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A rat genomic library was screened for tRNA genes with an unfractionated rat liver tRNA probe. About 70 clones containing tRNA genes were detected per rat genome. The organization of tRNA genes in five clones was analyzed by restriction endonuclease digestion, RNA-DNA hybridization and in vitro transcription with nuclear extracts from Xenopus oocytes. Evidence is presented suggesting that tRNA genes are distributed in the rat genome in small clusters spanning 1 to 2 kb and interspersed with large regions (minimum 8 to 20 kb) of non tRNA-coding DNA. The tRNA gene clusters were found to contain the sequences for a variety of tRNA species. Genes for a single isoacceptor, were found in more than one clone. The detailed study of one clone shows the repetition of a cluster of four tRNA sequences at a distance of about 8 kb. The arrangement of tRNA genes in rat appears to follow the irregular pattern of tRNA gene organization previously reported in Drosophila and Xenopus.

The development of the cerebral microvasculature of the rat was studied during three successive postnatal periods, namely: 1) neonatal period, i.e., 1 to 9 days after birth (capillary sprouting period); 2) myelinization period, i.e., 10 to 20 days; and 3) young adult period, i.e., 2 to 3 months. The survey covered structural aspects and distribution of binding sites for anionic or cationic probes and for albumin-gold complexes on the luminal surface of the microvascular endothelium. The salient results are: a) an extensive development of the endoplasmic reticulum of endothelial cells during the first period (presumably in relation with the production of basement membrane components); b) the high surface density of coated pits and coated vesicles that peaks during the myelinization period; c) the paucity of plasmalemmal vesicle and their differential distribution (their volume density is higher in the endothelium of arterioles than in that of capillaries and venules); d) the existence of an extensive smooth surface tubular system in the cytoplasm of endothelial cells, whose structural connections and functional significance remains to be established; and e) the presence of pericytes with elaborate interactions with endothelia in the early developmental periods. Labeling by perfused tracers indicates an uneven patchy distribution of binding sites for cationic ferritin (generally limited to the plasmalemma proper) and a more even distribution of binding sites for cationic and anionic hemeundecapeptides. Binding patterns did not change during the developmental periods studied. No binding sites were detected for albumin-gold complexes.

The ability of cyclic AMP to inhibit growth, cause cytolysis and induce synthesis of cyclic AMP-phosphodiesterase in S49.1 mouse lymphoma cells is deficient in cells selected on the basis of their resistance to killing by 2 mM dibutyryl cyclic AMP. The properties of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in the cyclic AMP-sensitive (S) and cyclic AMP-resistant (R) lymphoma cells were comparatively studied. The cyclic AMP-dependent protein kinase activity or R cells cytosol exhibits an apparent Ka for activation by cyclic AMP 100-fold greater than that of the enzyme from the parental S cells. The free regulatory and catalytic subunits from both S and R kinase are thermolabile, when associated in the holoenzyme the two subunits are more stable to heat inactivation in R kinase than in S kinase. The increased heat stability of R kinase is observed however only for the enzyme in which the catalytic and cyclic AMP-binding activities are expressed at high cyclic AMP concentrations (10(-5)--10(-4) M), the activities expressed at low cyclic AMP concentrations (10(-9)--10(-6) M) being thermolabile. The regulatory subunit of S kinase can be stabilized against heat inactivation by cyclic AMP binding both at 2-10(-7) and 10(-5) M cyclic AMP concentrations. In contrast, the regulatory subunit-cyclic AMP complex from R kinase is stable to heat inactivation only when formed in the presence of high cyclic AMP concentrations (10(-5)M). The findings indicate that the transition from a cyclic AMP-sensitive to a cyclic AMP-resistant lymphoma cell phenotype is related to a structural alteration in the regulatory subunit of the cyclic AMP-dependent protein kinase which has affected the protein's affinity for cyclic AMP and its interaction with the catalytic subunit.

Turkey erythrocyte membranes showed specific binding of [(3)H]epinephrine. The concentration of hormone required for half-maximal binding (30 muM) was the same as that required for half-maximal activation of the adenylate cyclase located in the same membrane preparation. The binding reaction at 37 degrees C reached completion during the first minute of incubation, which agrees well with the known rapidity of the biological response to catecholamines. Specific binding was abolished by heating the membranes 1 min at 100 degrees C. Chromatography of the bound (3)H, after its extraction from the membranes, indicated that the hormone had fully retained its chemical structure. Epinephrine binding was inhibited by the beta-adrenergic blocking agent propranolol, which also inhibited the activation of adenylate cyclase by the hormone. The specificity of phenethylamine derivatives in displacing [(3)H]epinephrine from the binding sites showed that a typical catecholamine receptor was responsible for the binding. Displacement of the bound hormone by analogs lacking the catechol group was more extensive at 37 degrees C than at 0 degrees C. Some of the analogs that displaced epinephrine from the binding site caused only a feeble activation of the adenylate cyclase, but were able to inhibit the activation of the enzyme by epinephrine. Thus, binding to a catecholamine receptor on a membrane preparation is an essential, but insufficient, condition to elicit a response.