Gone, 117 (1992) 193-200 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

193

GENE 06522

Structure and lens expression of the gene encoding chicken

pA3/Al-crystallin (Genomic sequence; murine; human; promoter; transcriptional regulation; primer extension)

J o a n B. M c D e r m o t t , Charlotte A. P e t e r s o n * a n d J o r a m Piatigorsky Laboratory of Molecular and Developmental Biology. National Eye Institute, National ,~nstitutes of Health. Bethesda. AID 20892 (U.S.A.)

Received by D.T. Denhardt: 13 December 1991; Revised/Accepted: 19 February/2 ! February 1992; Received at publishers: 31 March i992

SUMMARY

The ~A 1- and ~A3-crystallins are major polypeptides in the lenses of vertebrates. We present evidence that a single ~A3/A1 gone encodes these two proteins in the chicken. The ~A3/AI gene has been sequenced and its functional promoter identified in transfection experiments. The chicken ~A3/AI gone has the same structure as the human orthologue: six exons with standard splice sites and two alternative start codons from which the protein products are apparently translated. Northern analysis revealed an abundant 0.9-kb transcript in the lenses of l-2-day-old chickens and no detectable transcripts in the rest of the eye, brain, heart, kidney, fiver or skeletal muscle. The 5'-flanking sequence of the chicken ~A3/AI gene is very similar to that of the human and mouse genes, suggesting conservation of important putative regulatory sequences in addition to the TATA box. A thymidine-rich element (bp -218 to -163) and a potential AP-l-binding site (bp -264 to -258) are present within the chicken 5'-flanking region. A DNA fragment from -382 to + 22 of the chicken ~A3/A1 gene is sufficient to promote expression of the bacterial cat gene in transfected chicken primary lens epithelial cells, but not in transfected dermal fibroblasts. Moreover, the sequence from positions -382 to -143 of the chicken ~A3/AI promoter appears to be critical for proper transcription initiation and expression in the transfected lens cells.

INTRODUCTION

Crystallins are soluble proteins present in high concentration in eye lenses. There are four major classes of crysm

,

Correspondence to: Dr. J. Piatigorsky, N.I.H., Bldg. 6, Rm. 201, Bethesda, MD 20892 (U.S.A.) Tel. (30 I) 496-2764; Fax (301)402-078 !.

*Present address: University of Arkansas for Medical Sciences, Departments of Medicine and Biochemistry and Molecular Biology, Little Rock, AR 72205 (U.S.A.) Tel. (501)661-1202. Abbreviations: aa, amino acid(s); pA3/AI, pA3/Al-crystallin; ~A3/AI, gene encoding ~A3/AI; bp, base pair(s); CAT, chloramphenicoi acetyltransferase; cat, gone encoding CAT; kb, kilobase(s) or 1000bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; PLE, primary cultured lens epithelium; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/0.015 M Na3 .citrate pH 7.6; SSPE, 0.15 M NaCl/0.01 M NaH2PO4/0.001 M EDTA pH 7.0; tsp, transcription start point(s).

tallins in vertebrates: ~,/~, ? and b. In birds and reptiles almost all the y-crystatiins are absent and ~-crystallin, an enzyme crystallin, is hight~ expressed. Many other common enzymatic proteins also serve as crystallins in specific taxa (see Wistow and P~atigorsky, 1988). The speciesspecific refractive properties of lenses may be set up during development by the spatial regulation of expression of the crystallins. The/Lcrystallins represent approximately half of the soluble protein in adult chicken, as well as other vertebrate lenses; the BA3- and/~A 1-crystallin polypeptides (formerly B26 and/~19; Hejtmancik and Piatigorsky, 1983; Ostrer and Piatigorsky, 1980) are the most prevalent (Hejtmancik et al., 1985). Direct aa sequencing has shown that the ~A3-crystallin polypeptide is identical to the/~AI polypeptide with the exception of 17 N-terminal aa residues not present in/~A lcrystallin in bovine lenses (Berbers et al., 19~4). While the sequence of chicken pA3/A 1 cDNA showed that two start

