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R Koop

Publications and source records attributed to R Koop.

9 recordsLinked to original sources
PubMed2001

Transcription factor Sp3 is regulated by acetylation.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Previous analyses showed that, unlike Sp1, Sp3 fails to activate transcription in certain promoter settings. This is due to the presence of an inhibitory domain located between the second glutamine-rich activation domain and the DNA-binding domain. To further analyze the transcriptional properties of Sp3, we have expressed and purified recombinant Sp3 and Sp1 as epitope-tagged proteins from stable transfected insect cells. We found that Sp3 does act as a strong activator similar to Sp1 in an in vitro transcription assay using Sp1/Sp3-depleted HeLa nuclear extract. However, on the same promoter Sp3 is almost inactive when transfected into cells. Mutational studies demonstrate that a single lysine residue is responsible for the low transcriptional activity of Sp3 in vivo. We show that Sp3, but not a mutant of Sp3 that lacks this lysine residue, is highly acetylated in vivo. Our results strongly suggest that the transcriptional activity of Sp3 is regulated by acetylation. The consequences of acetylation for the activity of Sp3 are discussed.

Acetylation↗
PubMed1999

Rapid purification of intact minichromosomes over a glycerol cushion.

Negatively supercoiled plasmids can be assembled into dynamic minichromosomes using Drosophila embryo extract as a source of histones and chromatin assembly factors. However, analysis of such mini-chromosomes is often difficult due to the presence in the crude extract of a large excess of macromolecules and low molecular weight molecules including ATP. Several techniques have been used to partially purify the minichromosomes based on either sizing columns or centrifugation on sucrose gradients. We have developed a single-step method employing a 30 min ultracentrifugation through a glycerol cushion. In contrast to chromatin purified in sucrose-containing buffers, the minichromosomes obtained with this method are suitable for transcriptional analysis. This method is fast, quantitative, flexible, can deal with several samples simultaneously and leads to concentration of the chromatin. As centrifugation through glycerol yields chromatin free of ATP and several characterized chromatin remodeling complexes, this method should be useful for structural and functional studies in vitro.

Animals↗
PubMed1999

Two-step synergism between the progesterone receptor and the DNA-binding domain of nuclear factor 1 on MMTV minichromosomes.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.

Adenosine Triphosphatases↗
PubMed1998

Mechanisms of viral activators.

Adenovirus large E1A, Epstein-Barr virus Zebra, and herpes simplex virus VP16 were studied as models of animal cell transcriptional activators. Large E1A can activate transcription from a TATA box, a result that leads us to suggest that it interacts with a general transcription factor. Initial studies showed that large E1A binds directly to the TBP subunit of TFIID. However, analysis of multiple E1A and TBP mutants failed to support the significance of this in vitro interaction for the mechanism of activation. Recent studies to be reported elsewhere indicate that conserved region 3 of large E1A, which is required for its activation function, binds to one subunit of a multisubunit protein that stimulates in vitro transcription in response to large E1A and other activators. A method was developed for the rapid purification of TFIID approximately 25,000-fold to near homogeneity from a cell line engineered to express an epitope-tagged form of TBP. Purified TFIID contains 11 major TAFs ranging in mass from approximately 250 to 20 kD. Zta and VP16, but not large E1A, greatly stimulate the rate and extent of assembly of a TFIID-TFIIA complex on promoter DNA (DA complex). For VP16, this is a function of the carboxy-terminal activation subdomain. An excellent correlation was found between the ability of VP16C mutants to stimulate DA complex assembly and their ability to activate transcription in vivo. Consequently, for a subset of activation domains, DA complex assembly activity is an important component of the overall mechanism of activation.

Adenovirus E1A Proteins↗
PubMed1998

CIF150, a human cofactor for transcription factor IID-dependent initiator function.

