Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNA-protein crosslinking within whole cells or isolated nuclei, specific immunoaffinity isolation of crosslinked complexes via protein and identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte beta globin gene is characterized by about 1.5-2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyme genes, whereas HMG 1/2, E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletion of H1/H5 in beta globin sequences was verified by the 'protein image' hybridisation technique (1). The DNase I hypersensitive site located 5' upstream from beta globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantitative changes of protein pattern suggest transient local perturbation of the chromatin on transcription.
A refined map for the linear arrangement of histones along DNA in nucleosomal core particles has been determined by DNA-protein crosslinking. On one strand of 145-bp core DNA, histones are aligned in the following order: (5') H2B25,35-H455,65-H375,85,95/H488-H2B105,11 5-H2A118-H3135,145/H2A145 (3') (the subscripts give approximate distance in nucleotides of the main histone contacts from the 5'-end). Hence, the histone tetramer (H3,H4)2 and two dimers (H2A-H2B) are arranged on double-stranded core DNA in a symmetrical and rather autonomous way: H2A/H3-(H2A-H2B)-(H3,H4)2-(H2B-H2A)-H3/H2A. The primary organization was found to be very similar in core particles isolated from repressed nuclei of sea urchin sperm and chicken erythrocytes, from active in replication and transcription nuclei of Drosophila embryos and yeast and from somatic cells of lily. These data show that (i) the core structure is highly conserved in evolution and (ii) the overall inactivation of chromatin does not affect the arrangement of histones along DNA and thus does not seem to be regulated on this level of the core structure.
A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L micron3 myeloma cells, total cDNA was synthesized and amplified approximately 10(5) fold. A library containing 10(6) clones was established from 1/6 of the amplified cDNA. Screening of the library with probes for three genes expressed in these cells revealed a number of corresponding clones in each case. The longest obtained clones contained inserts of 1.5 kb length. No sequences originating from carriers or from rRNA was found in 14 randomly picked clones.
A high-resolution map for the arrangement of histones along DNA in the nucleosome core particle has been determined by a sequencing procedure based on crosslinking histones to the 5'-terminal DNA fragments produced by scission of one DNA strand at the point of crosslinking. The position of histones on DNA has been identified by measuring the length of crosslinked DNA fragments. The results demonstrate that each of the histones is arranged within several adjacent or dispersed DNA segments of a little less than 10 nucleotides in length. Histone-free intervals are located between these segments at the regular distances of about (1O). nucleotides from the 5' end of the DNA and are likely to face one side of the DNA helix. Histones appear to be arranged in a similar manner on both DNA strands and do not form "locks" around DNA. A linearized model of the core particle is proposed.The periodic structure of chromatin now appears to be well established (for review, see refs. 1 and 2). A repeat unit of chromatin, termed the nucleosome, is made up of an octamer aggregate of histones containing pairs of each of the four main types, H2A, H2B, H3, and H4, which together with histone HI (3) is complexed with DNA of a variable length (150-240 base pairs). The basic structural element of the nucleosome in all cell types seems to be a core particle containing the histone octamer and DNA of about 140 base pairs in length (4, 5). Recent studies of the core particle crystals suggest that its structure is a flat disc of dimensions 110 X 110 X 57 A; about 1.75 turns of DNA in the B form are wound around the protein core (6). The proximity of histones in chromatin has been revealed by histone-histone crosslinking experiments (for review, see ref. 1). Histones H3 and H4 appear to play a central part in nucleosome formation (7) as first suggested by Kornberg (8). In the nucleosome the DNA sites separated by 10-nucleotide intervals are exposed to the action of different nucleases (7, 9). As determined by methylation experiments (10, 11), histones are partly buried in the major groove and leave the minor groove of DNA well exposed.Here we report a high-resolution map for the arrangement of histones along the core DNA determined by a sequencing procedure (11) in which histones were crosslinked to DNA within the core (12). Crosslinking causes the specific scission of one DNA strand at the point of crosslinking and the attachment of only the 5'-terminal DNA fragment to histones. The size of the single-stranded DNA fragments crosslinked to each histone species has been measured to identify the position of histones on one strand of the core DNA from its 5' end. A fairly clear picture of the detailed organization of histones has emerged from this map. Preliminary results of this work have been published elsewhere (11).
MATERIALS AND METHODSCrosslinking Histones to DNA. The core particles were prepared from Hl-depleted chromatin of mouse Ehrlich ascites tumor cells as described (3, 11). The core particles (0.5 mg/ml) were methylated (abou...
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