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M Bellard

Publications and source records attributed to M Bellard.

35 records · Page 2Linked to original sources
PubMed1988

Analysis of a DNase I-hypersensitive site in transgenic Drosophila reveals a key regulatory element of Sgs3.

We have undertaken chromatin studies on transformed Drosophila strains carrying DNA sequences modified in the region of the DNase I (EC 3.1.4.5)-hypersensitive sites -750 and -600 base pairs upstream from the Sgs3 start site. Although both sites are developmentally specific, modifications in the -750 site have little or no effect on Sgs3-encoded transcript levels, whereas either deletion or replacement of sequences at the -600 site causes an important reduction in transcript levels. The element associated with the -600 site enhances Sgs3 transcription when displaced with respect to the start site. This combined approach has defined sequence elements necessary both for normal transcript levels as well as the chromatin structure characteristic of Sgs3 activity in vivo.

Animals↗
PubMed1986

Changes in the chromatin structure of Drosophila glue genes accompany developmental cessation of transcription in wild type and transformed strains.

Three Drosophila salivary gland glue genes show a dramatic transition in their DNAse I hypersensitive sites during the short period between the late third instar and the white prepupa, which correlates with the cessation of their transcription. In culture cells, where the genes are inactive, there is a chromatin configuration similar to that of prepupal salivary glands. In two transformed fly strains where the sgs3 gene is active at new chromosomal sites, including one in which 2.6 kb of sgs3 upstream sequences have been inverted, the same DNAase I hypersensitive sites and developmental transitions are seen over the same DNA regions. These results, together with the analysis of transformants carrying rearranged sgs3 genes, suggest that there is at least one distal DNAase I hypersensitive site associated with an element of regulation which may be exchanged between sgs genes.

Animals↗
PubMed1986

Steroid hormone dependence of four DNase I-hypersensitive regions located within the 7000-bp 5'-flanking segment of the ovalbumin gene.

Four DNase I-hypersensitive regions (I-IV) occur in the 5'-flanking region of the ovalbumin gene in hen oviducts. One is centered close to the cap site (position +1) of transcription and the others at -0.8, -3.3 and -6.0 kb. The correlation of hypersensitivity with transcription was determined for each region in oviducts of chicks, where expression of the ovalbumin gene can be controlled by administration and withdrawal of steroid hormones. DNase I-hypersensitive regions were mapped by the indirect end-labeling technique and ovalbumin mRNA levels were determined by the dot blot assay. Diethylstilbestrol (DES) induces the appearance of hypersensitive regions I-IV whereas progesterone induces regions I, II and IV, but not region III. Upon withdrawal, regions II and III, and most of regions I and IV, disappear. A weak zone of hypersensitivity in region I near the cap site persists during withdrawal and a new zone of hypersensitivity appears between regions I and II. There is strong correlation between hypersensitivity at regions I-IV and gene transcription; ovalbumin mRNA levels are high in stimulated chicks, where hypersensitive regions are present, and drop to very low levels in withdrawn chicks, where the hypersensitivity is absent. We suggest that proteins, perhaps hormone receptors acting together with tissue-specific factors, induce DNAse I-hypersensitive regions I-IV of the ovalbumin gene.

Animals↗
PubMed1986

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Hormonally induced alterations of chromatin structure in the polyadenylation and transcription termination regions of the chicken ovalbumin gene.

