Southwestern Blot Analysis Research Articles

Single-strand-DNA-binding factors specifically recognize the pyrimidine element in the chick α2(I) collagen gene promoter

A pyrimidine element with mirror repeats centered at position -192 bp of the chick alpha2(I) collagen promoter interacts with sequence-specific DNA-binding factors. These factors bind to only the pyrimidine strand of this region and have no affinity for the complementary purine strand. Binding activity is also seen with the double-stranded form of this element, but with less affinity than to the single-stranded pyrimidine species. Southwestern blot analyses have shown that proteins of 80 and 134 kDa in chick embryo fibroblast nuclear-extracts bind to the pyrimidine strand, whereas only a 134 kDa DNA-binding protein was found in chondrocyte nuclear extracts. The binding mechanism of these nuclear proteins with single-stranded DNA might be based on a non-B-DNA conformation of the pyrimidine element. The position of this binding site in the promoter region, its potential for adopting an unusual secondary structure and the presence of the 80 kDa binding factor in chick embryo fibroblasts, but not in chondrocytes, suggest a possible role for this factor in the expression of the alpha2(I) collagen gene.

DNA-Binding Activities in Streptococcus gordonii : Identification of a Receptor-Nickase and a Histonelike Protein

Extraction of Streptococcus gordonii cells with the mild chaotropic agent, LiCl, drastically decreased DNA transforming ability, had little effect on viability, and released both DNA nicking and binding activities. Both activities were Mg2+ and Ca2+ independent and were not competence specific. Southwestern blot analysis of the extract identified putative surface proteins of 56 kDa and 68 kDa in strain Challis and Wicky, respectively. Extracts also contained a 10-kDa DNA-binding protein, designated HSgo, that belongs to the eubacterial histonelike class of proteins.

Host factor with single-stranded DNA-binding activity involved in transcription from baculovirus polyhedrin promoter

This chapter describes the preparation of insect cell nuclear extracts, analyses of protein binding by gel retardation assays, UV cross-linking of DNA-protein complex, and southwestern blot analysis; and elucidates the protocol followed for the determination of its binding affinity to the template DNA. The affinity column purification of polyhedron promoter-binding protein (PPBP) is also described. PPBP exhibits both dsDNA and single-stranded (ss) DNA-binding activities. Despite being a host factor, its ability to bind to transcriptionally important motifs and to regulate transcription of the Baculovirus very late polyhedrin promoter make it an interesting model for studying protein–protein interactions at the promoter. The "minimal" polyhedrin promoter has been demonstrated to be an 18-bp region (-42 to -59) surrounding the transcription start point, whereas the sequences encoding the untranslated mRNA leader (-1 to -49) are required for the very late burst of polyhedrin expression. Two factors that interact directly with the polyhedrin promoter or its neighboring sequences are also identified.

RHox: A homeobox gene expressed in osteoblastic cells

Homeodomain proteins are characterized by a conserved domain with a helix-turn-helix motif. These proteins act as regulatory factors in tissue differentiation and proliferation. However, their role in the regulation of osteoblast differentiation is unknown. In this study we have identified and characterized a homeobox gene in osteoblast-like cells. This gene, termed rHox, was isolated from a cDNA library derived from rat osteoblast-like cells. The nucleotide sequence of the 1,375 base pair (bp) cDNA contains a noncoding leader sequence of 329 bp, a 735 bp open reading frame, and 312 bp of 3' noncoding sequence. Sequence comparison demonstrates that rHox is identical to the mouse Pmx gene (also called MHox) at the amino acid level and 90% homologous at the nucleotide level. Both Southwestern blotting and gel shift analyses indicate that rHox has potential to bind both the collagen I alpha 1 and the osteocalcin promoters. Transfection experiments using an rHox expression vector showed a strong repression of target promoter activity, regardless of whether the target promoters contained homeodomain binding response elements. These data suggest that rHox is a potent negative regulator of gene expression, although the specific role of rHox in bone gene regulation remains to be determined.

