Détermination de la fraction du génome codant pour les RNA ribosomiques et messagers dans le cerveau du rat adulte

Abstract

The proportion of the total genome coding for ribosomal and messenger RNA in adult rat brain has been estimated by specific hybrid formation with homologous DNA. The method of hybridization was that of Gillespie & Spiegelman (1965). The hybrid formation was optimum at 70 °C, in 4 × SSC (0.6 m-NaCl-0.06 m-sodium citrate) and kinetic studies showed that maximum hybridization was reached after 15 hours of incubation. Saturation curves of DNA with brain ribosomal RNA show that a plateau is reached when approximately 0.15% of the DNA has complexed with ribosomal RNA. Evidence for the specificity of the hybrid and for the absence of contaminating DNA-like RNA's or spurious hybrids is as follows: (1) the ribosomal RNA has been prepared from polysomes treated with ribonuclease in such conditions that only the messenger RNA is hydrolysed ( Jacob, Samec, Stévenin, Garel & Mandel, 1967); (2) transfer RNA's have been eliminated on Sephadex G200; (3) the saturation plateau is reached for low RNA input (15 to 20 μg RNA/ml.) as expected when only a few RNA species are present; (4) the base composition of the hybridized RNA is close to that of ribosomal RNA ((G+C)/(A+U) = 1.92). On the basis of the DNA content per diploid rat cell, one would estimate that about 6000 cistrons are complementary to the two types of ribosomal RNA. The significance of this multiplicity of cistrons coding for ribosomal RNA may be related either to a large number of nucleolar organizers in the genome of the rat, or to a redundancy of ribosomal cistrons in a given nucleolar organizer. The major difficulty for the estimation of the DNA fraction complementary to messenger RNA's is the direct determination of the specific activity of these RNA's. In the present work we have shown that, when isotopic equilibrium is reached, the specific activity of messenger RNA is equal to that of the alpha-phosphate atom of its precursors, the nucleoside triphosphates. 24 hours after intracisternal injection of 32P the specific activities of the brain acid-soluble nucleotides are indeed identical and nearly constant for several days. The establishment of saturation curves of DNA with RNA from brains of rats killed 24, 40 and 48 hours after the injection enabled us to calculate that 1.2% of the DNA was complementary to messenger RNA. In contrast to ribosomal RNA, the saturation curve represents, as expected, an addition of the saturation curves of many individual messenger RNA's. High concentration of input RNA (2 to 3 mg/ ml.) were needed to reach the plateau. The base composition of the hybridized RNA was DNA-like ((G+C)/(A+U) = 0.64). If the molecular weight of messenger RNA's is between 10 5 and 10 6, the number of cistrons coding for them can be estimated to be 50,000 to 500,000. This may be due to an effectively high number of proteins coded for in the various types of brain cells, or to a redundancy of the cistrons coding for messenger RNA. There is also the possibility that some of the DNA-like RNA's, the role and fate of which are not known yet, do not have a messenger function.