The distribution of immunoreactive corticotropin-releasing hormone (CRF) in the forebrain and pituitary of the frog Rana ridibunda was studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. Relatively high concentrations of CRF-like material were found in both chiasmatic and infundibular regions of the hypothalamus (352 ± 11 and 422 ± 36 pg, respectively). Large amounts of CRF were also found in neurointermediate lobe extracts. Standard curves of synthetic CRF and the dilution curves for hypothalamic or neurointermediate lobe extracts were parallel. After Sephadex G-75 gel filtration, CRF-like immunoreactivity eluted in a single peak, in the same position as synthetic ovine CRF. Reversed-phase high-performance liquid chromatography of the material purified on Sephadex G-75 revealed 5 components with CRF-like immunoreactivity. The major peak had a retention time of 22 min as compared to 25.4 min for ovine CRF and 36 min for rat CRF. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the frogs. The great majority of the CRF-positive perikarya were seen in the ventral region of the preoptic nucleus. A few scattered perikarya were also observed in the dorsal preoptic nucleus and in the retrochiasmatic region. Immunoreactive fibers were found in the infundibular nucleus and in various extrahypothalamic zones. CRF-containing neurons were apparently distinct from mesotocinergic and vasotocinergic neurons. A large number of immunoreactive nerve fibers were observed in the median eminence in close contact with the capillaries of the pituitary portal plexus and in the neural lobe. A few CRF-positive fibers were detected in the intermediate lobe, whereas the distal lobe was totally negative. These results show that the diencephalon and pars intermedia-nervosa of the frog contain a peptide immunologically related to mammalian CRF.

The main DNA polymerases present in cytoplasmic and nuclear soluble extracts of sevenday-old chick embryos have been purified. One of them, DNA polymerase I, mainly present in the cytoplasm has been obtained about 50-60°/, pure. Its molecular weight is 148000 by Sephadex filtration and its s value is about 7.5 S. This DNA polymerase is devoid of DNAase and RNAase activity. DNA polymerase 11, of low molecular weight (50000), sedimenting a t 3 S, is the main DNA polymerase present in the nucleus and has been obtained goo/, pure. The DNA polymerase I1 activity is also present in the cytoplasm. The purified cytoplasmic DNA polymerase I1 has a molecular weight of 27000 and appears as a monomer of nuclear DNA polymerase 11. The DNA polymerase I1 preparations, from either nucleus or cytoplasm, are devoid of nuclease activity and exhibit the same primer specificity; both are able to copy RNA strands in rAn * dTn hybrids whereas DNA polymerase I only copies the DNA strands in such hybrids.Three DNA polymerases have been purified from bacteria and two, probably the major enzymes, have been isolated from animal cells [l-51. The use of mutants in the biochemical study of DNA replication or repair has greatly contributed to the progressive understanding of the physiological role of these bacterial enzymes [6,7].I n the absence of similar methods, experimental approaches of this problem in animal cells are more difficult. Using purified enzymes, the study of their activity in the presence of various "primers" might furnish valuable information on their function in the cell. I n a preliminary note we have described the partial purification [8] and more recently the template requirements [9] of a lowAbbreviations. Following Chang and Bollum [l] in this paper the terms template, initiator and primer are used. Template = polynucleotide chain used for selection of the complementary deoxynucleotide triphosphate. Initiator = oligo or polynucleotide chain complexed with the template and containing a free 3' OH end. Primer = templateinitiator complex. rA, = polyadenylic acid; dT, = polythymidilic acid; dA, = polydeoxyadenylic acid; d(A-T), = alternating deoxyadenylate and deoxythymidilate chains; dI, = polydeoxyinosinic acid; dG, = polydeoxyguanydilic acid; dC, = polydeoxycytydilic acid; A C [~H ] P~~-~R N A = a~etyl-[~H]phenylalanyl-tRNA; Sephadex CM-50 = carboxymethyl-Sephadex (3-50.

Extracts of mouse hypothalamus made in acid/urea containing protease inhibitors were analyzed for somatostatin immunoreactivity after molecular sieve filtration on Sephadex G-50. Higher molecular weight (higher- M r ) somatostatin-like forms with apparent molecular weights of 15,000, 10,000, and 6000 could be identified, besides the molecular weight 1600 somatostatin. Immunological identities with somatostatin were unambiguously demonstrated by the analysis of the displacement curves in the radioimmunoassay. The M r 15,000, 6000, and 1600 species were purified by affinity chromatography on an anti-somatostatin immune serum covalent conjugate with Sepharose used as immunoadsorbant. After disulfide reduction by dithiothreitol, the size of the M r 15,000 and 6000 somatostatin-like species was assessed either by molecular sieve filtration or by polyacrylamide gel electrophoresis. The results indicated that the higher- M r somatostatin-like species isolated from the hypothalamus did not result from hormone polymerization by means of disulfide interchange. The processing in vitro of the 15,000 higher- M r form of somatostatin was achieved by proteolytic enzymes coeluted with this species during the fractionation of hypothalamic extracts. Under neutral pH conditions the intermediary higher- M r forms were generated together with the M r 1600 somatostatin-like species. This processing activity could be either strongly inhibited at acidic pH or in acid/urea medium or else eliminated by selective immunoadsorption of the 15,000 higher- M r form. Neither trypsin nor the γ subunit of 7S nerve growth factor was able to produce this processing, suggesting that enzymes with other kinds of specificity may be involved. It is concluded that somatostatin biosynthesis in the mouse hypothalamus may occur via a high- M r precursor that is processed into intermediary forms leading to the tetradecapeptide hormone.

Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea.

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