cDNA, PCR reactions were carried out in a 50-pl reaction, containing. 100 pg of plasmid DNAcontaining the cDNAinsert, 10 nm Tris-HC1, pH. 8.3, 50 m~ KC], ...
THEJOURNAL OF BIOLCXXCAL CHEMISTRY 0 1994 by The American Societyfor Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 14, Issue of April
10660-10667, 1994 Printed in U.S.A.
GATA-type Zinc Finger Motif-containing Sequences and Chorion Bombyx mod* Gene Transcription Factors of the Silkworm (Received for publication, November 15, 1993, and in revised form, January 10, 1994)
Joel R. Drevet, YasirA. W. SkeikyS, and Kostas Iatroug From the Department of Medical Biochemistry, Faculty of Medicine, the University of Calgary, 3330 Hospital Drive N. W., Calgary, Alberta, Canada T2N 4N1
To characterize a DNA-binding protein, BCFI, which contains all of the cis regulatory elements required for sex, regulatestheexpression ofsilkmothchoriongenes tissue, and stage-specific expression (Mitsialis et al., 1987). through binding to gene promoter elements identical to By using as a working model a pair of chorion genes, HcA/ those recognized by the GATA family of transcription B.12, that encode high cysteine (Hc) chorion proteins and are factors, we have carried out polymerase chain reaction expressed during the late stages of choriogenesis (Eickbush amplifications of Bombyx mori genomic DNA using de- and Kafatos, 1982; Iatrou et al., 1982, 1984; Rodakisand Kafagenerate primers derived from the conserved DNA bind- tos, 1982; Rodakiset al., 1982, 1984),we have previously idening domain of mammalian GATA factors. Two single copy tified two chorion gene promoter-specific DNA binding activigenes, BmGATAcu and BmGATAP, wereidentified, which ties, BCFI and BCFII, that are present in nuclear extracts of encode sequences containing GATA-type zinc finger mo- silkmoth follicular cells (Skeiky and Iatrou,1991b). These DNA tifs. The BmGATAP gene is expressed in follicular and binding factors were shown to be specific for Hc chorion gene BmS tissue culture cells, the two cell types that contain BCFI. No BmGATAcu gene transcripts were detectable in promoters (15 gene pairs), and their appearance andaccumuthe tissues that were tested. Upon overexpressionin Es- lation in follicular cell nuclei were found to coincide with the cherichia coli, a peptide encompassing the BmGATAP time of activation of the cognate (Hc) chorion gene promoters. These results suggested that factors BCFI and BCFII act as zinc finger motif was able to bind specifically to the BCFI recognition motif of the chorion gene promoters. A specific regulatory proteinscontrolling the expression of the Hc chorion genes. Factor BCFI was further shown to be a key polyclonal antibody directed against the zinc finger domain of BmGATAP was also used in gel retardation as- determinant for transcription preinitiationcomplex formation, says to confirm that factor BCFI is indeed encoded by because its presence was required for stable bindingof BCFII, the BmGATAP gene. Conceptual translation of a com- presumably via protein-protein contact(s) a n d o r local modifiplete cDNA clone encoding the BmGATAP protein re- cation of DNA conformation and subsequent assemblyof addivealed that this protein has a size similar to that an of tional factors on the Hc chorion gene promoters (Skeiky and immunoreactiveprotein,presumablyBCFI,which is Iatrou, 1991b). present in follicular cell extracts. The DNA sequence to which factor BCFI binds is the hexanucleotide motifAGATAA, which matches theconsensus binding site of the GATA family of zinc finger motif-containing During the terminal stages of silkmoth oogenesis, more than DNA-binding proteins (Orkin, 1992). The GATA class of zinc 100 different proteins are produced by the cells of the follicular finger proteins is characterized by a highly conserved DNA two repeats of the motif Cys-X,-Cysepithelium that surrounds the oocyte, secreted toward theoo- binding domain containing cyte, and cross-linked extracellularly to form the eggshell or X,,-Cys-X,-Cys and has a DNA recognition target chorion (Kafatos et al., 1977). Chorion protein synthesis (cho- [m]GATA[A/GI. Evidence for the relationship between BCFI riogenesis) occurs overa period of 4 days during pharate adult and the GATA family of proteins has come from DNA binding and competition assays, which have shown that the target sedevelopment and reflectsdirectly the temporallyregulated quences of BCFI and the avianGATA protein are interchangetranscriptional activation of the corresponding structural genes (Bock et al.,1982; Iatrou et al.,1982; Sweversand Iatrou,1992). able andthat the BCFI target sequence is able toform a stable An interesting featureof the genesencoding chorion proteins complex with a factor that is present in nuclear extracts of is their organization into pairs of divergently oriented tran- haemin-stimulated K562 tissue culture cells, presumably the scription units (Jones and Kafatos, 1980; Iatrou and Tsitilou, human GATA-1 protein (Skeiky and Iatrou, 1991b). Based on these observations, we have postulated thatBCFI 1983; Iatrou et al., 1984; Burke and Eickbush,1986; Spoerel et al., 1986; Hibner et al., 1988; Skeiky and Iatrou, 1990). These may be a protein containingGATA-type zinc finger motifs. Such units sharea short bidirectional promoter (300 2 30 bp),’ which a n assumption has led previously to theidentification of elt-1, a GATA-type protein of Caenorhabditis elegans (Spieth et al., 1991). GATA-related, single zinc finger motif-containing reguof latory proteins, whose DNA binding sequences contain a simi*This work was supported by the MedicalResearchCouncil Canada. The costsof publication of this article were defrayed inpart by lar WGATAR core motif, have also been found in Aspergillus the payment of page charges. This article must therefore be hereby nidulans and Neurospora crassa (Fu andMarzluff, 1990; Kudla marked “advertisement” in accordance with 18 U.S.C.Section1734 et al.,1990), suggesting that the DNA binding domain of GATAsolely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted related proteins is conserved from fungi to mammals. to the GenBankmIEMBL Data Bank with accession number(s1 L27451. In this paper, we report on the identification of two silkworm f Present address: Seattle Biomedical Research Institute, 4 Nicker- genes, BmGATAa and BmGATAP, which encode GATA-like son St., W., Seattle, WA. 6 To whom CorresDondence should be addressed. ”el.: 403-220-7536: polymerase chain reaction; kb, kilobaseb); RACE, rapid amplification of FA: 403-270-0737. The abbreviationsused are: bp, base pair(s);Hc, high cysteine; PCR, cDNA ends.
