Volume 6 number 11 1987

Current Eye Research

Nucleotide sequence for the cDNA of the bovine PB2 crystallin and assignment of the orthologous human locus to chromosome 22

David Hogg, Michael B.Gorin', Camilla Heinzmannz, Susan Zollman2, T.Mohandas3, Ivana Klisak2, Robert S. Sparkesz, Martin Breitman, Lap-Chee Tsui4 and Joseph Horwitz'

Mount Sinai Research Institute, Toronto, Ontario, Canada, 'Jules Stein Eye Institute, Los Angeles, CA and 2Department of Medicine, UCLA Center for Health Sciences, Los Angeles, CA, USA

Received on August 3, 1987; accepted on September 30, 1987

ABSTRACT We have identified and characterized two over- lapping bovine cDNA clones corresponding to the bovine crystallin pBp. The longer of the two clones, which contains the entire coding and 3' untranslated region a s well a s 54 nucleotides of the 5' untranslated sequence was used to identify and map an orthologous human gene, HupB2, to chromosome 22, q11.2-q12.2. As one other human p- crystallin, HupA3/AlI has been mapped to chromosome 17, our results indicate that, unlike the tightly linked cr cry stall ins, the human p-crystallins a re not syntenic within the genome.

INTRODUCTION The crystallins, which comprise greater than 90% of the water soluble protein of the eye lens, are thought to be critically important in maintaining lens transparency (1,2). In mammals, these polypeptides may be divided on the basis of immunological reactivity into three major classes: the a, p and 7-crystallins (3). A number of these proteins have been sequenced and their corres- ponding cDNAs or genes have been characterized (3). Mapping studies have localized the two human a- crystallins, CYA and aB, to chromosomes 21 and 16 respectively (4). In contrast to the dispersed a- crystallin family, the been shown to be tightly linked in man (5-7) and rat (8). In humans, six or seven highly homologous 7-crystallin genes (at least two of which are pseudogenes), have been shown to be clustered on the long arm of chromosome 2 (5,6), region q33-35

Less is known about the p-crystallins. This highly heterogenous group of proteins, ranging in molecular mass from 23 to 33 kilodaltons (Kd), comprises six to seven separate polypeptides in man, each of which bears a different N-terminal extension (9-17). The N-terminus has been thought

7-crystallin genes have

(7).

to determine the oligomerization or membrane binding properties of this group of crystallins (1,9,18). Furthermore, some members of this class, designated basic or pB crystallins, bear a C- terminal extension, while the acidic or pA crystallins do not (11).

Despite the considerable heterogeneity in the N-and C-terminal extensions of the 8-crystallins, their internal protein sequences are relatively conserved and show distant homology to the 7-

crystallins (12,13,19), a s do the gene structures of the two classes. Due to the greater hetero- geneity of the p-crystallins, we have suggested that they diverged earlier from a common progenitor sequence prior to the tandem duplication of the 7 -

crystallins (14,20). It is therefore possible that the p-crystallins will be found to be dispersed through the genome, in contrast to the tightly linked 7 family.

T o further our understanding of the p- crystallins we have been involved in the isolation and characterization of p-crystallin cDNAs and their corresponding genes. By using these a s probes we will be able to study the evolution of this gene family and investigate the role of p-crystallins in the genesis of familial cataracts. Here we report the isolation and sequence of a cDNA corresponding to the bovine p-crystallin pBp, and the assignment of the orthologous human gene, designated HupB2, to chromosome 22, q11.2-412.2.

MATERIALS AND METHODS The cDNA clones used in this study were isolated from the bovine cDNA library constructed by Gorin and Horowitz (21). Hybrid select translation was carried out essentially a s described (21). The cDNA

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0 IRL Press Limited, Oxford, England. 1335

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-1 1 -551 1 0 8 1 ACGG GCAC TCTC AGG GCTG GTGT CACT GGTC ACTC CTGC ACG GM CAG CCC GCC ATG GCC TCA GAT CAC CAG ACC CM GCG GGC AAG CCG

Met N a Ser Asp His G l n The G l n N a G l y L y s Pro

ttt ttt t t -t- t t tt) -t- --- ttt +tt --t tt t 1 tt) -- - I t I- -t --- ++++-I t 371 U G CCC CTC Mc CCC MG ATC ATC ATC TTT GAG CAG GAG Mc TTC CAG GGT CAC TCG CAT GAG CTC AAC Goo CCC TGC CCC AAC CTG AAG

G l n Pro Leu Assn P r o L y s 11. 11. 11. Phe G l u G l n G l u a n Oh. Gln G l y His Ser His G l u L.u Asn G l y Pro C y s Pro Aan Leu L y s

