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O Westergaard

Publications and source records attributed to O Westergaard.

At least 73 records · Page 4Linked to original sources
PubMed1985

A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin.

Topoisomerase I is associated with DNAase I hypersensitive sites in the nontranscribed spacers flanking the rRNA genes in Tetrahymena thermophila. The endogenous topoisomerase I introduces site and strand specific single-strand cleavages in the rDNA spacers in situ. The cleavages occur base specifically within a hexadecameric sequence element present in two or three direct repeats at the hypersensitive sites. The sequence specificity and polarity of the cleavage reaction are identical when the enzyme is reacted with naked rDNA, indicating that the repetitive element functions as a high-affinity topoisomerase I attraction site in the r-chromatin. The biological mechanism associated with this phenomenon appears to be widespread among eukaryotes, since the topoisomerase I recognition sequence is conserved in the rDNA spacers of phylogenetically remote organisms, such as fungi, slime molds, ciliates, and insects.

Animals↗
PubMed1984

Studies on transcription termination and splicing of the rRNA precursor in vivo in the presence of proflavine.

In isolated nucleoli from Tetrahymena thermophila, low concentrations of the intercalating agent proflavine inhibit both transcription termination and splicing of the rRNA precursor. Proflavine also exerts an in vivo effect on the process of transcription termination under conditions, where the growth rate is only slightly reduced. Thus, approximately 40% of the rRNA precursor molecules, accumulated in nucleoli during 60 min of treatment with the drug, are longer than the normal 35S rRNA precursor. R-Loop mapping of these longer precursor molecules isolated after 30 and 60 min of incubation demonstrates that the RNA polymerases have a 50 fold lower elongation rate in the spacer region than in the coding region. Proflavine in the given concentration is found to have no significant effect on the splicing of properly terminated precursor molecules. In contrast, none of the longer non-terminated molecules are found to be spliced. These results indicate that proflavine primarily affects the process of transcription termination and that the splicing event is inhibited due to the improper termination of the precursor molecule.

Acridines↗
PubMed1983

A site and strand specific nuclease activity with analogies to topoisomerase I frames the rRNA gene of Tetrahymena.

Exposure of macronuclear chromatin from Tetrahymena thermophila to sodium dodecyl sulfate causes an endogenous nuclease to cleave the extra-chromosomal rDNA at specific sites. All cuts are single-strand cleavages specific to the non-coding strand. Three cleavages map in the central non-transcribed spacer of the palindromic molecule at positions -1000, -600 and -150 bp with respect to the transcription initiation point. A fourth site is located close to the transcription termination point, while no cleavage is observed in the coding region. The position of each cleavage is in the immediate neighbourhood of DNAse I hypersensitive sites. Additionally, certain DNA sequence motifs are repeated in the region around the cleavages. Upon cleavage induction a protein becomes attached to the rDNA. Our results indicate covalent binding to the generated 3' end, in analogy to the aborted reaction of topoisomerase I.

Animals↗
PubMed1982

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena.

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.

Animals↗
PubMed1981

Nuclease-sensitive regions on the extrachromosomal r-chromatin from Tetrahymena pyriformis.

The extrachromosomal DNA coding for the ribosomal precursor in Tetrahymena contains a transcribed region with a size of 6 x 10(3) base pairs plus non-transcribed central and distal spacers. In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared. Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures. The specific DNA fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific 32P-labelled RNA probes. Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non-transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region. Specific regions on the r-chromatin are hypersensitive to DNase I. The first cleavage occurs in the non-transcribed central spacer region, while the second cleavage takes place in a region near the 3' end. The hypersensitivity of the central part of r-chromatin is also found by autodigestion in isolated nucleoli.

Base Sequence↗
PubMed1980

In vitro splicing of the ribosomal RNA precursor in isolated nucleoli from Tetrahymena.

