Characterization of an α-amanitin insensitive RNA polymerase from cauliflower

α-Amanitin insensitive RNA polymerase (polymerase I isolated from apical parts of the cauliflower inflorescence was highly stable for several months at − 18°. The DEAE-cellulose fraction was more effective in utilizing denatured DNA than native DNA as a template. Optimum pH for RNA synthesis was ca 7 in the reaction mixture with Tris-HCl or with Tris-maleate buffer. From the properties examined, it seems that DNA-dependent RNA polymerase I of cauliflower differs from other eucaryotic RNA polymerases.     

DNA-dependent in vitro synthesis of ribosomal proteins,protein elongation factors,and RNA polymerase subunit α: Inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) had been implicated in stringent control, we examied the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage λfus3 and λrif^d18. λfus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit α, in addition to genes for approximately 27 r proteins. λrif^d18 carries genes for EF-Tu, RNA polymerase subunits β and β^I, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2–0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit α, as well as rRNAs.     

Isolation of DNA-dependent RNA polymerase fromStreptomyces granaticolor and its binding to phage ϕ29 DNA

Partially purified DNA-dependent RNA polymerase ofStreptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits, represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage ϕ29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases fromEscherichia coli andBacillus subtilis.     

Structural Characterization of LNA and α-L-LNA Hybridized to RNA

Abstract

LNA and α-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we present a short overview of the structural results we have obtained for LNA:RNA and α-L-LNA:RNA hybrids. Specifically, we have shown that LNA acts as an A-type mimic, while α-L-LNA acts as a B-type mimic when built into oligonucleotides.     

Polypeptide profiles of chlorophyll · protein complexes and thylakoid membranes of spinach chloroplasts

In addition to the major chlorophyll · protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll $\text{a}\text{b}$ · protein and from the reaction centre complex of Photosystem II.Data are presented on the polypeptide profiles of purified digitonin-subchloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll · protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.     

Participation of β-carotene in reactivation of PSI of heptane-extracted spinach chloroplasts

A carotenoid requirement for photosystem I activity in spinach chloroplasts using extraction-reconstitution technique has been investigated. The transfer of electron from N,N,N,N-tetramethyl-p-phenylene diamine through the chloroplast photosystem to methyl viologen dye or to NADP⁺ was used as an assay of photosystem I activity. Extraction of lyophilized spinach chloroplasts with heptane at near 0°C removed almost all -carotene and reduced photochemical activities associated with photosystem I to a low level (about 15% of the original activity). Reconstitution of the extracted chloroplasts with -carotene completely restored photosystem I activity. The maximum rate of methyl viologen photoreduction in reconstituted chloroplasts occurred at an -carotene/chlorophyll molar ratio of 0.5. Cyclic phosphorylation mediated by phenazine methosulphate was partially restored. Xanthophylls (lutein, neoxanthin, violaxanthin), as components of chloroplast membranes, were not able to replace -carotene in reconstitution of chloroplasts and had essentially no effect on restoring photoreactions. On the basis of the P700/total chlorophyll ratio it can be assumed that extraction of lyophilized chloroplasts with heptane do not affect photosystem I reaction centre. Therefore it is possible that -carotene, removed during heptane extraction and belonging mainly to the antenna pigment pool of photosystem I, is effective in the restoration of photosystem I activity.Abbreviations chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - MV methyl viologen - PMS phenazine methosulphate - PQA plastoquinone A - PS I photosystem I - PS II photosystem II - TMPD N,N,N',N'-tetramethyl-p-phenylene diamine - Tricine N-tris(hydroxymethyl)methyglycine. D-1, D-10, D-50, D-144 represent chloroplast subfractions sedimented at 1000 × g, 10,000 g, 50,000 × g and 144,000 × g - s supernatant This paper is a partial fulfillment of the requirements for the Ph.D. degree of A.T. at Maria Curie-Skodowska University, Lublin.     

Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.     

Site specific inhibition by α-benzyl-α-bromomalodinitrile (BBMD) of electron transport in spinach chloroplasts

The addition of α-benzyl-α-bromomalodinitrile to different controlled states (non-phosphorylating [2]. phosphorylating [3], ATP-inhibited [4] and uncoupled) of photosynthetic electron transport to ferricyanide or benzoquinone demonstrate a significant inhibition in isolated spinach chloroplasts. α-Benzyl-α-bromomalodinitrile pretreatement of isolated chloroplasts or addition of α-benzyl-α-bromomalodinitrile at the onset of illumination completely abolished the O₂ evolving reaction. The level of the steady state fluorescence in intact chloroplasts showed a α-benzyl-α-bromomalodinitrile concentration-dependent increase. The gradual decrease in the reoxidation capacity of the reduced quencher, Q with increasing α-benzyl-α-bromomalodinitrile concentrations provides evidence for an additional inhibitory site for α-benzyl-α-bromomalodinitrile between the two photosystems.     

Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase μ (polμ), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polμ and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polμ. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polμ expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polμ is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polμ in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polμ provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polμ themselves.     

Characterization of 26S proteasome α- and β-type and ATPase subunits from spinach and their expression during early stages of seedling development

Three kinds of cDNAs encoding 26S proteasome subunits have been cloned from spinach (Spinacia oleracea L.). These genes, designated as SOPSC8, SOPSC1 and SOPRS7, encode an -type and a -type subunit of the 20S catalytic core, and an ATPase subunit of the 19/22S regulatory complex, respectively. The deduced protein sequences showed high sequence similarities to other proteasome - and -type and ATPase subunit proteins. Southern blot analysis indicates that there are additional members of these dispersed proteasome families in the spinach genome. These three subunit genes are expressed simultaneously during germination and reach a maximum one day after sowing followed by a decline. The expression of these genes also increases during cotyledon senescence.     

Characterization of a ts β′ mutant RNA polymerase ofEscherichia coli

Summary RNA polymerase isolated from ts XH56, a conditional lethal mutant unable to grow and synthesize RNA at 42°, was found to be temperature sensitive in vitro. The mutation affects the subunit as determined by mixed reconstitution of isolated subunits from wild-type and mutant enzyme. The mutant RNA polymerase is unstable; addition of glycerol stabilizes the enzyme and increases its activity on native DNA. In addition, the mutant enzyme is sensitive to high ionic strength. Both high temperature and high ionic strength do not affect chain elongation; thus, the mutation renders the enzyme unable either to bind to or melt out promotor sites. From these data we conclude that the subunit plays an important role in promotor selection.     

Regulation of RNA polymerase II activity in α-amanitin-resistant CHO hybrid cells

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to α-amanitin with mutant cells containing RNA polymerase II activity resistant to α-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing α-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to α-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the α-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975).A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in α-amanitin exhibited a 2–3 fold increase in the activity of the α-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the α-amanitin-inactivated polymerase II of wild-type sensitivity by ³H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of ³H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.     

Intramolecular distortion of the α-helical structure of polypeptides

Broadening of the infrared amide A, amide I and amide II bands of α-helical polypeptides has been observed for thermodynamically unstable α-helices. This spectroscopic fact can be explained now by the geometrical distortions of the backbone of the helical structure. Two models for distorted helices which include regular or irregular distortions of the angles of internal rotation of the main polypeptide chain have been considered. It is pointed out that the instability of α-helix is associated with irregular distortions of the polypeptide backbone.