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Histones are major structural components of the basic repeating subunit of chromatin, the nucleosome (1). Core histone sequences are remarkably conserved during evolution. Nonetheless, a number of histone variants are known (2-4). Synthesis of core histone variants is stage-specific during sea urchin development (3, 4), tissue-specific in mammals (5), and correlated with erythroid differentiation in Friend leukemia cells (6, 7). These observations suggest that nucleosome heterogeneity is related to the functional specialization of specific elements of chromatin. In this context, it may be important that mononucleosomes isolated from Drosophila chromatin contain large amounts of a histone-like protein that is not a standard core histone. This protein was first identified by Alfageme et al. (8), and designated D2, or "Drosophila 2."In this communication, we demonstrate that D2 is nucleosomal and histone-like, yet is readily distinguished from each of the four "standard" core histones. It is also conserved during the evolution of Drosophila. The curious features of D2 raise questions about histone evolution and the organization of histone genes in Drosophila.MATERIALS AND METHODS Purification of Nuclei. D. melanogaster embryo nuclei and adult head nuclei were prepared with three modifications of published procedures (9, 10): (i) buffer A of Hewish and Burgoyne (11), containing 1 mM EDTA, 0.2 mM ethylene glycol bis(f3-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), and 0.2 mM phenylmethylsulfonyl fluoride was used for the nuclear isolation; (ii) the crude nuclear pellet was washed in buffer containing 0.1% Triton X-100; (iii) the detergent-washed nuclei were then resuspended and recentrifuged three times in buffer lacking Triton X-100.Purification of Mononucleosomes. Nuclei were suspended in buffer A of Hewish and Burgoyne (11), made 4 mM in CaCl2, and digested at 250C to 25-29% acid solubility with staphylococcal nuclease (EC 3.1.31.1). Digested nuclei were lysed according to Noll and Kornberg (12). Digests were fractionated by sedimentation through linear (5-20%) sucrose gradients containing 0.5 M NaCl (13) or by electrophoresis in 5% acrylamide gels (14).Analysis of Nucleosomal DNA. DNA was extracted from pooled, sucrose-gradient-purified nucleosomes (15), omitting RNase treatment. DNA was released from electrophoretically purified nucleosomes with 1% NaDodSO4 and proteinase K at 100 ,ug/ml. DNA was electrophoresed on 5% acrylamide slab gels (16) and visualized by staining with ethidium bromide.Analysis of Nucleosomal Proteins. Gradient-purified nucleosomes were precipitated with 10 mM MgCl2 (17) and extracted with protamine/urea/acetic acid (18). The released chromosomal proteins were applied directly to acetic acid/ urea/Triton DF-16 gels (see below). Electrophoretically purified nucleosomes were electrophoresed into dialysis bags and extracted with 0.25 M HCO (8). The extracted proteins were analyzed by two-dimensional electrophoresis (see below). Purification of Histones. D. melanogaster embryo histones w...

An earlier paper (Snyder 1957) was concerned with the occurrence of obligate apomixis in several clones of Paspalum secans Hitchc. and Chase collected in the vicinity of Mayaguez, Puerto Rico, the type locality of the species.The clones investigated were found to be aposporic, with embryo development initiated by autonomous diploid parthenogenesis.Normal develop ment of the embryos and the formation of caryopses, however, were found to depend on fertilization of the polar nuclei and subsequent endosperm forma tion, i.e., the clones studied are pseudogamous.Meiotic behavior during megasporogenesis was not studied in detail, although large numbers of uni valent chromosomes were readily apparent at diakinesis and first metaphase. The primary megasporocytes commonly degenerate without dividing, but when meiosis does occur it was found to consist of two divisions resulting in the formation of a triad or tetrad of non-functional megaspores.A preliminary consideration of microsporogenesis in a number of clones revealed almost complete asyndesis at diakinesis and first metaphase, and the predominant production of dyads of microspores.The present study has been concerned with the details of meiotic behavior during microsporogenesis in several clones of the species. The nature and extent of the irregularities in meiosis are of interest in view of the pseudogamous characteristic of the materials, and also in relation to certain theories concerning mechanisms involved in the meiotic process.