Abstract
Using a synthetic peptide that encompasses the zinc finger domain of the eukaryotic transcription factor Sp1, we produced a number of monoclonal antibodies that specifically reacted with the target antigen. Analysis by competitive inhibition assay of five of the monoclonal antibodies revealed that they all recognized a dominant epitope in the synthetic peptide and reacted strongly to recombinantly synthesized β-galactosidase-Sp1 fusion polypeptide. To determine cellular distribution of Sp1-like molecules, cytoplasmic and nuclear proteins from human lung fibroblasts (HFL-1) and a human rhabdomyosarcoma cell line (A204) were immunoblotted and reacted with our antibodies. In addition to the well characterized 95 Kd and 105 Kd proteins, considered to be the authentic Sp1 polypeptide, a number of other cellular proteins reacted with these antibodies. Immunofluorescence staining of the cells with mAb to the zinc finger of Sp1 also revealed cell-specific differences in intracellular distribution of Sp1-like molecules. Both cytoplasmic and nuclear staining was readily observed in the rhabdomyosarcoma cells. In contrast, while some HFL-1 cells exhibited staining of only cytoplasm, both cytoplasmic and nuclear immunofluorescence was seen in others.
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Maniatis T, Goodbourn S, FischerJA: Regulation of inducible and tissue-specific gene expression. Science 236: 1237–1245, 1987
Ptashne M: How eukaryotic transcriptional activators work? Nature 335: 683–689, 1988
Jones NC, Rigby PWJ, Ziff EB: Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev 2: 267–281, 1988
Jones KA, Yamamoto KR, Tjian R: Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42: 559–572, 1985
Lee W, Haslinger A, Karin M, Tjian R: Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV-40. Nature 325: 368–372, 1987
Dynan WS, Sazer S, Tjian R, Schimke RT: Transcription factor Sp1 recognizes a DNA sequence in the mouse dihydrofolate reductase promoter. Nature319: 246–248, 1986
Ishii S, Kadonaga JT, Tjian R, Brady JN, Merlino GT, Pastan I: Binding of the Sp1 transcription factor by the human Harvey ras 1 proto-oncogene promoter. Science 232: 1410–1413, 1986
Dynan WS, Tjian R: Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell 32: 669–680, 1983a
Dynan WS, Tjian R: The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV-40 early promoter. Cell 35: 79–87, 1983b
Gidoni D, Dynan WS, Tjian R: Multiple specific contacts between a mammalian transcription factor and its cognate promoters. Nature 312: 409–413, 1984
Gidoni D, Kadonaga JT, Barrera-Saldana H, Takahashi K, Chambon P, Tjian R: Bidirectional SV-40 transcription mediated by tandem Sp1 binding interactions. Science 230: 511–517, 1985
Kadonaga JT, Tjian R: Affinity purification of sequencespecific DNA binding proteins. Proc Natl Acad Sci USA 83: 5889–5893, 1986
Briggs MR, Kadonaga JT, Bell SP, Tjian R: Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science 234: 47–52, 1986
Kadonaga JT, Carner KR, Masiarz FR, Tjian R: Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51: 1079–1090, 1987
Klug A, Rhodes D: ‘Zinc fingers’: a novel protein motif for nucleic acid recognition. Trends Biochem 12: 464–469, 1987
Courey AJ, Tjian R: Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutaminerich activation motif. Cell 55: 887–898, 1988
Courey AJ, Holtzman DA, Jackson SP, Tjian R: Synergistic activation by the glutamine-rich domain of human transcriptional factor Sp1. Cell 59: 827–836, 1989
Hasty DL, Beachey EH, Simpson WA, Dale JB: Hybridoma antibodies against protective and nonprotective antigenic determinants of a structurally defined polypeptide fragment of streptococcal M protein. J Exp Med 155: 1010–1018, 1982
Hasty DL, Mainardi CL: Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectins. Biochim Biophys Acta 709: 318–324, 1982
Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497, 1975
Dignam JP, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase If in a soluble extract from isolated mammalian nuclei. Nucl Acids Res 11: 1475–1489, 1983
Laemmli UK: Cleavage of the structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970
Johnson GD, Araujo GMCN: A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Method 43: 349–350, 1981
Mierendorf RC, Percy C, Young RA: Gene isolation by screening kgtll libraries with antibodies. Methods Enzymol 152: 458–469, 1987
Jackson SP, Tjian R:O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell 55: 125–133, 1988
Lewis CD, Clark SP, Felsenfeld G, Gould H: An erythrocyte-specific protein that binds to the poly(dG) region of the chicken β-globin gene promoter. Genes Dev 2: 863–873, 1988
Bodner M, Castrillo J, Theill LE, Deerinick T, Ellisman M, Karin M: The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 55: 505–518, 1988
Bos TJ, Bohmann D, Tsuchie H, Tjian R, Vogt PK: V-jun encodes a nuclear protein with enhancer binding properties of AP-1. Cell 52: 705–712, 1988
Baeuerle PA, Baltimore D: Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NFkB transcription factor. Cell 53: 211–217, 1988
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Katai, H., Terato, K. & Raghow, R. Generation of monoclonal antibodies to the zinc finger domain of the eukaryotic transcription factor Sp1. Mol Cell Biochem 101, 73–81 (1991). https://doi.org/10.1007/BF00238440
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DOI: https://doi.org/10.1007/BF00238440