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D Doenecke

Publications and source records attributed to D Doenecke.

At least 91 records · Page 5Linked to original sources
PubMed1985

Structure of a duck H3 variant histone gene: a H3 subtype with four cysteine residues.

A duck recombinant DNA phage library was screened for H3 histone genes, and the sequence of a variant H3 gene, which appears not to be part of a histone gene cluster, has been determined. As derived from the nucleotide sequence, this gene codes for a 135-amino acid (aa) protein (as any other H3) and shows 10 aa substitutions compared with most published H3 structures. Six of these aa changes are based on one nucleotide (nt) substitutions in arginine codons. This results in three new histidines and, in addition to the highly conserved cysteine at position 110, three more cysteines are found in this H3 histone subtype.

Amino Acid Sequence↗
PubMed1984

Conserved dyad symmetry structures at the 3' end of H5 histone genes. Analysis of the duck H5 gene.

The duck H5 histone gene and its flanking DNA have been isolated and sequenced. S1 nuclease mapping reveals that transcription starts 149 nucleotides upstream of the initiation codon and that the site of polyadenylation is located 200 nucleotides downstream of the termination codon. A comparison with the chicken H5 gene demonstrates that the 3' non-translated segment of the polyadenylated H5 mRNA carries two conserved dyad symmetry sequences. The first potential hairpin is located directly after the termination codon of the H5 gene and is highly conserved, whereas the second stem and loop structure maps shortly upstream of the polyadenylation site and shows a homology block at the central part of this inverted DNA repeat.

Amino Acid Sequence↗
PubMed1984

Differential response of avian red blood cell nucleosomes to heparin.

In avian erythrocyte chromatin, heparin interacts differentially with H1, H5 and the nucleosomal core histones. In non-erythroid cells, a partial extraction of H2A, H2B and H1 yields H3/H4/DNA complexes and particles of unchanged nucleosomal composition. The assay system for this heparin effect includes sucrose gradients, formaldehyde fixation and cesium chloride gradient centrifugation. A comparison of avian erythrocyte nucleosomes with chromatin subunits from precursor cells shows that H5 interferes with the heparin effect whereas a removal of H5 renders the core histones accessible to the polyanion.

Anemia↗
PubMed1979

Modification of DNA in chromatin with methyltransferase from Haemophilus influenzae Rd.

The accessibility of DNA in nucleosome dimers (as a model of the chromosomal chain of nucleosomes) was determined by means of modification methylases from Haemophilus influenzae Rd. Using these enzymes, the rate of modification of nucleosome dimers is about one fifth the rate observed with protein-free DNA from chromatin subunit dimers. Methylated DNA sites in nucleosome dimers are readily accessible to micrococcal nuclease. The analysis of the fragment pattern of nucleosomes after methylation and mild nuclease treatment reveals that the methylated sites are predominantly located in the internucleosomal linker DNA. Polylysine binding experiments further support this interpretation. This compound preferentially interacts with the nucleosomal core DNA and protects it against internal cleavage. It neither affects the degradation of methylated sites drastically nor does it inhibit the methylation of nucleosome dimers. Thus, a combination of protection, cleavage and modification is proposed as a useful tool for the analysis of the structure of chromatin.

Adenine↗
PubMed1978

Digestion of chromosomal proteins in formaldehyde treated chromatin.

Treatment of chromatin subunits (nucleosome monomers) with formaldehyde results in the formation of cross-links between DNA and histones and between histones and histones. Digestion of chromosomal proteins with proteinase K does not lower the protein/DNA weight ratio below 0.08 to 0.1 as determined by cesium chloride gradient centrifugation of the digestion product from formaldehyde-treated nucleosomes. In addition to proteinase K, formaldehyde-treated nucleosomes were tested for accessibility to trypsin and pronase. The CsCl gradient patterns show, that pronase digestion and proteinase K treatment yield similar results. Trypsin treatment of control and formaldehyde-treated nucleosomes shows, that the sites which are accessible for trypsin in native nucleosomes, are blocked after formaldehyde treatment. Analysis of the CsCl gradient peak fractions in polyacrylamide gels shows, that the reliability of DNA fragment size determinations depends on the completeness of deproteinization.

Animals↗
PubMed1977

Properties of the partially purified activated glucocorticoid receptor of rat liver. Binding to chromatin subunits.

