Deoxyribonuclease I sensitivity of the ovomucoid-ovoinhibitor gene complex in oviduct nuclei and relative location of CR1 repetitive sequences

The location of CR1 middle repetitive sequences within or near the boundaries of the ovalbumin DNase I sensitive domain has suggested that CR1 sequences may play a role in defining transition regions of DNase I sensitivity in hen oviduct nuclei. We have examined this apparent relationship of CR1 sequences and transitions of chromatin structure by determining the DNase I sensitivity in oviduct nuclei of a 47-kilobase region that contains five CR1 sequences and the transcribed ovomucoid and ovoinhibitor genes. We find that three of the CR1 sequences occur within a broad transition region of decreasing DNase I sensitivity downstream of the ovomucoid gene. Another CR1 is in a region of decreased DNase I sensitivity within the ovoinhibitor gene. The fifth CR1 sequence is in a DNase I sensitive region between the two genes but which is less sensitive to DNase I digestion than the region immediately upstream from the ovomucoid gene. Thus, the CR1 sequences occur within regions of reduced relative DNase I sensitivity, suggesting that CR1s could facilitate the formation of a chromatin conformation that is less sensitive to DNase I digestion. Unexpectedly, the noncoding strand of sequences within and immediately adjacent to the 5' end of the actively transcribed ovomucoid and ovalbumin genes was less sensitive to DNase I digestion than their respective coding strands.     

Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion

The chromatin structure encompassing the lysozyme gene domain in hen oviduct nuclei was studied by measuring the partitioning of coding and flanking sequences during chromatin fractionation and by analyzing the nucleosome repeat in response to micrococcal nuclease digestion. Following micrococcal nuclease digestion, nuclei were sedimented to obtain a chromatin fraction released during digestion (S1) and then lysed in tris(hydroxymethyl)aminomethane-(ethylenedinitrilo)tetraacetic acid-[ethylenebis(oxyethylenenitrilo)]tetraacetic acid and centrifuged again to yield a second solubilized chromatin fraction (S2) and a pelleted fraction (P2). By dot-blot hybridization with 14 specific probes, it is found that the fractionation procedure defines three classes of sequences within the lysozyme gene domain. The coding sequences, which partition with fraction P2, are flanked by class I flanking sequences, which partition with fractions S1 and P2 and which extend over 11 kilobases (kb) on the 5'side and probably over about 4 kb on the 3' side. The partitioning of class II flanking sequences, which are located distal of class I flanking sequences, is different from that of class I flanking sequences. Coding sequences lack a canonical nucleosome repeat, class I flanking sequences possess a disturbed nucleosome repeat, and class II flanking sequences generate an extended nucleosomal ladder. Coding and class I flanking sequences are more readily digested by micrococcal nuclease than class II flanking sequences and the inactive beta A-globin gene. In hen liver, where the lysozyme gene is inactive, coding and class I flanking sequences fractionate into fractions S2 and P2.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA and chromatin structure of the human alpha 1 (I) collagen gene

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.     

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes.

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.