DNA-cellulose Affinity Chromatography Research Articles

Organization of Mitochondrial-Nucleoids in BY-2 Cultured Tobacco Cells

The molecular organization of mitochondrial-nucleoids (mt-nucleoids) isolated from BY-2 cultured tobacco (Nicotiana tabacum L.) cells was analyzed. Fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) revealed that isolated mt-nucleoids retained the compact structural organization as that observed in vivo. Treatments of mt-nucleoids with DNase I, proteinase K and 2 M NaCl resulted in the disappearance, partial swelling, or complete dispersion of their DAPI-fluorescence, respectively, whereas treatment with RNase A did not affect their compact appearance. These results suggest that the compact structure of mt-nucleoids is maintained by electrostatic interaction between DNA and proteins. When mt-nucleoids that were treated with micrococcal nuclease (MNase) were analyzed by agarose gel electrophoresis, a ladder-like electrophoretic pattern of DNA, whose minimum fragment size was about 75 bp, was observed. This result suggests the presence of nucleosome-like, repetitive structures in the mt-nucleoids. Treatment of mt-nucleoids with increasing concentrations of NaCl gradually solubilized various proteins, including those with DNA-binding activities, which was accompanied by gradual dispersion of the mt-nucleoid structure. This result suggests that a variety of DNA-binding proteins with differing degrees of binding strength are present in the mt-nucleoids, and are involved in the compact organization of DNA-protein complexes at various levels. Detailed analyses of mt-nucleoids by Southwestern blotting, SDS-DNA PAGE, and DNA-cellulose affinity chromatography revealed the presence of many different DNA-binding proteins in the 2 M NaCl-soluble fraction of mt-nucleoids, which are expected to be involved in the compact organization of mt-nucleoids at various levels.

Insoluble Aggregates and Protease-resistant Conformers of Prion Protein in Uninfected Human Brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.

Flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and combines with the Jak kinase inhibitor AG490 to achieve cytotoxic synergy

Up-regulated signal transducers and activators of transcription (STAT)-mediated signaling is believed to contribute to the pathogenesis of a variety of solid and hematologic cancers. Consequently, inhibition of STAT-mediated signaling has recently been proposed as a potential new therapeutic approach to the treatment of cancers. Having shown previously that the pan-cyclin-dependent kinase inhibitor flavopiridol binds to DNA and seems to kill cancer cells via that process in some circumstances, we evaluated the hypothesis that flavopiridol might consequently disrupt STAT3/DNA interactions, attenuate STAT3-directed transcription, and down-regulate STAT3 downstream polypeptides, including the antiapoptotic polypeptide Mcl-1. SDS-PAGE/immunoblotting and reverse transcription-PCR were used to assess RNA and polypeptide levels, respectively. DNA cellulose affinity chromatography and a nuclear elution assay were used to evaluate the ability of flavopiridol to disrupt STAT3/DNA interactions. A STAT3 luciferase reporter assay was used to examine the ability of flavopiridol to attenuate STAT3-directed transcription. Colony-forming assays were used to assess cytotoxic synergy between flavopiridol and AG490. Flavopiridol was found to (a) disrupt STAT3/DNA interactions (DNA cellulose affinity chromatography and nuclear elution assay), (b) attenuate STAT3-directed transcription (STAT3 luciferase reporter assay), and (c) down-regulate the STAT3 downstream antiapoptotic polypeptide Mcl-1 at the transcriptional level (reverse transcription-PCR and SDS-PAGE/immunoblotting). Furthermore, flavopiridol, but not the microtubule inhibitor paclitaxel, could be combined with the STAT3 pathway inhibitor AG490 to achieve cytotoxic synergy in A549 human non-small cell lung cancer cells. Collectively, these data suggest that flavopiridol can attenuate STAT3-directed transcription in a targeted fashion and may therefore be exploitable clinically in the development of chemotherapy regimens combining flavopiridol and other inhibitors of STAT3 signaling pathways.

