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P Caiafa

Publications and source records attributed to P Caiafa.

At least 37 records · Page 2Linked to original sources
PubMed1994

Histone-induced condensation of rat testis chromatin: testis-specific H1t versus somatic H1 variants.

Due the likely role of H1 histone variants in inducing the formation of folded DNA filaments with different stabilities, the condensing capacity of the testis-specific H1t versus the somatic variants was tested. Circular dichroism analyses of rat testis H1-depleted oligonucleosomes (5-2kbp) revealed that H1t, which appears in germ cells during the meiotic prophase of mammalian spermatogenesis, exerts the lowest condensing effect as compared to the other variants. The distribution of H1 subtypes among different chromatin fractions was also investigated and gave evidence that H1t is more abundant in chromatin regions which are more sensitive to DNAase I digestion.

Animals↗
PubMed1994

Nuclear matrix-associated poly(ADPribosyl)ation system in rat testis chromatin.

The presence of poly(ADPR)polymerase in the third level of rat testis chromatin, i.e., in stripped chromatin loops and nuclear matrix, was assessed using enzymatic assays, activity blots, and Western blots. The distribution and the size of ADPribose polymers associated to proteins of the same nuclear fractions was analyzed after incubation of intact isolated nuclei with [14C]- or [32P]NAD+. Short ADPribose oligomers, not larger than 3 residues, were found to be associated to tightly bound chromosomal proteins, which resisted extraction by 2 M NaCl, whereas longer oligomers (8-13 residues long) were associated to loosely bound chromosomal proteins. The identity of ADPribose-protein conjugates was determined by autoradiographic analysis of nuclear protein extracts. Tightly bound histone-like proteins appear to be ADPribosylated both in stripped chromatin loops and in nuclear matrix.

Animals↗
PubMed1993

Inhibition of CpG methylation in linker DNA by H1 histone.

H1 exerts a specific in vitro inhibitory effect on enzymic DNA methylation. The experiments reported in this paper were undertaken in order to assess whether the lower methylation level found in internucleosomal DNA compared to core DNA is the in vivo consequence of the well-known localization of this histone in the linker region, as opposed to a possible deficiency of CpG dinucleotides in linker DNA. The methyl-accepting ability of H1-depleted oligonucleosomes from human placenta and of the corresponding core particles were assayed by addition of purified DNA methyltransferase, using S-adenosylmethionine as the methyl group donor. We have found that approx. 80% of newly-incorporated methyl groups are localized in linker DNA, which is indeed a good potential substrate for enzymic DNA methylation. Addition of quasi-physiological amounts of H1 to H1-depleted oligonucleosomes markedly reduced their methyl-accepting ability, while exerting a re-condensing effect on these particles, as revealed by the distortions of their circular dichroism spectra.

Chromatin↗
PubMed1993

Effect of H1 histone isoforms on the methylation of single- or double-stranded DNA.

The loosely and tightly bound H1 histone isoforms were shown to exert, on the in vitro methylation of linker DNA in H1-depleted oligonucleosomes, inhibitory or activating effects respectively similar to those previously shown in the methylation of Micrococcus luteus dsDNA. When assayed on the enzymic methylation of Micrococcus luteus ssDNA, addition of the tightly bound one resulted in a stimulation similar to that exerted on double-stranded bacterial DNA or on linker DNA from mammalian chromatin, while the loosely bound isoform had no effect whatsoever. The transformation of the "typical" loosely bound H1 isoform into its tightly bound counterpart can be visualized as being an essential event in the modulation of DNA methylation process in eukaryotic chromatin.

Cell Nucleus↗
PubMed1991

Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones.

After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid-soluble tightly-bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct H1-, H2A-, H2B-, H3- and H4-like components, the 'digested matrix' being however characterized by the absence of tightly-bound H1. These tightly-bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H1 components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H1 has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.

Chromatin↗
PubMed1991

Histones and DNA methylation in mammalian chromatin. Differential inhibition by histone H1.

Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of double-stranded DNA (from Micrococcus luteus) by human placenta DNA methyltransferase. The absence of EDTA during the histone renaturation procedure abolished--at least in the 'physiological' range of the histones/DNA ratio--the inhibition. The H1 component was responsible for this inhibition, no effect being exerted by the other histones. H1 preparations were more effective if renatured in the presence of EDTA--90% inhibition being reached at a 0.3:1 (w/w) H1/DNA ratio. It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the histones, although this effect could also be shown with the 'inactive' H2a, H2b and H3 components, and was instead less evident with histone H1. The restriction to histone H1 of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin.

Animals↗
PubMed1990

Tightly-bound form of poly(ADP-ribose)polymerase in the higher order of chromatin organization.

A tightly-bound form of poly(ADP-ribose)polymerase is present, within the third level of rat testis chromatin structure, both in the loops and in chromatin matrix. When chromatin matrix was extensively digested with DNAaseI, only little residual enzymatic activity remained in the insoluble fraction, the extent of DNA hydrolysis being well correlated to the progressive loss of the poly(ADP-ribose)polymerase activity. These findings suggest that the tightly-bound form of the enzyme is not an intrinsic protein component of chromatin matrix but is only indirectly located in this structure, being rather associated to the attachment points of loop DNA on the matrix.

Animals↗
PubMed1988

Localization, in human placenta, of the tightly bound form of DNA methylase in the higher order of chromatin organization.

In human placenta, the DNA of all subfractions of the third level of chromatin organization exhibits similar values of the methylcytosine-to-cytosine ratio. The tightly bound form of DNA methyltransferase is mostly recovered in the 'stripped loop' fraction, although, on the basis of the DNA content, the 'stripped loops' and the 'stripped matrix' appear to possess a similar amount of the enzyme. DNA methyltransferase activity is instead totally absent from the 'digested matrix', i.e., from the fraction remaining after digestion of the 'stripped matrix' with DNAase I. Upon addition of exogenous DNA methyltransferase, however, the DNA of this fraction, which is only 1% (in weight) of the total chromatin DNA and which has a length of approx. 9 kbp, can readily undergo methylation.

5-Methylcytosine↗
PubMed1988

Tightly-bound non-histone proteins in different nucleosome-like subpopulations from pig kidney chromatin.

By differential sucrose gradient centrifugation of pig kidney chromatin in the presence or absence of Na-EDTA and under varying ionic strength conditions, three nucleosome-like subpopulations with different buoyant densities can be obtained. These particles, on the basis of their histones and HMG protein pattern, of the 5-methylcytosine level of their DNA and of the RNA polymerase activity associated with them, can be considered as originating from chromatin fractions differently involved in gene expression. Two-dimensional electrophoresis of the tightly-bound non-histone proteins shows a distinct pattern for each subpopulation, such protein components being notably present in restricted numbers but in high amounts in the subpopulation which was apparently derived from condensed heterochromatin.

Animals↗
PubMed1988

Do tightly-bound chromatin proteins play a role in DNA methylation?

When chromatin matrix, "stripped" from its loosely-bound components by extraction with 3 M NaCl, is extensively digested with DNAase I, a fraction is obtained, which carries no endogenous DNA methyltransferase activity but which is a good substrate for externally added enzyme. Under the same conditions, protein-free DNA isolated from this fraction can instead hardly be methylated, this different behaviour pointing to a role of DNA-tightly-bound proteins in favoring or promoting the catalytic action of the enzyme. A similar stimulation of enzymatic methylation could also be shown when, in the presence of this same fraction, single stranded Micrococcus luteus DNA was incubated with placental methyltransferase, using S-adenosylmethionine as a methyl donor. This finding can be correlated to the existence, in chromatin loops, of small regions which resist digestion by DNAase I also after high-salt removal of their loosely-bound components (presumably because of the presence of tightly-bound proteins) and whose DNA is characterized by high methylation levels and, at the same time, by high relative content of thymine.