The activated glucocorticoid receptor from rat liver has been purified over 3000-fold by repeated adsorption to phosphocellulose. The partially purified receptor-triamcinolone complex is stable for sever weeks at -75 degrees C in the presence of 0.1% bovine serum albumin, and sediments at 2.9 (+/- 0.1) S in sucrose gradients containing 0.15 M Na Cl. The Stokes' radius of the partially purified receptor is 26.5 A, and the frictional ratio is 1.14, indicating that at near physiological salt concentration the activated receptor is slightly elongated and has a molecular weight of 33 500. These preparations of receptor are free of exo- and endonucleases and bind to DNA and chromatin, as well as to the native chromatin subunits, the nucleosomes. Removal of histone H1 and further digestion of the nucleosomal DNA to the core size of about 140 base pairs indicate that the integrity of the nucleosome structure determines the extent of interaction with the glucocorticoid receptor. Although the binding of receptor to unfractionated rat liver chromatin is more efficient than to chicken erythrocyte chromatin, the nucleosomes of both tissues bind equal amounts of receptor. Therefore, the factors responsible for this tissue difference do not reside in the nucleosomes, but rather in a higher order structure of the chromatin or in that part of the genome which is not organized as nucleosomes.

Animals↗
PubMed1977

Binding of polylysine to chromatin subunits and cleavage by micrococcal nuclease. A comparison of accessible sites.

Native chromatin and chromatin subunits (nucleosomes) were titrated with polylysine and digested with micrococcal nuclease and deoxyribonuclease I at individual lysine/nucleotide ratios. In contrast to earlier reports, which had been obtained using mechanically sheared chromatin, a comparison of the sites accessible for micrococcal nuclease and polylysine reveals that polylysine does not preferentially protect the micrococcal-nuclease-susceptible sites in chromatin. Similar results were obtained in digestion experiments with DNase I. From the experimental data presented we conclude that polylysine does not preferentially bind to the internucleosomal DNA, which is the prime target site for micrococcal nuclease, but rather to the total nucleosomal DNA moiety.

Animals↗
PubMed1976

Movement of histones in chromatin induced by shearing.

Methylation of accessible DNA within chromatin by restriction modification methylases from Haemophilus influenzae was used to detect movement of histones along the DNA strand during chromatin manipulation. Methylation at different stages of chromatin preparation was followed by titration of the nucleoprotein with ploy(D-lysine), digestion of chromosomal proteins with pronase and analysis of the DNA-poly(D-lysine) complex in steep cesium chloride gradients. Comparison of the specific radioactivities in the peak fractions of the free DNA and the DNA-poly(D-lysine) complex, respectively, reveals that lateral movement of histones, relative to specific sites in the DNA marked by restriction methylases, occurs during manipulation and fragmentation of chromatin.

Animals↗
PubMed1976

Cesium chloride gradients of chromatin after treatment with micrococcal nuclease.

Cesium chloride equilibrium density centrifugation shows that treatment of rat liver nuclei with low concentrations of micrococcal nuclease for extremely short periods of time results in the appearance of chromatin fractions of low protein/DNA ratio and even free DNA. The DNA of these chromatin fractions is shorter than the DNA moiety of one chromatin subunit. The amount of high buoyant density material is decreased with increasing digestion time. We conclude that this material belongs to the minor chromatin fraction which is not organized according to the subunit model.

Animals↗
PubMed1975

Protein content of chromatin fractions separated by sucrose gradient centrifugation.

When sheared chromatin is centrifuged in a steep sucrose gradient, two broad peaks are resolved. DNA extracted from both fractions has approximately the same molecular weight. The basis for this fractionation seems to be differential aggregation. The slowly sedimenting material shows a lower protein/DNA ratio than the rapidly sedimenting chromatin as judged by equilbrium density centrifugation in CsCl after formaldehyde fixation or under nonionic conditions. After selective removal of histone fland further shear, most of the slowly sedimenting chromatin material appears as free DNA in steep cesium chloride gradients. The data are consistent with several recent reports concerning the subunit structure of chromatin.

Cells, Cultured↗
PubMed1975

The nature of protein association with chromatin.

The reality of the nonrandom distribution of histones along the chromatin strand was investigated in several ways. It does not appear to derive from histone exchange during shearing and is evident in chromatin fixed with formaldehyde prior to shearing. Endogenous or Escherichia coli polymerase are preferentially associated with regions of chromatin with a low protein/DNAratio. Although RNA polymerase and histones are fixed to chromatin after formaldehyde treatment with high efficiency, only a minor fraction of non-histone protein is fixed under similar conditions. Even after washing in high salt to minimize adventitious association, most remaining non-histone protein fails to be fixed. The utility of this approach for defining chromosomal proteins is discussed.

Cells, Cultured↗