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Thymidylate synthetase has been purified from cultures of Escherichia coli infected with bacteriophages T4 or T5, with the T4 enzyme being purified to at least 50% of homogeneity, and both enzymes being resolved from the corresponding host enzyme. The molecular weights are 58,000 for the T4 enzyme and 55,000 for the T5 enzyme, as estimated by gel filtration and confirmed for the T4 enzyme by sucrose gradient analysis. Disc gel electrophoresis of the T4 enzyme in sodium dodecyl sulfate gives a single band with a molecular weight of 29,000, suggesting that the enzyme is composed of two subunits. Kinetic analysis of the inhibition of the T4 enzyme by 5-fluorodeoxyuridylate (FdUMP) gives results similar to those earlier reported for the T2 and T6 enzymes. Inhibition is competitive with respect to deoxyuridylate (dUMP) if the enzyme is not preincubated with inhibitor, but a brief preincubation of enzyme and inhibitor in the presence of 5, 10-methylenetetrahydrofolate generates a pattern of noncompetitive, stoichiometric inhibition. FdUMP remains bound to the enzyme through gel filtration chromatography, consistent with various observations that this inhibitor is covalently bound. However, the enzyme-inhibitor complex is dissociated by treatment with sodium dodecyl sulfate prior to chromatography. Moreover, in contrast to studies on thymidylate synthetase from other sources, oxidation of tetrahydrofolate by FdUMP-inhibited enzyme could not be detected. Inhibition of the T5 enzyme by FdUMP is not stoichiometric, and the enzyme-inhibitor complex is readily dissociated by gel filtration. These findings suggest that there are significant differences in mechanism of FdUMP binding by thymidylate synthetases of different origins. Inhibition of the T4 enzyme by trifluoromethyldeoxyuridine 5′-monophosphate (F₃dTMP) follows the kinetics of stoichiometric inhibition, but data from both gel filtration and enzyme-inhibitor titration indicate that the enzyme binds 12–13 times as much F₃dTMP as FdUMP, suggesting that most of the F₃dTMP is bound at noncatalytic sites.     

APOBEC3G Polymorphism as a Selective Barrier to Cross-Species Transmission and Emergence of Pathogenic SIV and AIDS in a Primate Host

Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary “arms-race” between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary “arms-race” hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.     

The establishment of the North-American mosquito fish,Gambasia holbrooki,in Syrian inland waters)

Abstract

The North-American mosquito fish, Gambusia holbrooki (Girard, 1859), came to Syria in 1929 but only recently spread over large parts of the country. Between 1977 and 1979 Gambusia was studied in various water bodies throughout Syria. The conditions For its successful establishment and its effect on populations of the indigenous cyprinodont fish Aphanius mento (Heckel, 1843) were surveyed. Out of 31 localities where Gambusia and/or Aphanius occurred 17 were inhabited by both species, 11 only by Aphanius and three only by Gambusia. A comparison of these localities indicated that Gambusia is better adapted to high salinity and polluted water whereas Aphanius tolerates stronger currents. Possible negative effects of Gambusia on the indigenous aquatic fauna of Syria are briefly discussed.     

Biochemical events essential to the recombination activity of Escherichia coli RecA protein. II. Co-dominant effects of RecA142 protein on wild-type RecA protein function

