Gene, 78 (1989) 225-233

Elsevier

225

GEN 03002

Structure of the bovine eye lens ys-crystallin gene (formerly /?s)

(Recombinant DNA; nomenclature; exon/intron distribution; cDNA-library; phylogenetic tree; chromosome

mapping)

Geert L.M. van Rensa, Jos M.H. Raats”, Huub P.C. DriessenC, Margot Oldenburg b, Juul T. Wijnen b, P. Meera Khanb, Wilfried W. de Jong” and Hans Bloemendal”

a Department of Biochemistry, University of Nijmegen, Ndmegen (The Netherlands): b Department of Human Genetics, Sylvius Laboratories, University of Leiden, Leiden (The Netherlands) Tel. (71)276090, and c Department of C~st~~Io~aphy, Birbeck College, London WCIE 7HX, (U.K.) Tef. (1)58~6622

Received by H. van Ormondt: 16 August 1988 Revised: 8 November 1988

Accepted: 13 February 1989

SUMMARY

The organization of a number of crystallin genes has already been resolved. One of the remaining genes of which the structure was hitherto unknown is the 7~ gene (formerly /is). We determined the complete sequence of the bovine ys-crystallin-coding gene, apart from the middle region of the first intron. Since it contains three exons and two introns, we conclude that the former &, also at the gene level is y-crystallin-like. However, it is located on chromosome 3, in contrast to other y genes which occur in tandem on the human c~omosome 2.

INTRODUCTION

In the mammalian lens there occur three major families of crystallins (a, /? and y). They comprise 80-90x of the water-soluble lens protein fraction and are immunologically distinct. a Crystallin con- sists of two types of subunits, whereas /?- and y-

C~~e~~~~e~ce to: Dr. H. Bloemendal, P.O. Box 9101,650O HB Nijmegen (The Netherlands) Tel. (80)514254; Fax. (80)553450.

Abbreviations: aa, amino acid(s); BDF, bovine dimeric repeat; bp, base pair(s); cDNA, DNA complementary to RNA; ys, gene (DNA) coding for ys crystallin; kb, 1000 bp; LMT, low melting temperature; nt, nucleotide(s); poly(A) + RNA, polyadenylated RNA; SDS, sodium dodecyl sulfate.

crystallins are composed of six to seven related pol~eptides (Bloemend~ et al., 1981; 1982; Piatigorsky, 1984; Wistow and Piatigorsky, 1988). The B and y crystallins form a superfamily of related proteins, which are apparently derived from a common ancestral gene (Driessen et al., 1981; Schoenmakers et al., 1984; Quax-Jeuken et al., 1985a).

The tertiary structures of some y crystahins have been elucidated by crystallographic analysis (Blundell et al., 1981; Wistow et al., 1983; Sergeev et al., 1987) and three-dimensional structure of /?-crystallins has been modeled from the structure of the y-crystallins (Wistow et al., 1981; Slingsby et al., 1988). The chains are folded into two extremely

0378-I 119/89/$03.50 0 1989 Bsevier Science Publishers B.V. (Biomedical Division)

226

similar domains, each consisting of two ‘Greek key’ motifs, suggesting the occurrence of two duplications of a primordial B/y-gene. The tertiary structure is directly related to the gene organization of the p- and y-crystallin superfamily. Each of the four predicted structural motifs of the ~-f~ily is encoded by a separate exon (Inana et al., 1983), whereas the y-crystallin domains comprising two motifs, are encoded by only one exon (Schoenmakers et al., 1984).

An exceptional member of this superfamily is ys (formerly ~Js). It is, like the other ‘J crystallins, a monomeric protein, in contrast to the @ crystallins which associate in various combinations to form low- or high-M, aggregates (Bloemendal, 1981; Piatigorsky, 198 1). The /J-crystallins have blocked N-terminal extensions of various lengths, which are probably involved in dimer formation or interaction with other polypeptides (Berbers et al., 1983; Slingsby et al., 1988). The absence of an N-teeing extension in y-crystallins, which have a free N-termi- nal aa residue, may therefore explain their mono- meric behavior. The former fis-crystallin has been designated as a P-crystallin because of its blocked N terminus and its iso-electric point, which is in the /Z-crystallin range (Van Dam, 1966).

The complete nucleotide sequence coding for bovine ys-crystallin has been determined from a cDNA clone (Quax-Jeuken et al., 1985a). It contains 177 aa residues, has a M, of 20 773 and a blocked N-terminal serine. Comparison of this sequence with /I- and y-crystallins, and construction of phylogenetic trees suggested that ys is more related to the monome~c y-crystallins than to the ohgomeric /3-crystallins.

To definitely determine whether ys indeed belongs to the y-crystallins rather than to the /J crystallins, or to refute this assumption, we decided to elucidate the organization of the ys gene. With this in mind we cloned for the first time a ys gene and performed a nucleotide sequence analysis.

It has been shown previousIy (Den Dunnen et al., 1985b) that the members of the human y-gene family are all located on chromosome 2. We now wanted to determine the chromosomal localization of the human ys gene to establish whether ys is linked to the y genes or is located on another chromosome, like the dispersed /I genes (Lubsen et al., 1988; Wistow and Piatigorsky, 1988).

MATERIALS AND METHODS

(a) Isolation of a full-length ys cDNA probe

Using poly(A) + RNA from polysomes obtained from the outer cortex of calf lens (Bloemend~ et al., 1966) we constructed an oligo(dT)-primed cDNA library as described by Huynh et al. (1985). Approxi- mately 8 x lo5 plaques were screened to obtain a full-length cDNA clone of ys. Screening was done according to the method of Church and Gilbert (1984).

