Multiple origins of transcription for the human placental lactogen genes

Transcriptional activity of the three human placental lactogen (hPL) genes was compared in vitro and their relation to the hPL RNA species obtained from term placenta was analyzed. We used in vitro transcription system containing the HeLa cell crude extract as the source for RNA polymerase II and initiation factors. Gene fragments of identical length of 0.96 kilobase pair made from hPL1, hPL3, and hPL4 each contained the 5' flanking sequence of 497 base pairs, the first exon, the first intron, and a portion of the second exon. More than 90% transcripts of the hPL1 template were 470 nucleotides long, indicating that transcription was initiated at the proposed cap site. hPL3 and hPL4 genes generated heterogeneous RNA products of about 430, 470, 520, and 680 nucleotides suggesting that multiple start points were recognized for RNA synthesis in vitro. alpha-Amanitin sensitivity of transcription indicated that the DNA-dependent RNA synthesis was carried out by RNA polymerase II. These results show that hPL1, hPL3, and hPL4 genes have functional promoters and multiple initiation sites for transcription. Primer extension analysis of the 5' termini of hPL RNA isolated from term placenta shows that 82-83% of the transcripts are initiated at a region 29 base pairs downstream from a "TATA" sequence. This origin is observed in vitro for the transcript 470 nucleotides long. An additional upstream initiation region (-53) accounts for 8% of transcripts in term placenta and corresponds to the origin for the in vitro transcript of 520 nucleotides. At least three other sites 15, 23, and 39 base pairs downstream from the major cap site are functional in vivo. The initiation site at +40 is utilized preferentially for transcription from hPL3 and hPL4 genes in vitro. We have mapped the different transcription origins on hPL genes.