In the accompanying paper, RecA142 protein was found to be completely defective in DNA heteroduplex formation. Here, we show that RecA142 protein not only is defective in this activity but also is inhibitory for certain activities of wild-type RecA protein. Under appropriate conditions, RecA142 protein substantially inhibits the DNA strand exchange reaction catalyzed by wild-type RecA protein; at equimolar concentrations of each protein, formation of full-length gapped duplex DNA product molecules is less than 7% of the amount produced by wild-type protein alone. Inhibition by RecA142 protein is also evident in S1 nuclease assays of DNA heteroduplex formation, although the extent of inhibition is less than is observed for the complete DNA strand exchange process; at equimolar concentrations of wild-type and mutant proteins, the extent of DNA heteroduplex formation is 36% of the wild-type protein level. This difference implies that RecA142 protein prevents, at minimum, the branch migration normally observed during DNA strand exchange. RecA142 protein does not inhibit either the single-strand (ss) DNA-dependent ATPase activity or the coaggregation activities of wild-type RecA protein. This suggests that these reactions are not responsible for the inhibition of wild-type protein DNA strand exchange activity by RecA142 protein. However, under conditions where RecA142 protein inhibits DNA strand exchange activity, RecA142 protein renders the M13 ssDNA-dependent ATPase activity of wild-type protein sensitive to inhibition by single-strand DNA-binding protein, and it inhibits the double-strand DNA-dependent ATPase activity of wild-type RecA protein. These results imply that these two activities are important components of the overall DNA strand exchange process. These experiments also demonstrate the applicability of using defective mutant RecA proteins as specific codominant inhibitors of wild-type protein activities in vitro and should be of general utility for mechanistic analysis of RecA protein function both in vitro and in vivo.     

Intranuclear rodlets in woodchuck thyroid follicular cells

Summary Intranuclear rodlets are seen in thyroid follicular cells of woodchucks fixed during summer and fall. They are not observed in winter and spring animals. A functional significance of these structures is suggested.Supported in part by PHS 05429-13-5 (P.P. Krupp, Department of Anatomy) and PHS R01 10254 (E.A. Sims, Metabolic Unit, Department of Medicine)     

Attractiveness qualifies the effect of observation on trusting behavior in an economic game

Recent studies show that subtle cues of observation affect cooperation even when anonymity is explicitly assured. For instance, recent studies have shown that the presence of eyes increases cooperation on social economic tasks. Here, we tested the effects of cues of observation on trusting behavior in a two-player Trust game and the extent to which these effects are qualified by participants' own attractiveness. Although explicit cues of being observed (i.e., when participants were informed that the other player would see their face) tended to increase trusting behavior, this effect was qualified by the participants' other-rated attractiveness (estimated from third-party ratings of face photographs). Participants' own physical attractiveness was positively correlated with the extent to which they trusted others more when they believed they could be seen than when they believed they could not be seen. This interaction between cues of observation and own attractiveness suggests context dependence of trusting behavior that is sensitive to whether and how others react to one's physical appearance.     

Seasonal ultrastructural variations in pinealocytes of the woodchuck,Marmota monax

The ultrastructure of the pinealocyte in the woodchuck, Marmota monax, was studied during the four seasons of the year. Fall cells have a fairly uniform cytoplasmic density, organelles consistent with synthetic and/or secretory activity and rather extensive pericapillary and intercellular spaces. Many winter pinealocytes are nearly devoid of ribosomes and granular endoplasmic reticulum but contain lipid droplets associated with mitochondria. Pericapillary and intercellular spaces are minimal. Spring glands have the greatest variation in cytoplasmic density with intercellular and pericapillary spaces similar to that seen in fall glands. Cells containing electron dense cytoplasm have Golgi zone associated, secretory granules, free ribosomes, short sections of granular endoplasmic reticulum and dense bodies. Cells with a more electron lucent cytoplasm are similar to the most frequently observed summer pinealocytes which have numerous Golgi zones but few associated secretory granules. Microtubules are prominent in the cytoplasm of these cells, the plasma membranes are smooth and intercellular and pericapillary spaces are minimal. A yearly rhythm or cyclic activity of the pinealocyte is suggested.     

Microarray-based gene expression analysis of hepatocellular carcinoma

Microarray studies have successfully shed light on various aspects of the molecular mechanisms behind the development of hepatocellular carcinoma (HCC), such as the identification of novel molecular subgroups and the genetic profiles associated with metastasis and venous invasion. These experiments, mainly comprising genome wide profiling, potentially represent the basis of novel targeted therapeutic strategies in HCC. In response, we summarize the multiple reported expression profiles in HCC associated with HCC development, novel subgroups, venous invasion and metastasis.