Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-6

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>position 40295005–40295504), rat (chromosome 10 position 59889637–59890136) and mouse (chromosome 11 position 72611373–72611872) was obtained by BLAST searching of genomic databases via the ENSEMBL genome browser . The 500 bp upstream of the ATG start codon of each gene was aligned using CLUSTAL. Alignment equivalent to -68/+149 of the human core promoter is shown. Sequence identity to the human P2Xsequence over this region was 99.1%, 83.4%, 76.8%, and 75.9% for chimp, dog, mouse and rat respectively. "." indicates nucleotides identical to the human P2Xsequence. Dashes indicate positions where gaps have been inserted in order to maintain the alignment. The transcription start site of the human P2Xgene determined in this study is indicated as +1. Potential transcription start sites predicted by PROSCAN [17] are indicated as arrows. In each species, potential NF-1, Sp1a and Sp1b binding sites (indicated by boxes) are present in equivalent positions to the human P2Xcore promoter

Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-2

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>nces used for primer extension analysis (Pet1-4) are indicated by horizontal arrows. End points of plasmid deletion constructs in this region are indicated by "p" and number above the sequence. Sequence numbering is relative to the transcription start site indicated as +1. The translation initiation ATG is boxed. The EMBL/Genbank accession number of this sequence is

Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-3

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>and unlabelled mutated O-NF1-mut (lane 5). Complexes supershifted by pre-incubation with an NF-1 antibody (NF-1Ab, lane 2) are marked "SS". "NS" indicates non specific binding complexes, the level of which varied between individual nuclear extractions (lanes 1 and 3). "F" indicates free probe. B) Supershift assays using Sp1 (lanes 2 and 5) and Sp3 (lanes 3 and 6) specific antibodies. C) Competition experiments in the absence (lanes 1 and 5) or presence of excess of unlabelled competitors: Lanes 2 and 6, an excess of unlabelled O-Sp1a or O-Sp1b probe respectively. Lanes 3 and 7, an excess of unlabelled mutated oligonucleotide O-Sp1a-mut and O-Sp1b-mut respectively. Lanes 4 and 8, an excess of a consensus Sp1 oligonucleotide (Promega, E323A)

Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-4

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>F-1 and Sp1 binding sites of the proximal promoter region were assayed for promoter activity in PMA-treated MEG-01 cells (sequences of mutations are shown in Table 2). Shaded symbols represent mutated binding sites. Values are presented as means ± S.E of at least three independent experiments and are normalized against the wild type p-190/+149 construct for panels A to C and the wild type construct p-4407/+365 in panel D. A) Effects of mutations in the construct p-190/+149. B) Effect of mutation after removal of the potential NF-κB binding site. C) Effect of mutation after removal of the transcription start site that is utilized by smooth muscle cells [15]. Dotted vertical arrow (TSS) represents the transcription start site determined in this study. D) The Sp1a, Sp1b and NF-1 sites were mutated in the original full length 4.7 kb reporter construct p-4407/+365 to generate the triple mutated construct p-4407/+365(mut4). Promoter activities were determined in MEG-01 cells either treated (+) or untreated (-) with PMA

Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-1

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>atment of cells with 10 nM PMA. Data are means of absolute firefly luciferase activity of three independent transfections. B) Luciferase activity of deletion constructs in PMA treated MEG-01 cells. TSS, transcription start site. PT depicts a polypyrimidine tract located from -1801 to -1405 bp. Values are presented as means of at least three experiments and normalized against the construct p-4407/+365. Names of plasmid constructs correspond to the 5' and 3' ends of the insert relative to the transcription start site e.g. construct p-4407/+365 extends from 4407 bp 5' to 365 bp 3' of the transcription start site

Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-0

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>led to poly(A) RNA from PMA-treated MEG-01 cells and extended with reverse transcriptase. Extension products for Pet1, Pet3 and Pet4 that equate to the same transcription start site are indicated with arrows. (M), molecular mass marker. B) Pet3 extension reaction (lane 6) run in parallel with a sequencing ladder. The location of primers is depicted in Figure 3

Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-5

<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>pitated with antibodies to Sp1 (lane 4), Sp3 (lane 5), NF-1 (lane 6), TFIIB (lane 7), or in the absence of antibody (lane 3). PCR was performed with specific primers for the , and promoter and for the coding region as a negative control. A sample representative of the total input chromatin (input DNA lane 1) was included in the PCR analysis. Lane 2 shows the supernatant of the 'unbound' sample. PCR products were between 282 and 983 bp in length. PCR cycle numbers were 31 for the , and promoter and 36 for the coding region. Primer sequences are given in Table 3