Calf Thymus DNA-cellulose Research Articles

Different populations of progesterone receptor–steroid complexes in binding to specific DNA sequences: effects of salts on kinetics and specificity

We previously reported evidence for two subpopulations of several classes of steroid receptors that could be distinguished by their requirement of a low molecular weight factor ( M r=700–3000 Da) for binding to nonspecific, calf thymus DNA–cellulose [Cavanaugh, A. H. and Simons Jr., S. S., Journal of Steroid Biochemistry and Molecular Biology, 48, 433–446 (1994)]. This factor appeared to be enriched in (NH 4) 2SO 4 precipitates of nuclear extracts. Using human progesterone receptors (PRs) and biologically active DNA sequences in a modified avidin/biotin-coupled DNA (ABCD) binding assay, we now report a factor-mediated increase in PR binding to specific DNA sites that was indistinguishable from that seen with nonspecific sites. The main advantages of this modified assay are that both kinetic and equilibrium binding of receptor-steroid complexes to DNA can be directly monitored in solution. The ability of either Sephadex G-50 chromatography or sodium arsenite to prevent that binding which is increased by added factor supported the existence of PR subpopulations that are independent of the acceptor DNA sequence. The factor was found, surprisingly, to be low concentrations (≥5 mM) of (NH 4) 2SO 4, which anomalously is partially excluded from Sephadex G-10 columns, and can be mimicked by some salts but not sodium arsenite. Kinetic analyses demonstrated that the mechanism of action of salt was to accelerate the rate of binding of PR. Salt also had a much greater effect on the nonspecific binding of PR, such that the ratio of specific to nonspecific DNA binding was greatest at elevated salt concentrations (∼75 mM) that afforded submaximal levels of PR binding to specific DNA sites. Further analysis of the DNA-bound receptors revealed that the smaller, A-form of PR is preferentially bound to specific DNA sequences both in the presence and in the absence of various salt concentrations. Thus, the differences in DNA binding of PR±salt do not correlate with the preferential binding of A or B isoform. The unequal behavior of PR subpopulations and/or isoforms for binding to specific DNA sequences offers added mechanisms for selective transcriptional regulation of genes in intact cells.

Properties of nuclear and cytosolic ecdysteroid receptors from an epithelial cell line from Chironomus tentans

Recently ecdysteroid receptors have been identified in extracts of an epithelial cell line of the dipteran insect Chironomus tentans. These proteins have been shown to bind with high affinity to DNA and preferably to known ecdysteroid response elements. DNA-binding assays were performed under excess of DNA-binding sites in comparison to ecdysteroid receptors. Thereby, a proportion of about 20% of total ecdysteroid receptors in cytosolic extracts and an average of 50% of nuclear receptors could be demonstrated to bind to DNA. Ecdysteroid receptors from nuclear and cytosolic extracts displayed different S-value patterns. Cytosolic extract showed maximal ecdysteroid binding at S-values of about 4.4 S and greater than 8 S regardless of salt concentration in the buffer. Nucleic ecdysteroid receptors displayed a single peak of ecdysteroid binding at about 5 S. Furthermore, 0.4M NaCl could not completely elute nuclear ecdysteroid receptors from calf thymus-DNA cellulose columns whereas cytosolic receptors could almost be completely eluted with buffers of this ionic strength. This suggests a higher affinity to DNA for nuclear ecdysteroid receptors. These findings indicate that ecdysteroid receptors in this insect cell line may require activation step(s) similar to those discussed for vertebrate steroid receptors.

High level expression and purification of herpes simplex virus type 1 immediate early polypeptide Vmw110.

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) polypeptides. This paper reports the construction of a baculovirus vector which expresses large amounts of Vmw110, the product of IE gene 1. The expressed protein has been purified to near homogeneity and has a mobility on SDS polyacrylamide gels identical to that of Vmw110 produced during HSV-1 infection. Characterisation of its properties indicated that it forms dimers and perhaps higher order oligomers in solution and that the purified protein binds to both single stranded and double stranded calf thymus DNA cellulose columns. However, filter binding experiments were unable to detect any stable association of Vmw110 with DNA in solution.