10660
10661
GATA Danscription Factors for Silkworm Chorion Genes proteins, andon the cloning of BmGATAp cDNAfrom a Bombyx mori follicular cell cDNA library. We show that BmGATAP is expressed in the tissues where the BCFI DNA binding activity is found and present data suggesting that theproduct of the BmGATAp gene is factor BCFI.
-10
MATERIALSANDMETHODS Animals, Nuclear Extracts, and Nucleic Acid PreparationsSilkglands obtained from silkworm strain C108 (Iatrou et al., 1980) were used as thesource of genomic DNA.Tissues isolated from a hybrid strain obtained from the Forest Pest Management Institute (Sault Ste. Marie, Ontario, Canada) were used to prepare total nucleic acid. A hybrid Japanese strain and strain 703 were used to construct the follicular cDNA libraries. Larvae and pupae were maintained at 25 "C, and ovarian follicles were dissected from day 6-8 pupae. DNA, total nucleic acid, and nuclear protein extracts were prepared as described previously (Iatrou et al., 1980; Skeiky and Iatrou, 1987; 1990, 1991a, 1991b). Oligonucleotides-The double-stranded oligonucleotide I used in bandshift experiments was previously described (Skeiky and Iatrou, 1991b).The forward and reverse primers used in the amplification of B. mori genomic DNA were designed from the published amino acid sequences of the human, mouse, and chicken GATA proteins (compiled and compared in Zon et al., 1990). The forward primer was 5'-GCE-
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AGATAA T G
FIG.1. Chorion gene promoter DNA-binding proteins. Panel A, the 3.8-kb EcoRI(E) fragment of genomic DNA containing the chorion ~GA~AGlTG~CTlGT~ACGTlkl\~CTlTG~CTlGG~ACTlGC~ACGTlAgene pair HwVB.12. Arrows indicate transcriptional orientations; open C[ACTlGC-3', andthe reverse primer was 5'-GCTCTAGA[AGTlC- boxes, introns; dotted boxes, exons. The line below the genes depicts the 260 bpof the common promoter element that can be excised bydigestion C~AG]CA~ACGT]GC~AG]?T~AGlCA~ACGTlAC~ACGTlGG~AGlTC~AGTICC-3'. For specific BmGATAp amplifications within the DNA binding with HindIII (D) and HincII ( C ) , and the positions recognized by the domain, the forward primer (FP1) was 5'-GCGGAATTCGAG'ITGGCG- two DNAbinding factors, BCFI (square) andBCFII (circle)(Skeiky and GAG'ITITTCACTGAGGGG-3', and the reverse primer (RP1)was 5'- Iatrou, 1991b).Contact points for the factors, determined from DNaseI GCGAAGCTTCATGGTGAGAGGTCGGlTGACG-3'.For the extension footprinting (bracketed areas) and methylation interference (asterisks) of the BmGATAp cDNA toward the 5' extremity, the RACE technique assays, are indicated on the sequence shown below. P indicates the (Frohmann et al., 1988) was employed using a specific internal Bm- position of a PstI site that was used in subsequent experiments for the generation of a 170-bp probe used in gel retardation assays. Panel B , the GATAp primer (RP2), 5'-CGCAAGCTTGTAGTGGCCGGTGCsimilarity between the BCFI target sequence and the consensus hexCGTCGCGGTG-3'. To facilitate cloning of the amplified products, an- anucleotide motif recognizedby all of the members of the GATA family chor sequences (italics) containing chosen restriction sites (XbaI, of DNA-bindingproteins. EcoRI, or HindIII, underlined) were added 5' of the forward and the reverse primers. PCR Amplification Conditions-The initial PCR reactions were per- vidson, 1976).Transfers of genomic DNAand RNA to nylonmembranes formed in a final volume of 100 pl containing 0.5 p~ each of the two (Hybond-N+,Amersham Corp.) werecarried out using 10 x SSC and 2.0 degenerate primers, 200-600 ng of genomic DNA, 10 nm Tris-HC1, pH nm NaOH, respectively. Randomlyprimed, 32P-labeledfragments (lo8lo9 cpdpg) were used as hybridization probes. Hybridizations were 8.3, 50 nm KC1, 3.5 n m MgCl,, 0.01% gelatin (w/v), 200p~ each of the four dNTPs, and 2.5 units of Taq DNApolymerase(Perkin-Elmer).After performed for 12 h at 65 "C in 300 m~ NaCl, 50 m~ sodium phosphate, a denaturation step of 4 min at 94 "C, amplifications were carried out pH 7.0, 5 m~ EDTA, 10 x Denhardt's solution, 10% (w/v)dextran sulfate, 1%(w/v) SDS,1mg/ml yeast RNA, and 5 x lo6 cpdml. Following for 5 cycles of 94 "C/1 min, 45 "C/1 min, 72 "C/1 min, followed by 30 cycles of 94 "C/1 min, 55 "C/1min, 72 "C/1min. Extension periods were hybridizations, the membranes were washed a t 65 "C, 2 times, for 15 increased by 2 s after each cycle. Ten-pl aliquots of the final PCR min with each of 2 x, 0.5 x, 0.2 x, and 0.1 x SSC containing 0.1% SDS mixtures were analyzed on a 2% agarose gel. Amplifiedfragments were and autoradiographed for 16h at -70 "C using x-ray film (Kodak,XARthen digested using the appropriate anchor restriction enzymes and 5). DNA Sequencing and Analysis-Dideoxynucleotide sequencing was gel-purifiedby binding to and elution from a DEAE-cellulose membrane performed using double-stranded plasmid DNA and Sequenase (U. S. (Schleicher & Schuell) before being cloned into the vector pUC18. Biochemical Corp.) as described elsewhere (Chen and Seeburg, 1985). For the amplification of the DNA binding domain ofBmGATAp cDNA, PCR reactions were carried out in a 50-pl reaction, containing The sequence of the 2.1-kb cDNA insert of clone BmGATAp was ob100 pgof plasmid DNAcontaining the cDNAinsert, 10 nm Tris-HC1, pH tained after sequencing a library of ExoIII-deleted clones generated in 8.3, 50 m~ KC], 0.01% gelatin, 2.5 m~ MgCl,, 200 p each of the four the plasmid pBluescript-SK' (Stratagene) as described by the manufacdNTPs, 0.05 p~ each of the two primers, and 1 unit of Taq DNA poly- turer. Sequence comparisons and conceptual translations of the Bmmerase (Life Technologies, Inc.) for 30 cycles of 94 "C/1 min, 55 "C/1 GATAa and BmGATAp clones were performedusing the Pustell-Kafamin, 72 "C/1 min. Ten-pl aliquots of the final PCR mixtures were ana- tos (International Biotechnologies, Inc.) sequence analysis software package. lyzed by electrophoresis in a 2% agarose gel. Construction and Screening of Follicular cDNA Expression The extension of the cDNAclone toward the 5' extremity was achieved by using the RACE protocol as described originally elsewhere Libraries--Two follicular cDNA libraries were used in this study. The (Frohmann et al., 1988).Briefly, a specific BmGATAp primer (RP2, see first library