Interaction of a low mobility group protein, LMG 160, with deoxyribonucleic acid

A fraction of low mobility group (LMG) nonhistone protein designated LMG 160 was isolated from rat liver chromatin by preparative gel electrophoresis and its interaction with DNA was studied using thermal denaturation and DNA–cellulose affinity chromatography techniques. The results showed that LMG 160 with an isoelecteric point of 5–5.5 was bound to DNA and decreased its melting temperature. Increasing ionic strengths decreased this effect. DNA–cellulose affinity chromatography showed the affinity of LMG 160 to double stranded DNA was higher than that to single stranded DNA, since it required 0.6 M NaCl for elution. The results suggest that LMG 160 protein preferentially binds to double stranded DNA destabilizes it and the binding is electrostatic.

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg 2+-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg 2+-independent.

Mechanism of the Activation of Proteinase Inhibitor Synthesis by Systemin Involves β-Sheet Structure, a Specific DNA-Binding Protein Domain

We analyzed a tertiary structure of systemin, the first identified polypeptide plant hormone, using two-dimensional NMR spectroscopy. From these data and molecular dynamics calculations we concluded that the peptide can adopt a Z-like-β-sheet structure, which has previously been found in many specific DNA-binding proteins. Using DNA-cellulose affinity chromatography, we showed that systemin binds strongly to DNA. We suggest that the specific systemin-DNA interaction, particularly in a promoter region of the proteinase inhibitors, could effect gone expression and thus explain the biological activity of systemin.

Turtle oviduct progesterone receptor: radioligand and immunocytochemical studies of changes during the seasonal cycle

In order to determine the regulation of the oviduct progesterone receptor inChrysemys picta, radioligand binding studies were performed to determine changes in the high and lower affinity binding sites during the seasonal cycle. Lower affinity sites were present in both cytosolic and nuclear fractions during the cycle and peaked during the peri-ovulatory/early luteal periods. The high affinity sites, present exclusively in the nuclear fraction, increased following the preovulatory peak in plasma estradiol, remained elevated during the early luteal phase following the post-ovulatory peak in progesterone, and declined to non-detectable levels just before egg-laying. DNA-cellulose affinity chromatography showed that both high and low affinity binding sites were integral to both progesterone receptor B and A isoforms. Western blot analysis confirmed the binding studies and showed that PR-B (115 kDa) was present in greatest amounts during the peri-ovulatory and luteal periods, whereas PR-A (88 kDa) increased during those periods and was present following egg-laying. Immunocytochemical analysis revealed increased progesterone receptor immunostaining from the winter to the peri-ovulatory period in the three major zones (luminal epithelium, submucosal glands and the myometrium) following the preovulatory peak in estradiol, a decrease in all three zones, especially the myometrium, in the late luteal period following the post-ovulatory peak in progesterone, and an increase again during fall recrudescence. Competition studies demonstrated that progesterone was the most effective competitor followed by pregnenolone, R5020 and deoxycorticosterone. RU 486 does not bind to the high affinity site, but binds quite well to the lower affinity site. This study suggests that progesterone receptor isoforms in the turtle oviduct may be under the regulation of changing estrogen/progesterone ratios during the cycle.

Putative steroid-binding receptors and nonreceptor components and testicular activity in the lizard Podarcis sicula sicula.

Labelled testosterone- and oestradiol-binding molecules have been found in the cytosol and nuclei of lizard testes. DNA-cellulose affinity chromatography was used to separate putative sex-steroid-binding receptors (adhering molecules) and nonreceptor components (nonadhering molecules). A putative androgen receptor (Kd: 10(-10) mol l-1; 3-9 fmol g-1 tissue) was found mainly in the nuclei of testicular cells when actively undergoing spermatogenesis. This suggests that, as in higher vertebrates, testosterone is implicated in spermatogenetic step regulation (meiosis and spermiogenesis) in lizard testis. In the cytosol, testosterone-binding molecules (Kd: 10(-9) mol l-1; 384-784 fmol g-1 tissue) with several properties of androgen-binding proteins are present from autumn to spring. The behaviour of these molecules is consistent with the role assigned to androgen-binding proteins as androgen reservoir. A putative oestrogen receptor is present throughout the sexual cycle, except during the culmination phase (breeding). The putative oestrogen receptor may be involved in the regulation of the first spermatogenetic step (spermatogonia multiplication) and in the induction of post-reproductive refractoriness. This phase is present in temperate-zone lizards. These studies show that the evaluation of sex-steroid-binding molecules is useful in considering the relationships between sex hormones and spermatogenetic activity in the testes of lizards.