A microscale analytical batch Chromatographic method for detecting soluble viral DNA-binding proteins in crude extracts

We describe a micro-method for determining the presence in crude cellular extracts of soluble proteins which can bind to immobilized DNA, using the DNA-binding protein of human adenovirus as an example. Batch chromatography of radiolabeled proteins is performed in microcentrifuge tubes containing 50 μ1 packed volume of commercially available denatured calf thymus DNA-cellulose. Eluted single-stranded DNA-binding proteins are then visualized by fluorography following gel electrophoresis. The batch procedure gives yields of adenovirus DNA-binding protein which are comparable to those obtained with a mini-column of similar adsorbent volume. The scale of the procedure makes it convenient for simultaneously analyzing multiple samples.

Characterization of HU-like protein from Bifidobacterium longum

A HU-like protein (HBI) of Bifidobacterium longum was purified and characterized. HBI is heat-stable and acid-resistant, and has a molecular weight of about 9.1 kDa as estimated by its mobility on electrophoresis. HBI is intermediate in basicity (p I 9.8) between the HU-1 and HU-2 proteins of Escherichia coli, and is dissociated from a calf thymus DNA-cellulose column at 300–400 mM NaCl. Its amino acid composition shows many similarities with that of E coli HU. The NH 2-terminal amino acid sequence of HBl also shows significant similarities to the consensus sequence deduced from the sequences of eleven HU-like proteins from prokaryotic sources. Chemical crosslinking analysis indicated that the HBI protein predominantly forms a homotypic dimer.

Human c- erbA protein expressed in Escherichia coli: changes in hydrophobicity upon thyroid hormone binding

The human c- erb Aβ gene sequence was inserted in an Escherichia coli expression vector plasmid. The E. coli cells transformed with this plasmid produced proteins with molecular masses of 52 and 50 kDa. These products bound 3,5,3′-triiodo-L-thyronine (T 3) with an affinity constant of 4.3 × 10 9 liter/mol. The order of affinity for iodothyronine analogs was triiodothyroacetic acid > T 3 > 3,5,3′- triiodo- d-thyronine > l-thyroxine . Affinity labeling experiments showed that the 50 kDa protein was covalently labeled with [ 125I]T 3, and this was competed by triiodothyroacetic acid, T 3, and l-thyroxine (from potent to weaker competitor). The c- erb A protein bound to calf thymus DNA-cellulose and the binding was inhibited by 0.3 M KCl or 10 mM pyridoxal 5′-phosphate. Aqueous two-phase partitioning studies showed that the c- erb A product became less hydrophobic upon T 3 or triiodothyroacetic acid binding. The same finding was obtained when T 3 bound to partially purified rat liver nuclear thyroid hormone receptor. However, thyroxine binding globulin became more hydrophobic upon T 3 binding. Since the T 3 molecule partitioned preferentially into the upper polyethylene glycol-rich phase, the alteration of partitioning behavior of thyroxine binding globulin was explained by a simple additive effect of T 3. In contrast, the alteration of partitioning behavior of the c- erb A product or receptor reflected a conformational transition upon T 3 binding. The c- erb A protein expressed in E. coli showed various characteristics similar to classical thyroid hormone receptor and may be useful in studying the structure and function of the thyroid hormone receptor.

Requirement of protein co-factor for the DNA-binding function of the human ski proto-oncogene product

We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody. p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity. This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells. By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids.