194 codons are present at the appropriate positions for translation of both proteins from a single transcript (Peterson and Piatigorsky, 1986), Southern analysis raised the possibilities that either two functional genes or one functional gene and one pseudogene are present in the chicken genome (Hejtmancik and Piatigorsky, 1983). We show here that a polymorphism in the chicken genome explains the Southern-blot data, and we conclude that only one ~A3/AI gene exists in the chicken. We report the complete sequence of the chicken ~A3/A1 gene and find that its structure is similar to its human orthologue (Hogg et at., 1986). The ~A3/AI gene contains six exons: the first two exons each contain a start codon and encode the N-terminal arms of /~A3- and/~Al-crystallin polypeptides; the remaining four exons each encode one 'Greek key' structural motif (Blundell et ai., 1981) of these proteins. Extralenticular expression of the taxon-restricted enzyme crystallins has been demonstrated in many cases while the ubiquitous ~-,/~- and ~-crystallins were thought to be strictly lens-specific (Wistow and Piatigorsky, 1988; Piatigorsky, 1989; De Jong et al., 1989). Recent experiments have shown, however, that both 0cA-crystallin (Kato et al., 1991a) and ~B-crystallin are present in many different tissues (Dubin et al., 1989; Bhat and Nagineni, 1989; lwaki et al., 1989; Kato et al., 1991b), where they have an unknown function in normal and stressed cells (Ingolia and Craig, 1982; Klemenz et al., 1991). Transcripts coding for /~-crystallins, which may be distantly related to microbial stress proteins (Wistow, 1990), have also been detected outside the lens (Clayton et al., 1988; Head et al., 1991). In the present report detectable amounts of [3A3/AI mRNA were found only in the lens, where a single transcript was present at high levels. This together with other RNA and protein accumulation data for/IA3- and/1AIcrystallin (Ostrer et al., 1981; Hejtmancik et ai., 1985) suggest0" ,hat transcriptional regulation plays a major role in cc,v' ::hlg the expression of these proteins. We have thus bc;~,.~ ~, bttldy the putative regulatory sequences within the . -,:-r~,~,ngsequences of the chicken ~A3/A1 gene and show here that a promoter fragment from positions -382 to + 22 is sufficient to direct expression of the bacterial cat reporter ~:ene i,~ transfected primary cultures of embryonic chicken ien~ cgi~s but not in transfected dermal fibroblast cells. Conservation of selected regions of the 5'-flanking sequence of the chicken, mouse and human genes suggests the presence of several regulatory element ~ whtch can now be investigated in detail. RESULTS AND DISCUSSION

(a) Southern analysis Previous eDNA and protein sequence data on the bovine (Berbers et al., 1984), human (Hogg et al., 1986),

chicken and mouse (Peterson and Piatigorsky, 1986) ~A3and flA 1-crystallins have indicated that these two polypeptides are derived from a single mRNA by using separate AUG translation start codons. However, our earlier Southern-blot analysis of genomic DNA from White Leghorn chickens showed that both 5' and 3' eDNA probes hybridize to two bands ofBglI-digested DNA, suggesting that there might be separate genes for the flA3- and flAl-crystallin polypeptides (Hejtmancik and Piatigorsky, 1983). To determine whether the two Bgll bands hybridizing to the flA3/A1 eDNA could be due to restriction fragment polymorphism, DNA was isolated from individual chicken embryos, digested with Bgll and analyzed by Southern hybridization with a 600-bp Pstl fragment spanning exon 1. Individual samples produced either one or both bands; combining samples with single bands reproduced the double band pattern (Fig. 1). We conclude that a Bgll restriction site polymorphism exists in this chicken strain and that there is only one chicken flA3/AI gene. (b) Gene structure and sequence Two BamHI fragments of chicken genomic clone 3/~A3/ A1.160 were sequenced. The chicken pA3/AI gene has the same exon-intron structure reported for the human (Hogg et al., 1986) and mouse (Inana et al., 1983; Peterson and Piatigorsky, 1986) genes. The sequence (Fig. 2) of the coding region matches the repor'~ed chicken mRNA sequence

A

B

C

B+C

kb

23.1 9.4 m

6.64.4Fig. i, Southern hybridization of Bgll-restricted genomic DNA from individual chickens, Genomic DNA prepared from 7-day embryonic chicken bodies was extracted and precipitated by standard procedures. Samples were cleaved with Bgll, electrophoresed, and transferred to a nylon membrane by the Southern method, The membrane was hybridized overnight at 68°C in 6 x SSC/I.0% SDS with a 600-bp Pstl genomic fragment labeled with [ a-32P]dCTP using the Nick Translation Kit (U.S. Biochemical, Cleveland, OH). The membrane was washed in 3 x SSC/0.5% SDS at 68°C. Lanes: I, 10 pg DNA, individual A; 2 and 3, 5 pg DNA from individuals B and C, respectively; 4, 5 pg each from individuals B and C.