+-- I I I - I t t l t t l t I - +t I I+- I - ttt I t1 - tt I ttt tt t t l tt- -tt -t tt - t t I ttt -I ttt +-I 1 2 7 / GAG ACT CGC GTG GAG AAG GCG GGC TCC GTC CTG GTG CAG GCT GGA CCT TGG GTG GGC TAT GAG CAG GCC Mc TGC MA u;G WU; CAG TTC

G l u Thr G l y V a l G l u L y e N a G l y Ser V a l L.u V a l Cln Als G l y P r o T r p V a l G l y T y r G l u G l n N a Asan Cys L y s G l y G l u G l n Phe --- I I I -I- I -I- I -I- -I- -I- -1 - -I- --- - 1 - - 1 - --- . . . . , . . . . . 50 I

t ) t t+t t I I tt tt- ttt -- --- --+ t t l - -t- - t - -t- I I I -t- - tt+ t I t t-t ttt t 2 1 7 1 GTG TTT GAG AAG GGT GAG TAC CCC CGC TGG GAC Tco TGG ACC AGC U T CU; AGG ACG GAC TCG CTC AGC TCC CTG AGG CCC ATC A M GTG

V a l Phe G l u L y s G l y G l u Tyr Pro Arg ~ r p Asp Ser Trp Thr Ser Ser Arg Arg Thr Asp Set Leu Ser S e r Leu Arg Pro Ile L y s V a l --- ---

- - ttt t t l +-I t t t ttt -t t t l t t + - I tt I I - t+l - I 1 I-- --I ttt 1 t -+ - -- --- 3071 GAC AGC CAG GAG U T MG ATC ACC CTG TAT GAG AAC CCC Mc TTC ACG GGG MG MG ATG GAG GTG ATA GAC GAT GAC GTG CCC AGC TTC CU:

Asp Ser G l n G l u His L y e Ile Thr Leu T y r G l u Asn P r o Asn Phe Thr G l y L y e L y s Met G l u V a l Ile Asp Asp Asp V a l Pro Ser Phe H i 8 --- -I- - I - --- --- - 1 - ---

+-- ++I - - I I t1 +tl +-I ttt It- I I t1 ttl - tt t tt+ tt- (tt +t- -tt -t -tI --I t-t -- ttl I ttt t-I 4 0 0 1 W C CU: GGC TAC CAG GAG MG GTG TCT TCC GTG CGT GTG CAG AGC GGC ACG TGG GTT GGC TAC CAC TAC CCC GGC TAC C W GCG CTG CAG TAC

Ala His G l y T y r G l n G l u L y s V a l Ser S e r Val Arg Val G l n Ser G l y Thr T r p V a l G l y T y r G l n T y r Pro G l y T y r Arg G l y Leu G l n T y r --- - I - I I --- -I- I I -I- -I- -I- -I- I -I- --- --- --- --- --- --- - I - - 1 - ---

+I t- ++I II I +I- It- It1 I +I1 I -I +- + I I I Ill I1 I - + I ++I I 4 9 3 / CTG CTG GAG AAG GGC GAT TAC M G GAC AGC GGT GAC TTT GGG GCC CCC CAG CCC CAG GTG CAG TCC GTG CGC CGC ATC CGG GAC

L y s Asp Ser G l y Asp Phe G l y N s Pro G l n Pro G l n Val G l n Ser Val Arg Arg Ile Arg Asp Isu LEU G l u L y s G l y M p Tyr I

-- - - -- 5 7 7 / ATG CAG TGG CAC CAG COG GGC GCC TTC CAC CCC TCC AGC TAG TGC CCC TCC CTT GCC TCC TTC C U Goo GCT GGC CTC TGC CCA GCA TCC

Met Gln T r p His G l n Arg G l y N a Phe Ria Pro Sac 8.1

670/ U C CAG ACA TCC TGG AGA GTG AAT MA GTG TGA CTT GCA ACT T [POLY A - 7 4 1 [C-15]

0

Left margin - nucleotidm number: I. - >I indicates position 5' to AUG initiation 8ite "t" indicates position 3' to AUG initiation mite

Shaded boxes : Numbers indicate the amino acid residua (Xu) indicsta disparity of the &rived &no acid with

the d n o acid reported from p.pti& #*qu*nClng N and C terminal 8ewntm (non-corresponding) 0 llotifs 1 and 3 (corresponding)

mtifs 2 and 4 (correaponding) Unahaded box: 0 MTMA polyadanylation 6ipn.l sequence [G-81, [C-151 : Sequences generated during cDNA mynthesis and insertion into Pat1 site of pBR322.