Differences in nuclear thyroid hormone receptors among species

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3′-triiodo- l-thyronine (T 3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T 3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 ± 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T 3 at 22° for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 ± 1.47 × 10 5 M −1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 ± 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T 3-receptor complex using trypsin α-chymotrypsin, elastase, and papain produced distinct T 3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Helicase, DNA-binding, and immunological properties of replication-defective simian virus 40 mutant T antigens

Simian virus 40 T antigen (TAg) exhibits nonspecific and origin-specific DNA binding (ori binding) and ATPase and helicase activities, all of which are related to its roles in viral DNA replication. We have characterized some of the properties of four replication-defective but transformation-competent mutant TAgs, C6-2, T22, C11, and C8A. C6-2 and T22 TAgs were each previously determined to lack ori-binding properties, while C11 TAg was reported to lack ATPase activity. The C8A TAg did not exhibit defects in either ori-binding or ATPase functions. We have analyzed additional aspects of these mutant TAgs pertaining to their helicase, DNA-binding, and immunological properties. With the exception of the C11 TAg, all the other TAgs exhibited helicase activity. The lack of helicase activity by C11 TAg was consistent with its previously shown inability to hydrolyze ATP or to replicate viral DNA. These results therefore show that ori-binding and helicase activities are separate functions of TAg. Wild-type and mutant TAgs bound with similar efficiency to either native or denatured calf thymus DNA-cellulose, indicating no marked differences in their nonspecific DNA-binding properties. We also tested the binding of wild-type and mutant TAgs to a monoclonal antibody, PAb 100, that was previously shown to recognize an extremely small class of TAg that may represent a unique conformational form of the protein. Interestingly, while less than 10% of the wild-type, C6-2, C11, and T22 mutant TAgs were recognized by PAb 100, more than 60% of the C8A mutant TAg was bound by this antibody. Therefore, although no defect in biochemical function was observed with the C8A TAg, its deficiency in viral DNA replication may be related to an unusual conformation, as detected by its dramatically increased recognition by PAb 100. These results show that the helicase activity of TAg is not required for its transformation function.

Interaction of the 89K murine cytomegalovirus immediate-early protein with core histones.

The conditions that permit the interaction of immediate-early proteins of murine cytomegalovirus (MCMV) with DNA were studied. Chromatography of extracts from infected cells on MCMV DNA cellulose and calf thymus DNA cellulose showed that pp89, the regulatory major immediate-early protein, interacts with DNA and dissociates at salt concentrations between 0.3 and 0.6 M NaCl. pp76, a cleavage product of pp89, and additional minor ie 1 proteins eluted already at low ionic strength. Cellular DNA-binding factors were required for association of pp89 with DNA. These factors were identified as core histones. Chromatography of IE proteins on histone-Sepharose in the absence of DNA revealed a high-binding affinity that was resistant to 2 M NaCl. These results suggest that pp89 has no direct DNA-binding activity. A role for an amino acid sequence homology in the N-terminal region of pp89 with histone H2B in the pp89-histone-DNA interaction is discussed.

Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.

The product of the Drosophila segmentation gene Krüppel was produced in cultured insect cells using the baculovirus expression system. When a cloned Krüppel cDNA sequence was inserted into the viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately equal to 72,000 was observed in the nuclei of infected cells. Antibodies were raised against this protein and used to detect Krüppel in Drosophila embryos. Characterization of the Krüppel protein extracted from infected cells showed that it is tightly bound to the nucleus, it binds to calf thymus DNA-cellulose, and it is phosphorylated. These results support the hypothesis that Krüppel is a regulatory protein that acts by binding DNA.

Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions.

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor.

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.

A subclass of polyomavirus middle tumor antigen binds to DNA cellulose.