We have studied the chromatin structure of a 16-kb region of the chicken genome containing the 3'-terminal 2 kb of the ovalbumin pre-mRNA coding sequence and the 14-kb segment located immediately downstream from the main mRNA polyadenylation site. Using the indirect end-labelling technique, four major and two minor DNase I-hypersensitive regions were found in the oviduct chromatin, whereas they were not present in liver, kidney or erythrocyte chromatin. The first hypersensitive region (region A) was present in chromatin of oviducts from laying hen and estrogen- or progesterone-stimulated immature chicks, in which the ovalbumin gene is expressed, but not in the chromatin of 'acute withdrawn' chicks where the gene is no longer transcribed. Region A spans 1.3 kb, from 7.2 to 8.5 kb downstream from the ovalbumin gene capsite (position +1), and encompasses the 3' moiety of the last exon including the major polyadenylation signal and polyadenylation site located at +7546 and +7564, respectively. Region A also contains a minor polyadenylation signal present at +7294 and the corresponding polyadenylation site at +7368. Two putative termination sequences at +8445 and +8483 are also found at the 3' extremity of region A in a 170-bp DNA segment within which 90% of the ovalbumin primary transcripts apparently terminate. Two minor hormone-independent DNase I-hypersensitive regions (a1 and a2) located at +8.6 and +8.8 kb are also specific to oviduct chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

A close association between sites of DNase I hypersensitivity and sites of enhanced cleavage by micrococcal nuclease in the 5'-flanking region of the actively transcribed ovalbumin gene.

The organization of chromatin was analysed in a segment of the chicken ovalbumin gene extending 6.5 kb upstream from the start site of transcription. Nuclei of chicken oviduct cells and of erythrocytes, and preparations of 'naked' DNA were digested with DNase I and with micrococcal nuclease. The locations of specific nuclease cleavage sites were determined by analyzing the fragments obtained with an indirect end-labeling technique. In oviduct nuclei there are four regions of DNase I hypersensitivity centered at approximately 0.15, 0.80, 3.2 and 6.0 kb upstream from the mRNA cap site. DNase I hypersensitive regions are absent from the 5'-flanking regions of erythrocyte nuclei. Micrococcal nuclease cleavage sites were found that are unique to oviduct nuclei and others that are enhanced in oviduct nuclei, relative to erythrocyte nuclei and to naked DNA. The locations of these micrococcal nuclease cleavage sites are closely associated with the DNase I hypersensitive regions. Nuclease hypersensitivity in the 5'-flanking region of oviduct nuclei reflects alterations in chromatin structure that are specifically correlated with gene expression. Our results suggest the presence at hypersensitive regions of specific proteins which alter the chromatin structure, making the DNA more accessible to nuclease attack.

Animals↗
PubMed1982

Disruption of the typical chromatin structure in a 2500 base-pair region at the 5' end of the actively transcribed ovalbumin gene.

We examined the chromatin organizations of approximately 3 kb of DNA in the 5'-end flanking region of the ovalbumin gene in chicken erythrocyte and oviduct cell nuclei. With specific DNA probes and an indirect end-labeling technique, we analysed the pattern of the DNA fragments obtained after micrococcal nuclease digestion and generated comparative maps of the nuclease cuts. This region of the chicken genome displays a "typical" chromatin arrangement in erythrocyte nuclei, with nucleosomes apparently positioned at random. In contrast, in oviduct nuclei, the same region has an "altered" chromatin structure, and lacks a typical nucleosomal array. The existence of specifically positioned proteins and of alterations in the DNA secondary structure in this region of the oviduct chromatin is suggested by comparison of the nuclease cleavage maps which reveals specific changes: disappearance of nuclease cuts present in "naked" and erythrocyte chromatin DNAs, and appearance of new cuts absent from these DNAs.

Animals↗
PubMed1981

Clustering of RNA polymerase B molecules in the 5' moiety of the adult beta-globin gene of hen erythrocytes.

Nuclei were prepared from mature and immature hen erythrocytes and incubated for RNA synthesis in the absence or in the presence of Sarkosyl. The in vitro labelled synthesized RNA was hybridized to specific 5' and 3' fragments of the chicken adult beta-globin gene to investigate the possible presence of RNA polymerase molecules bound to this gene in the form of transcriptional complexes. Surprisingly, such RNA polymerase B molecules were found located preferentially in the 5' end moiety of the beta-globin genes of mature erythrocytes, although they are apparently evenly distributed along the beta-globin genes of immature polychromatic erythrocytes. The significance of these observations with respect to (1) preferential DNaseI sensitivity of "genes which have been transcribed" and (2) control of transcription in eukaryotic cells is discussed.