was constructed using total RNA from follicular cells. An above and Fig. 3) was used to reverse-transcribe poly(A)' follicular cell oligo(dT),, primer carrying a XhoI anchor sequence at its 5' end was RNA (2 pg) using "Superscript" reverse transcriptase (Life Technolo- used to generate the firstcDNA strand asreported previously elsewhere gies, Inc.) as recommended by the manufacturer. The first-strand cDNA (Brown and Kafatos, 1989), except that methylated dCTP was used instead of dCTP. Self-primed second-strand synthesis was carried out ) 2 units of terminal transferase was then tailed using dATP (200 p ~ and and EcoRI adaptors were ligated to the double-stranded cDNAs. Fol(Boehringer Mannheim) for 10 min at 30 "C. Second-strand synthesis was initiated using an oligo(dT),, primer and Taq DNA polymerase for lowing digestion with EcoRI and XhoI, the cDNAs were cloned into 20 min at 42 "C. PCR reactions were finally carried out for 35 cyclesof EcoRI/XhoI-digestedA ZAP11 vector (Stratagene). The resulting recom94 "C/1 min, 50 "C/1 min, 72 "C/2 min. Following electrophoresis, the binant phage DNA was packaged using the Epicurean packaging examplified fragment was purified and subcloned into vector pBluescript tracts (Stratagene) and used to infect XL1-Blue host cells. Following (Stratagene). amplification and screening, plasmids were recovered fromhybridizing HybridizationsSouthern hybridizations wereperformed as de- phage using the Stratagene in vivo excision protocol.T h e second cDNA library was a gift from Dr. N. A. Spoerel (University of Connecticut, scribed previously (Iatrou et al., 1980) using 5 pg of genomicDNA Farmington, CT). It was constructed from ovarian poly(AY RNA (strain digested with EcoRI, HzndIII, or EcoRI plus HindIII. For Northern hybridizations, RNA samples were loaded onto a methyl mercuric hy- 703). Double-stranded cDNAs were ligated to NotYEcoRI adapter-linkdroxide-agarose gel as described originally elsewhere (Bailey and Da- ers (Invitrogen librarian kit) and cloned into the EcoRI site of the A g t l l
GATA Danscription FactorsSilkworm for
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FIG.2. PCR amplifications and amino acid comparisons B. of mori and GATA-type zincfinger motifs.Panel A, B. mori genomic DNA (lane 1) was amplified witha set of degenerate primers as described in the text. Lanes 2-4 show control amplifications in the absence of the DNA template (lane2), in thepresence of only one of the two primers (lane3), or in theabsence of primers (lane4 ) . Lane 5 contains aTaq polymerase efficiency control (product derived from a reaction employing phageA DNA and corresponding primers). The sizes of the fragments amplifiedfrom silkworm DNA(showna t left) were determined by reference to DNAmarkers (shown at right) run in lane M.The position of the primers is indicated by a n asterisk. PanelB, amino acid sequence comparisons of the consensusGATA zinc finger domain, withthe conceptually translated silkworm fragment, BmGATAa, and part of the sequence of the BmGATAP clone isolated from a B. mori follicular cell cDNA library (see alsoFig. 4). The cysteine residues of the two zinc finger domains of each sequenceare shown in larger,bold 1etters.The locations of the degenerate primers used to amplify the BmGATAa and BmGATAP zinc finger domains are shownby the two lines above the GATA consensus sequence. vector using E. coli Y1090 as the host. Libraries were screened using preparation were used to immunize a rabbit via subcutaneous injecrandomly primed, 32P-labeled probes (2 x los c p d m l ) . Approximately tions, according tothe protocol of Harlow and Lane (1988). For immu250,000 plaques from each library were screened for the presence of nodetection ofBmGATAP in total protein extracts of follicular cells, BmGATAP cDNA clones. proteins were solubilized in urea/SDS buffer as originally described DNA Binding and Binding Competition Assays-Protein-DNA bind- elsewhere (Koelle et al., 1991). SDS-polyacrylamide gel electrophoresis ing assays were performeda s previously described (Skeiky and Iatrou, gels were then electroblotted onto nitrocellulose membranes(Bio-Rad) 1990, 1991a)using 2.5 x 10' cpmCerenkov of end-labeled double- a s recommended by the manufacturer. The membranes were blocked probed with the BmGATAP antiserum for 1 orgg with 5% Blotto (Sigma) and stranded oligonucleotide I and 2 pgof follicular nuclear extracts 5 of bacterial extract containing the fusion protein. Protein-DNA com- h a t room temperature. After washing for 10 minsuccessively in TNT plexes were analyzedby polyacrylamide gel electrophoresisas reported buffer (10mM Tris-HCI, pH 8.0,150m~ NaCI, and 0.05% Tween 20) plus previously (Skeiky and Iatrou,1990, 1991a). In the competition experi- 0.1% bovine serum albumin and TNT buffer plus 0.1% bovine serum ments and unless otherwise stated,50-fold a molar excessof nonradio- albumin plus 0.1% Nonidet P-40 (Sigma), blots were incubated in TNT active double-stranded oligonucleotide or the PstUHindIII restriction buffer plus 0.1% bovine serum albumin and "'1-protein A (30 pCi/gg; 1) International Chemicals and Nuclear Corp.)a t 1-2 x lo6 c p d m l for 1 fragment of the HcAB.12 promoter (Skeiky and Iatrou, 1991b) (Fig. was includedin the binding reactions before the additionof the extracts. h. Blots were washed as described above and autoradiographedfor 24 h at -70 "C. For the analysis of antibodybinding-mediated"supershift"experiments, 4-1.11 aliquots of a rabbit preimmune or immune serum were RESULTS added to the binding reaction after 10-min a incubation of the radioactive probe with the extracts. Identification of GATA-like Sequences in the Genome of BomOverexpression of FusionProteins a n d Generation of Polyclonal byx mori-Fig. lA summarizes resultsobtained from the initial Antibodies-The EcoRVHindIII fragment containing the PCR-amplicharacterization of the two DNA-binding proteins, BCFI and fied fragment of BmGATAP cDNAobtained using primers FP1 and RP1 (Fig. 3) was subcloned into pBluescript and into the expression vector BCFII, which bind to the promoter of a model Hc chorion gene pGEX-2T, in a manner allowing synthesis of P-galactosidase and glu- pair (HcAB.12) (Skeiky and Iatrou, 1991b). The similarity betathione S-transferase fusion proteins, respectively. E.coli strain DH5a tween the BCFI target sequence and thatof the GATA family of was transformed with each construct and induced to overexpress the DNA-binding proteins is illustrated in Fig. 1B. fusion proteins at 30 "C for 24 h. Total bacterial protein was subseTwo degenerate primers located within the highly conserved quently isolateda s originally described elsewhere (Desplan et al., 1985). The glutathione S-transferase-BmGATAP fusion protein (44 kDa) was DNA binding domain of the GATA factors (Fig. 2B ) were used to excised a s a band from a preparative SDS-polyacrylamide gel (12.5%, PCR amplify B. mori genomic DNA. Four major fragments ranging in size from 150 to 500 bp (Fig. 2 4 ) were obtained, of thecrude 19:1, acrylamidehisacrylamide), and100-pgaliquots
