IDENTIFICATION OF NOVEL CHICKEN ESTROGEN RECEPTOR-ALPHA MESSENGER-RIBONUCLEIC-ACID ISOFORMS GENERATED BY ALTERNATIVE SPLICING AND PROMOTER USAGE

Using the rapid amplification of complementary DNA ends (RACE) methodo logy we have identified three new chicken estrogen receptor-ol (cER al pha) messenger RNA (mRNA) variants in addition to the previously descr ibed form (isoform A). Whereas one of the new variants (isoform B) pre sents a 5'-extremity contiguous to the 5'-end of isoform A, the two ot her forms (isoforms C and D) are generated by alternative splicing of upstream exons (C and D) to a common site situated 70 nucleotides upst ream of the translation start site in the previously assigned exon 1 ( A). The 3'-end of exon le has been located at position - 1334 upstream of the transcription start site of the A isoform (+1). Whereas the ge nomic location of exon 1D is unknown, 700 bp 5' to this exon were isol ated by genomic walking, and their sequence was determined. The transc ription start sites of the cER alpha mRNA isoforms were defined. In tr ansfection experiments, the regions immediately upstream of the A-D cE R alpha mRNA isoforms were shown to possess cell-specific promoter act ivities. Three of these promoters were down-regulated in the presence of estradiol and ER alpha protein. It is concluded, therefore, that th e expression of the four different cER alpha mRNA isoforms is under th e control of four different promoters. Finally, RT-PCR, S1 nuclease ma pping, and primer extension analysis of these different cERa mRNA isof orms revealed a differential pattern of expression of the cERa: gene i n chicken tissues. Together, the results suggest that alternative B'-s plicing and promoter usage may be mechanisms used to modulate the leve ls of expression of the chicken ER alpha gene in a tissue-specific and /or developmental stage-specific manner.