Animals↗
PubMed1980

Differential nuclease sensitivity of the ovalbumin and beta-globin chromatin regions in erythrocytes and oviduct cells of laying hen.

We have monitored the differential nuclease sensitivity of defined regions of the chicken genome in different cells using a method which combines restriction enzyme digestion and blotting to diazobenzyloxymethyl (DBM)-paper (see Ref. 11). By using different specific probes and by scanning the bands on the autoradiograms, it is possible to compare on the same blot the digestion patterns of similar-sized fragments from different regions of the genome corresponding to "active" and reference "inactive" genes. We have demonstrated the preferential sensitivity to DNaseI and micrococcal nuclease digestion of the ovalbumin gene region in hen oviduct chromatin. The beta-globin gene region (containing both an adult and an embryonic gene) is also preferentially digested by DNaseI in hen mature erythrocyte nuclei, but at a lower rate than the ovalbumin gene region in oviduct. These observations raise the possibility that there may be several types of preferential nuclease sensitivities, all characterized by increased rates of digestion but to different levels, the highest corresponding to the very actively transcribing genes.

Animals↗
PubMed1978

Nucleosome structure.

Electron microscopic and biochemical results are presented supporting the following conclusions: (1) Two molecules of each histone H2A, H2B, H3 and H4 are necessary and sufficient to form a nucleosome with a diameter of 12.5 +/- 1 nm and containing about 200 base pairs of DNA. (2) H3 plus H4 alone can compact 129 +/- 8 DNA base pairs into a sub-nucleosomal particle with a diameter of 8 +/- 1 nm. In such a particle the DNA duplex is under a constraint equivalent to negative superhelicity. (3) Chromatin should be viewed as a dynamic structure, oscillating between a compact structure (the nucleosome) and more open structures, depending on the environmental conditions.

Animals↗
PubMed1976

Subunit structure of simian-virus-40 minichromosome.

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 +/- 2 nucleosomes, each containing 190 -- 200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 15% of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190 - 200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a 'core' containing a DNA segment of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone H1 is involved in the folding of chromatin fibers.

Animals↗
PubMed1976

Biochemical evidence of variability in the DNA repeat length in the chromatin of higher eukaryotes.

Biochemical evidence is presented which confirms that the DNA repeat length in micrococcal nuclease (spleen endonuclease, nucleate 3'-oligonucleotidohydrolase, EC 3-1-4-7) digests of Chinese hamster ovary chromatin is shorter than that of rat liver chromatin [J.L. Compton, R. Hancock, P. Oudet, and P. Chambon (1976) Eur. J. Biochem., in press]. A survey of available cells has shown that the DNA repeat length of the chromatin of higher eukaryotes varies widely. A value of 196 base pairs was found for cells of all mature tissues, regardless of the source of the tissue, whereas smaller values were found for cells of actively dividing tissues and larger values were found for a genetically inactive cell. Although the DNA repeat length of the chromatin of cells in culture was usually shorter than 196 base pairs, there was no general correlation between the size of the chromatin DNA repeat length and the rate of cell division or the functional state of the cell in culture. Examination of extensive micrococcal nuclease digests suggests that the chromatin subunits of all of the higher eukaryotic cells we have studied contain a core with approximately 140 base pairs of DNA.

Animals↗
PubMed1976

Stability of nucleosomes in native and reconstituted chromatins.

The stability of nucleosomes of SV40 minichromosomes extracted from infected cells or reconstituted by association of SV40 DNA and the four histones H2A, H2B, H3 and H4 was studied as a function of the ionic strength. As a measure of the stability of the nucleosome, we followed the disappearance of the nucleosomes from the original chromatin and their appearance on a "competing" DNA. We show here that the DNA and the histone components of the nucleosomes do not apprecially dissociate below 800 mM NaCl. At 800 mM and above, the histone moiety of the nucleosomes can dissociate from the DNA and efficiently participate to the formation of nucleosomes on a "competing" DNA.

Animals↗