-
GATA Danscription Factors for Silkworm Chorion Genes
BrnGATAcx
BrnGATAB
10663
genes. The identity in the relative migration of the hybridizing bands of the genomic DNA and the cloned BmGATAP fragment - " following digestion with EcoRI and XhoI confirmed that the cloned BmGATAP sequence was derived from a genomic DNA fragment. To deduce whether one of the two genomic fragments might be derived from the gene that encodes factor BCFI, the two cloned sequences were used as probes for RNA isolated from q4.0 follicular, Bm5 tissue culture, and silkgland cells (Fig. 3B). While no hybridization was seenwith the BmGATAa probe (not shown), BmGATAP mRNA was found to be present infollicular and Bm5 cells, the two cell types that contain factor BCFI, but not in silkgland cells, a tissue that lacks BCFI (Fig. 3B, left I panel 1. The size of the hybridizing RNA was approximately 3.0 B.rnorr genornlc DNA P kb. These results rule out BmGATAa as the gene thatencodes factor BCFI and leave open the possibility that thisfactor may be encoded by the BmGATAp gene. S Isolation and Characterization of BmGATAP cDNA-To obtain a cDNA clone for BmGATAP, a second follicular cell cDNA library was screened using as probe a BmGATAP cDNA frag3.0ment generated by PCR using reverse-transcribed follicular cell mRNA as the template anda set of specific primers encom41.25 passing the zinc finger domain ofBmGATAP. The screening produced several cDNA clones of variable lengths, none of BmGATAO ACTIN A 3 which extended beyond the first zinc finger domain, presumFIG.3. Detection of BmGATA sequences in silkworm genomic ably due to a stable secondary structure of the mRNA. To exDNA and RNA. Panel A, Southern hybridizationsof B. mori genomic tend the BmGATAP cDNA sequence toward the 5' end, the 5' DNA digested with EcoRI ( E ) ,XhoI (X),and EcoRIIXhoI (EX)using as RACE technique was used, from which a PCR-amplified fragprobes the BmGATAa fragment (left)and a fragment encompassingthe ment (0.87 kb) was subcloned, sequenced, and used to reconzinc finger domain of the BmGATAP sequence (right). Following hybridization with the BmGATAa probe and autoradiography, the blot stitute a longer cDNA clone (Fig. 4) whose size (2,828 bp) apwas stripped and rehybridized with the BmGATAP probe. Lane P shows proximated the size of the hybridizing RNA band seen in the the hybridization pattern obtained with the plasmid containing the Northern analysis (3.0 kb) (Fig. 3B). subcloned 4.0-kb EcoRIIXhoI (EX)of the BmGATAP gene analyzed on The conceptual translation of the extended cDNA clone rethe same gel and hybridized with the BmGATAP probe. Panel B , Northvealed that the sequence starts with an open reading frame ern hybridizations of B. mori RNA extracted from follicular (lane F), Bm5 culture (laneB ) , and silkgland cells(lane S ) using theBmGATAP with the first putative methionine codon located 10 bp downand the B. mori cytoplasmic actin A3 probe. The size of the hybridizing stream from the beginning of the cDNA clone. A primer extenbands (arrowhead) wasdeduced from the migration of RNA size sion reaction (not shown) carried out using primer (Fig. RP3 4) markers. showed a single extension product of 139 bp. Thus, thecloned cDNA is 58 bp shorter than thecorresponding mRNA, and the cloned, and sequenced. Upon conceptual translation, only the full size cDNA is 2,886 bp. A stop codon, found a t position 1535, 350-bp amplified product showed extensive similarity (70% is followed downstream by 1.3 kb of 3' untranslated sequences identity) (Fig. 2B) to the consensus amino acid sequence of the containing two putative polyadenylation signals. The 1527-bp zinc finger domain of the GATA-type factors (Zon et al., 1991). open reading frameof the cloned cDNA (starting from the first The homology encompassed the two repeats of the zinc finger methionine codon) encodes a 509 amino acid polypeptide of a motif Cys-X,-Cys-X,,-Cys-X,-Cys, which constitutes the bind- predicted size of 57 kDa. ing motif of the GATA factors. The B. mori sequence, termed No pronounced similarities to anypreviously described proBmGATAa, also contained stop codons (not shown) within the teins were found outside the zinc finger domain of the Bmspacer sequence that separates the two zinc finger motifs. GATAP protein. Prominent features of the protein (Fig. 4) inThus, as is the case for the mammalian and avian GATA-1 clude a putative nuclear localization signal located genes (Hannon et al., 1991; Tsai et al., 19911, each zinc finger immediately downstream of the second zinc finger and consistmotif of the BmGATAa gene isencoded by a different exon. ing of a stretch of basic residues (boldface); putative serine and A follicularcDNA library was screened with the cloned Bm- threonine phosphorylation sites (circled residues in italics)for GATAa fragment, and a 4.0-kb clone was isolated. The clone MAP kinase, cdc2 kinase, kinase A, and kinase C; a tract of 8 was sequenced, and the regions encoding zinc finger motifs arginine residues (double underlined) near the carboxyl exwere aligned and compared with the consensus GATADNA tremity of the protein, which may be involved in protein-probindingdomain and to BmGATAa (Fig. 2 B ) . This new se- tein interactions;a GCN-4/GCR-like acidic domain (dotted unquence, termed BmGATAp, showed closer similarity to the derline) upstreamof the DNA binding domain;a putative Oct-2 DNA binding domainof the consensus GATA protein (76% iden- activation domain (underlined) within the