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When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E3RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H3RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H3RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.

Methods are described for in situ hybridization of ribosomal DNA from Xenopus laevis, labelled in vitro with 125iodine, to mitotic and lampbrush chromosomes from Triturus cristatus carnifex. The hybridization reaction was carried out in a mixture containing 50% formamide, 4 X SSC, 0.1 M KI, at 37 degrees C, or in 2 X SSC, 0.1 M KI at 65 degrees C. Autoradiographs of mitotic metaphases from 2 males showed labelling over the middle of the short arm of one chromosome IX in each metaphase. In some cases, a region near the end of a longer chromosome was also labelled. In a lampbrush preparations, labelling was confined to a region identified as about 53 units, near the middle of the short arm of both halves of bivalent IX. The usefulness of the technique and the significance of the labelling of only 1 of the 2 chromosomes IX in mitotic preparations are discussed.

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a “diffuse” stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.