIV provirus induction in lymphocytes from infected individuals. If confirmed in a larger number of patients, such non-inducibility might serve as a predictor marker of progression of the disease.

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