C-terminal portion tity at the amino acid level) than to BmGATAa. However, as of the BmGATAP protein. The functional relevance of these was thecase for BmGATAa, the BmGATAP zinc finger repeats motifs remains to be determined. were separated by a 480-bp sequence containing multiple stop BmGATAp Encodes the Hc Chorion Promoter DNA Binding codons, suggesting that theisolated clone was genomic rather Factor BCFI-To find out if the DNA binding domain of Bmthan cDNA (see also below). GATAP recognizes the binding site for factor BCFI, a PCR The two cloned sequences, BmGATAa and BmGATAp, were fragment encompassing the BmGATAP zinc finger domain was used as probes for B. mori genomic DNA digests (Fig. 3A). In generated using primers FP1 and RP1 and the cloned Bmeach case, a single hybridizing band was observed suggesting GATAP cDNAas template(Fig. 4). The amplified fragment was that BmGATAa and BmGATAP are encoded by single copy subcloned in frame with the lacZsequence of pBluescript and
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160
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140
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230
220
210
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CCTCGACGACTCGCTCTTCGCCGCCGACCCCGGGCTCCGCTCCACCGACGGCGTCTACCTGCGCCGGAGTCCCGAGCT
L 300
290
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360
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TCGCACGACCATCACCACCACCTCGAGCAAGGCGGAGGCGGCAACGGCGCGGCGAGCAGGTGTACAGCGGTGCGCCTTCA
S 480
470
460
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440
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430
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630
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A
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L
@
G
C
C
v
620
ACACGCGGTTCGGCGGCGCCITCGCCTGCGCCCGCCGGAGCACGATCGGCGGTCGCGGGTCGCATCTTCCGTGTTGTGM T R G S A A P O P A P A G A R S A V A G R I F R V V N 710
700
690
680
670
660
650
720
TGATATTTACGCGGCGATGGGGATACACAATATGTTTATCGATGCGGACCTCGGCGGCGTGATTACACCAGGC~CGGAC
D I 740
Y A 730
A
M
G
I
H
N
M
F
I
D
A
D
L G 770
G
V
-
I @ ) P G F ---------ppl---------
G
L FIG. 4. The DNA and amino acid se7 760 750 quences of the BmGATAp cDNA TCGGCAAAGTGAAGGGTGGAGGCAAGCAGGTGTTGAGCTAACGTTTATGCGCACAGTCGAGTTGGCGGAGTTTTTCACT clone. The nucleotide sequence of the G K V K G G R Q A G V E L T F M R T V E~ L .A E ~ ~ F . . FT. 2828-bp BmGATAPclone is shown together with the amino acid sequence encoded by it. The nucleotide sequence between positions l and 775 (terminating at the point indicated by the vertical arrowhead) has been obtained through the 5' RACE reaction, while the remaining sequence was that of the longest cDNAclone isolated from the follicular cell cDNA library. Putative polyadenylation signals present in the 3'-untranslated region are GAAACCAAAGAACAGTAATATTAAGGCPGACAGAAGCGCTAAGGCAGTACAGCGAGCGTCTCCGCGGGCATC~GATGGA underlined.Amino acidresidues in circled K P K N S N I K A D R S A K A V Q R A @ P R A S R W R italics indicate putative phosphorylation 1280 1270 1260 1250 1240 1230 1220 1210 sites. The zinc finger motif-containing GAGCATATTGGATGCGTCGCCGCTCTCCGCCGCGCCTGGGCTACTACGTGCAGTCGGCGGAGGGCATGAAGCTGGAGGAG DNA binding domain is shadowed, and A Y W M R R R O P P R L G Y Y V Q S A E G M K L E E the cysteine residues belonging to each 1290 1360 1350 13401300 1330 1320 1310 CCGCAGCACCACGTCATGTACATCGGAGTGCCGGAGCTGGAGCTGGGACCAGACACAAGGACACGACGCCGCACCACGACCACTCC zinc finger are boxed. A putative nuclear P Q H H V M Y I G V P E L G P D T R T R R 3 @ T @ T E localization signal is indicated in boldface at the edge of the carboxyl finger. Puta-1400 1390 1380 1370 1440 1430 1420 1410 tively functional peptide motifs are also GCCGTGTCGCTCGCCAGCTGAGACCTCGCGACCAACTCCAAACATCATCATMTMTTAGTACG~GTTGTGTATAm J C R @ P A E T S R P @ P N I I I I I S T F V V Y I C shown as follows: a GCNUGCR-like acidic 1520 1510 1490 1480 1470 1460 1450 domain (dotted underline); an1500 Oct-2-like GTACGTGCGTGATAGAGCTCCCTCGCTCGGACCGCCGTCGCCGTCGCCGTCGCCGCTCGCCGCCC~TACACGTPCA~ activation domain (underline);a tract of S P P K Y T F T T C V I E L P R S D basic residues (double underline). Dashed 1550 1540 1530 9580 1600 1590 arrows indicate the positions and orientaAGACATGATTACATTTAGCC~TCTATATATTMTG~TATGCATATACATAGGTGTATACTCGTATTATAACGTP R H D Y I tions of primers used for the generation of 1680 16101670 1660 '1620 1650 1640 1630 the PCRprobe (FP1 and RPl), the 5' ACTGTAACATAATAATAACTAAGmGCGCGGGGAGCGGCGGCTGGTCGGGCGACGCTAAATGTPGTmMTG~G RACE reaction (RP2), the primer ex1760and 1750 1740 1730 1720 1710 1700 1690 tension analysis (RP3). GTTCGATTAGGGTCCPGTPACITGTTACCGATATCATTAAACCTATGAGACTGTGCATTMCCATTC~AACGTGCATTC
*
1840 1830 1820 18101770 1800 1790 1780
CTAAATAGATAGCTA~~TATATACAAT~AATTAGATTTAAGACG~T~CCCGCTGTPGTAGTAGAT 1920 1910 1900 1890 1880 1870 1860 1850
TCGGTCITCACTTTCAAGTGTTGATGA~TATGAACTCCATGACITGCAGAAACATTCCATGAATCGATTCMTTAGT 2000 1990 1980 1970 1960 1950 1940 1930
TCGTAGAACTCGTACGAGTATGCACTCGCACTCGCACTCGCACTCGCACTGGCACTCGCACTCGCACTCGCGCGCAGCGT 2080 2070 2060 2050 2040 2030 2020 2010
C C C T C G C C G T G C A G T G T C G C A T C G ~ T A C T C G T A T C C G T 2160 2150 2140 2130 2120 2110 2100 2090
GTAmTCAAAATPGTCTTAmACCACGCTCTCTAATTACCATTAAGTMGTMCATAC~CAAAC~C~CTACT 2240 2230 2220 22102170 2200 2190 2180 CAATATATAATATTGAAGCCATCATTGTATTGATATTGTTCTGTPATTATATTATCAATATGTGTACGGGAAGCTACACG 2310 2300 2290 2280 2270 2260 2250 2320
ACATG~AAATACAATACTTCM~ATTATTATCATATTTTATATATTTAGAGACTATGCAT~~TTATATTAA 2400 2390 2380 2370 2360 2350 2340 2330
TATAATTATTTCCTCTCITAACAAGGTGGACCGAATTATTMTA~ACCCMG~CTCACTMGTGGCTTAGTTATACT 2410
2420
2430
2440
2450
2460
2470
2480
~TAGAG~TT~CATMTATGTCATGTCATTTTGGGGGCACCTGTTGGGCTCGGATAAGATAATATGTTCTTCG 2490
2500
2510
2520
2530
2540
2550
2560
CGTCCTGCTGGTGGATPGAGATGAAATGGTCCCCACTGCTTACTCTT~TTMTTTATACGATAGATGCATTTAAATT 2570
2580
2590
2600
2610
2620
2630
2640
ATGATCACCGCAACAGA~ATATGTTATGTTATGTTAAATAGATTCTGAGACACGGACGCGC~GATATA~M~
2720 2710 2700 2690 2680 2670 2660 2650 TAATGAAGTCGTAGGmAmAAAGAAGCPTCGCCAAATAGTAGGTACGTGTAGGCTGTGGACGCCCGCGCCGCCGCGC 2800 2790 2780 2770 2760 2750 2740 2730
CGTAGGGACGACTCGAGTCTACGTCGATCGCGATCTGTACATATTAG~AAGGTGTC~GTACA~CTATT~GTPCT 2820 2810
A T T A T A A A A C P
GATA Danscription Factors for Silkworm Chorion Genes
66 50 D
t
S
10665
