Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis最新文献

A histone H4-specific methyltransferase was purified 80–100-fold from nuclei of calf lymphocytes and from calf thymus. Some biochemical properties of the enzyme are described. The enzyme transfers in vitro methyl groups from $\text{S-}\text{adenosylmethionine}$ preferentially to the lysine residue 20 of histone H4. This is the major in vivo methylation site of H4. DNA-bound or nucleosomal H4 is not methylated in vitro. We have used methylated and unmodified H4 (in the presence of sufficient quantities of the other core histones) for nucleosome reconstitution in vitro and have not found significant differences in the efficiencies of assembly.

Ribosomal proteins from Artemia salina have been separated in a two-dimensional acrylamide gel system and assigned to the small and large ribosomal subunits. Poly(A)-containing RNA was prepared from dormant cysts and from polysomes of 30 min, 1 h, 5 h and 12 h embryos and hatching larvae. The mRNA from different stages was translated in a wheat-germ lysate and its template activity for ribosomal proteins was analyzed. It was observed that mRNA activity for ribosomal proteins is stored in the cytoplasm of dormant cysts and that it is found associated with polysomes of 30 min and of later stages.

The DNA methylase activity present in embryos and cultured cells of Xenopus laevis resembles DNA methylase from mammalian tissues. Little or no activity is found in mature germinal vesicles, though nuclear activity rises rapidly after fertilization. This rise may result in part from a relocation of cytoplasmic enzyme.

A previously described synthetase system of Escherichia coli that utilizes ribonucleoside triphosphates has been purified extensively and shown to consist of an apoenzyme and three protein factors. The apoenzyme itself was revealed to be polynucleotide phosphorylase. The conditions under which the latter — an enzyme incorporating nucleoside diphosphates — is converted to a system catalyzing the uptake of nucleoside triphosphates have been studied in detail with respect to primer requirements, the influence of triphosphates on diphosphate utilization and vice versa, and the possibly regulatory effect of the guanosine di- and triphosphates. The fully supplemented enzyme system (polynucleotide synthetase) incorporates GTP only in the presence of ATP, producing a polynucleotide with an A : G ratio near unity.

Repair of ultraviolet-light-induced DNA damage in a highly pathogenic Gram-negative bacterium, Vibrio cholerae, has been examined. All three strains of V. cholerae belonging to two serotypes, Inaba and Ogawa, are very sensitive to ultraviolet irradiation, having inactivation cross-sections ranging from 0.18 to 0.24 m²/J. Although these cells are proficient in repairing the DNA damage by a photoreactivation mechanism, they do not possess efficient dark repair systems. The mild toxinogenic strain 154 of classical Vibrios presumably lacks any excision repair mechanism and studies of irradiated cell DNA indicate that the ultraviolet-induced pyrimidine dimers may not be excised. Ultraviolet-irradiated cells after saturation of dark repair can be further photoreactivated.

Within 96 h after initial isoproterenol administration, DNA replication and cell cycling were activated, as reflected in the bimodal distribution of nuclear fluorescence determined by flow-microfluorometric technques. A group of proteins, the cetyltrimethylammonium bromide extractable nuclear proteins (CTAB-proteins), isolated from electrostatically sorted nuclei of rat salivary glands, was shown by staining and autoradiography after two-dimensional electrophoresis to undergo differential synthesis during various phases of the in vivo cell cycle after isoproterenol administration. Stained chromatographs revealed quantitative differences in protein synthesis. Gel autoradiography was a more sensitive technique than staining for detecting nuclear protein synthesis during cell cycling. As observed in the autoradiographs of the CTAB-proteins, isoproterenol initiated two distinct periods of protein synthesis in the salivary gland cell cycle: one during the 2C population (G₀/G₁), and one during the 4C population (G₂/M). Protein synthesis after isoproterenol administration was much more dramatic in the 2C (isoproterenol) population, where 22 additional spots were observed, than in the 4C (isoproterenol) population, where five new spots were seen. There was less radioactive incorporation in the 4C (isoproterenol) population. Two spots ‘a’ and ‘b’ that demonstrate differential protein synthesis in stained gel chromatographs and gel autoradiographs were shown to have electrophoretic mobilities, molecular weights and amino acid compositions highly similar to those of HMG1 and HMG2, respectively. A positive correlation could also be drawn between quantitative levels of ‘a’ and ‘b’ and their levels of incorporation during cellular activity with HMG (high mobility group) proteins. For example protein ‘b’ (HMG2) was consistently more abundant in proliferating cell populations than in the quiescent ones. Autoradiographic patterns of the CTAB-proteins indicated that proteins ‘a’ and ‘b’ were synthesized during the $\text{G}{}_0\text{G}{}_1$ phase of the cell cycle, as were the majority of CTAB-proteins.

