Mutability of bacteriophage M13 by ultraviolet light: Role of pyrimidine dimers

Summary The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag⁺ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.     

Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.     

T lymphocytes responding to Mls-locus antigens are Lyt-1+, 2− andI-A restricted

We have investigated primary and secondary responses of mouse splenic T cells to strong mixed lymphocyte stimulating antigens controlled by theMls locus using MHC-identical mixtures of cells. Our studies show that strong primaryMls-locus specific responses involve recognition of self I-A antigens, since BUdR and light suicide or F1 into parent radiation bone-marrow chimeras both demonstrate a preference of unprimed F1 T cells to respond to Mis-locus antigens associated with one parent's MHC antigens. Furthermore, conventional anti-I-A antisera and monoclonal anti-I-A antibody both inhibitMls-locus responses in an MHC-specific manner. Finally, as is typical of T cells responding to I-A antigens or to nominal antigens associated with self I-A,Mlslocus responses are mediated by Lyt-1⁺, 2^– cells. One striking finding in these studies was the very high frequency of cells capable of responding to Mls-locus antigens, the highest being 1/300 splenic T cells. This plus evidence for recruitment during primaryMls-locus responses may account for reports of a lack ofI-A restriction in secondary anti-Mls locus responses to strong Mls-locus antigens, a finding with which we concur. The possibility that these secondary responses between noncongenic strains of mice may be directed at other genetic loci is also discussed. These experiments leave open the question of the biological role of theMls-locus and of the very large number of T cells reactive to it.Abbreviations used in this paper MHC Major histocompatibility complex - MIg Mouse immunoglobulin - MLC Mixed lymphocyte culture - TCGF T-cell growth factor     

Polarized fluorescence studies of electrically oriented DNA-dye solutions

Transient changes have been recorded in each of the four polarized components of fluorescence, when dilute solutions of dye-tagged DNA are subjected to short electric pulses. The directions of the absorption and emission transition moments, and hence of the plane of the dye molecules, relative to the DNA geometry have been estimated for eleven dyes. Data obtained for ethidium bromide and five acridine derivatives are consistent with the intercalation model generally accepted for these dyes. In addition, it is shown that neutral red, acridine red and probably auramine O also bind with their molecular planes essentially perpendicular to the long helical axis. The remaining two, hydroxystilbamidine and the bibenzimidazole derivative Hoechst 33258, give rise to effects which indicate that these molecules bind in such a manner that the absorption and emission transitions are closely associated with the grooves of the DNA helix.     

Inotropic effects and changes in sodium and calcium contents associated with inhibition of monovalent cation active transport by ouabain in cultured myocardial cells

Cultured monolayers of spontaneously contracting chick embryo ventricular cells were perfused with culture medium containing ouabain. Contractile state was monitored by an optical-video system recording amplitude and velocity of cell wall motion. Positive inotropic effects of 2.5 x 10(-7) to 10(-6) M ouabain were manifest within 1.5-2 min, and reached a stable plateau within 5-6 min. The inotropic effect was fully reversed within 5 min after washout of ouabain. Inhibition of uptake of 42K+ (or the K+ analog 86Rb+) and efflux of 24Na+ occurred 1.5-2 min after exposure to ouabain. The degree of inhibition of transport was closely related to the magnitude of the positive inotropic effect throughout the ouabain concentration range 10(-7) to 10(-6) M. After washout of ouabain from monolayers, the monovalent cation active transport rate returned to normal within 1 min. Thus, both the onset and offset of inotropic action of ouabain were closely related temporally to inhibition of the sodium pump. Exposure to ouabain caused significant increases in exchangeable Na and Ca contents that appeared to be developed within 5 min. These data support the hypothesis that inhibition of monovalent cation active transport by ouabain is causally related to the development of positive inotropy and are consistent with modulation of Ca content by intracellular Na+ via the Na+-Ca2+ exchange carrier mechanism.     

Restriction cleavage maps of coliphages 186 and P2

Summary The restriction enzymes BamHI, BglII, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, BglII-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and XhoI-1; b) P2, BamHI-3, BglII-2 EcoRI-3, HindIII-0, PstI-3, XbaI-1 and XhoI-0. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.     

The effects of taurine and hypotaurine on in vitro fertilization in the golden hamster

Taurine and hypotaurine were examined for their efficacy in replacing sperm motility factor (SMF), prepared from bovine adrenal cortex, for in vitro fertilization in the golden hamster. Combinations of these amino acids at concentrations of 0.001, 0.01, 0.1, and 1 mM together with 16 μM isoproterenol (a catecholamine β-agonist) were added to the sperm incubations. After three hours of sperm preincubation, oviductal eggs were added to the sperm suspensions and examined for penetration and stage of fertilization after three or five hours of culture. At 0.001 mM, neither taurine or hypotaurine was capable of maintaining motility of hamster sperm for four to 4½ hours or of inducing fertilization. With all other concentrations, both amino acids were found to maintain motility of sperm as well as SMF. Hypotaurine stimulated motility to a greater extent than taurine and both required isoproterenol for the greatest motility. A low proportion of cumulus-free ova were fertilized when sperm were preincubated with either amino acid alone over the range of 0.01 to 1 mM; however, over 80% fertilization was consistently obtained when isoproterenol was also present during sperm incubation. Proportions of ova fertilized with taurine or hypotaurine present during sperm preincubation were comparable to those achieved with SMF. The possibility that taurine or hypotaurine is the sperm motility factor is discussed. After three hours of sperm/egg incubation, a lag in the early events of fertilization was observed in experimental groups treated with one of the amino acids (0.01 mM) alone compared with groups treated with isoproterenol present. However, if sperm/egg incubation was extended from three to five hours, no increase in number of eggs penetrated was found. Therefore, the delay observed at three hours was considered a function of fewer numbers of capacitated sperm present in the absence of isoproterenol rather than of the need for an extended capacitation time.     

Enhancement of rat brain tyrosine aminotransferase activity by cortisol

Administration of cortisol to rats enhanced brain tyrosine:2-oxoglutarate aminotransferase activity by a mechanism excluding stabilization, but possibly involving enhanced synthesis, of the enzyme.     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.