Symbols . b o w nudeofid. mquencr: "-" Nucleotide homology with beta N - A 1 sequence "t" Nucleotide homology with Imta N - A 1 sequence and with

"I" Nucleotide homology with the corresponding motif the correaponding motif aequence.

sequence . Symbols below the &rived d n o acid sequence:

"---" lvliino acid homology with beta A3-A1 sequence "-+-" Amino acid homology with k t a N - A 1 sequence and with

the corresponding motif sequence " I " Amino acid homology with the corresponding motif

sequence . [ P o l y A - 7 4 1 : 74 nucleotides of 3' polyadenylate region

Figure 1. Sequence of cDNA of bovine crystallin pB2 (pBp).

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clones were either sequenced directly from the parent pBR322 vector, or subcloned into the vector pSP64 and then sequenced. All sequencing was performed using the Maxam-Gilbert technique (22). Predicted restriction endonuclease sites were confirmed by direct digestion.

cleaved genomic DNA isolated from a hybrid somatic mouse-human panel. Southern and Northern gel analyses were carried out using standard methods (14,231.

In situ hybridization to normal chromosomes followed typical protocols (24,25), using tritiated probes of specific activity of approximately 5 x lo7 cpm/pg. Slides were exposed for seven days and silver grains on or touching chromosomes were counted.

Hybridization was carried out with Hind III-

RESULTS The cDNA clones, 5C4 and llC8, were initially selected based upon the relative abundance of their respective mRNAs. These two clones were among a group of cDNA clones that gave a positive signal when hybridized with a labelled cDNA generated from total lens poly-A+ mRNA. In hybrid selection experiments these two cDNAs selected a mRNA which encodes a 25 to 30 Kd polypeptide. Neither of the cDNAs cross-hybridized with the previously described bovine p25/23 cDNA (21) (herein referred to a s pA3/A1) and hence they were further characterized.

Figure 1 shows the nucleotide sequence of clone llC8. This cDNA contains an open reading frame which encodes a protein of 205 amino acids with a calculated molecular mass of 23,317, a s well a s 54 basepairs (bp) of 5’ and 91 bp of 3’ untranslated sequences. A polyadenylation signal, AATAAA, present 73 bp downstream of the TAG termination codon i s itself followed 16 nucleotides later by a poly dA tail. In contrast, it is clear that 5C4 represents a partial cDNA lacking about

Comparison of the polypeptide sequence predicted by llC8 with previously described bovine p-crystallins revealed almost complete identity with the 8-crystallin pBp, hereafter called pB2, which had been characterized using protein sequencing methods by Driesen et a1 (13). Disparity between our data and that of Driesen et a1 is noted fo r amino acids #27, 31, 35, 36, 37 and 39 (see Fig.1). It is likely that the differences between the nucleotide-derived peptide sequence and the protein sequence derived by Driesen et a1 represent simple transpositional e r ro r s occurring at the time of amino acid sequencing. Though the overall amino acid composition remains unchanged, the observations of Driesen et a1 (13) comparing the

A B

28

18

Figure 2. Northern analysis of fetal lens and HeLa cell RNA. Total cellular RNA (10 ug) of fetal lens (lane A) and HeLa cells (lane B) was hybridized with labelled cDNA insert of the clone llC8. The positions of 285 and 18s RNA are noted.

250 bp of 5’ sequences present in llC8. Hence, after initial restriction endonuclease mapping and sequencing, further characterization of this clone was not performed.

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3.4

A B C D E F G H

Figure 3. Southern analysis of some representative mouse-human somatic cell hybrid lines. 10 ug of genomic DNA from each hybrid cell line or parental line digested with Hind 111 was hybridized with labelled cDNA insert of the clone llC8.

Lane A represents DNA from hybrid clone 84-30; lane B, 84-34; lane C, 84-35; lane D, 84-37; lane E, 84-38; lane F, 84-39; lane G, mouse parental DNA and lane H, human parental DNA. See Table 1 for a full description of hybrid lines.

crystallins require some alteration. Specifically, revision of the residues noted above increases the homology of the bovine p-crystallins pB2 with pB1 from 35% to 45% within the f i rs t motif, and from 33% to 40% within the f i rs t motif in the case of pB2 vs pA3/A1 (an acidic p-crystallin). The change from glutarnine to histidine in residue 31 requires one to postulate that all three bases of this codon have been altered in the evolution of bovine pB2 with respect to the bovine, rat and chicken pB1. Otherwise the revised sequence confirms and strengthens the observations regarding the stability and conservation of the p-crystallin family .