overexpressed in E. coli as a P-galactosidase fusion protein. Bacterial extracts containing the P-galactosidaselBmGATAP fusion protein (18 kDa) (Fig. 5A) were prepared and testedin bandshift experiments for their ability to generate complexes with the BCFI target sequence (Fig. 5B 1. A complex was formed (lane 21, which could be competed out by double-stranded oligonucleotide I or the HcA5.12promoter fragment (lanes 3 and 4 ) but not by nonspecific competitors (not shown). Therefore, the BmGATAP zinc finger domain can bind the targetsequence motif AGATAA in vitro. To find out whether the BmGATAP gene product is factor BCFI, a polyclonal antibody was raised against theBmGATAP zinc finger domain expressed as a glutathione S-transferase fusionprotein (glutathione S-transferaselBmGATAP). The specificity of the immune serum was assessed through a Western blot of protein extracts from bacteria expressing glutathione S-transferase or the P-galactosidaselBmGATAPfusion protein. As shown in Fig. 5C, the immune serumrecognized both glutathione S-transferase and the j?-galactosidaseBmGATAP fusion protein. This antiserum wasalso used in a Western blot analysis of follicular cell protein extracts (Fig. 5 0 ) . A major 66-kDa and a minor 50-kDa polypeptide were detected by this assay (lane F).Consistent with the absence of an RNA hybridization signal withthe BmGATAp probe (Fig. 3B ), no immunoreacting proteins were detectable in extracts prepared from silkgland cells (lane S). Finally, the immune serum was added to a bandshift reaction (Fig. 5 E ) and was shown to recognize BCFI bound to oligonucleotide I by the generation of a new, “supershifted” complex (lane 8).The appearance of a second complex at higher antiserum concentrations (lane 9 )is probably due to thebinding of additional antibody molecules to the first one. DISCUSSION
To investigate thefactors that areinvolved in the transcriptional regulation of silkmoth chorion genes, we have previously 1 2 3 4 5 6 7 8 9 1 0 characterized DNA-binding proteins, whose presence in follicFIG.5. The structural relationships between BmGATAP and ular cell nuclei correlates with the restricted expression of Hc BCFI. Panel A, electrophoretic analysis of extracts isolated from bacteria overexpressing the 18-kDaP-galactosidaselSmGATAP fusion pro- chorion genes (Skeikyand Iatrou,1991b). The major DNAbindtein (lane 3,arrowhead). Control lanes contain protein samples(5 1.18) ing activity identified, BCFI, was shown to recognize a DNA from untransformed bacteria(lane 1 ) or from bacteria transformed with sequence identical to that recognized by the GATA family of the expression vector alone (lane 2 ) . Panel B , bandshift assays using DNA-binding proteins (reviewed by Orkin (1992)). oligonucleotide I a s probe and protein extracts (E)from bacteria overThe fact that two functionally unrelated DNA binding factors expressing theP-galactosidaselSmGATAP fusion construct.Oligonucleotide I was incubated with no protein extract (lane 1 ) or with 5 pg of recognize identicalsequences does not automatically imply bacterial protein extract (lanes 2 4 ) in thepresence (+) or absence (-) of that thetwo proteins are structurally related. However, in the a 50-fold molar excess of competitor DNA (C). Competitor DNAs were case of the GATA family of transcription factors, the recent oligonucleotide I (I) or the HcA/B.12 promoter fragment (PI, respeccharacterization of representatives from fungi (Fu and Marztively. Control lanes 5 and 6 contain bandshift reactions performed with luff, 1990; Kudla et al., 1990), amphibians (Zon et al., 1991), oligonucleotide I and extracts of untransformed bacteria and bacteria transformed with pBluescript vector, respectively. Panel C, character- nematodes (Spieth et al., 19911, yeast (Cunningham and Cooization of the immune serum. Five pg of protein extractsfrom bacteria per, 1991), and plants (Baltz et al., 1992) has shown that all of overexpressing glutathione S-transferase (lanes 1 ) or the them contain a highly conserved, zinc finger-type DNA binding P-galactosidase/BmGATAP fusion protein (lanes 2 ) were loaded onto a 12% SDS-polyacrylamide gel electrophoresis. set A of samples (left)was motif, which recognizes the core sequence [A/T]GATA[G/A]. stained with Coomassie Brilliant Blue, while a second set (right) was Therefore, the DNA binding properties of all GATA-type protransferred onto a nylon membrane and incubated with the serum teins appear to havebeen conserved through evolution. Based (1/1000 dilution) of a rabbit immunized with the glutathione on these observations, we have hypothesized that factor BCFI S-transferaseBmGATAP fusion protein. The glutathione S-transferase of B. mori could be a member of the GATA family ofDNAprotein (27.5 kDa) and the P-ga1actosidaseBmGATAP fusion polypepbinding proteins. tide (18 kDa) are indicated by the solid and open arrowheads, respectively, and are theonly species reacting with the immune serum. Panel We have now identified two silkworm genomic sequences, D,Western blot analysis. Twenty pg of follicular (F)and silkgland ( S ) BmGATAa and BmGATAp, which contain the two C2C2zinc cell proteins were loaded onto a 15% SDS-polyacrylamide gel electrophoresis, transferred onto a nylon membrane, and incubated with the finger motifs characteristic of the GATA family of proteins. We immune serum (l/200 dilution) and’2sI-labeled protein A. A major 66kDa and a minor 50-kDa polypepetides (arrowheads)are recognized by the antiserum in follicular cell protein extracts. Panel E , supershift assay. Two pg aliquots of follicular nuclear protein extracts were incubated with the “‘P-labeled oligonucleotide I (1 ng, 10.000 cpm; lanes 2-10). The position ofthe DNAcomplex with factor BCFI is indicated by the solid arrow. Lane1 contains the labeledoligonucleotide I incubated in theabsence of extracts. Lanes 3 and 4 contain competition reactions
performed with a 20-fold (lane 3)or 50-fold (lane 4 ) molar excess of unlabeled oligonucleotide I. Lanes 6-9 contain 0.25 pl(lanes 6 and 8)or 0.5 pl (lanes 7 and 9 )of preimmune serum (lanes 6 and 7 ) or immune serum (lanes 8 and 9 )added to the reaction mixtures at the endof the incubation period. The open arrowheads indicate theslow supershifted complexes generated by the immune serum.