We examined the binding of polyomavirus large (L-T)-, middle (M-T)-, and small-tumor antigens to DNA cellulose. At pH 6.0, the majority of L-T bound to calf thymus DNA cellulose, while little or no small tumor antigen was retained under these conditions. Unexpectedly, a small but reproducible proportion of M-T bound to both native and denatured DNA cellulose. M-T encoded by polyomavirus mutant dl 8, which expressed shortened L-T and M-T, bound to DNA, indicating that the deleted sequences are not required for DNA binding. Also, M-T from transformed BMT-1 rat cells, which synthesize exclusively this polyomavirus tumor antigen, bound to DNA, indicating that its binding is not due to association with other polyomavirus-encoded proteins. Using the DNA fragment immunoassay, we found that, under conditions in which L-T bound specifically to DNA fragments containing viral regulatory sequences, no viral DNA fragments were bound by M-T. The existence of distinct subpopulations of M-T that differ in their DNA-binding properties was indicated by rebinding experiments in which M-T that had bound to DNA cellulose rebound very efficiently, while that which had not been originally retained by DNA cellulose rebound poorly. Furthermore, the M-T-pp60 c-src complex did not bind to DNA cellulose. These data suggest that polyomavirus M-T is heterogeneous, consisting of populations of molecules that differ in their interactions with DNA cellulose.

Interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with RNA and synthetic polyribonucleotides.

The interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with RNA and synthetic polynucleotides has been examined by using receptor from rachitic chicken intestine. Total intestinal RNA inhibited the binding of receptor to calf thymus DNA-cellulose with an efficiency equivalent to single-stranded DNA. A comparison of the inhibitory activity of several polyribonucleotides gave the following order of activities: poly(I) = poly(G) greater than or equal to double-stranded DNA greater than single-stranded DNA = poly(U). Poly(A), poly(C), and the ribonucleoside monophosphates AMP, GMP, CMP, and UMP had minimal activity. A preference for single-stranded homopolymers was observed [i.e., poly(I) and poly(U) were active, whereas poly(I):poly(C) and poly(U):poly(A) were not]. The ability of nucleic acids to displace receptor from DNA-cellulose was also measured. Both poly(G) and poly(I) were more active than double-stranded DNA in this assay. Furthermore, differences were noted between intestinal RNA fractions separated on the basis of poly(A) content. Receptor also bound to immobilized intestinal RNA and polynucleotides. The KCl concentration necessary to disrupt binding to a given polynucleotide generally paralleled the activity of that molecule in DNA-cellulose inhibition and displacement assays. These results suggest that the 1 alpha,25-dihydroxyvitamin D3 receptor can interact with RNA as well as DNA.

The interaction in vitro of the intermediate filament protein vimentin with synthetic polyribo- and polydeoxyribonucleotides.

In a continuation of our studies on the association of the intermediate filament protein vimentin with unfolded ribosomal subunits, rRNA and various naturally occurring RNAs and DNAs, we have investigated the binding of vimentin to a variety of synthetic polyribo- and polydeoxyribonucleotides. The vimentin-binding potentials of the different nucleic acids were determined in competition with 28S rRNA from Ehrlich ascites tumor cells. The reaction products were analysed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Among the homopolynucleotides tested, poly(rG), poly(rX), poly(rI), poly(rs4U) and poly(dG) were very good competitors, whereas poly(dC) was moderately so. Poly(rA), poly(rU), poly(rC), poly(dA), poly(dU), poly(dT) and poly(dI) were very weak competitors. Annealing of the active homopolymers to their complementary homopolymer strands resulted in a reduction of the vimentin-binding potential; however, the resulting duplexes still had substantial vimentin-binding capacity. A similar reduction was observed when the mononucleotides of the active homopolymers were randomly copolymerized with their complementary nucleotides or incorporated into strictly alternating, double-stranded DNAs. DNA-type copolymers, duplexes and alternating copolymers containing the nuclear bases adenine, thymine and uracil were moderate competitors, but, in general, they were significantly more active than the corresponding homopolymers. This result, together with those of the analysis of a series of random heteropolyribonucleotides, indicates that there is some influence of the base sequence and/or secondary structure of the nucleic acids on their vimentin-binding capacities. Whereas the minimum length of oligo(dG) for efficient vimentin binding was between 11 and 18 nucleotides, (dT)18 was still incapable of interacting with vimentin. In order to obtain information on the salt stability of vimentin-nucleic acid adducts, we have also performed affinity chromatographies on single-stranded calf thymus DNA-cellulose and on rRNA and homopolyribonucleotides covalently coupled to agarose. Although because of unspecific secondary interaction of vimentin with the matrix, these nucleic acid-substituted absorbents could not be used for this purpose under non-denaturing conditions, in the presence of 6M urea a relationship between the nucleic acid substitution and the salt stability of vimentin binding was observed. Finally, the nucleic acid-binding properties of vimentin are compared with those of cytosolic steroid hormone receptors, particularly with those of the androgen receptor.