sequence of the bovine cDNA pllC8 with the human gene HupA3/Al reveals a high degree of homology between the 5’ regions of the sequences encoding motifs 2 and 4, a s shown in Fig. 1. Homology is also noted between nucleotide and predicted amino acid sequences in the 5’ ends of motifs 1 and 3,

Comparison of the nucleotide and amino acid

although not to the same degree. We were able to predict the exon-intron boundaries between motifs in pB2 by comparison with the HupA31Al gene structure (see Fig.1). Whether the C-terminus i s encoded by a separate exon is unknown at this time. Previous studies of crystallin structure (15,19) suggest that there may be a small discrepancy corresponding to two amino acids between the splice junctions and protein motifs.

Before using the bovine cDNA to map its presumed human counterpart, we f i rs t established that a lens-specific transcript homologous to the bovine cDNA sequences was present in the human eye lens. To this end, human fetal lens RNA was subjected to Northern analysis using the bovine cDNA a s probe. As shown in Fig.2, a transcript of about 800 bp is present in the eye lens but not in RNA extracted from HeLa cells. This confirms that a human gene orthologous to pB2 is transcriptionally active in the lens.

Chromosome mapping was performed by Southern

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Table I . Segregat ion o f t h e 6 C r y s t a l l i n Gene w i t h Human Chromosomes i n Mouse-Human Somatic C e l l Hybr ids

A. 6 C r y s t a l l i n B. Human Chromosomes Hybr id Clone 1 2 3 4 5 6 7 8 9 1 0 11 12 13 1 4 15 16 17 1 8 1 9 20 21 22 X Y

84-2 + + + - + + + + + - - + + - + + + + + + + - + - -

84-5

84-20

+ +

- + + - + + - t - - - - ( + ) t + - + - + - + + + + - ( + ) t + + - + t - - ( + ) ( + ) + + - - + + - + + + ( + ) -

84-26 + + - + + + + + + - + - + + + + + + + + + + + - +

84-30 + - - + + ( + ) t - - - + - ( + ) t + - - + + - ( + ) + + - - 84-34 + - - - + - + + t - + + - - + - - + - ( + ) + - t - - 84-37 + - - - + + + + (+) - - + + - t - - t - - + + + - - 84-38 + + - + + ( + ) + t + - + - + + + + - + t + ( + ) - + - -

84-3

84-4

+ + + + - + + + - + - - + + + - + t + + - - - -

+ + + t - + + + - + - - + + + - + + + + - - - - 84-7 - - - + + - + + + - - + + - + - + + - - + - - - -

84-1 3

84-21

84-25

84-27

84-35

84-39

No. o f Discordant Hybr ids 7 8 9 6 3 8 8 8 8 9 7 8 6 5 1 0 7 9 8 5 7 7 0 6 7

A.

B. Assignment o f human chromosomes t o mouse-human somatic c e l l hybr ids .

Resu l ts o f Southern h y b r i d i z a t i o n o f mouse-human somatic c e l l h y b r i d s u s i n g t h e bov ine cDNA as probe. I + ' and ' - I i n d i c a t e t h e presence o r absence o f human 6 - c r y s t a l l i n sequences.

o f t h e human chromosome i n g r e a t e r t h a n 30% o f metaphases analyzed, '(t)' i t s presence i n 10-30% o f metaphases and ' - I i t s absence.

' + I i n d i c a t e s presence

analysis of human-mouse hybrid cell lines and by hybridizing bands may thus be due to restriction direct in situ hybridization of human chromosomes. Clone llC8 detected a single band of 7.0 kilobases (Kb) in mouse DNA digested with Hind 111; in human DNA a strongly hybridizing band was present at 3.4 Kb with weaker bands at 9.6, 7.0 and 4.8 Kb. A s demonstrated in Fig.3, the human bands (with the exception of the 7.0 band, which i s presumably hidden under the strong 7.0 mouse band) segregate together in the human-mouse hybrids. The weakly

endonuclease cleavage within a single gene, or may represent one or more homologous sequences syntenic to the gB2 gene. Use of several other restriction endonucleases failed to provide additional information (data not shown). Recent cloning studies have in fact established the existence of two human pB2 genes (26); our data suggest, therefore, that these two genes are linked.