GATA ll-anscription Factors for Silkworm Chorion Genes
10666 CORSenSUS
GRECVN~hTATPL~GTGHYLQJACGLYH~~N~LIKPKRRLS~GTQQLNCOTSTTTL~~GDPV~AffiLYYKLH~LT~KEGIQTRNR
GATA-1
D
**
* * *
*
***
V
* **
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* ** *
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K MIISK LV
R
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* **
* ***
S
T
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L
L
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G
M GATA-I GATA-3
S
GATA-I
NS
E l t -1 BmGATM
D
T
VHN IH
S H
Q
I F
HHA
N
M
V
S
M
I
S
I
VLS
KPQN QK T
KQR
c
K P
E V
M T
T
E E
N
NGP
R N
GE H
F
K E
VQET
F
TAS
A R
K D DS
K
FIG.6. The DNA binding domains of the BmGATAp protein and othermembers of the GATA family. The DNA binding domainsof the known vertebrate GATA-type factors (human, mouse, chicken, and Xenopus GATA-1 (Zon et al., 1991); human, chicken,andxenopusGATA-2 (Zon et al., 1991); human, mouse, chicken, andXenopusGATA-3 (Zon et ai., 1991); and mouse GATA-4 (Arceci et at., 19931),the C. etegans Elt-1 protein (Spiethet al., 1991), and BmGATAP have been aligned withthe consensus sequenceof the GATA binding domain(Zon et at., 1991). Residues below of the GATA-I sequences, multiple substitutions thatoccur a t single sites the consensus sequence indicate amino acid substitutions. In the case are listed in senarate ~ ~ ~ s . ~ ~ e rbelow i s kthe s consensus indicate residues that areconserved in GATA-I, GATA-2, and GATA-3 but have diverged in at least one of the other sequencesthat are compared.
have focused our attention on the BmGATAp gene, because its tissue-specific expression is consistent with the distribution of the Hc chorion gene promoter binding factor BCFI that was investigated in a previous study (Skeiky and Iatrou, 1991b). Four lines of evidence suggest that theBmGATAp gene encodes factor BCFI. First, the BmGATAp gene is expressed in follicular and Bm5 tissue culture cells, the two cell types in which faetor BCFI is present. Second, a fl-galactosidas&mGATA/3 ftusion protein made in E. coli is able to bind specifically the BCFI target sequence in vitro. Third, a polyclonal antibody directed against the BmGATAp zinc finger domain recognizes the complex formed by factor BCFI present in follicular cell nuclear extracts and its target promoter sequence. Finally,the same antibody recognizes a 66-kDa protein, which is presentin follicular but not in silkgland cell protein extracts. Amino acid sequence comparisons between the zinc finger motifs of BmGATAp, the C. elegans GATA-type protein, Elt-1 (Spieth et al., 1991), and the vertebrate GATA-1,GATA-2, GATA-3, and GATA-4 subfamilies, which can be distinguished on the basis of conserved residues within their DNA binding domain (Zon et al., 1991; Arceciet al., 1993), revealed that the GATA-type factors can be classified into two groups (Fig. 6). The first one has as members factors GATA-1,GATA-2, and GATA-3. The second one encompasses Elt-1, BmGATAP, and GATA-4. It appears that the members of the first group are relatively recent offshoots of an ancestral GATA-typesequence. GATA-4, BmGATAp, and Elt-1, on the other hand, appear to have diverged from a progenitor GATA gene sequence to a similar extent. Interestingly, many of the amino acid substitutions that distinguish the two groups of sequences from each other have occurred at conserved positions; out of the 29 positions that distinguish the members of the first group from those of the second one(asterisks in Fig. 6), 50%(14129) are shared by at least two members o f the second group, while 20% (6/29) are shared by all of them. At this moment, we have no information regarding the site of expression of the BmGATAa gene. Considering, however, that BmGATAa encodes a GATA-type motif, it islikely that itsprotein will also prove to be a transcription factor. Although BmGATAa and BmGATAp were clearly shown to be encodedby single copy genes, low stringency hybridizations (not shown) have revealed the presence of additional, faintly hybridizing bands suggesting that, as is the case with mammals and birds (Ho et al., 1991;Yamamoto et al., 19901, a muftigene family of GATA-related sequences may also exist in the silkworm genome. This i s also supported by the immunological detection of the minor (50 kDa) polypeptide in follicular cell extracts.