195

-896

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-796 -696 -$96 -496 -396 -296 -196

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-1498 -1396 -1296

-1196 -1096 -996

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GOC~GTCCCCATGGGCOOATGGAAGG~&TG2'CC~CGCA2qlCCC2GCG2C2CAGG~~C~~CC~~ P V P 14 G P W K C>Geee TGNIq: zGGe~G~L~CCC262v1~~ ' C A C C & G & G G 2 ~ C T G T A C C A ~ A G ~ C ~ C ~ A ~ ~&~A~A~¢~ T ~ &GGCAGAG~GGGC/t.CGGACCAGeAGGCACGAGAGC TATGAAGGCGG2~GI'GGGTA~A~ ~ ~ & ~ ~ A ~ ~ ACAS[GGC(:AG2TCA'~GTAGGGAAceAAGAGGGAGAGGAM~C&TAC&TAAG2GGGCAAA~C C C ~ A ~ ~ & ~ ¢ ~

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Fig. 2. Nucleotide sequence o f the chicken ~A3/AI gene. T w o 8 a m H l fragments derived from genomic clone 2~A3/AI. 160 (Peterson and PJatigorsky, 1986) were subcloned into pBiuescriptKS + (Stratagene, La Jolla, CA). The inserts in plasmids pJM3 and p J M 4 were sequenced using a series of exonuclease Ill digestion plasmids (Promega, Madison, W l ) and spec~c oligo primers. Sequence reactions were performed on double-stranded D N A with the Sequenase 2.0 kit (U.S. Biochemical, Cleveland, OH). T h e fragment assembly and D N A analysis programs o f the G C G package (Devereux et ,~l., 1984) were used for all sequence analysis. The BamHl and original EcoRl cloning sites are marked 'b' and 'e', respectively. The tsp is marked by a bent arrow; two overlapping polyadenylation signals are underlined. The translated sequence appears below the six exons; the two A U G start codons are hexed; an asterisk marks the stop codon. Silent nt substitutions from the reported c D N A sequence (Peterson and PiatJgorsky, 1986) at bp 2634, 2670 and 3520 are underlined. Potential Spl-binding sites at bp - 1086 and -892, a potential AP-l-binding site at bp - 264, a T-rich sequence from bp - 218 to -168 and the T A T A box are underlined. G e n B a n k accession No. M88460.

196 primer extension and S1 nuclease analysis (Hogg et al., 1986). Although Gorin and Horwitz (1984) tentatively placed the bovine cap site 33 bp upstream of the AUG based on primer extension analysis, we suggest that their eDNA clone pfl, which starts 5 bp upstream of the AUG codon, was a full-length eDNA and that all flA3/A1 transcripts contain an unusually short 5' untranslated region of about 5 bp. Some sequences of interest in the 5'-flanking region of the chicken ~A3/A1 gene are a long thymidine-rich (T-rich) tract between bp - 163 and -218, an AP-l-binding site at

(Peterson and Piatigorsky, 1986) except for three silent base substitutions. A total of six exons make up the coding region, the first two encoding an N-terminal arm and the last four each encoding one of the 'Greek key' structural motifs of the proteins (Fig. 2). The two AUG codons in the first two exons and the splice site positions are precisely conserved between all species. The tsp found by primer extension by Peterson and Piatigorsky (1986) for the mouse and chicken genes predict a 5' untranslated region of 5 bp, while that of the human gene was predicted to be between 3 and 7 bp on the basis of the tsp position determined by . .

CHICKEN - 4 0 5

C&CAGCAC&CGGGGC,qGGGGG&CCCGC'IW~AGCG&TGCTCCCCAGATGGTGCCG~~CCACC~CCC~C~C~CC

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-135

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. . . . GCCC~&TAAJ~.&GCTCO,GCCGCTG/~GT&GGC/~,GACIP,T G & C & ~ G ~ G ~ T A C C ~ C ~ A ~

Fig. 3. Comparison and analysis of" the 5'-flanking regions of the chicken, mouse and human ~A3/A! genes. The sequence for the mouse was obtained from the Ndel-BamHI fragment of genomic clone ,~M/]23Cr2 (Inana et al., 1983). Sequencing and computer analysis were performed as in Fig. 2. The mouse and human upstream sequences were aligned with the chicken upstream region used in the promoter functional analysis described in section d. The aligned regions were found using the Gap and Bestfit programs of the GCG package (Devereux et al., 1984). Identical nt are connected by vertical lines. Exon 1, the TATA box and regions of sequence similarity are boxed. The potential AP-l-binding site and T-rich element of the chicken are underlined. The polypurine stretches in Region A of the mouse and human are shaded.