Different Composite Regulatory Elements Direct Expression of the Human α Subunit Gene to Pituitary and Placenta

To identify elements of the human alpha subunit gene necessary for cell-specific expression, we generated an array of block mutations spanning approximately 400 base pairs (bp) of promoter proximal region and examined them using transient transfection analysis in pituitary (alpha T3) and placental (BeWo) cell lines. Comparison of promoter activity in the two cell types revealed both common and unique elements required for transcription in pituitary and placenta. Two strong elements, the cyclic AMP response element (CRE) and the upstream regulatory element (URE), regulate expression of the alpha subunit gene in BeWo cells. In contrast, promoter activity in alpha T3 cells requires an array of weaker elements. These include the CREs, the URE, as well as two previously described elements, pituitary glycoprotein hormone basal element (PGBE) and gonadotrope-specific element (GSE), and two new elements we designated as the alpha basal elements 1 and 2 (alpha BE1 and alpha BE2). These new elements reside between -316 and -302 bp (alpha BE1) and -296 and -285 bp (alpha BE2) of the human alpha subunit promoter and bind distinct proteins designated alpha BP1 and alpha BP2, respectively. Southwestern blot analysis revealed that alpha BE1 specifically binds 54- and 56-kDa proteins. Additional studies disclosed several potential interactions between proteins that bind the CRE and proteins that occupy PGBE, alpha BE1, and alpha BE2, suggesting that gonadotrope-specific expression occurs through a unique composite regulatory element that includes components of the placenta-specific enhancer.

Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin α-subunit gene

Our previous studies demonstrated that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (-151 to -135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (-223 to -190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA-protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.

A POU homeo domain protein related to dPOU-19/pdm-1 binds to the regulatory DNA necessary for vital expression of the Drosophila choline acetyltransferase gene

Expression of the choline acetyltransferase (ChAT) gene in Drosophila melanogaster is responsible for production of the neurotransmitter acetylcholine and is necessary for viability. In previous studies, we have shown that the regulatory region for normal ChAT expression is large and composed of multiple regulatory elements (Kitamoto et al., 1992; Kitamoto and Salvaterra, 1993). In this study, using various lengths of 5' flanking DNA fused to wild type ChAT cDNA, we have defined a 0.3 kilobase (kb) region of the cis-regulatory DNA, which is essential for restoring viability of Cha lethal mutants. DNase I footprinting analysis of this 0.3 kb DNA revealed a protected 22 bp sequence that contains an octamer-like motif (ATTCAAAT) with one base difference from the consensus octamer motif (ATGCAAAT). Electrophoretic mobility shift assays and Southwestern blot analysis confirmed the presence of specific binding factor(s) for the 22 bp sequence in embryo nuclear extracts, and competition studies established the importance of the octamer-like motif for high-affinity binding. Using the 22-mer as a probe, we have isolated a cDNA clone encoding the Drosophila POU homeo domain protein, dPOU-19/pdm-1, whose target genes and specific binding sequences have not been identified. We propose that vital expression of the Drosophila ChAT gene is regulated by a member of the dPOU-19/pdm-1 putative transcription factor family.

A Single HMG Domain in High-Mobility Group 1 Protein Binds to DNAs as Small as 20 Base Pairs Containing the Major Cisplatin Adduct

Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.

Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.

Transcriptional regulation by growth hormone (GH) represents the culmination of signal transduction pathways that are initiated by the cell surface GH receptor and are targeted to the nucleus. Recent studies have demonstrated that the activated GH receptor can stimulate Stat1, a cytoplasmic transcription factor that becomes tyrosine phosphorylated and translocates to the nucleus, where it can interact with specific DNA sequences to modulate gene expression. GH also has been found to induce protein binding to a portion of the rat serine protease inhibitor (Spi) 2.1 gene promoter that is required for GH-induced transcription of Spi 2.1. Using GH-deficient hypophysectomized rats as a model, we show that GH treatment rapidly and potently induces both nuclear Spi 2.1 mRNA expression in the liver and specific nuclear protein binding to a 45-bp segment of the Spi 2.1 gene promoter. A GH-inducible gel-shifted complex appears within 15 min of systemic hormone administration and can be inhibited by an antiphosphotyrosine monoclonal antibody but is not blocked by a polyclonal antiserum to Stat1, Stat3, or Stat4, even though the nucleotide sequence contains two gamma interferon-activated sequence-like elements that could interact with STAT proteins. By Southwestern (DNA-protein) blot analysis, approximately 41- and 35-kDa GH-inducible proteins were detected in hepatic nuclear extracts with the Spi 2.1 DNA probe. Thus, a GH-activated signaling pathway stimulates Spi 2.1 gene expression through a unique mechanism that does not appear to involve known members of the STAT family of transcription factors.

