IN A PREVIOUS paper where the incorporation of radioactive tracers into the constituents of brain polysomes was investigated (JACOB, SAMEC, STEVENIN, GAREL and MANDEL, 1967), acid-insoluble radioactivity of [3H]orotic acid, [32P]phosphate and [14C]leucine was detected in low sedimenting material. On the other hand, light cytoplasmic ribonucleoprotein (RNP) particles, either ribosomal precursors (JOKLIK and BECKER, 1965; MCCONKEY and HOPKINS, 1965; HENSHAW, REVEL and HIATT, 1965; GIRARD, LATHAM, PENMAN and DARNELL, 1965; RISTOW and KOHLER, 1966; HOGAN and KORNER, 1966 ; BISHOP, 1966) or informosomes (BELITSINA, AJTKHOZHIN, GAVRILOVA and SPIRIN, 1964; SPIRIN and NEMER, 1965) have been described in various animal cells.In the present work, results will be presented showing that light RNP particles are present in brain-cell cytoplasm and that they are associated with the endoplasmic reticulum.
M E T H O D SThe methods of injection of radioactive precursors, preparation of microsomes and of 50 and 30 S ribosomal subunits, release and sedimentation analysis of RNA have all been described previously (JACOB el al., 1967).Analysis of light RNP particles by gradient centrifugation. The microsomal pellet was gently suspended in buffer A (0.01 M-tris-HCI, pH 7-4; 0.025 M-KCI; 0.0025 M-MgCI,). Unless otherwise specified, 0.7 rnl were used per three rat brains. A solution of 11 % (w/v) sodium deoxycholate (DOC) was added to a final concentration of 1 per cent. Small aggregates were sometimes apparent and they were removed by a centrifugation at 6000 revimin for 10 min. The suspension was layered on top of a linear sucrose density gradient, 15-30%, 14 ml each. Centrifugation was carried out at 53,800 g (23,000 rev/min) for 15 hr. Twenty-five to thirty fractions were collected after piercing the bottom of the tube and analysed for optical extinction at 260 mp and acid-insoluble radioactivity.Harvesting of light RNP particles ,for RNA analysis. Sucrose density gradients were performed as above, fractions were collected and extinction was measured. The fractions of the 20-60 S region of the gradient (see results) were pooled and the particles were sedimented by a 2-hr centrifugation at 65,000 rev/min (269,000 g) in an R65 head of a Spinco L2-65. The pellets were suspended in 1 ml per nine rat brains. Non-radioactive ribosomal RNA was added as a carrier and extraction of RNA was carried out by the technique already used for polysomes (JACOB et al., 1967).
R E S U L T SDistribution of extinction at 260 mp. When centrifugation is carried out under the conditions described in the Methods section, three regions can be recognized along the gradient (Figs. 1 and 2). At the bottom of the tube, a region of relatively high extinction contains within about ten fractions the light polysomes and 80 S monomeric ribosomes. The heavier polysomes are pelleted. Near the top of the Abbreviations used: DOC, sodium deoxycholate; RNP, ribonucleoprotein.
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