197 -264 and Spl-binding sites at -891 and -1086. GenBank searches of the sequences surrounding the T-rich tract did not uncover a homologous stretch with any repetitive elements or pseudogenes. Stretches of T residues in the coding strand have been found to increase expression of several genes in yeast (Struhl, 1985; Lue et al., 1989; Lorch et al., 1990), especially in the presence of other activating elements further upstream. The AP-l-binding site (Lee et al., 1987) 50 bp upstream of the T-element is well positioned to affect transcription of this region. There are long T-rich tracts in comparable locations in the promoters of duck T-crystallin (Kim et al., 1991) and squid S-crystallin (Tomarev et al., 1992). These crystallins are expressed at very high levels in the lenses of their respective species, perhaps partly because the T-rich element in each is working in concert with other elements to increase transcription. Comparison of the chicken and human flA3/AI gene promoters revealed a highly conserved region (Region A, Fig. 3) of about 72 bp within 140 bp of the tsp. This striking similarity prompted us to sequence an NdeI-BamHI fragment of the mouse flA3/A 1 promoter from the genomic clone AMf123Cr2 (Inana et al., 1983). The three promoters contain a G + C-rich box and TATAAA sequence around nt positions -47 and -30, respectively, and Region A at position -141 in chicken, -126 in mouse, and -125 in human (Fig. 3). The sequence identity between Region A in chicken and mouse or human is 70% (51 of 73 bp), higher than the identity of exon 1 between chicken and mouse (507/o, 18/36); or chicken and human (53%, 19/36). Between mouse and human the conservation of Region A (86% identity, 62/72) approaches that of exon 1 (89%, 32/36). Thus, the sequence conservation of Region A is consistent with its having a role in expression of ltA3/AI (Fig. 3). Region A of the mouse and human contains a particularly purine-rich tract (shaded region). The available sequences of the mouse and human promoters upstream of Region A align well (Fig. 3), with the insertion of four gaps longer than 2 bp. The boxed regions show sequence identities of 73-79?/0, reflecting a lower rate of substitution than that found for introns or gene-flanking sequences of mouse and human, which generally share 63 % sequence identity (Soares et al., 1985; Aarts et al., 1989; Den Dunnen et al., 1989). The chicken sequence upstream of Region A shows no areas of relatedness to either mouse or human, which may reflect the distant evolutionary relationship between these species. (c) Northern analysis Hejtmancik etal. (1985) have shown that flA3/AI mRNAs are the dominant fl-crystallin mRNAs in the lenses of 19-day embryonic chickens. RNA samples were prepared from lens, the eye after lens removal, forebrain, hindbrain, heart, kidney, liver and skeletal thigh muscle of 1-

and 2-day-old chickens. A single intense ~AJ/A1 hybridization signal at about 920 bp was detectable in 1 #g lens RNA, but undetectable in 10 #g of RNA from other tissues (Fig. 4). In another experiment the lens hybridization band was still evident at 0.05 #g of RNA (not shown), indicating that ~A3/AI transcripts must be present at levels at least 200-fold higher in lenses than in other tissues at this stage of development. It remains possible that expression of this gene occurs in non-lens tissue either earlier or later in development than has been examined here. (d) Analysis of promoter function To identify a functional promoter of the flA3/A ! gene, we tested a series of[3A3/Al promoter/CAT plasmids in transfected primary lens epithelial (PLE) cells of embryonic chickens. Using PCR, we constructed plasmids containing chicken promoter sequences (bp - 382 to + 22 and -143 to + 22) between the BamHI and Xhol sites ofpBLCAT3 (Lubnow and Schutz, 1987) so that the flA3/Al-crystallin AUG codon was in frame with that of CAT. The larger fragment functioned as an active promoter in the transfected lens cells, as judged by the stimulation of CAT ac-



r-

e-

kb

9.5 --

7.5-

4.42.4-1.4-

-/~A3/Al-Crystallin

Fig. 4. Northern analysis of tissue RNA. Selected tissues were collected from 1- and 2-day-old chickens and homogenized in RNAzol B (Cinna/ Biotecx, Friendswood, TX) to isolate total RNA. Glyoxal-treated RNA (10pg from each non-lens tissue and 1 #g from the lens) was electrophoresed in a 1.2% agarose gel in 10 mM phosphate buffer, pH 6.5, essentially as in Thomas (1983), and transferred to ~ nylon membrane. A 400-bp ApaI-Kpnl flA3/AI eDNA fragment was labeled by nick translation and hybridized as in Fig. 1. The membrane was washed twice with 2 × SSPE/0.1% SDS at room temperature and twice with 0.1 x SSPE/ 0.1% SDS at 55°C. RNA kb ladder (Gihco BRL, Galthersburg, MD) on the left. The membrane was probed a second time with a 2.0-kb Pstl fragment of chicken [3-actin eDNA (Cleveland et al., 1980), showing the quality and relative loading of the RNA in each lane.

198 Relative CAT Activity

PLE -382

AP-1

T-rich

RegionA

TATA

Fibrobi~sts

cat

4.76~:1.74 0.594-0.25 -143 :.,.

