1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.
TWO species of form A (or I) RNA polymerase have been identified in eucaryotic cells and there is evidence that this a-amanitin-insensitive activity exists as two discrete pools: a pool of 'free' activity, which is identified by its ability to transcribe poly [d(A-T)] in the presence of actinomycin in vitro, and a pool of enzyme in the form of a transcription complex ('engaged') which is unaffected by inhibitors of initiation of RNA synthesis.I. The principles underlying and the practical application of the technique used to define the pool of 'free' RNA polymerase activity have been analysed in considerable detail. On the basis of actinomycin titrations of poly[d(A-T)]dependent activity in isolated organelles, it is concluded that a pool of 'free' RNA polymerase A activity exists in mammalian nuclei which, under certain circumstances, is lost from nuclei during their isolation. The evidence presented suggests that nucleoli, resolved from nuclei by the classical sonication technique, contain form A polymerase exclusively in the transcription complex form.2. Different techniques used to solubilise RNA polymerase activity from nucleoli are shown to give rise to different proportions of the two formA RNA polymerase species (A1 and AII, as defined by their differential elution from phosphocellulose) : whereas low-ionic-strength extraction gives rise to form AII, high-salt, sonication extracts contain predominantly the form A1 enzyme. It is shown that the sonication technique results in the conversion of form A11 to form AI. By a careful appraisal of the products of these procedures and a novel polymerase solubilisation technique, it is concluded that RNA polymerase A11 is the 'engaged' form of the enzyme found in the transcription complex.3 . Making use of the finding that the 'free' form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity has been characterised without the requirement for solubilisation techniques which might result in the conversion of one form to another: the 'free' species is shown to be form A1 RNA polymerase.4. These conclusions that two discrete pools of formA RNA polymerase activity contain different species of the enzyme are briefly discussed in the light of other published information concerning their subunit structures and their potential role in the expression of the ribosomal RNA coding sequences.The form A (or type I) DNA-dependent RNA polymerase activity is considered to be localised primarily within the nucleolus [l] and is thought to be responsible for the expression of the ribosomal RNA coding sequences in eucaryotic cells [2,3]. It has been known for some time that at least two species of form A polymerase may exist : this apparant heterogeneity was first described for an activity derived from rat liver which was resolved into two components by phosphoEnzyme. RNA polymerase or nucleoside triphosphate: RNA nucleotidyltransferase (EC 2.7.7.6).cellulose chromatography [4]. These activities are referred to as forms A1 and A11 according to their order...
1. Radioactive orotic acid, uridine and adenosine were administered to rats by intracisternal injection. The effects of the size of the dose, the specific radioactivity and time on the incorporation into the RNA of unfractionated nuclei of brain tissue were examined to establish appropriate conditions for studies of the relative activities in vivo of the various sorts of brain nuclei fractionated by zonal centrifugation. Uridine is incorporated more efficiently than either orotic acid or adenosine. 2. With [(3)H]uridine as precursor the astrocytes in zone (II) contain the highest radioactivity except at the beginning of the experiment when the neuronal nuclei of zone (I) are more highly labelled. This fraction utilizes [(14)C]orotic acid more readily than the nuclei of zone (II). The astrocytic nuclei of zone (III) show a general resemblance to those of zone (II). Considerable differences between the incorporations into the two types of oligodendrocyte nuclei in zones (IV) and (V) are observed. 3. The relative synthetic activities of the major types of brain nuclei in vitro and in vivo are discussed.
On the bas~s of obse~afions on carcinogenesis in ~in'o by turnout v'.ma~ez, chem~c~ds and irradiation Sach~ ~ ] ] ha~ suggested that flue development of a visible ~urnour in ~i~o requires (a) a change m the control mechanisms for cetl replication (b) fixmion of this change in the ce~ so that it can be tran~rn~t~ted as a hered.hary properly and (c) growth of a sin~e turnout celt 5nlo a ~sib]e ruinous. He has further naggested that cheraical carcinogens may. reduce ch~.n~es ~n ce]flular contlo] mech ~an~s,ms by interacI,~ng with DNA wh~]e adrM~t~ng that the primary interaction might be with some other ~ntrace~ular consti~ent or d~rect-]y wi~ the cell membrane. Fixation of the/runsformed ~tate ~ postulated T,. be depen,denl on ce~ replication oeemrlng soon after She imfia~ interaction between flue .c~cinogen and the cell ~2 5]. Some evidence cons~stent wifla such a mechanism h~s re cemly been presented by Stewart and Ma~ee |6] who have ~hown flaat sin~e doses of dhnethy.initrosamine, sufficienI ~o haduee kidney turnouts "an rats; are followed w~thin a weekby ~tirnulat~on ofDNA syntheg, s mad ce~
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