We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone.
RNA was extracted from fetal calf skin by two different procedures, using phenol or guanidine hydrochloride. Poly(A)-rich RNA was separated by oligo(dT)-cellulose affinity chromatography and was further fractionated by sucrose density gradient centrifugation. When translated in an optimized wheat germ extract cell-free system, unfractionated guanidine-hydrochloride-extracted poly(A)-rich RNA directed the synthesis of two collagenase-sensitive protein bands, while phenolextracted poly(A)-rich RNA with a sedimentation coefficient higher than 25 S was the only fraction to direct the same synthesis. On the basis of their electrophoretic mobility on a sodium dodecylsulfate/ urea/polyacrylamide gel, these proteins were identified with procollagen stl(1) and procollagen a2. Inhibition of translation by phenol-extracted poly(A)-rich RNA with a sedimentation coefficient lower than 25 S was also observed.Guanidine-hydrochloride-extracted poly(A)-rich RNA from fetal calf skin directed the synthesis of three distinct collagenase-sensitive proteins in the micrococcal-nuclease-digested rabbit reticulocyte cell-free system ; these seemed to correspond to procollagen a1 (I), procollagen a2 and procollagen a1 (I1 I).Several different types of collagens are known to compose the resistant framework which ensures the physical properties of connective tissues (for review, see [l]). Collagen mRNA has been isolated from various connective tissues, for example, tendon [2,3], bone [4-71, lung [8] and fibroblast culture [3,9-131. It has been found to be monocistronic [14] and its M, has been estimated to be 1.5 x lo6 for each of the three polypeptide chains composing the precursor of the collagen molecule. This large mRNA has been translated in different endogenous [15] or exogenous cellfree systems such as the wheat germ [4-7,111, the rabbit reticulocyte lysate [3,8,10,12] and the Krebs ascites tumor [9] to yield precursors of a1 and a2 chains.Skin is a much more complex tissue since it is composed of a parenchyme of ectodermal origin (the epidermis and the epidermal derived appendages) and a connective tissue. Its fibrous framework predominantly consists of types I and 111 collagen in measurable concentrations [16,17], as well as other types of collagens in trace amounts. Skin, therefore, represents a potential source of several mRNA species.Skin is a most interesting tissue for studying the genetic expression of the various collagen species since the proportion of type I11 to type I collagen undergoes changes during development, wound healing [16,17] and various pathological processes. In order to approach the control mechanism regulating such changes, suitable techniques have been devised for extraction and translation of skin mRNA. This paper compares the efficiency of the phenol method compared to the guanidine hydrochloride method for skin RNA extraction. The isolated RNAs, purified by oligo(dT)-cellulose affinity chromatography and sucrose gradient ultracentrifugation, were translated in two different cell-free systems. The pres...
Two RNA polymerase activities (RNA polymerase A and B) were solubilized by ammonium sulfate extraction of nuclei from human placenta. Both purified activities were completely dependent upon added DNA. RNA polymerase A was insensitive to -amanitin, whereas RNA polymerase B was inhibited by this compound. The enzymes differed in their responses to salt conditions. The template specificities of RNA polymerase B were studied using native and denatured DNA from different sources as well as synthetic double-stranded polydeoxynucleotides. Both denatured calf-thymus DNA and poly [d(i-C)-d(I-Q] were excellent templates. Several kinds of phage DNA were not used as templates unless denatured. The inhibitory effect of various semi-synthetic derivatives of rifamycin SV were investigated for possible inhibitory effects on RNA polymerase B. One of these compounds, AF/013, inhibited RNA synthesis at a concentration of 20 g/m/. Structural studies on RNA polymerase B indicated the presence of three large subunits with molecular weights of about 200000, 180000 and 150000. Lösliche RNA-Polymerase-Aktivitäten aus menschlicher Plazenta: Strukturelle und funktionelle Eigenschaften Zusammenfassung: Aus den Kernen der menschlichen Plazenta wurden durch Extraktion mit Ammoniumsulfat zwei RNA-Polymerase-Aktivitäten (RNA Polymerase A und B) gewonnen und gereinigt. Beide Aktivitäten waren völlig abhängig von zugesetzter DNA. RNA-Polymerase A war insensitiv gegenüber -Amanitin, während RNA-Polymerase B durch diese Verbindung inhibiert wurde. Beide Enzyme unterscheiden sich in ihrer Abhän-gigkeit von Salzkonzentrationen. Die Matrizen-Spezifität von RNA-Polymerase B wurde geprüft. Folgende Polynucleotide wurden untersucht: native und denaturierte DNA von verschiedenen Organismen, synthetische doppelsträn-gige Polydesoxynucleotide. Denaturierte Kalbsthymus-DNA und poly[d(I-C) · d(I-C)] waren ausgezeichnete Matrizen. Phagen-DNA stimulierte erst nach Denaturierung die RNA-Synthese. Der inhibierende Einfluß von einigen semisynthetischen Derivaten von Rifamycin SV auf die Aktivität der RNA-Polymerase B wurde getestet. Eine Verbindung aus dieser Reihe, Rifamycin AF/013, inhibierte die RNA-Synthese bei einer Konzentration von 20 g/m/. Untersuchungen über die strukturellen Eigenschaften der RNA-Polymerase B zeigten die Anwesenheit von mindestens drei Untereinheiten mit folgenden Molekulargewichten: 200000, 180000 und 150000.
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