0.76,0.32 0.604-0.12

Fig. 5. Relative CAT activity of lens and fibroblast cell extracts transfected with reporter plasmids driven by pA3/A 1 promoter fragments. Diagrammatic representations of the promoter elements are shown. Methods: Plasmid constructions: Reporter plasmids carrying the cat gene driven by a pA3/AI promoter fragment were made in pBLCAT3 (Labnow and Schutz, 1987) using PCR-generated fragments. Two genomic fragments were amplified (from bp - 382 to + 22 and from bp - 143 to + 22) from pJM4. The 5' oligos (5'-GGGGATCCGCTGCAGCGATGCTCCCCAGAT and 5'-TGGATCCGACCATGGGGATGAGCTGCTGAA, respectively) carried a BamHl site and the 3' reverse oligo (5'-GCTCCTCGAGACAGCTGCTTCGCCCATGTCAT) carried an Xhol site. Twenty cycles of amplification were performed using ,,he standard conditions described with the AmpliTaq kit (Perkin Elmer Cetus, Norwalk, CT) with l-min segments at 94°C, 55°C and 72°C. The amplified products were muitimerized before digestion with BamH! + Xhol. Isolated fragments were iigated into pBLCAT3 digested with Bam Hl + Xhol. Sequencing of the inserts in both directions confirmed that the/IA3/AI translation initiation was in frame with CAT and that the insert sequences were not mutated. Cell culture and transfection: Primary lens explants were plated from 12-day-old chicken embryos as previously described tBorras et al., 1988) except that lenses were trypsinized at 37°C for I min before and after disruption with a pipette, Eight lenses were plated per 60 mm dish in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS) and 50 pg/mi of gentamicin. Fibroblasts were prepared by trypsin digestion of dorsal skin in Hanks' F-10 medium for 30 min, disrupted with a pipette, pelleted with FBS and resuspended in the above culture medium. Three 60-mm dishes were plated per skin flap. Cells were washea and fed with the above medium 48 h after plating and transfeeted 3 or 4 h later. Each dish was cotransfected with 10/~g of test plasmid and 1 #g of pTB! (Borras ot al., 1988), an internal control for transfection efficiency which carries the lacZ gene driven by the Rous sarcoma virus LTR (long terminal repeat). Transfection was done by the Ca. phosphate precipitation method for adherent cells, without glycerol shock (Segal, 1986). The plates were washed twice and fed with the above medium 16 h later. Cells were washed twice with Dulbecco's phosphate-buffered saline 24 h later, collected from the dishes, lysed in 250 mM Tris. HCI, pH 7.5, by repeated freezing and thawing, and the extract supernatant collected for enzyme assay. Enzyme assays: ~-Galactosidase activity was measured for 5 pl extract in a total volume of 0.5 mi of 100 mM Na. phosphate, pH 7.5 (Nielsen et al., 1983). Cell extracts were heated to 65°C for 15 rain and assayed for CAT activity according to Neumann et al. (1987) with [~H]acetyl coenzyme A (New England Nuclear, Boston, MA). The rate of CAT activity was normalized to p-galactosidase activity for each dish. CAT activity was calculated relative to the promoterless vector pBLCAT3, set to 1.0. The average and standard deviation of the relative CAT activity of all dishes for each treatment are shown. For -382/CAT in lens, n = 17; in fibroblasts, n = 12. For -143/CAT in lens, n = 18; in fibroblasts, n = 6.

tivity over the promoterless vector. By contrast, cells transletted with the -143/CAT plasmid did not show higher CAT activity than the cells transfected with the promoterless plasmid (Fig. 5). Sequences between bp -382 and + 22 increased CAT activity about fivefold over background in PLE cells derived from chicken 12-day embryonic lens. No activity was detected in primary cultures of chicken embryonic dermal fibroblasts with any of the plasraids. Since promoter fragment bp - 382 to + 22 was active, whereas promoter fragment bp - 143 to + 22 was not (Fig. 5), some sequence(s) between bp - 3 8 2 and -143 must confer or enhance activity in lens cells. Primer extension analyses (Fig. 6) shows that transcription from the -382 to + 22 fragment initiates at two nt around the expected tsp, while transcription of -143 to + 22 initiates weakly at the TATA box. It is possible that the absence of CAT activity obtained from PLE cells transfected with the -143 to + 22 construct is due partly to translation from the longer transcript beginning at the start codon at + 1 (see Fig, 6), producing a polypeptide out of frame with the CAT enzyme, In any event, it appears that proper transcriptional initiation depends on sequences between bp - 382 and - 143. This area includes the potential AP-l-binding site at -264 and the T-rich element from bp - 163 to -218. The region from bp - 71 to -140 which shows conservation with the mouse and human genes is