In conclusion,this paperconstitutes the first report of GATAtype proteins in insects. As with other GATA-type factors isolated from a wide variety of organisms ranging from vertebrates to fungi, no sequence conservations with other known proteins have been found outside the DNA binding domain of the BmGATAfl protein. Considering that no clear conservation exists a t the level of the regulatory pathways in which the GATA factors are involved (see, for example, Ito et al. (1993) and Arceci et al. (1993)), it is likely that asi s the case with the homeodomain motif, the GATA domain will be foundto represent a DNA binding motif common to gene regulators serving a wide variety of functions. Acknowiedgments-We thank Dr. N. Spoerel for the A gtll follicular expression library, Drs. N. Mounier and J. C. Prudhomme for the B. mori actin A3 gene clone, Dr. L. Swevers for critical input, G. Mack for advice with the Western analysis, R. Kadonaga andN.Svelka for expert technical support, and the Forest Pest ManagementInstitute (Forestry Canada) for providing one of the silkworm strains used in this study. REFERENCES Areeci, R. J., King,A.A. J., Simon, M. C., Orkin, S . H., and Wilson, D. B.(1993)Mo.iot. Cell. Bid. 13, 2235-2246 Bailey, J. M., and Davidson, N. (1976)Anal. Biochem. 74 75-85 Baltz, R., Domon, C., Pillay,D. T. N., and S ~ i ~ e t z , A . ( l 9 9 2 ) PJ.~2,713-721 n~. Bock. S. C., nemeier, D. C., Mester, K., Wu, M., and Goldsmith, M. R.11982)Proc. Natl. Acad. Sci. U. S. A. 79, 1032-1036 Brown, N. H., and Kafatos, F.C. 119881J. Mol. Biol. 203,425-437 Burke, W. D., and Eickbush, T.H. (1986)J. Mol. Bioi. 190,343356 Chen, E. I., and Seeburg, P.H. (1985)DNA 4, 165-166 Cunningham, T.S., and Cooper, T.G . (1991)Mol. Cell. Biol. 11,6205-6215 Desplan, C., Theis, J., and O'Farrell, P.H.(1985)Nature 318,630435 Eickbush, T. H., and Kafatos, F. C.(1982)Cell 19,633-643 Fmbmann, M. A., Dush, M. K., and Martin, G . R. (1988) P m . Natl. Acad. sci. U. S. A. 85,8998-9002 Fu, Y.H., and Marzluff, G. A. (1990)Mol. Cell. Biol. 10, 1056-1065 Hannon. R.,Evans. T., Felsenfeld. G., and Gould, H. 11991)Proc. Natl. had.sei, U. S.'A. &, 300&-3008 Harlow, E., and Lane, D. (19883 in Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, New York Hibner, B. L.,Burke, W. D., Zeeanidou, R., Rodakis, G. C., and Eickbush, T.H. (2988)Deu. Bioi. 125,425-131 Ho. 1. C.. Vorhees. P. Marin. N.,Karuinski-Oakley,B., %ai, S . E, Orkin, S . H., and Leiden, J. M. (1991) EMbO J. IO, 1187-1192 Iatrou, K., and Tsitilou, S . G. (1983)EMBO J. 2, 1431-1440 Iatmu, K., Tsitilou, S. G., Goldsmith, M.R., and Kafatos, F. C. (1980) Cell 2% 654-669 Iatmu, K, Tsitilou, S. G., and Kafatos, F.C. (1982)J. Mol. Bid. 167.417-434 Iatrou. K., l'sitilou, S. G., and Kafatos, E C. (1984)Proc. NatL. A d . Sei. U. s. A. 81,.4452456 Ito, E., Toki, T.,Ishihara, H., Ohtani, H., Gu, L., Yokohama, M., Engel, J. D., and Yamamoto, M.(1993)Nature 562, 46-68 Jones, C. W., and Kafatos, F. C.(1980)Cell 22,855-867 Kafatos, F. C., Regier, J. C., Mazur, G. D., Nadel, M. R., Blau, H. M., Petri, W. H.. Wyman, A. R., Gelinas, G. E., Moore, P. B., Efetratiadis, A., Vournakis, J. N., Goldsmith, M, R., Hunsley, R., Baker, B., Nardi, J., and Koehler, M. (1977) in Results and problems in Cell Differentiation (Beennann, E., edf Vol. 8, pp. 45-145, Springer-Verlag, Berlin Koelle, M. R., Talbot, W. S., Segraves, W. A,, Bender, M. T.,Cherbas, P., and Hognew, D. S. (1991)Cell 67.59-77
GATA Panscription Factors for Silkworm Chorion Genes Kudla, B., Caddick, M. X.,Langdon, T., Martinez-Rossi, N. M.,Bennet, C. F., Sibley, S., Davies, R. W., and Arst, J. H. N. (1990) EMBO J. 9, 1355-1362 Mitsialis, S.A., Spoerel, N. A,, Leviten, M., and Kafatos, F. C. (1987) k .Natl. Acad. Sci. U.S. A. 84, 7987-7991 Orkin, S. H. (1992) Blood 80,575481 Rodakis, G . C., and Kafatos, F. C. (1982) Proc. Natl. Acad. Sci. U.S. A. 79,35513555 Rodakis, G. C., Moschonas, N. K., and Kafatos, E C. (1982) Mol. Cell. Biol. 2, 554-563 Rodakis, G . C., Lecanidou, R., and Eickbush, T.H. (1984)J. Mol. Euol. 20,265-273 Skeiky, Y.A. W., and Iatrou, K. (1987) J. Biol. Chern. 262, 6628-6636 Skeiky, Y.A. W., and Iatrou, K. (1990) J. Mol. Biol. 213, 53-66 Skeiky, Y.A. W.,and Iatrou, K. (1991a) J. Mol. Bid. 218,517427
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Skeiky, Y. A. W., and Iatrou, K. (1991b)Mol. Cell. Biol. 11, 1954-1964 Spieth, J., Shim, Y. H., Lea, K, Conrad, R., and Blumenthal, T.(1991) Mol. Cell. Biol. 11, 4651-4659 Spoerel, N. A,, Nguyen, H. T., Eickbush, T. H., and Kafatos, F. C. (1986) J. Mol. Biol. lSo,23-35 Swevers, L., and Iatrou, K. (1992) Deu. Biol. 160, 12-22 %ai, S.F., Strauss, E., and Orkin, S. H. (1991) Genes & Deu. 5, 919-931 Yamamoto, M., KO,L. J., Leonard, M. W., Beug, H., Orkin, S. H., and Engel, J. D. (1990) Genes & Deu. 4, 1650-1662 Zon, L. I., %ai, S. F.,Burgess, S.,Matsdaira, P., Bruns, G. A. P., and Orkin. S. H. (199O)Proc. Natl. Acad. Sci. U.S. A. 87,668-672 Zon, L. I., Mather, C., Burgess, S.,Bolce, M. E., Harland, R. M., and Orkin, S. H. (1991) Prm. Natl. Acad. Sci. U. S. A 10, 10642-10646
