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...

Approximately 30-40% of Drosophila virilis DNA complementary to cloned Drosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as the D. melanogaster core histone genes in the plasmid cDm500: H2B H3 H4 H2A. Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30-40% of D. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution of Drosophila. The ancestors of modern Drosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in the virilis and melanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes of D. virilis and D. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently, D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes of D. virilis and D. melanogaster are greater than 25% divergent.(ABSTRACT TRUNCATED AT 250 WORDS)

In embryonic nuclei of DrosophiZa viriZis, 45% of the DNA is satellite, and 50% of the Hl histone is phosphorylated. In polytene salivary gland nuclei, < 1% of the DNA is satellite, and < 10% of the Hl is phosphorylated(1). The decreased Hl phosphorylation in these non-dividing cells could be correlated either with the under-replication of satellite DNA, or with the absence of mitotic activity. To distinguish between these alternatives, we analyzed the Hl histones in adult head cells, which contain the diploid level of satellite DNA, and are predominantly non-dividing. Nearly half of the Hl in these cells is phosphorylated. Thus, the decreased phosphorylation of Hl in polytene cells is correlated with the under-replication of satellite DNA and not with cell replication. The phosphorylated Hl's migrate 4% slower than the unphosphorylated Hl's on SDS-acrylamide gels. The mobility difference may arise because the phosphorylated and unphosphorylated Hl's have different conformations in SDS. This putative conformational difference could be essential to the compaction of satellite DNA into heterochromatin. INTRODUCTIONWe have previously proposed that phosphorylated HI histones compact the

No abstract

No abstract