therefore insufficient to direct proper transcriptional regulation. It should be underscored that neither the AP-1binding sequence nor the T-rich sequence is present in the mammalian ~A3/AI genes, and further studies are necessary in order to determine whether these sequences are important for transcriptional control of the chicken ~A3/A1 gene, Crystallin mRNAs and proteins accumulate to high levels in the lens, probably through several mechanisms including enhanced transcription, stabilization of mRNA, and inhibition of proteases. Whether the high transcriptional activity in the lens, which is the hallmark of crystallin expression, is due to a single transcription factor, group of factors, or special configuration of chromosomal DNA is unknown (see Piatigorsky and Zelenka, 1992). The only other jff-crystallin promoter studied to date is that of the chicken BBI gene (Roth et al., 1991). There is little sequence similarity between the pA3/AI and pBI promoters, and the ~A3/A1 promoter does not contain the functional elements which Roth et al. (1991) found to be important for transcription of the ~BI gene. Continuing analysis of crystallin transcriptional regulation may reveal some common mechanisms of control or demonstrate that different regulatory pathways are possible or even necessary !'~ creating the fine-tuned distribution of lens crystallins.

199

ae

I--

I--

eo

CJ

O ~

,~ L3

~

GATC nt

--103 2~

w

o.

8

--73 --72

(3) flA3MI transcripts are found at high levels in chicken lenses at the time of hatching. Northern blots failed to detect flA3/AI mRNA in the rest of the eye, brain, heart, kidney, liver and skeletal muscle, suggesting that little if any flA3/AI mRNA is present in these non-lens tissues. (4) A fragment from position - 3 8 2 to + 22 fused to bacterial cat gone is transcriptionally active in transient transfection assays in embryonic chicken lens cells and not in dermal fibroblasts. The sequence between -382 and -143 appears critical for proper initiation from and activity of the flA3/A1 promoter in the transfected lens cells.

ACKNOWLEDGEMENTS

We are grateful to Dr. Christina Sax for her help with the transfection experiments, and Dr. Peter Good and Ms. Barbara Norman for their advice and reagents throughout this work. We also thank the former and present members of this laboratory, especially Drs. John Klement, Robert Dubin and Cynthia Jaworski for many helpful discussions; Dr. J. Fielding Hejtmancik, who originally isolated the chicken flA3/A1 clones; and Mrs. Dawn Chicchirichi for secretarial assistance.

-4e 4, +8 I , I GGGCC&GCAGCGCCCTATAAAAGCTGAGCCGCTGAAGTAGGCAGA~GACATG Fig. 6. Primer extension of transfected and mock-transfected PLE cells. Primer extension reactions were performed essentially as in McKnight et al. (1981), using [ 732p]dATP end-labeled oligos to the antisense strand ofcat. About 106 cpm of probe was annealed at 65 °C for 5 min aad 50 °C for 15 min to RNA collected from transfected PLEs. Hybridized samples were incubated 45 rain at 42°C with 5 units AMV reverse transcriptase (Gibco BRL, Gaithersburg, MD) in 1.25 mM Tris (pH 8.3)/10 mM DTT/ ! mM dNTPs (U.S. Biochemical, Cleveland, OH)/8 mM MgCl:/20 units RNasin (Promega, Madison, WI). Samples were precipitated and resuspended in formamide/dye buffer and electrophoresed in 8% polyacrylamide/8 M urea sequencing gels. The same unlabeled oligo was used to sequence the antisense strand of the -382/pBLCAT3 vector by the dideoxynucleotide sequencing method as described in Fig. 2. GATC, antisense strand sequence; mock transfection and transfection with -382/ CAT, 30/~g RNA; transfection with -143/CAT and pBLCAT3, 40/~g RNA. Product sizes are indicated in nt in the right margin. The sense strand sequence is shown at the bottom. The expected tsp is indicated with an arrow, and sites corresponding to the major bands for -382/CAT and -143/CAT are boxed and marked with an asterisk, respectively. The minor bands at 144 nt and 175 nt for -382/CAT are unmar|:ed and may represent minor tsp for this construct.

(e) Conclusions (1) The chicken genome contains a single flA3/A1 gone containing a BglI restriction polymorphism site. (2) The structure of the chicken pA3/A 1 gone shows the same correspondence of protein motifs and exons as the human and mouse homologues. The promoters of these three species share a conserved 72-bp region which we call Region A.