Promoter regulation of a differentially expressed gene in the human colonic epithelial cell lines HT29-18 and HT29-18-C 1

Gene A4 is transcriptionally activated upon enterocyte differentiation of the human colonic epithelial cell line HT29-18 and its highly differentiated subclone HT29-18-C 1 [Oliva et al., Arch. Biochem. Biophys. 302 (1993) 183–192]. To characterize the mechanisms regulating the differential transcription of A4, we analyzed its immediate 5′-flanking region for regulatory elements. Promoter-linked transfection experiments of progressively deleted A4 5′-flanking sequences fused to the bacterial cat reporter gene suggest the presence of one negative and two positive DNA elements within the first 371 bp of the A4 promoter ( pA4). DNase I footprint and electrophoretic mobility shift assays demonstrate that one positive element which contains the core binding sequence for the transcription factor, Spl, mediates an equal level of transcription in the two cell types. The second positive element, localized between nucleotide positions −169 and −152, contains a sequence previously unrecognized as a transcription factor-binding site. This element mediates a twofold increase in the activity of pA4 in HT29-18-C 1, as compared to HT29-18. Furthermore, nuclear extracts obtained from HT29-18-C 1 contain a higher binding activity for this element than those from HT29-18. Southwestern blot analysis suggests that the protein interacting with this element has an estimated molecular mass of 50 kDa. We conclude that this protein may be involved in the differential regulation of A4 in these intestinal cell lines.

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter.

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.

Association of USF and c-Myc with a helix-loop-helix-consensus motif in the core promoter of the murine type II beta regulatory subunit gene of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase.

Previous studies showed that the core promoter of the mouse cAMP-dependent protein kinase regulatory subunit type II beta (RII beta) gene was composed of two functional elements. One element was GC rich and bound the Sp1 transcription factor. The second element contained a helix-loop-helix (HLH)-motif. Each element conferred transcriptional activity when inserted upstream of a reporter gene, chloramphenicol acetyltransferase and transfected into mouse NB2a neuroblastoma cells and Chinese hamster ovary (CHO) cells. The core promoter was further characterized by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factors and reduced the transcriptional activity of constructs containing the chloramphenicol acetyltransferase reporter when transfected into these cells. The results showed that the central nucleotides as well as the adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and interference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription factors were forming complexes with the HLH-consensus motif. The results suggest that RII beta transcription may be regulated, in part, by USF and c-Myc in NB2a and CHO cells.

Structural and functional organization of herpes simplex virus DNA polymerase investigated by limited proteolysis.

The 1235 residue herpes simplex virus DNA polymerase is a prototype alpha-like DNA polymerase and also an antiviral drug target. To investigate its organization, we mapped favored cleavage sites for seven proteases and identified three major classes of stable proteolytic fragments: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipitation experiments, the first two classes of fragments remained associated; thus, cleavage in the center of the protein did not resolve structurally separate domains. In contrast, the 12-kDa C-terminal fragments did not remain associated with other fragments, suggesting a small separable C-terminal domain. The 70-85-kDa N-terminal fragments contained 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destroy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. The C-terminal fragments bound the viral polymerase processivity factor, UL42. The results map activities to regions of herpes simplex virus polymerase and suggest a model for its organization that may be pertinent to other DNA polymerases.

Induction of differentiation in the cultured F9 teratocarcinoma stem cells by triterpene acids.

The effects of the triterpene acids, ursolic acid and oleanolic acid, on the differentiation of F9 teratocarcinoma stem cells were studied. These agents caused the morphological change of F9 cells into endoderm cells, as did retinoic acid (RA). Moreover, expression of laminin B1, type IV collagen and retinoic acid receptor beta (RAR beta) increased in ursolic- and oleanolic-acid treated F9 cells. Since these agents are structurally similar to the glucocorticoid hormone, we studied the effects of dexamethasone, a synthetic glucocorticoid, on F9 cells. Dexamethasone also induced the morphological change and altered the expression of laminin B1, type IV collagen, and RAR beta in F9 cells. In addition, transcription of glucocorticoid receptor was detected after treatment with these three agents. According to Southwestern blot analysis, a 94-kDa protein, thought to be a glucocorticoid receptor, was detected in F9 cells treated with these agents. In a gel-shift assay, we identified protein factors binding to the glucocorticoid-responsive element (GRE) in the nuclear proteins from F9 cells treated with ursolic or oleanolic acid. The binding activity of the GRE-binding protein disappeared on the addition of unlabeled GRE oligonucleotide. Taken together, these results suggest that UA and OA can induce the differentiation of F9 cells and may regulate the expression of differentiation-specific genes, probably by forming a complex with the glucocorticoid receptor or its analogous nuclear receptor.