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat.

Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)2D3 receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [3H]-1,25(OH)2D3 is noted in both the logarithmic growth phase and at confluence (Nmax 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. KD was 10(-11) M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)2D3 receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest Nmax in the proliferating cartilage. 1,25(OH)2D3 in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10(-12) M and dose-dependent inhibition at 10(-10) M and below. Inhibition was specific and not seen with 24,25(OH)2D3 or dexamethasone. Growth phase-dependent 1,25(OH)2D3 receptor expression and effects of 1,25(OH)2D3 on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.

Effect of vitamin D on growth cartilage cell proliferation in vitro.

Disturbed calcification of the growth plate and stunting is a frequent finding in vitamin D-deficiency rickets, vitamin D-dependency rickets and renal osteodystrophy, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of Ic, 25-dihydroxy vitamin D3 [1,25(OH)2D3] receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In tibial growth plates and in primary cultures of tibial growth cartilage of male Sprague-Dawley rats (80 g) specific binding of [3H]-1,25(OH)2D3 was noted. Scatchard analysis revealed the presence of a single class of non-interacting binding sites. Kd was 10(-11) M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA-cellulose affinity chromatography. By immunohistology, growth cartilage cells (rabbit tibia) were shown to express nuclear 1,25(OH)2D3 receptors, most prominently in the proliferative and early hypertrophic zone. This corresponds to binding data which showed highest binding of 1,25(OH)2D3 in the logarithmic growth phase (12,780 molecules/cell versus 4,538 molecules/cell in confluent cells) in primary cultures of growth plate chondrocytes. In the presence of delipidated fetal calf serum 1,25(OH)2D3 had a biphasic effect on cell proliferation and density, i.e. stimulation at 10(-12) M and dose-dependent inhibition at 10(-10) M and below. Inhibition was specific and not seen with 24 (R), 25-dihydroxy-vitamin D3 or dexamethasone. Growth phase-dependent 1,25(OH)2D3 receptor expression and specific effects of 1,25(OH)2D3 on chondrocyte proliferation in vitro point to a role for vitamin D in the homeostasis of growth cartilage of the rat.

Defining nucleic acid-binding properties of avian retrovirus integrase by deletion analysis

Integration of retroviral DNA into the host genome requires the activity of retrovirus-encoded integration protein IN. We expressed Rous sarcoma virus (RSV) IN, 286 amino acid residues in length, by using in vitro transcription, followed by in vitro translation in rabbit reticulocyte lysate. The nucleic acid-binding activity of in vitro-translated IN was assessed by using DNA-cellulose affinity chromatography and poly(U)-Sepharose affinity chromatography and by sedimentation analysis in the presence or absence of DNA. In vitro-translated RSV IN exhibited nucleic acid-binding activity similar to that of IN purified from avian myeloblastosis virus. To identify regions of IN which bind to nucleic acids, several deletions of RSV IN were generated. The NH2-terminal 26 amino acids, including the two His residues of a His-Cys box, were not necessary for IN nucleic acid binding with any of the substrates tested. The substrates included native calf thymus DNA, poly(U), and a double-stranded linear DNA molecule with RSV long terminal repeat sequences at its termini. The COOH-terminal region (residues 178 to 286) of IN bound quantitatively (greater than 90%) to poly(U) and to single-stranded circular phi X174 DNA but did not exhibit the double-stranded linear DNA-binding ability of the entire IN molecule.

Synthesis of a 60 kD nuclear DNA binding protein induced by cytosine arabinoside in the HL 60 leukemic cell line

Cytosine arabinoside (ara-C) is being employed at low dosage as differentiative rather than a cytotoxic agent in the therapy of leukemias. We have analyzed nuclear proteins from HL 60 leukemic cells treated with ara-C and have observed increased expression of a 60 kD protein in a dose-dependent fashion. This protein is actively synthesized, as assessed by labeled methionine incorporation. Using DNA cellulose affinity chromatography we could also demonstrate DNA binding properties of the 60 kD protein.

Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. I. Purification and some properties of AP DNA endonucleases in rat liver chromatin

Three kinds of apurinic/apyrimidinic (AP) DNA endonuclease, APcI, APcII, APcIII, were purified from rat liver chromatin through 1M KCl extraction, DEAE-trisacryl ion exchange chromatography. Sephadex G-150 gel filtration and AP DNA cellulose affinity chromatography. Activities of the purified APcI, APcII and APcIII were 62.5, 83.3 and 52.0 EU/mg of protein, respectively. Molecular weights of APcI, APcII and APcIII, each consisting of a single polypeptide, were 30,000, 42,000 and 13,000, and isoelectric points of them were 7.2, 6.3 and 6.2, respectively. Three enzymes showed different substrate specificities; APcI acted only on AP DNA, and APcII acted on both AP DNA and UV DNA, while APcIII acted on 3'-methyl-4-monomethylaminoazobenzene (3'-Me MAB) DNA adduct as well as AP DNA and UV DNA. These results indicate that three kinds of AP DNA endonuclease present in rat liver chromatin have structural and functional diversities.

Heterogeneity and properties of transformation of corticosteroid receptors in spinal cord and hippocampus

The central nervous system contains two classes of corticoid receptors, named types I and II following terminology accepted for the kidney. Phenotypically, type I sites are differentiated into a corticosterone (CORT)-preferring species (Ia) and a mineralocorticoid receptor (Ib). These populations were tentatively compared in the spinal cord and hippocampus. Using [ 3H]dexamethasone (DEX) and selective blockage of sites, we have observed that type II receptors were comparable in both tissues, while Ia was almost exclusive of the hippocampus. Saturation analysis using [ 3H]DEX demonstrated that type Ia was a low affinity receptor ( K d ≈ 2–5 nM while type II was a higher affinity site ( K dII < K dI). Using [ 3H]CORT, or [ 3H]aldosterone (ALDO), as ligand, preferential labeling of type I sites was achieved, always showing higher concentrations in the hippocampus. Therefore, [ 3H]DEX seems a ligand of choice to visualize types Ia and II receptors. Another difference noted between the spinal cord and hippocampus, pertained to the sensitivity towards the enzyme RNAse A, which increases heat-induced transformation of the bound receptor, according to the results of DNA cellulose affinity chromatography. In these experiments, type I sites of both spinal cord and hippocampus, plus type II of hippocampus, showed sensitivity towards the enzyme, whereas type II of the spinal cord was refractory to RNAse A enhancement of transformation. These results indicate that the dynamics of transformation is different among receptors showing similar affinity and competition, suggesting further heterogeneity due to receptors themselves, or to tissue factors regulating their biochemical properties.

EVIDENCE THAT 1,25 DIHYDROXYVITAMIN D3, IS BIOACTIVE IN THE FETAL SHEEP INTESTINE

Intestinal calcium binding protein (iCaBP) is the best known molecular expression of the hormone 1,25 dihydroxyvitamin D (1,25) and its synthesis in response to 1,25 requires the presence of specific 1,25 receptor macromolecules (1.25R). In rats, the developmental appearance of the 1.25R, iCaBP and its responsiveness to 1,25 occur around weaning. To test the hypothesis that in the fetal sheep, where there is evidence for a role of fetal 1,25 in mineral metabolism, there would be prenatal appearance of intestinal 1,25R and iCaBP, we isolated cytosolic intestinal 1,25R from samples of fetal (F:n=8) and maternal (M:n=5) small intestinal mucosa at 138d of gestation (term=145d). 1,25R were characterized by sedimentation coefficient (Sed), Scatchard analysis of saturation binding data and DNA-cellulose affinity chromatography (DNAcell). Gel filtration of F and M intestinal mucosal cytosols revealed a high affinity calcium binding protein in the 12,000 molecular weight region that demonstrated decreased anionic charge when bound to calcium (Ca) with the following elution profile on DEAE-A25 chromatography: (-Ca:elution at 0.2M NaCl / +Ca:elution in void volume). The purified apoprotein exhibited an acidic PI of 5.2 on isoelectric focusing. Conclusion: These data suggest that 1,25 is biologically active in the ovine fetal intestine.