REFERENCES Aarts, H.J.M., Jacobs, E.H.M., Van Wi,igen, G., Lubsen, H.H. and Schoenmakers, J.G.G.: Different evolution rates within the lensspecific p-crystallin gone family. J. Mol. Evol. 28 (1989) 313-321. Berbers, G.A.M., Hoekman, W.A., Bloemendahl, H., de Jong, W.W., Kleinschmidt, T. and Braunitzer, G.: Homology between the primary structures of the major bovine fl-crystailin chains. Eur. J. Biochem. 139 (1984) 467-479. Bhat, S.P. and Nagineni, C.N.: :cB subunit of lens-specific protein acrystallin is present in other ocular and non-ocular tissues. Biochem. Biophys. Res. Commun. 158 (1989) 319-325. Blundell, T.L., Lindley, P.F., Miller, L., Moss, D.S., Slingsby, C., Tickle, IJ., Turneli, W.G. and Wistow, G.J.: The molecular structure and stability of the eye lens: X-ray analysis of 7-crystallin !I. Nature 289 (1981) 771-777. Borras, T., Peterson, C.A. and Piatigorsky, J.: Evidence for positive and negative regulation in the promoter of the chicken bl-crystallin gone. Develop. Biol. 127 (1988) 209-219. Clayton, R.M., Head, M.W., Sedowofia, S.K.A. and Peter, A.: p-Crystallin transcription in embryonic chick retina cells. In: Piatigorsky, J., Shinohara, T. and Zelenka, P. (Ed~.), Molecular Biology of the Eye: Genes, Vision, and Ocular Disease. Alan R. Liss, New York, NY, 1988, pp. 239-247. Cleveland, D.W., Lopata, M.A., MacDonald, R.J., Cowan, N.J., Rutter, W.J. and Kirschner, M.W.: Number and evolutionary conservation of a- and fl-tubulin and cytoplasmic fl- and ~,-actin genes using specific cloned cDNA probes. Cell 20 (1980) 95-105. De Jong, W.W., Hendriks, W., Mulders, J.W.M. and Bloemendahl, H.: Evolution of e)e lens crystailins; the stress connection. Trends Biochem. Sci. 14 (1989) 365-368. Den Dunnen, J.T., Van Neck, J.W., Cremers, F.P.M., Lubsen, ~,TH. and

200 Schoenmakers, J.G.G.: Nucleotide sequence of the rat ?-crystallin gene region and comparison with an orthologous human region. Gene 78 (1989) 201-213. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Dubin, R.A., Wawrousek, E.F. and Piatigorsky, J.: Expression of the murine ,,B-crystallin gene is not restricted to the lens. Mol. Cell. Biol. 9 (1989) 1083-1091. Gorin, M.B. and Horwitz, J.: Cloning and characterization of a cow beta crystallin cDNA. Curr. Eye Re:,. 3 (1984) 939-948. Head, M.W., Peter, A. and Clayton, R.M.: Evidence for the extralenticular expression of members of the p-crystaUin gene family in the chick and a comparison with 6.crystallin during differentiation and transdifferentiation. Differentiation 68 (1991) 147-156. Hejtmancik, J.F. and Piatigorsky, J.: Diversity ofp-crystallin mRNAs of the chicken lens. J. Biol. Chem. 258 (1983) 3382-3387. Hejtmancik, J.F., Beebe, D.C., Ostrer, H. and Piatigorsky, J.: 6- and 8" CrystaUin mRNA levels in the embryonic and posthatched chicken lens: temporal and spatial changes during development. Develop. Biol. 109 (1985) 72-81, Hogg, D., Tsui, L.-C., Gorin, M. and Breitman, M.L.: Characterization of the human/).crystailin gene HupA3/AI reveals ancestral relationships among the/~7-crystallin superfamily. J. Biol. Chem. 261 (1986) ! 2420-12427. lnana, G., Piatigorsky, J., Norman, B., Slingsby, C. and Blundell, T.: Gene and protein structure of a fl-crystallin polypeptide in murine lens: relationship of exons and structural motifs. Nature 302 (1983) 310-315. Ingolia, T.D. and Craig, E.A.: Four small Drosophilaheat shock proteins are related to each other and to mammalian alpha-crystallin. Prec. Natl. Acad. Sci. USA 79 (1982) 2360-2364. lwaki, T., Kume-lwaki, A., Liem, R.K. and Goldman, J.E.: uB-crystallin is expressed in non-lenticular tissues and accumulates in Alexander's disease brain. Cell 57 (1989) 71-78. Kate, K., Shinohara, H., Kurobe, N., Goto, S., Inaguma, Y. a,~d Ohshima, K.: Immunoreactiv¢ aA crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. Biochim. Biophys. Acta 1080 (1991a) 173-180. Kate, K., Shinohara, H., Kurobe, N., Inaguma, Y., Shimizu, K. and Ohshima, K.: Tissue distribution and developmental profiles of immunoreactive aB crystallin in the rat determined with a sensitive immunoassay system. Biochim, Biophys. Acta 1074 (1991b) 201208. Kim, R.Y., Lietman, T., Piatigorsky, J, and Wistow, GJ.: Structure and expression of the duck a-enolase/¢-crystallin-encoding gene. Gene 103 (1991) 193-200. Klemenz, R., Frohli, E., Steiger, R.H., Schafer, R. and Aoyama, A.: aB-Crystallin is a small heat shock protein. Proc. Natl. Acad. Sei. USA 88 (1991) 3652-3656. Lee, W., Mitchell, P. and Tjian, R.: Purified transcription factor AP-I interacts with TPA-inducible enhancer elements. Cell 49 (1987) 741752. Lorch, Y., Lue, N.F. and Kornberg, R.D.: Interchangeable RNA polymerase i and II enhancers. Proc. Natl. Acad. Sci. USA 87 (1990) 8202-8206.

