Characterization of the chicken and rat DCoH gene domains using an improved ligation-mediated PCR method.

The bifunctional protein DCoH (dimerization cofactor of HNF-1) is a transcriptional coactivator of homeodomain proteins and a cytoplasmic enzyme. Here we report on the isolation, nucleotide sequence and genomic structure of the chicken and rat DCoH genes. They have an unusual first exon coding for a single amino acid.

Genomic structure of the POU-related hepatic transcription factor HNF-1 alpha.

Hepatocyte Nuclear Factor-1 alpha (HNF-1 alpha) is a vertebrate transcriptional regulatory protein which plays a prominent role in the activation of a large family of hepatocyte-specific genes. It is the prototype of a new subfamily of POU-related proteins important in embryogenesis and cell-type specification. Here we report on the genomic structure and DNA sequence of HNF-1 alpha of the hen.

Hepatic nuclear factor 1 alpha (HNF-1 alpha) is expressed in the oviduct of hens and interacts with regulatory elements of the lysozyme gene.

HNF-1 alpha is a nuclear transcriptional regulatory protein required for the expression of a variety of liver-specific genes. This factor was previously considered liver-specific but later shown to be expressed also in a few other mammalian tissues. Here we report on the occurrence of HNF-1 alpha in the avian oviduct.

Partial overlapping of binding sequences for steroid hormone receptors and DNaseI hypersensitive sites in the rabbit uteroglobin gene region.

Four DNaseI hypersensitive (HS) chromatin regions were found in the uteroglobin locus located at -3.7, -2.4, -0.

DNase I-hypersensitive sites in the chromatin structure of the lysozyme gene in steroid hormone target and non-target cells.

The pattern of DNase I-hypersensitive sites in the chromatin domain of the lysozyme gene was investigated in several organs and cell-types of the chicken. In the cluster of hypersensitive chromatin sites framing the gene, different classes of sites could be discerned: A subset was common to essentially all cells examined except for erythrocytes. Thus several highly nuclease susceptible structures exist around the gene even in its repressed state.

The DNase I sensitive domain of the chicken lysozyme gene spans 24 kb.

We have determined the DNase I sensitive chromatin domain of the lysozyme gene in the hen oviduct. When nuclei were digested with DNase I, about 14 kb of upstream and 6 kb of downstream sequences in addition to the 4 kb long transcribed region were preferentially degraded. The transcription start site is located near the center of the approximately 24 kb long sensitive domain.

Alternative sets of DNase I-hypersensitive sites characterize the various functional states of the chicken lysozyme gene.

The structural organization of chromatin is thought to determine the state of differentiation and activity of eukaryotic genes. Local interruptions of the regular nucleosomal array, the so-called DNase-hypersensitive sites, may indicate regions of the genome which play a critical part in regulation of differential gene activity. We present here two new observations on the chromatin structure of the chicken lysozyme gene, which strongly support a regulatory function for these sites.

Positioning of nucleosomes in satellite I-containing chromatin of rat liver.

The location of nucleosomes on rat satellite I DNA has been investigated using a new approach. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonuclease III and nucleases S1. From the total population of core DNA fragments the satellite-containing fragments were isolated by molecular cloning and the complete sequence of 50 clones was determined.

Nuclease-hypersensitive sites in the chromatin domain of the chicken lysozyme gene.

We have examined the chromatin structure of a 22 kilobase-pair chromosomal region containing the lysozyme gene in laying hen. Nuclease-hypersensitive sites were probed with DNAase I by using an indirect end-labeling technique. Eight DNAase I-hypersensitive sites could be mapped in the flanking regions of the gene in oviduct cells, in which the gene is expressed.

Different repeat lengths in rat satellite I DNA containing chromatin and bulk chromatin.

The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.

Non-random arrangement of nucleosomes in satellite I containing chromatin of rat liver.

The location of nucleosomes on the nucleotide sequence of rat satellite I DNA was investigated using micrococcal nuclease, exonuclease III, and restriction nucleases as tools. Hae III cleaved the satellite DNA containing chromatin very preferentially in the linker region. Nucleosomes were found predominantly in three defined positions on the 370 bp satellite I monomer unit.

Nucleotide sequence of a highly repetitive component of rat DNA.

A highly repetitive component of rat DNA which could not yet be enriched by density gradient centrifugation was isolated with the help of the restriction nuclease Sau3AI. This nuclease converted the bulk of the DNA to small fragments and left a repetitive DNA component as large fragments which were subsequently purified by gel filtration and electrophoresis. This DNA component which was termed rat satellite DNA I is composed of tandemly repeated 370 bp blocks.

The non-histone proteins of the rat liver nucleus and their distribution amongst chromatin fractions as produced by nuclease digestion.

The search for proteins involved in maintaining higher order chromatin structures has led to a systematic examination of the non-histone proteins (NHP) of rat liver nuclei in the context of nuclease digestion studies. 40-45% of the 3H-tryptophan labelled NHP originally present could be removed by extensive washing in a "physiological" buffer, incubation at 37 degrees C with or without nuclease and a further wash step. Nuclei at this stage had a remarkably constant NHP content (ca.

Characterization of restriction nuclease prepared chromatin by electron microscopy.

Chromatin was solubilized from rat liver nuclei by digestion with the restriction nuclease EcoRI or HaeIII in the presence or absence of EDTA and sodium chloride. The samples were investigated by electron microscopy after positive and negative staining with uranyl acetate under a number of conditions. Depending on the salt concentration during solubilization the chromatin appeared as beads on the string or in more compact form.

Prepartation of soluble chromatin and specific chromatin fractions with restriction nucleases.

By digestion of rat liver nuclei with EndoR HaeIII, EndoR EcoRI, and EndoR Bam and subsequent lysis of the nuclei approx. 90%, 40%, and 45%, respectively, of the chromatin were solubilized. The plateau values of solubilization are in agreement with a model in which the chromatin strands are crosslinked and/or attached to a supporting structure.

Specific cleavage of chromatin by restriction nucleases.

Digestion of mouse and rat liver nuclei with a restriction nuclease from Bacillus subtilis (Bsu) is examined in continuation of previous work from this laboratory (Pfeiffer et al., 1975, Nature 258, 450). The finding of more than 95% C in the 5'-termini of the DNA fragments generated during digestion with Bsu shows that the participation of endogenous nucleases in Bsu digestion is extremely small.

Nuclease digestion in between and within nucleosomes.

In the course of digestions of rat liver nuclei with micrococcal nuclease the size of the nucleosomal DNA is shortened by 50-60 nucleotide pairs from starting lengths of about 200, 400, 600, 800, etc. nucleotide pairs in the monomeric and oligomeric nucleosomes, respectively. Acid soluble DNA material is created relatively slowly as compared to the rate of formation of subnucleosomal material.