Chicken oviduct progesterone receptor: Location of specific regions of high-affinity binding in cloned DNA fragments of hormone-responsive genes

We have used a DNA-cellulose competitive binding assay to measure the extent of displacement of the chicken oviduct progesterone-receptor complex from calf thymus DNA-cellulose by purified cloned fragments of genomic DNA. Several DNA fragments from hormonally responsive genes coding for egg-white proteins were found to be efficient competitors for either crude or partially purified receptor complexes when compared with calf thymus DNA. Data obtained with deletion mutations constructed in vitro allowed delineation of a specific region necessary for strong competition, located 250–300 bp upstream from the mRNA startsite of the oval-bumin gene. Sequence homologies with this 5′-upstream region were observed in other fragments of the ovalbumin, conalbumin, ovomucoid, X and Y genes, which were also efficient competitors. Based on a comparison of such sequences of homology, a consensus sequence that may constitute a region binding progesterone-receptor complex has been constructed: ATC TT CCATT T ATCTG T GTTGTA. The results suggest that specific double-stranded DNA sequences are recognized by. the oviduct progesterone-receptor complex in vitro, and are relevant to the question of whether specific DNA sequences are directly involved as genomic binding sites for steroid receptors.

Effects of RNA and ribonuclease on the binding of estrogen and glucocorticoid receptors from MCF-7 cells to DNA-cellulose.

Effects of RNA and ribonucleases on the binding of estrogen- and glucocorticoid-receptor complexes from MCF-7 cells to calf thymus DNA-cellulose have been studied under cell-free conditions. Exogenous ribonucleases can increase binding of estrogen- and glucocorticoid-receptor complexes to DNA-cellulose. Purified RNA derived from MCF-7 cells can inhibit estrogen- and glucocorticoid-receptor binding to DNA-cellulose. Under low salt conditions, RNA appears to be associated with estrogen-receptor complexes from MCF-7 cells. These observations taken together suggest that there is an interaction between steroid hormone-receptor complexes and RNA. The implications of this interaction are discussed.

Study of androgen-binding cytosol proteins from rat prostate: Purification of androgen receptor

The crude 8 and 4 S androgen-binding cytosol fractions from castrated rat prostate have been fractionated by sequential chromatography on calf thymus DNA-cellulose and rat prostate DNA-cellulose. This procedure yields one specific DNA-binding 8 S protein fraction (A1 b) showing selective affinity toward prostate DNA, one nonspecific DNA-binding protein fraction (A2 b) which binds to both calf thymus DNA and rat prostate DNA, and a non-DNA-binding fraction (A1 a) from the 8 S cytosol fraction. The 4 S cytosol fraction does not interact with DNA from either source. Studies of these androgen-binding fractions indicate that only fraction A1 b shows significant specific binding to [ 3H]dihydrotestosterone. The [ 3H] dihydrotestosterone-A1 b complex manifests active target tissue-specific translocation into the nucleus. The complex also stimulates transcription of DNA in vitro in an RNA polymerase II reaction as compared with the other androgen-binding cytosol fractions. The employment of selective DNA-affinity chromatography of the androgen-binding cytosol proteins results in considerable simplification of the molecular complexity of the crude 8 S fraction, with a partial purification of the active androgen receptor from prostate cytosol.