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. 2005 Sep 15;19(18):2122–2137. doi: 10.1101/gad.1339905

Figure 6.

Figure 6.

The Drosophila p53 gene contains an internal promoter. (A) GeneRacer PCR on Drosophila p53 mRNA. Schema of the Drosophila p53 gene: (black) noncoding sequence; (white) coding sequence. Total RNA extract from Drosophila embryos was treated according to the manufacturer's protocol to allow amplification of capped mRNA. The reverse transcription was performed by using primer DRE8. The first PCR was performed using the GeneRacer primer-1 and the Drosophila p53-specific primer DRE51. One microliter of the first PCR was used to perform a nested PCR using the GeneRacer primer-2 and the p53-specific primer DRE52. The product of the second PCR was analyzed on a 1% agarose gel, and the DNA was stained with ethidium bromide. After cloning and sequencing, the bands (1) contained sequences from two additional exons (exon A and B) upstream of the previously described exon 1 of the Drosophila p53 gene. The upper band contained a splice variant. Band 2 contains sequences from the previously described exon 1 of Drosophila p53. (★) Nonspecific bands. (B) The Drosophila p53 gene contains an internal promoter. The reverse transcription was performed using primer DRE8 on total Drosophila embryo RNA extracts. A first PCR was performed using primers derived from exon A (DFA) or from exon 1 (DF1) and the reverse primer DRE51 followed by a semi-nested PCR using primer DFA or DF1 with primer DRE52. The arrows indicate the PCR fragments corresponding to Drosophila p53. Fragment dp53n corresponds to the Drosophila p53 mRNA not spliced at intron B and initiated at the first site (1) of initiation of transcription. Fragment dp53L contains the Drosophila p53 mRNA initiated at the first site (1) of initiation of transcription with splicing of intron B. Fragment dp53 contains the Drosophila p53 mRNA initiated at the second site of transcription initiation. (C) Schema of the Drosophila p53 protein isoforms. The Drosophila p53 gene can encode, theoretically, three different protein isoforms. The splice form dp53n may encode a truncated form of Drosophila p53 containing only the N-terminal end (123 amino acids). The form dp53L may encode a Drosophila p53 of 495 amino acids containing a large N-terminal domain. The form dp53 encodes the published Drosophila p53 protein. (D) In vitro translation of dp53L. The dp53L cDNA was fused to a Flag-tag and cloned in a T7-driven promoter expression vector. dp53L protein was produced by in vitro translation using a reticulocytes lysates kit and revealed by Western blot using anti-Flag antibody.