Lubnow, B. and Schutz, G.: CAT constructs with multiple unique restriction sites for the functional analysis of eucaryotic promoters '-.zzd regulatory elements. Nucleic Acids Res. 15 (1987) 5490. Lue, N.F., Buchman, A.R. and Kornberg, R.D.: Activation ofyeast RNA polymerase II transcription by a thymidine-rich upstream element in vitro. Prec. Natl. Acad. Sci. USA 86 (1989) 486-490. McKnight, S.L., Gavis, E.R. and Kingsbury, R.: Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell 25 (1981) 385-398. Neumann, J.R., Morency, C.A. and Russian, K.O.: A novel rapid assay for chloramphenicol acetyltransferase gene expression. BioTechniques 5 (1987) 444-447. Nielsen, D.A., Chou, J., Mackrell, A.J., Casadaban, M.J. and Steiner, D.F.: Expression of a preproinsulin-/]-galactosidase gene fusion in mammalian cells. Prec. Natl. Aead. Sci. USA 80 (1983) 5198-5202. Ostrer, H. and Piatigorsky, J.: p-Crystallins of the adult chicken lens: relatedness of the polypeptides and their aggregates. Exp. Eye Res. 30 (1980) 679-689. Ostrer, H., Beebe, D.C. and Piatigorsky, J.: p-Crystallin mRNAs: differential distribution in the developing chicken lens. Develop. Biol. 86 (1981) 403-408. Peterson, C.A. and Piatigorsky, J.: Preferential conservation of the globular domains of the/IA3/A l-crystallin polypeptide of the chicken eye lens. Gene 45 (1986) 139-147. Piatigorsky, J.: Lens crystallin and their genes: diversity and tissue-specific expression. FASEB J. 3 (1989) 1933-1940. Piatigorsky, J. and Zelenka, P.: Transcriptional regulation of crystallin genes: cis elements, trans-factors and signal transduction systems in the lens. In: Wassarman, P.M. (Ed.), Adv. Dev. Biochem., JAI Press, Greenwich, 1992, pp. 21 !-256. Roth, HJ., Des, G.C. and Piatigorsky, J.: Chicken ,0Bl-crystallin gene expression: presence of conserved functional polyomavirus enhancerlike and octamer binding-like promoter elements found in non-lens genes. Mol. Ceil. Biol. 11 (1991) 1488-1499. Segal, S.: Calcium phosphate transfection of nonadherent and adherent cells with purified plasmids. In: Davis, L.G., Dibner, M.D. and Battey, J.F. (Eds.), Basic Methods in Molecular Biology, Elsevier Science Publ., New York, 1986, pp. 286-289. Scares, M.B., Schon, E., Henderson, A., Karathanasis, S.K., Care, R., Zeidin, S., Chirgwin, J. and Efstratiadis, A.: RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon. Mol. Cell. Biol. 5 (1985) 2090-2103. Struhl, K.: Naturally occurring poly (dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Prec. Natl. Acad. Sci. USA 82 (1985) 8419-8423. Thomas, P.S.: Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Methods Enzymol. 100 (1983) 255-266. Tomarev, S.I., Zinovieva, R.D. and Piatigorsky, J.: Characterization of squid crystallin genes: comparison with mammalian glutathione Stransferase genes. J. Biol. Chem. 267 (1992) 8604-8612. Wistow, G.: Evolution of a protein superfamily: relationships between vertebrate lens erystallins and microorganism dormancy proteins. J. Mol. Evol. 30 (1990) 140-145. Wistow, G.J. and Piatigorsky, J.: Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu. Rev. Biothem. 57 (1988) 479-504.

A1-crystallin.

The beta A1- and beta A3-crystallins are major polypeptides in the lenses of vertebrates. We present evidence that a single beta A3/A1 gene encodes th...
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