Analysis of the discordancy of the 3.4, 4.8

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5-

q

10 i51

20 -

15-

i o - 5 -

II I I I 111 I I I 1111 I I l l I I I 1 I I I 1 I I P q P q P q P q P q P q P q P

13 14 15 16 17 18 19 20 21 22 X

CHROMOSOMES

Figure 4. Histogram of in situ hybridization results. A sharp peak is noted.

and 9.0 Kb bands in the panel of mouse-human hybrids allowed assignment of the orthologous human genes to chromosome 22 (see Table 1).

The histogram corresponding to the in situ hybridization studies (Fig.4) confirms the assignment of the HupB2 genes to chromosome 22 without suggestion of another locus on a second chromosome. Figure 5 shows the detailed hybridization to chromosome 22 and allows sublocalization of the gene to qll.2-12.2. While the possibility of more than one locus in this region cannot be excluded by these studies, the data a re again consistent with linkage of the two pB2 genes (26).

DISCUSSION The localization of the human p-crystallin gB2 genes to chromosome 22, qll.2-12.2 establishes that the p-crystallins a re not clustered in the genome.

Figure 5. Diagram of detailed in situmapping of HupE2. Assignment of the gene i s to q11.2-12.2.

The pB2 genes, however, appear to be linked, suggesting that they may have undergone a duplication event following the dispersion of the

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8-gene family. It is not possible to determine at this time if both genes are transcriptionally active. Further studies are required to extend our understanding of this locus.

The identification of a new probe and the mapping of the human pB2 genes expands our ability to examine the crystallin genes a s possible sites for mutations which would lead to congenital cataracts. There a re undoubtedly multiple loci responsible for the many forms of inherited cataracts which have been described (27). Genetic linkage of inherited cataracts has been found with the Duffy blood group on chromosome 1 in one family (28,29) and to the haptoglobin locus on the long arm of chromosome 16 in a family with autosomal dominant posterior polar cataracts (30). More recently, close genetic linkage has been found between a dominant hereditary cataract and the human 7-crystallin gene cluster on chromosome 24 (31). However, a direct association between a specific crystallin gene defect and cataract has remained elusive. probes for the 8-crystallin we have described here provide important tools for investigating the molecular basis of cataracts.

The development of molecular

ACKNOWLEDGEMENTS D.H. is a research fellow of the Medical Research Council of Canada.

CORRESPONDING AUTHOR: Michael B. Gorin, M.D. Ph.D., Clinical Branch of Ophthalmic Genetics, National Eye Institute - N.I.H. Building 10, Room lON226, Bethesda, MD 20892

3Division of Medical Genetics, Harbor-UCLA Medical Center, Torrance, CA, USA and 4Hospital for Sick Children, Toronto, Ontario, Canada

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den Dunnen J.T., Moormann, R.J.M., and Schoenmakers, J.G.G. (1985) Rat p-crystallins are internally duplicated and homologous to 7- crystallins. Bioch. et Biophys. Acta. - 824,

Driessen, H.P.C., Herbrink, P., Bloemendal, H. and de Jong, W.W. (1981) Primary structure of the bovine p-crystallin Bp chain. Eur. J.Biochem. 121, 83-91. Hogg, D., l'sui, L.-C., Gorin, M., and Breitman, M. (1986) Characterization of the Human 8-crystallin Gene HupA3/Al Reveals Ancestral Relationships among the 8 7- crystallin Superfamily. J. Biol. Chem., 261, -

Wistow, G., Slingsby, C., Blundell, T., Driessen, H., De Jong, W. and Bloemendal, H. (1981) Eye Proteins, The Three-Dimensional Structure of 8-crystallin Predicted from Monomeric 7-crystallin. FEBS Letters, - 133, 9- 16. Hejtmancik, J.F., Thompson, M.A., and Piatigorsky, J.( 1986) cDNA and Deduced Protein Sequence for the pB1-Crystallin Polypeptide of the Chicken Lens. J. Biol. Chem., 261, 982-987. P e t e r s 0 n i . A . and Piatigorsky, J. (1986)

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B'likndell, T . , Lindley, P., Miller, L., Moss, D., Slingsby, C., Tickle, I., Turnell, B. and Wistow, G. (1981) The Molecular Structure and Stability of the Eye Lens: X-Ray analysis of 7-crystallin 11. Nature, 289, 771-777. Meakin, S. ,Breitman, r a n d Tsui, L.C. (1985) Structural and evolutionary relationships among five members of the human 7-crystallin family. Mol. Cell. Biol. 5, 1408-1414. Gorin, M.B. and Horwitz, J. (1984) Cloning and Characterization of a Cow p-Crystallin cDNA. Current Eye Res., 3, 939-948. Maxam, A.M. and Gilbert, W. (1980) A new method for sequencing DNA. Methods Enzymol.

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