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

The acidic proteins extracted by 0.4 M NH₄Cl and 50% ethanol from ribosomes from Saccharomyces cerevisiae, wheat germ, Artemia salina, Drosophila melanogaster, rat liver and rabbit reticulocytes have been studied comparatively in several structural and functional aspects. All the species studied have in the ribosome two strongly acidic proteins with $\text{p}\text{I}$ values not greater than pH 4.5, which appear to be monophosphorylated in the case of S. cerevisiae, A. salina, D. melanogaster and wheat germ. Rat liver proteins are multiphosphorylated, as possibly are those from reticulocytes. The molecular weight of these acidic proteins as determined by SDS electrophoresis ranges from around 13 500 to 17 000 and, except in the case of yeast, of which both proteins have the same molecular weight, the size of the two proteins in the other species differs by approx. 1000–2000. In general, the size of the proteins increases with the evolutionary position of the organism, as seems to be the case with the degree of phosphorylation. From an immunological point of view the ribosomal acidic proteins of eukaryotic cells are partially related, since antisera against yeast protein cross-react with proteins from wheat germ, rat liver and reticulocytes. Bacterial proteins L7 and L12 are very weakly recognized by the anti-yeast sera. Anti-bacterial acidic proteins do not cross-react with any of the protein from the species studied. The proteins from all the species studied are functional equivalents and can reconstitute the activity of particles of S. cerevisiae deprived of their acidic proteins.

Nuclear triiodothyronine-binding proteins (NTBP) which are at present considered as a nuclear receptor for triiodothyronine (T₃) have been partially purified (about 100-fold) from rat liver nuclei by Sephadex G-100 gel filtration and DEAE-Sephadex chromatography and incubated with [¹²⁵I]-T₃([¹²⁵I]T₃-NTBP complexes). Their ability to bind to DNA and chromatin was analyzed by using in the first case rat liver or calf thymus DNA either soluble or coupled to Sepharose 4B and in the second case expanded chromatin preparations from residual chromatin after NTBP extraction (extracted chromatin) or from whole nuclei (total chromatin). [¹²⁵I]T₃-NTBP complexes could be rapidly and totally bound to DNA; chromatin binding was slower and less efficient on a DNA weight basis, particularly with total chromatin. In both cases, binding presented similar characteristics: it was highly sensitive to concentration of KCl and divalent cations; it depended upon the presence of reducing agents and probably the maintenance of a proper conformation of the receptor molecule; it did not need the presence of bound hormone; it was inhibited by ethidium bromide, actinomycin D and heparin. DNA binding of [¹²⁵I]T₃-NTBP complexes was similar with eukaryotic DNAs (rat, calf), reduced with Escherichia coli DNA, synthetic poly[d(A-T)] and heat-denatured DNA, and almost non-existent with poly(dA) or yeast RNA. No saturation of rat liver DNA could be detected, suggesting a capacity higher than 130 pmol NTBP/mg DNA. Saturation curves were observed in only three experiments out of six using total chromatin preparations and suggested capacities about 50-fold higher than the physiological concentrations of NTBP in nuclei (0.5 pmol/mg DNA). Furthermore, NTBP binding was significantly lower with spleen chromatin preparations as compared to liver ones. Thus, the T₃ receptor appears as a DNA binding protein; it is suggested from our in vitro chromatin binding studies that DNA could be the major locus of NTBP binding in chromatin. The extent of DNA binding in chromatin is limited by other chromatin components in a more pronounced manner with the least modified chromatin preparations and with chromatin preparations from spleen, a tissue poorly responsive to thyroid hormones. Nevertheless, another chromatin localization of the T₃ receptor cannot be excluded.

Synthesis of carbamyl phosphate synthetase was undetectable in fetal rat liver at 16 days gestation but by 4–5 days after birth (11–12 days later), this single protein accounted for approx. 5% of total liver protein and roughly 1% of total liver protein synthesis. Likewise, translatable mRNA coding for the enzyme was absent from 16-day fetal livers but then rapidly accumulated reaching maximum levels just after birth. The in vitro primary translation product of carbamyl phosphate synthetase mRNA corresponded to a higher molecular weight biosynthetic precursor of the enzyme; peptide maps obtained from the precursor synthesized both in vivo and in vitro and from the mature enzyme made in vivo were the same. When livers of neonatal rats were perfused with collagenase and further treated to yield a preparation of freshly dispersed hepatocytes highly active in general protein synthesis, a procedure which took about 45 min to complete, biosynthesis of carbamyl phosphate synthetase was found to be completely absent in these cells. The mRNA coding for the enzyme, however, could be extracted from the dispersed hepatocytes and was actively translatable in vitro, at levels approximately 75% of those for mRNA obtained from intact liver. Repression of biogenesis of carbamyl phosphate synthetase in dispersed hepatocytes, therefore, must involve a mechanism which shifts the mRNA coding for the enzyme out of active polysomal complexes and renders it further untranslatable in vivo but not in vitro.

Total human DNA was fractionated from the three types of Cs₂SO₄ gradient used to prepare satellites I, II and III. Three satellite DNAs were found: satellite I with a mean buoyant density of 1.688 g/ml comprising about 1.3% of the total, satellite II with a mean buoyant of 1.696 g/ml comprising about 1% of the total and satellite III with a mean buoyant density of 1.699 g/ml comprising about 2.2% of the total. The buoyant densities of these satellites after purification were 1.686, 1.694 and 1.697 g/ml, respectively. A preparation with the attributes of satellite IV was isolated from the shoulder region of a satellite III preparative gradient. In situ hybridization using complementary RNA showed that the three satellites were located predominantly on chromosomes 9, Y, 15 and 1. Satellite II also showed marked hybridization to chromosome 16. Satellites I and III and II and III cross-hybridized to each other but satellites I and II did not. On the basis of our hybridization data, we suggest that some of the same sequences which comprise satellite III also comprise satellites I and II.

DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was $\text{0.53 ± 0.08}\text{μm/min}$. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.

The in vitro transcription of chloroplast DNA (ctDNA) is studied using a DNA-protein complex isolated from spinach plastids. The RNA products are compared to the in vivo synthesized ctRNA by competition for hybridization. At least 80% of the in vitro RNA sequences are present in vivo. Modifications of ionic strength or introduction of heparin in the reaction medium has an important effect on the transcriptional activity of the complex. Furthermore, the length of the RNA chains increases with increasing ionic strength and amount of heparin. The RNA products are analysed by Southern hybridizations to EcoRI ctDNA fragments. Changes in the ionic strength or in the amount of heparin modify heterogeneously the transcription of the various DNA regions. The quantitative distribution of transcripts among the ctDNA fragments is used as evidence for the selectivity of the transcription. The activity of the DNA-protein complex is much more resistant to high ionic strength than an heterologous transcription system using Escherichia coli RNA polymerase and ctDNA. This latter system transcribes less ctDNA fragments.

A sea urchin ribosomal DNA 1.9 kilobase BamHI fragment adjacent to the 5′ end of the 18 S gene has been mapped with the restriction enzymes, XhoI, EcoRI, SmaI and HinfI. A 270 basepair fragment which most likely contains the 5′ end of the presumed primary transcript of rRNA was identified by hybridization of [³²P]DNA fragments to total nuclear RNA separated on methylmercury hydroxide gels and bound to diazobenzyloxymethyl paper. Under these denaturing conditions the size of Lytechinus variegatus precursor rRNA was determined to be 7.2 kilobases (33 S).

When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coli, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch.

The degradation of a mixture of four 5′-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1–0.5 mg/I) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.