DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells.

Affinity chromatography on single-stranded and double-stranded DNA-cellulose indicates that 12 proteins previously identified from herpes simplex virus type 2-infected cells, ranging in molecular weight from 28 X 10(3) to 186 X 10(3), bind to DNA-cellulose. The DNA-binding proteins found in infected cells differed in relative binding strengths for denatured DNA-cellulose. The virus specificity of these DNA-binding proteins was further studied by comparison with DNA-binding proteins isolated from mock-infected cells, and by immunoprecipitation of infected-cell DNA-binding proteins with antisera specific for viral antigens. The promise this technique holds for the purification and study of polypeptides involved in virus DNA replication, recombination, or repair is discussed.     

Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum.

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH₂[³²P]O₄ and [³H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.     

The effect of 5-bromodeoxyuridine on the synthesis of nuclear and cytoplasmic DNA-binding proteins isolated from sea urchin embryos.

Newly synthesized DNA-binding proteins were isolated from the nuclei and, separately from, the cytoplasm of sea urchin mofula stage embryos. The presence of 5-bromodeoxyuridine during embryogenesis did not appear to alter the synthesis of either class of DNA-binding proteins. This result tends to argue that cell differentiation in early embryos is not regulated by differential synthesis of DNA-binding proteins. Sea urchin mofulae synthesize a broad range, by molecular weight, or cytoplasmic DNA-binding proteins which dissociate from sea urchin DNA-cellulose at relatively high salt concentrations (0.6-2.0 M NaCl). The most prominant of these apparently high-binding-affinity proteins has an approximate molecular weight of 33,000.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

DNA-binding proteins present in varicella-zoster virus-infected cells.

DNA-binding proteins present in varicella-zoster virus-infected cells were identified by DNA-cellulose chromatography of radioactively labeled cell extracts. Seven virus-specific proteins, ranging in molecular weight from approximately 175,000 to 21,000, showed affinity for single- or double-stranded DNA or both. These proteins include the varicella-zoster virus major capsid protein, a phosphorylated tegument protein, and a 125,000-molecular-weight species which may be analogous to the major DNA-binding protein of herpes simplex virus. We also identified a number of DNA-binding phosphoproteins by these procedures. Finally, protein blot studies were carried out to determine whether these proteins bind preferentially to virus rather than to host cell DNA.     

DNA-binding proteins in xeroderma pigmentosum fibroblasts.

DNA-binding proteins in human fibroblasts were examined by chromatography on DNA-cellulose columns. By successive chromatography on columns containing native, denatured, and UV-irradiated DNA-cellulose respectively the proteins binding to different types of DNA could be studied. Elution of the columns with sodium chloride followed by polyacrylamide gel electrophoresis allowed several DNA-binding proteins to be identified. All of the major DNA-binding proteins were present in strains of xeroderma pigmentosum cells respectively deficient in excision-repair and post-replication repair of ultraviolet-induced damage.     

Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.

We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.     

Isolation of early viral proteins from poxvirus-infected chick embryo fibroblasts by DNA-cellulose chromatography and inhibition of their synthesis by chicken interferon

Up to seven early poxvirus-specific proteins have been isolated from vaccinia-WR-infected and cowpox-virus-infected chick embryo fibroblasts by affinity chromatography on native DNA-cellulose columns. The proteins have been characterized by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by nonequilibrium pH-gradient electrophoresis. The molecular weights of the viral proteins were determined by comparison with proteins of known molecular weight and are comparable to several of the vaccinia-WR-specific DNA-binding proteins isolated previously from infected L-929 cells by Solosky J. M., Esteban M. and Holowczak J.A. [J. Virol. 25, 263-273 (1978)]. The viral proteins binding reversibly to native DNA have been classified as immediate early viral gene products. Synthesis of cowpox-virus-induced early DNA-binding proteins is inhibited in chick cells pretreated with homologous interferon at a concentration of 500--1000 units/ml.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

DNA-binding activity of retinoblastoma protein is intrinsic to its carboxyl-terminal region

The retinoblastoma (RB) gene encodes a nuclear phosphoprotein with a molecular weight of 110,000 (pp110RB) associated with DNA-binding activity. This sequence-nonspecific DNA binding activity was further studied by Southwestern and DNA-cellulose chromatography using purified fusion proteins expressed in Escherichia coli. Three fusion proteins, containing amino acids 612-775, 776-928, and 612-928 of pp110RB, bound to DNA; the binding affinity of the latter was approximately 20-fold higher than those of either smaller region. Other regions of pp110RB had no detectable binding activity, indicating that the carboxyl-terminal region of the RB protein is the major domain responsible for interacting with DNA. Since several potential phosphorylation sites reside within this region, isoforms of RB protein from cellular lysates with various degrees of phosphorylation were compared with respect to their DNA-binding affinity. The hyperphosphorylated form was eluted from DNA-cellulose columns at 0.1-0.3 M NaCl, whereas the hypophosphorylated form appeared in the eluates only at salt concentrations of 0.4-0.7 M, implying that phosphorylation of RB protein may affect its DNA-binding activity. That pp110RB can bind DNA intrinsically, and that this activity can be modulated by phosphorylation, is consistent with the proposed regulatory role of the RB protein in cell growth and differentiation.     