The macronuclear rRNA genes of Tetrahymena thermophila contain an 0.4 kb intervening sequence in the 26S rRNA coding region. The sequence is represented within the primary transcription product. We demonstrate in this paper that the enzyme activities necessary for the endonucleolytic cleavage as well as for the ligation of the transcript are associated with the isolated. The intervening sequence is excised as an unique molecule, which is stable in vitro. About 50% of the in vitro synthesized RNA is processed. Faithful in vitro transcription occurs in the presence of the divalent ions Mg2+, Mn2+ and Co2+ while processing takes place only in the presence of Mg2+. The absolute requirement for Mg2+ in the excision reaction enables us to synthesize labelled pre-rRNA in the presence of Mn2+ or Co2+. The synthesized RNA can be used as a substrate in studies of th processing enzymes in vitro.

Animals↗
PubMed1979

Effect of lucanthone (miracil D) on transcription of ribosomal RNA genes from Tetrahymena in vivo and in vitro.

Addition of lucanthone (1-5 mug/ml) to cultures of Tetrahymena results in a preferential inhibition of the synthesis of ribosomal RNA. Transcriptional studies with isolated nucleoli from Tetrahymena demonstrate that the endogenous RNA polymerases of the r-chromatin (chromatin form of rDNA) do not recognize the normal termination and move into the spacer region distal to the terminator in the presence of lucanthone. This is shown by hybridization of the transcript synthesized in the presence of the drug to restriction fragments of rDNA. Lucanthone seems specific in its action on termination as it does not inhibit the elongation process on the chromatin. Among various DNA-binding drugs tested only lucanthone and proflavine are found to cause repression of the termination. The data obtained suggest that the reduced synthesis of rRNA in lucanthone-treated eukaryotic cells is due to lack of reinitiating RNA polymerases possibly caused by improper termination.

Animals↗
PubMed1979

Termination of transcription in nucleoli isolated from Tetrahymena.

Correct termination of transcription of the rRNA gene in nucleoli isolated from Tetrahymena is dependent on a protein factor with a molecular weight between 50,000 and 100,000. At low ionic strength the endogenous RNA polymerase synthesizes a transcript identical in size to the precursor rRNA extracted from cells. In the presence of ammonium sulfate, however, the RNA polymerase reads through the normal termination point as demonstrated by size and hybridization studies of the transcript. After ammonium sulfate treatment, rDNA associated with chromosomal proteins (r-chromatin) can be separated from the termination factor by differential centrifugation. The endogenous RNA polymerase on the salt-treated r-chromatin is no longer able to recognize the normal terminator even at low ionic strength. Normal termination properties can be reconstituted by complementation with intact nucleoli or with a protein factor extracted from nucleoli.

Animals↗
PubMed1978

Transcriptional properties of nucleoli isolated from Tetrahymena.

Nucleoli can be isolated from Tetrahymena in a yield of 30-60%. The isolated nucleoli contain rDNA (at least 90% pure) and have a protein to DNA ratio of 30:1. The endogenous RNA-polymerase activity of the r-chromatin has the following properties: (i) The in vitro transcript has a maximal size identical to the in vivo 35S rRNA precursor, demonstrating correct termination on the gene, (ii) 79% of the in vitro transcript is complementary to cDNA of 17S and 25S rRNA which is close to the theoretical maximum for the 35S rRNA precursor, (iii) the elongation rate of the endogenous RNA-polymerase molecules is 9-12 nucleotides/sec, (iv) an average of 4-16 active RNA polymerases are associated with each rDNA molecule depending upon the preparation.

Animals↗
PubMed1978

Effect of cycloheximide on maturation of replicative intermediates into high-molecular-weight DNA in Tetrahymena.