The transcription factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated factors (TAFs), with the exception of dTAF(II)150. We previously reported the identification of CIF, an essential cofactor for TFIID-dependent transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAF(II)150, and present biochemical evidence that this factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAF(II)150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAF(II)135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

Amino Acid Sequence↗
PubMed1994

A rapid luciferase transfection assay for transcription activation effects and stability control of estrogenic drugs in cell cultures.

A rapid assay system for measuring the potential of estrogenic drugs is introduced. Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc. The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug. The assay system was used to measure the stability of the drug diaqua-[1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine] platinum(II) sulfate, containing an estrogenic ligand and reactive platinum. Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active. It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.

Antineoplastic Agents↗
PubMed1994

1-Carbamoylalkyl-2-phenylindoles: relationship between side chain structure and estrogen antagonism.

The 2-phenylindole system has proved to be a versatile structure for the design of potent antiestrogens, especially when functional groups have been introduced into the alkyl side chain in position 1. In analogy to steroidal structures such as ICI 164,384 a number of 2-phenylindoles with carbamoylalkyl and aminoalkyl side chains were synthesized. They bind to the calf uterine estrogen receptor with relative binding affinities between 2.1 and 21 (estradiol = 100). The antiestrogenic effect of these compounds was demonstrated by the inhibition of transcriptional activity which was measured in a new luciferase assay with the EREwtc luc as reporter plasmid. The derivative with a methyl-n-propyldodecanamide side chain (4h) antagonized the effect of estradiol (10(-9) M) completely at concentrations of 10(-7) M and higher. As a sensitive model for quantification of estrogenic and antiestrogenic effects in vitro we used HeLa-cells cotransfected both with the reporter plasmid and estrogen receptor expression vectors HEG0 and HE0. In cells transfected with these vectors transcriptional activity was strongly dependent on side chain structure. With mutated receptors we were able to show that this activity was mainly due to TAF-1 whereas TAF-2 remained silent. When we studied the effect of some of the new compounds in vivo using the mouse uterine weight assay, we observed a correlation between transcriptional activity in transfected HeLa cells and estrogenic effects in mice. Two of the 1-carbamoylalkyl-2-phenylindoles (4f, 4h) proved to be "pure" antiestrogens both in vitro and in vivo. In estrogen-sensitive MCF-7 breast cancer cells, they strongly inhibit cellular growth. Some of the IC50-values were close to 10(-8) M.

Animals↗
PubMed1983

[Significance of nonsaponifiable constituents of dietary fats on the bioactivity of vitamin E].

In experiments with male Wistar rats the influence of the non-saponifiable constituents of dietary fats: dl-alpha-tocopherol (60 ppm), dl-gamma-tocopherol (480 ppm), ubiquinone (96 ppm) and beta-sitosterol (3035 ppm) on the tocopherol status was investigated, considering the fatty acid composition of the tested fats. For a test period of eight weeks the animals were fed isoenergetic diets containing three types of dietary fats: corn oil (60% PUFA), a kind of "stripped corn oil" (60% PUFA) and butter (nearly 5% PUFA). Independent of the PUFA-content of the diet, the tocopherol supplementations were able to stabilize the erythrocyte membrane; the calculated hemolysis rates were about 2%. The absence of tocopherols in the diets ("stripped corn oil", butter) caused an increase of the hemolysis rate up to 70% after two weeks. The original amounts of tocopherols in corn oil tended to minimize the hemolysis. Ubiquinone and beta-sitosterol did not reduce the hemolysis rates when they were applied without tocopherols. With respect to creatine-phosphokinase activity, creatine and creatinine excretion the results were similar. Plasma and erythrocyte levels of alpha- and gamma-tocopherol were determined in all groups and discussed in connection with the other examined parameters of tocopherol status. The ultimate result of this experiment is that the content of tocopherols in dietary fats is not always adequate to keep vitamin E status normal, especially if polyunsaturated fatty acid content is high in the diet. Reflecting the vitamin E adequacy of dietary fats, not only alpha-tocopherol but also gamma-tocopherol should be much more considered than previously.

Animals↗