Volume 15 Number 3 1987 Nucleic Acids Research The rat a1-fetoprotein gene: characterization of the 5'-flanking region and tandem organization with the albumin gene Mario Chevrette, Michel Guertin, Bernard Turcotte and Luc Bdlanger* Centre de recherche en cancdrologie de l'Universite Laval, L'H6tel-Dieu de Quebec, Quebec GIR 2J6, Canada Submitted November 20, 1986 A 0 10 20 30 40 5' ALBUMIN 88~~~~~~~~~1 7 - AFPAF- 50 60 kb 3, We have constructed a Sprague-Dawley rat genomic library in X EMBL4. The library (nonamplified) was screened with rat AFP and albumin cDNAs. Thirty-three positive clones were characterized by restriction mapping and Southern analysis. The rat AFP gene spans =19 kb and it is located = 15 kb downstream from the albumin gene, with the same polarity of transcription. Transcription initiation sites on the AFP gene were mapped by Sl protection at the marked nucleotides in the sequence 5'-ACAGTA-3'. Several stretches of DNA were found strongly conserved in the 5'-flanking region of rat, mouse (1) and human (2) AFP genes. The TATA box is conserved at -27/-32 bp, and a CAT box (5'-CCAAT-3') at -117/-119 bp on the noncoding strand. A 7-bp inverted repeat flanks the CAT box and another CCAAT pentamer (inverted on the noncoding strand) conserved at -60/-65 bp. A consensus gluco- corticoid receptor binding sequence 5'-TGT&CT-3' is present at -164/-166 bp. The segment at rat positions -224 to -203 also contains a glucocorticoid receptor binding hexamer (inverted on the noncoding strand), and a direct repeat similar to the trans activator binding octamer 5'-ATTTGCAT-3' conserved in regulatory regions of immuno- globulin, histone and other genes (3 and references therein). Thus, a 88230-bp region adjacent to the cap site may play an important role in the expression and glucocorticoid repression (4) of the AFP transcription unit. The TATA/CAT and octamer domains may be involved in the binding of transcriptional activators; perhaps glucocorticoid receptors repress the AFP gene by interfering with CAT- or octamer-binding activators. B r -895 00AAACAGrA ATGCACATAT CCAACCAAAA C9CTtGAACA CATAAAAOAA r -945 AACTCC0ACT tATACTTTAO ATOCCCACir rGAATrTTTTr TITACAAGTT r -795 T0CCATTCTr 90T9AT*9TAT AGArAACTTA AACAAACTCT CACAGACICC r -745 CX..AACC8AG MU9M8M9A 0GA7CATGA9 CCT09C0CCAG TCA0ACTA r -695 TATCA0TCCT C0TTArTTA0. TGTTA0CCAC ATrAICCAAr r0CCTCAGAC r-645 rTCCTCGArC TATArCC r -628 AT GCATCTGTCA CATACATGCA G*A TACGCT h -732 ... .. C..AG..A.A a -600 ..... A TT.. r -597 TTGGCACACG ACA8TAGAAr GGGAACTCGT 9CATCAG C TATGIGCTCT -707 .T A.IA A...CT.G.. C. *-568 ........ .....I.... C.C r -548 AAGTTG6CTA 9ciTAAACCA GACACCAAAC ACAAAA 9TAAA8ACA -654 9...TCGC.T r TACcT.A G. -19 ...9. ..T r A.A ....88 r -503 CCCCCT9GAC CAAr GG9TTTGACC G98 AATA8 h -604 ..... AIACAATA A6IGAAA.. C ..C.9I.AC... C. * -469 ...... G..0. ...(-88). C AC. r -471 CAACIGATAr A80A89AA TACACCCACr GAGCAAGCAG CTCAACACT *-3542. .C 9... ...... CU. * -44z T..... C....AT A T.1 .....A.....T^..IC .C. r -422 CAGAC AIAC 9L99CAGC6CC TAGCATGCAA CATGCACAt 8tct9ct8*t h-422 ..9C C ... A 6.C. I.U9 -3094 G .0.... 6T r -373 tlIlRTITA AACGAAFTAA IGCA^IT GGAGCTACAT ACTAAGCCCG n -384 ..A G..C.I .A ..... .I. ACAl..AT.GC. .CC AT .. m -259 ...C ..T. ... 9... r -326 CIATAIITIC TIrATGCcTG IAIAC^A I T cTTcAG AAATAAAATA h -334 C.C ...... T.AA C.. Al ... G. G..T a-207 .*.. G C. T9.0 G r -276 AATC6T*C9T CAGCACCT GGCAGATACTrc T 9T99GA6A G9T9T9TT h -29U .A TC* G a -26* .9.. ....T... C r -Z89 9C999990 c 9096' 9GCA0 TTG9CAAGGT T T* 8TTTI8 n -240 I.AC.W., ...l...... 1AI .....A. AAAATAAG. ..CA... ..CA * -21d A r -190 TCITITCCAC ICGAACTG FCTIr 0988T0CAIA98 ATCAGACTGA n -19U ..........Gr. C.CA ..... A:C...... .. ..... ::::::::A :C::A.A. 5-i88 .......... ....... C r -140 CCCIIC I GT AAT TlFECPAAAMIcc CTAACTTCM CAT^AUAGA n -140 ..9 .8*88*6 ..CT..C.T C A -1 8 ... .. ... r - 91 AAA98 TAACATGTT 6CCCAC8G986 6l*CTA6 T T8086*900 h _ 9U TACC.1 ..... C.A....G. A A A AA: . :::........G:::.. a 89 T.G.. .r ...8 G r - 45 CACITAAAAA GCO fWrA-AA GAACTTCAGC GCTACTGCTC cACATATCCG n - 40 TG..C G . At 83 AC r * 6 6C0TCTACCA C09TCT906 *tq *q 88q c88 989 9c 9tq "9 r 49 tq9 agegee tee stt tcc tt c tt cte ctetat9 ct ttt r d get 9 cc ae9 qt9 ctq C#c ae 9g9 ttt 99 et r . 1*27 969T 8AT6C TT9 6T9 8C0C G66 1338 Limited, Oxford, England. 1 338 0 1 R L Press Limited, Oxford, England.