PMC:1920263 / 18603-20663 JSONTXT

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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1920263","sourcedb":"PMC","sourceid":"1920263","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1920263","text":"Characterization of the transcriptional start sites of APOBEC3G by 5′-RACE\nTo identify the transcriptional start sites (TSS) of APOBEC3G (A3G) in A3.01 T cells, we performed 5′-rapid amplification of cDNA ends analysis (RACE) with A3G-specific primers (see Figure 1). Agarose gel electrophoresis resolved the nested PCR products into three bands of different electrophoretic mobility with a dominant middle band (Figure 2). For each band, the DNA was cloned and sequence analysis of six or seven individual transformants was performed. We observed that the transcriptional start sites of the A3G gene were located between 58 and 361 nt upstream of the ATG start codon (Figure 1). Although TSS were variable and most sites were only detected once among the 19 clones analyzed, one TSS was identified in six individual clones. This TSS was located 66 nt upstream of the start of the published A3G mRNA sequence (GenBank™ accession number NM021822) and we defined this position as the major transcriptional start site of the A3G gene.\nFigure 1. Sequence of the A3G promoter and the downstream region. The first 1000 bp upstream of the major TSS are shown in lower case, the first 800 bp of the transcribed sequence are shown in upper case. Introns are removed, but their positions are indicated. Arrows refer to transcriptional start sites and their observed frequency is given by the numbers above. The primer binding sites for 5′-RACE analysis and cloning of the luciferase reporter constructs are underlined and the names of the primers are annotated. Gray and black arrowheads define the regions designated E1 and E2 which were cloned into vector pGL3-Promoter and used as EMSA probes. The ATG start codon and the identified Sp1/Sp3 transcription factor binding site are shown in bold.\nFigure 2. 5′-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5′-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.","divisions":[{"label":"Title","span":{"begin":0,"end":74}},{"label":"Figure 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