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Functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures.

1992; Elsevier BV; Volume: 267; Issue: 15 Linguagem: Inglês

10.1016/s0021-9258(19)50071-0

1083-351X

D.E. Zhang, Jeffrey P. Rabek, Chung‐Cheng Hsieh, Carlos A. Torres‐Ramos, John Papaconstantinou,

Folate and B Vitamins Research

Analysis of Mouse AFP Enhancers 10677 was important to elucidate the DNA-protein interactions of the AFP enhancers and their role in enhancer function.To do this we have sequenced the entire enhancer region (from -1009 to -6983 bp), assayed the enhancers I, 11, and I11 as well as MerI, 11, and I11 activities in primary fetal liver culture, and performed DNase I protection analyses to identify the binding sites in MerI using normal fetal liver and adult liver nuclear extracts.We also report on experiments to define the role of the trans-acting factors and cis-acting binding sites of MerI in augmenting AFP gene expression, using site-specific and deletion mutations. MATERIALS AND METHODSConstructing Plasmids for CATAssay and Substitution and Deletion Mutagenesis-The p(-1009)AFPcat (BSD-CAT) and pAFPcat were as reported previously (9,15), which contain AFP gene -1009 to +37 bp and -6983 to +37 bp upstream of the CAT gene, respectively.As shown in Fig. 1, pEnI(-1009)AFPcat contains AFP enhancer I DNA fragment (BamHI (-3835 bp) to BamHI(-1010 bp)) at the 5' end of AFP promoter (-1009 to +37 bp).The pEnII(-1009)AFPcatcontains AFP enhancer I1 DNA fragment (BamHI (-5331 bp) to BamHI(-3836 bp)) at the 5' end of AFP promoter.The pEnIII(-1009)AFPcat contains AFP enhancer 111 DNA fragment (PuuII(-6981 bp) to BamHI(-5332 bp)) at the 5' end of AFP promoter.The orientation of all these enhancer fragments in the CAT constructs are as in the AFP genome.For substitution mutagenesis, the XbaI-EcoRI minimum enhancer region I (MerI) fragment from pUIC19 was inserted into M13mp19, mpMerI.Three substitution mutants mpMIA, mpMIB, and mpMIC, in which the mutation sites correspond to the sites of nuclear protein protection regions Ia, Ib, and IC on the AFP MerI were prepared by oligonucleotide-directed mutagenesis of the mpMerI with synthetic oligonucleotides that paired with 12 nucleotides of the wild-type sequence on either side of 6-8-nucleotide target sequence.The base sequence for each substitution (Fig. 3) consisted of a XhoI recognition sequence.The mutations were confirmed by XhoI restriction endonuclease site analysis and dideoxy sequencing.All the substitution mutant fragments were isolated by digestion with BamHI and KpnI and used to replace the wild-type BamHI and KpnI fragment in the pMerI(-1009)AFPcat.In pMID(-1009)AFPcat sequences from -2317 to -2275 bp of the MerI enhancer fragment were deleted.This construct was generated by digestion with the slow form of Ba131 exonuclease (International Biotechnologies, Inc.) from the XhoI site in pMIB(-1009)AFPcat.