Effect of the 90 kDa heat shock protein, HSP90, on glucocorticoid receptor binding to DNA-cellulose

Glucocorticoid receptors in the IM-9 human lymphoblastoid cell line were affinity labeled with [3H]dexamethasone 21-mesylate and activated to a DNA-binding form by filtration through a Bio-Gel A-1.5m column. The 90 kDa heat shock protein, HSP90, was identified by labeling IM-9 cells with 35S-methionine at both 37 degrees C and 42 degrees C and purified to near homogeneity by sequential chromatography through DE52 and hydroxyapatite. Addition of purified HSP90 to activated, affinity labeled glucocorticoid receptors in a molecular ratio of 16 to 1 inhibited the binding of the receptors to DNA-cellulose. HSP90 did not affect the binding of other proteins to DNA-cellulose, indicating that the inhibitory effect of HSP90 was specific for the glucocorticoid receptor. These results suggest that HSP90 may associate with the glucocorticoid receptor, masking its DNA-binding site and thereby inhibiting receptor interaction with DNA.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.     

Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins

Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.     

Dynamics of DNA-binding activity of cytoplasmic proteins in autoproteolysis

The quality proteolysis changing of DNA-binding proteins of cytosol mice liver was studied by affinity chromatography on DNA-cellulose. It is shown that neutral proteolysis leads to the second peak of DNA binding.     

Existence of a form of SV40 T antigen incapable of interacting with DNA

The interaction of SV40 T-antigen and viral DNA was studied by using adsorption of DNA-protein complexes on nitrocellulose filters. The T-antigen purification procedure included ion-exchange chromatography on DEAE-cellulose, selective adsorption of cellular proteins on single-stranded DNA-cellulose, chromatography on heparin-Sepharose and removal of cell proteins by an immunosorbent. Only the latter step allowed to remove the contamination of cellular DNA-binding proteins, judging from the reaction of T-antigen neutralization by specific antibodies. It was shown that T-antigen and cellular DNA-binding proteins interact with SV40 DNA at different values of pH, namely ah 6,0-6,4 and 7,9, respectively. The T-antigen obtained was passed through a column with native DNA-cellulose at pH and ionic strength values optimal for interaction with DNA. The bulk of T-antigen (30-40%) did not bind to native thymus DNA and did not interact with SV40 DNA. It is assumed that this fraction is a form of T-antigen, which undergoes structural or functional changes during specific interaction with viral or cellular DNAs.     

DNA-binding proteins in yeast: Effect of growth phase and mitochondrial function

DNA-binding proteins of the yeast Saccharomyces cerevisiae have been examined by DNA-cellulose chromatography with the expectation that they should represent, in part, a subclass of those proteins which bind to or interact with the chromosomes in vivo. After a high speed supernatant of a deoxyribonuclease-treated cell lysate is passed through a column of calf thymus DNA-cellulose, the DNA-binding proteins are eluted with a discontinuous salt gradient. The DNA-binding proteins, which show a broad distribution in size when examined by electrophoresis on polyacrylamide slab-gels in the presence of sodium dodecyl sulfate, represent about 0.2–0.3% of the cell's protein corresponding to about 5 × 10⁹-molecular weight of protein per haploid cell. Our data demonstrate quantitative and qualitative changes in the spectrum of DNA-binding proteins which may be correlated with changes in growth rate, stage of the growth cycle and phenotypic (repressed versus derepressed) and genetic alterations in mitochondrial function (grandes versus petites). The largest change which we have noted in the spectrum of DNA-binding proteins is between glucose-grown log-phase grande cells and grande cells in stationary phase. In many of the comparisons made, a number of specific DNA-binding proteins are seen to vary by as much as 5–10-fold. From estimates of the number of molecules of a DNA-binding protein present in the cell, we conclude that the system we have described is capable of detecting less than 100 molecules per yeast cell; within the range of the level of the lac represser in Escherichia coli.     

Binding of microtubule proteins to DNA: specificity of the interaction.

Tubulin is detected among the DNA-binding proteins when an extract from fibroblasts is chromatographed on DNA-cellulose. Further purification of the colchicine-binding activity shows that purified tubulin from fibroblasts does not bind to DNA. Depolymerized brain microtubule proteins show a high affinity for DNA. The fraction bound is composed of tubulin and microtubule-associated proteins. Experiments with fractionated microtubule proteins indicate that tubulin-free microtubule associated proteins bind to DNA, while tubulin free of microtubule-associated proteins does not. Microtubule-associated proteins bind better to eukaryotic than to phage DNA suggesting a specificity of the interaction.     

Mitochondrial DNA-binding proteins that bind preferentially to supercoiled molecules containing the D-loop region of Xenopus laevis mtDNA

From the bulk of the Xenopus laevis mitochondrial proteins insoluble in 1% Triton X-100 + 1M NaCl, we have isolated, by DNA-cellulose chromatography, a protein fraction enriched in DNA-binding proteins. This fraction contains proteins showing a specific affinity for supercoiled DNA molecules containing the mitochondrial DNA displacement-loop region, as measured by filter binding and competition assays.     

The bacteriophage T4 regulatory protein gpunf/alc binds to DNA in the absence of RNA polymerase.

DNA-cellulose chromatography and two-dimensional gel electrophoresis have been used to demonstrate the DNA-binding capacity of bacteriophage T4 gpunf/alc. The unf/alc protein does not bind to DNA via an association with RNA polymerase; gpunf/alc was shown to bind to DNA after separation from RNA polymerase and other large proteins by Sephadex chromatography.     

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins--including numerous keratins characterized only recently--are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family.