Replicative intermediates of discrete size (approximately 41 S) are observed in the eukaryotic organism Tetrahymena pyriformis, when the organism is grown under defined physiological conditions. The intermediates (believed to represent replicons) are synthesized and accumulated over longer periods of time (less than 90 min), if the cells are treated with low concentrations of cycloheximide. Under these conditions the rate of total DNA synthesis is only slightly inhibited (less than 15%), while maturation of intermediate DNA into high-molecular-weight DNA is completely blocked (greater than 98%). Cycloheximide appears to inhibit the maturation process more specifically than other protein synthesis inhibitors. Studies of the accumulated intermediates on alkaline buoyant density gradients demonstrate that initiation of new putative replicons occurs during treatment with cycloheximide.

Animals↗
PubMed1976

Accumulation of replicative DNA intermediates in Tetrahymena after excision-repairable damage to DNA.

Inhibition of DNA synthesis in the eukaryotic organism Tetrahymena pyriformis by thymine starvation results in the formation and accumulation of a distinct class of DNA during the first hour after re-initiation of the synthesis. This DNA, when synthesized in the presence of 5-bromodeoxyuridine (BrdUrd), bands as a distinct peak at an intermediate density of (see article) in a neutral CsCl buoyant density gradient. Following short pulses of BrdUrd, the DNA of intermediate density accounts for up to 90% of the newly synthesized DNA and binding assays on nitrocellulose filters show the presence of single-stranded regions. Alkaline buoyant density and alkaline velocity gradients clearly demonstrate the presence of non-covalently linked newly synthesized fragments with an average length shorter than that of the parental strands. Pulse experiments show that the DNA of intermediate density is progressively converted to fully hybrid (light - heavy) DNA molecules. It is therefore suggested that in response to damage caused by thymine starvation, a replicative DNA intermediate accumulates at the growing point. A similar phenomenon has also been observed after irridation with ultraviolet light. The effect of the protein synthesis inhibitor cycloheximide on the formation on the intermediate and the fully hybrid material is also described.

Animals↗
PubMed1976

Tetrahymena ribosomal RNA gene chromatin is digested by micrococcal nuclease at sites which have the same regular spacing on the DNA as corresponding sites in the bulk nuclear chromatin.

Synchronised cells of Tetrahymena pyriformis GL were labelled with 3H thymidine at a stage in the cell cycle when only the mitochondrial and extrachromosomal nucleolar ribosomal DNAs were replicating. In this way it was possible to prepare nuclei labelled selectively in the DNA of the ribosomal RNA genes. Since the ribosomal RNA cistrons of these cells are also very active in serving as a template for transcription, experiments were performed to test whether these genes are organised upon a nucleoprotein subunit structure of the kind that has been found in the total chromatin of a wide range of eukaryotic cell types. Tetrahymena macronuclei were prepared labelled uniformly in their DNA with 32P and labelled only in their nucleolar ribosomal DNA with 3H. Both the ribosomal genes and the bulk chromatin were then degraded in situ using micrococcal nuclease. The DNA fragments resulting from mild digestion were analysed on gels to reveal an identical DNA degradation pattern within both the ribosomal and bulk chromatins. It is concluded that the nucleoprotein structure of nucleolar rRNA cistrons posesses a periodic repeat along the DNA which is identical to that found in the substructure of unfractionated chromatin.

Animals↗
PubMed1975

Iolation and charcterization of a DNA-binding non-histone protein from Tetrahymena pyriformis.

Three proteins (A, B and C) that bind specifically to single-stranded DNA have been isolated from the eukaryotic organism Tetrahymena pyriformis. Their molecular weights are 47 000, 41 000 and 32 000. The amino acid composition of the A protein indicates that it is a non-histone protein and sucrose gradient centrifugation shows that it binds to bacteriophage M13 DNA and to oligo (dT)100 in a cooperative manner. The exonucleolytic degradation of oligo (dT)100 is prevented when it is bound to the A protein. The effect of A protein on the exonucleolytic reaction confirms the cooperative manner of binding of A protein binding to oligo (dT)100 and shows that this process may be prevented by high ionic strength. The A protein seems to be without enzymatic activity.

Amino Acids↗