Post-translational and transcriptional regulation of polyamine biosynthesis in Escherichia coli

1. 1. Ornithine and arginine decarboxylases (ODC and ADC) of Escherichia coli are inhibited post-translationally by antizyme and ribosomal proteins S20 and L34. 2. 2. The inhibition of either enzyme is relieved when excess of the other decarboxylase is added. 3. 3. Using this approach, in vitro as well as in vivo, we demonstrate that the extent of the post-translational inhibition of ODC and ADC in E. coli is at least 65 and 50%, respectively. 4. 4. The inhibited enzyme levels increase even further upon exposure of cells to polyamines. 5. 5. The post-translational mode of regulation can counteract a 4-fold increase of ODC protein in the cell. 6. 6. The negative transcriptional regulation of ODC and ADC expression by polyamines is mediated by transcription factors and not by direct polyamine effects on the promoters of their genes. 7. 7. Three proteins interacting with the ODC promoter region were found by southwestern blot analysis.

Identification and characterization of the human transforming growth factor-alpha initiator.

Eukaryotic transcription requires promoter proximal elements. For class II promoters, two such elements are the TATA box and the initiator. The promoter for the human transforming growth factor-alpha (TGF alpha) gene has been shown to lack a TATA box, yet initiate transcription at a unique site. We have identified an approximately 13-basepair sequence between -5 and +8 as a new promoter element. We call this element the TGF alpha initiator based on the following observations: 1) it is located at the transcription initiation site; 2) the promoter activity is largely reduced by either deletion or mutation of the element; and 3) mutations result in initiation upstream of the authentic start site; the TGF alpha initiator directs site-specific initiation. An electrophoretic mobility shift assay demonstrated that a protein(s) in nuclear extracts forms complexes with the TGF alpha initiator. This interaction is sequence specific and depends on nucleotides that are critical for the promoter activity in vivo. Two polypeptides, 105 and 95 kilodaltons, were detected by Southwestern blot analysis on the basis that they specifically interact with the TGF alpha initiator. The larger polypeptide, TIBP-1, was subsequently purified by a matrix-immobilized TGF alpha initiator sequence and was shown to possess the TGF alpha initiator-specific mobility shift activity and an ability to interact with the initiator when immobilized on a membrane. In summary, we identified and characterized the TGF alpha initiator, a proximal element that is important for the accurate transcription of the TGF alpha gene, and the 105-kilodalton protein that interacts with this element.(ABSTRACT TRUNCATED AT 250 WORDS)

A 43-kDa nuclear tobacco protein interacts with a specific single-stranded DNA sequence from the 5'-upstream region of the Agrobacterium rhizogenes rolC gene.

We identified in the nuclear extract of tobacco seedlings a 43-kDa DNA-binding protein RCS2 that interacted with the 5'-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid. DNA-protein gel shift and competition assays demonstrated that RCS2 bound to single-stranded DNA in a sequence-specific manner. A five-base direct repeat is important for the DNA binding of RCS2. South-Western blot analysis was employed to determine the size of RCS2 which appears to be approx. 43 kDa.

Multiple silencer elements are involved in regulating the chicken vimentin gene.

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

A novel cDNA, priBc, encoding a protein with a Zn(II)2Cys6 zinc cluster DNA-binding motif, derived from the basidiomycete Lentinus edodes

A cDNA clone (designated priBc) was isolated from a primordial cDNA library of the basidiomycete, Lentinus edodes ( Le). The priBc clone consisted of 2628 bp encoding 565 amino acids. As was expected, the priB transcript was abundant in primordia, while preprimordial mycelia and mature fruiting bodies contained lower levels of this Le transcript. The deduced PRIB protein (64kDa) contained a ‘Zn(II)2Cys6 zinc cluster’ DNA-binding motif. PRIB was produced in Escherichia coli using the bacteriophage T7 expression system. Southwestern blot analysis revealed that PRIB binds to the DNA fragment containing the upstream region of priB.