Binding Sites↗
PubMed1987

Tightly bound non-histone proteins: distribution of tissue-specific components in the chromatin organization.

We have investigated the distribution of tissue-specific tightly bound non-histone proteins in the first and third levels of chromatin organization. The proteins of this class have been extracted from whole chromatin and chromatin fractions prepared from pig liver or kidney. The tissue-specific proteins have high molecular mass (ranging from 135 KDa to 70 KDa in liver, over 135 KDa in kidney) and in kidney a more basic isoelectric point. These proteins are mainly located outside the core particles, and are instead present only in the chromatin matrix, become more intense after extensive digestion of the matrix with DNAase I.

Animals↗
PubMed1986

5-Methylcytosine levels in nucleosome subpopulations differently involved in gene expression.

Sucrose density gradient centrifugation in the presence or absence of Na-EDTA and at different ionic strengths allows one to obtain well-defined nucleosome subpopulations the DNA of which, examined by gas chromatography-mass spectrometry, is in all cases hypermethylated as compared to spacer regions, but to a different extent for the different subpopulations. The various nucleosomes differ also in their content of histones and of high-mobility-group proteins, as well as in the levels of RNA polymerase activity associated with them. Such data suggest that these nucleosome subpopulations originate from chromatin fractions differently involved in gene expression.

5-Methylcytosine↗
PubMed1986

Distribution of tissue-specific tightly bound non-histone proteins in the first level of repeating chromatin structures.

Non-histone proteins, tightly bound to DNA, have been extracted from whole chromatin and core particles prepared from pig liver or kidney. We have investigated by bidimensional slab gel electrophoresis the distribution of this protein class in the first level of repeating structure of chromatin. Our results reveal that non-histone proteins tightly bound to DNA are a heterogeneous protein class. Some of them, particularly in the core particles, appear to be essentially the same in both tissues, though having differences in their isoelectric point, which may be attributed to postsynthetic modifications. We have calculated that this protein class is associated to only 10% of nucleosomes, these nucleosomes having, on the average, one protein molecule for each core DNA. The tissue-specific proteins have high molecular mass (ranging from 135 kDa to 70 kDa in liver, over 135 kDa in kidney) and, in kidney, a more basic isoelectric point. These proteins are mainly located outside the core particles; they could be situated in the spacer regions and/or be involved in determining higher levels of chromatin organization.

Amino Acids↗
PubMed1985

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion.

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.

Animals↗
PubMed1985

Distribution of tissue-specific tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion.

The tissue specificity of a particular class of non-histone chromatin proteins characterized by strong bonds to DNA, tightly bound non-histone chromatin proteins (t.b. NHCp), was investigated. In connection with its possible role in the cell differentiation, its distribution in chromatin fractions obtained by digestion with DNAase II and precipitation with MgCl2 was studied. It was shown by bidimensional gel electrophoresis that some proteins of this class are tissue specific for pig liver compared to kidney chromatin. For both tissues, most tissue specific proteins were found to be peculiar to the chromatin fraction that remains insoluble after prolonged digestion with DNAase II. The proteins found in this chromatin fraction are common to the nuclear matrix proteins and, considering the chromatin organization, may be involved in the attachment point for loops in the axial matrix structure. It is possible that proteins of this class, directly or indirectly, play an important role in the regulation of transcription and differentiation in eukaryotes, by modulating the supercoiled state of DNA.

Animals↗
PubMed1981

Tightly bound non-histone proteins in nucleosomes from pig-liver chromatin.

Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine. By this method non-histone proteins have been found to be associated with the core particles. Proteins tightly bound to the core particle DNA (i.e. dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71000 to 20000. They show, furthermore, a peculiar amino acid composition. Other tightly bound proteins have been shown to be present only in the spacer regions. The existence of two different classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are compatible or incompatible, respectively, with the simultaneous binding of the histone octamer.

Animals↗