Structure and sequence of the promoter area and of a 5' upstream demethylation site of the estrogen-regulated chicken vitellogenin ii gene

Abstract

We sequenced 487 base pairs (bp) covering the 5' end of chicken vitellogenin II gene and 840 bp of its 5' end-flanking region. This region includes a MspI/HpaII restriction site which becomes undermethylated in chicken liver upon estrogen treatment. Southern blot analysis of restriction enzyme digests of total DNA confirms that this undermethylation site is the one situated at -611 bp from the cap sequence. This site is flanked at nucleotides -567, -629, and -667 by stretches of DNA very rich in A + T. We used S1 nuclease mapping as well as a primer extension procedure to map the transcription starting point of the vitellogenin II gene. We found a 5'CATAAAA3' box between nucleotides -32 and -26, and between nucleotides -77 and -69 a sequence (5'TTGAGAATT3') homologous to the bacterial RNA polymerase-binding site. A similar sequence (5'TGTTTACATAAA3') is also found between nucleotides -101 and -90. A comparison of the sequence of the primer-extended DNA with the 5' end of vitellogenin II gene revealed the presence of two small exons of 53 and 21 bp between nucleotides +1 and +53, and +169 and +189, respectively, and two small introns of 115 and 100 bp between nucleotides +54 and +168, and +190 and +290, respectively. The first AUG at nucleotide +14 is in front of an open reading frame. We found that 77% of the amino acids coded by the first exon are hydrophobic, a feature compatible with a signal sequence of a secreted protein such as vitellogenin.