A proton NMR study at 360 MHz and 500 MHz was carried out on the tetranucleoside triphosphate d(TAAT) at a temperature of 27°C. Extensive decoupling experiments allowed a complete and unambiguous spectral assignment. The data are interpreted in terms of the N and S deoxyribose pseudorotational ranges. From the observed proton-proton coupling constants it is calculated that (a) the populations of deoxyribose S-form are as follows: dT(1)-, 85%; -dA(2)-, 97%; -dA(3)-, 81%; -dT(4), 64%; and (b) the g⁺ populations (backbone notation) along the exocyclic C4′-C5′ bond in -dA(2)-, dA(3)- and -dT(4) are 82%, 86% and 78%, respectively. From these values, combined with chemical shift considerations, it is concluded that the central -dA(2)-dA(3)- part of the molecule occurs preferentially as a mixture of two right-handed single-helical conformations, denoted S-S and S-N, in a ratio of approximately 8 : 2. This situation closely mimics that found for the 3′-end of d(A-A-A) (Olsthoorn, C.S.M., Bostelaar, L.J., Van Boom, J.H. and Altona, C. (1980) Eur. J. Biochem. 112, 95–110). Similarly, the conformational behaviour of the dT(1)-dA(2)- and -dA(3)-dT(4) terminals appears roughly identical to that displayed by the corresponding dinucleoside monophosphates. The molecule as a whole does not show signs of cooperativity of stacking.

Using either DEAE-cellulose chromatography or pH 4–6 isoelectric focusing, we have separated mouse skeletal muscle neutral RNAase II-inhibitor complex into two fractions (designated α and β).para-Hydroxymercuriphenylsulfonate-induced dissociation/inactivation of the inhibitor yields free RNAase II enzyme fractions with differing pH profiles, CM-cellulose chromatographic behavior and reactivity with the RNAase II inhibitor of human placenta. However, the free RNAase fractions react equally with purified inhibitor from skeletal muscle and are not separable by pH 8–9.5 isoelectric focusing. These data suggest that mouse skeletal muscle has two heterogeneous forms of RNAase II. Additionally, heterologous RNAase II inhibitors may be used as investigational tools when probing neutral RNAase II heterogeneity.

$\text{N-}\text{Ethylmaleimide}$, a sulfhydryl-specific reagent, strongly inhibits AMV reverse transcriptase by specifically interfering with the template-binding site of the enzyme. However, the kinetics of inhibition differ widely with the composition and structure of the templates employed. The copying of templates with multiple 3′-hydroxyl termini appeared to be more susceptible to $\text{N-}\text{ethylmaleimide}$ treatment, suggesting that the reagent may interfere with initiation of DNA synthesis. The ability of a template bound to enzyme prior to $\text{N-}\text{ethylmaleimide}$ treatment to protect against inactivation of copying of other templates also, implies a common binding site for the different templates. Template exchange experiments demonstrated competition between activated calf thymus DNA and rA_n · dT_12–18 for binding to the enzyme. Thus, templates varying widely in composition and conformation appear to bind at a common site on reverse transcriptase. The experimental data also show suggestive evidence for small but finite differences in the requirements for optimal binding for templates of different structures.

The presence of interstrand crosslinks in the DNA of cis-diamminedichloroplatinum(II) (cisplatin)-treated Chinese hamster cells was demonstrated by the formation of a species of hybrid-labelled DNA in cells that had been density and radioactively prelabelled with a combination of [³H]thymidine and 2′-deoxy-5-bromouridine (BrdUrd) prior to treatment. The extent of crosslinking and of total binding of platinum to the DNA increased linearly with dose over a wide range of concentrations of cisplatin. Simultaneous measurement of the size of co-isolated ¹⁴C-labelled DNA from untreated cells permitted an estimate of the size of the crosslinked DNA and hence of the proportion of bound platinum involved in a crosslinking reaction. No increase in the proportion of crosslinking reactions occurred during several hours post treatment incubation of cells when crosslinks were measured by this technique, in contrast to other studies using different techniques to measure DNA interstrand crosslinking in cisplatin treated cells (see following paper, Pera, M.F., Jr., Rawlings, C.J., Shackleton, J. and Roberts, J.J. (1981) Biochim. Biophys. Acta 655, 152–166). The enforced high concentrations of cisplatin required by this method to detect crosslinks precludes studies of their possible repair.