FIG. 2.

(A) In the upper part of the panel, the three transcripts starting from the P2 promoter, the PCR primers, and the 147-bp and the 255-bp (*) PCR products are represented; black boxes indicate exons common to the three transcripts; grey boxes indicate exons common only to aspartyl-β-hydroxylase and junctate. Oligo(dT) RT-PCR was performed with total RNA isolated from human adult normal tissues or cell lines with the e2F/e3R (lanes a to g) or the e2F1/e5R (lanes h and i) primers and analyzed with agarose gel electrophoresis (lower part of panel). Lanes: a, kidney; b, brain; c, adrenal gland; d, liver; e, heart; f, skeletal muscle; g, RD cell line; h, C2C12-GM cells (C2C12 cells cultured in growth medium in the presence of 10% fetal calf serum); i, C2C12-DM cells (C2C12 cells cultured in differentiation medium in the presence of 2% horse serum and 10 μg of insulin/ml); M, pUC Mix Marker 8 (Fermentas). (B) Analysis of 5′ RACE products of AβH-J-J exon 2 starting transcripts. cDNAs were synthesized from RD cells (lane m) or human adult skeletal muscle (lane n) total RNA by using the e5R primer. After addition of a poly(G) tail, PCR was performed with the gene-specific e3R primer. One-fifth of the nested PCRs, after a procedure performed with a gene-specific e2R primer, was analyzed by gel electrophoresis. Lanes j to l, control reactions from RD cell RNA performed in the absence of reverse transcriptase, terminal deoxynucleotidyl transferase, and both, respectively. (C) Nucleotide sequence of RACE products, obtained by direct sequencing or cloning. The P2 transcription initiation site (+1), the primers used for the last PCR, and the translation start site (ATG) are indicated.