Detection of human cytomegalovirus-induced DNA-binding proteins on Western blots

DNA-binding proteins have been isolated from human embryonic fibroblast cells infected with human cytomegalovirus strain AD169 by native and denatured DNA-cellulose affinity chromatography and Polymin P precipitation. The DNA-binding proteins separated by polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose sheet were identified by a modified ELISA reaction. Six DNA-binding proteins were found in infected cells, their molecular weights range from 52K to 18K.

Purification and subunit structure of recBC DNase from Escherichia coli harboring a recB and recC genes-inserted plasmid.

recBC DNase of Escherichia coli has been purified from the transformant, HB101/pFS11-04 (recB+ recC+), by successive ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, hydroxyapatite chromatography, DNA cellulose affinity chromatography, and second DEAE-cellulose chromatography. The purified enzyme was obtained in an overall yield of 3%. The enzyme protein appeared as a single pure component on native polyacrylamide gel electrophoresis. The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The results show that recBC DNase consists of two nonidentical subunits with molecular weights of 125,000 and 135,000, and isoelectric points of 5.6 and 5.7, respectively.

Characterization of estrogen receptors in the hamster brain

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol ( K d = 10 −9 M), limited capacity (30–50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7–8S region of a 5–20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.

Androgen and estrogen receptors in perinatal ferret brain

Using DNA-cellulose affinity chromatography and either 3H-labeled dihydrotestosterone, testosterone, or estradiol, we qualitatively and quantitatively analyzed the androgen- and estrogen-binding activities present in four regions of male and female ferret brain at prenatal, early and late neonatal, and adult ages. The cytosolic androgen- and estrogen-binding activities in ferret brain at all ages studied were qualitatively similar in both sexes and in all brain regions and exhibited characteristics which resemble those of androgen and estrogen receptors from other species, including rodents and nonhuman primates. A developmental analysis indicated that high levels of both androgen and estrogen receptors were present in the hypothalamus-preoptic area as early as 5 days before birth. A significant, transient decline in concentrations of estrogen receptors (approximately 5-fold) occurred in anterior hypothalamus-preoptic area and mediobasal hypothalamus at 12 days of age in both males and females; this phenomenon has not been observed in any other species studied to date. The observed ontogeny of androgen receptors correlates with the known ability of testosterone, acting over postnatal days 5 to 20, to cause coital masculinization in ferrets, whereas the observed postnatal dip in estradiol receptor concentrations correlates with the inability of estradiol to cause coital masculinization or defeminization of receptive behavior in this species.

Isolation and characterization of a novel nucleic acid binding protein from calf lenses

The transitional process of lens cellular differentiation is accompanied by several unique morphological and biochemical changes. Pyknosis or apoptosis of the nucleus involves extensive degradation of genetic materials. In an attempt to search for a gene product responsible for such a regulatory process, we have adopted DNA-cellulose affinity chromatography to enrich the specific binding protein. A binding protein was isolated by high salt (0.8M KCl) wash of the lens polysomal fraction and purified to apparent homogeneity by DNA-cellulose affinity column and chromatofocusing. The nucleic acid binding protein has an apparent molecular weight of 36,000, designated as regulatory factor 36 (RF-36), as determined by SDS/PAGE. Amino acid composition analysis indicated that RF-36 contains high proportions of glycine, alanine, characteristic of the core heteronucleus RNP proteins. Comparative immunological studies with other DNA binding protein antigen (e.g. helix destabilizing protein) suggest the existence of some common overlapping determinant. However, when monoclonal anti-RF-36 was used as immunoprobe, no cross immunoactivity was detected between these homologous binding proteins, suggesting some antigenic diversity among these two nucleic acid binding proteins from different organisms.