(b) Primer extension

To determine the 5’ part of the mRNA, a primer extension was carried out with a 16-nt primer, based on cDNA sequences, which was supplied by Prof. J.H. van Boom (University of Leiden). Reactions were done as described by Qua-Jeuken et al. (1985a).

(c) Model building of ys crystallin

The structure of ys was modeled on 711 according to methods described elsewhere (Quax-Jeuken et al., 1985a).

(d) Construction and screening of a partial gene library

To isolate the ?/s gene a partial bovine gene library was constructed. An approx. IO-kb fraction of a complete BamHI digest of c~omosomal DNA was ligated into vector arms prepared as described by Loenen and Brammar (1980). For in vitro packaging of the recombinant phages a commercial kit (Amersham International, U.K.) was used. The infection was done on Escherichia cob K802, and the library was screened according to the method of Church and Gilbert (1984) with the cDNA probe ys I1 (See RESULTS AND DISCUSSION, SeCtiOn a).

(e) Construction of a physical map

A physical map of the y.r gene was determined after ligation into apUCl8 plasmid vector. Enzymes, as described in the legend to Fig. 3, were used according to the instructions of the supplier

221

(Boeh~nger-M~nh~~; BRL). Coding sequences were mapped by Southern blotting and hybridization with a nearly full-length cDNA probe of ys and two (5’ and 3’) PstI fragments of pBLBs as specified under RESULTS AND DISCUSSION, section a.

(f) Nucleotide sequence analysis

The cDNAs and the coding and flanking se- quences of the 7s gene were subcloned with the appropriate enzymes in M13mpl0, M13mpl1, M13mp18 or M13mp19. The chain-termination method (Sanger et al., 1980) was used to sequence the Ml3 inserts. Both strands were sequenced com- pletely. Computer programs were used to record, edit and translate the nucleotide sequences (Staden, 1977).

(g) Cell hybrids and genetic analysis

Hester-human somatic cell hybrids derived from fusions with the Chinese hamster line a3 or E36 (Quax et al., 1985), were used in the present study (see Table I, footnote b).

RESULTSANDDISCUSSION

(a) The complete sequence of the ys cDNA clone

A partial sequence of ys cDNA has been published previously (Quax-Jeuken et al., 1985a). Unfortu- nately this clone, named pBL@, lacked the sequence coding for the five N-terminal residues and all the 5’-noncoding sequences. To determine the complete sequence of the ys cDNA and to acquire a clone long enough to map the 5’-noncoding sequences in the gene, we constructed a cDNA library in lgtll (Huyng et al., 1985). The library was screened (Church and Gilbert, 1984) with the [a-32P]dATP- labeled 230-bp 5’ PstI fragment of the previous clone pBL/Is (Qua-Jeuken et al., 1985a). Several positive clones were isolated and sequenced. The longest one (~~10) comprised 12 bp corresponding to the first exon, the position of which was inferred by homology with the known p and y genes. From this clone a 5’ 93-bp EcoRI-RsaI fragment was isolated to rescreen the cDNA library. This enabled us to isolate a clone

(ysl 1) which contained, besides the sequence corre- sponding with the complete fust exon, a 5’-non- coding 39-nt stretch (nt 85-123; Fig. 4).

A primer-extension experiment was performed to determine the few 5’ nt still lacking in the ysll cDNA clone. The synthetic 16nt primer used was complements to the ys mRNA (Quax-Jeuken et al., 1985a). After hyb~dization and extension, nt 80-84 (see Fig. 4A) could be read on the autoradio- graph (Fig. 1). From the cap site downstream, the first 4 nt could not be read on the sequence ladder. They were deduced from the gene clone (RESULTS AND DISCUSSION, section d) to complete the cDNA sequence. The total length of the mRNA, excluding the poly(A) tail, is 658 nt. This is in good a~eement with the earlier estimated length of the ys mRNA on Northern blots (Quax-Jeuken et al., 1984).

:a -t aQQ

tC” aaa

Fig. 1. Autoradiograph of a bovine poly(A)+mRNA sequencing

gel. The 5’ sequence of the cDNA of ps was determined with

bovine mRNA (50 fig/ml) as a template and a synthetic 16 nt

primer (Quax-Jeuken et al., 1985a) complementary to the mRNA

sequence. The sequence ladder of the 5’ mRNA dideoxy A, G,

C and T reactions (a, g, c, t) is shown twice. The interpretation

is given on the right margin next to the sequence ladder.

228

Fig. 2. Stereo view of the complete computer-derived model of ys protein on the basis of the 1.6-A coordinates of bovine $1 crystallin using the program FRODO, as described previously (Jones, 1978).

(b) Predicted tertiary structure of pcrystallin

The sequence of ys can easily be modeled by computer-graphic methods into the four-motif struc- ture of yII-crystallin (Blundell et al., 1981; Wistow et al., 1981; Summers et al., 1984). In the core of ys the four t~ptoph~s occupy the same central posi- tions as in the $1 structure. A stereo view of the complete computer-graphics derived model of bovine ys, viewed approximately perpendicular to the plane containing the three pseudo two-fold axes relating the motifs and the domains, is shown in Fig. 2. The N-terminal domain is on the left. The N-terminal arm of four residues is shown in an extended form and is probably too short, compared with those of the oligomeric /I crystallms (four to 14 times as long) to have a significant effect on aggre- gation

(c) Isolation of the p gene

Soused-blot analysis of bovine genomic DNA with the yscDNA (ys 11; RESULTS AND DISCUSSION,

section a) revealed a single hybridization band after

digestion with BarnHI (not shown). This finding suggested that the complete sequence of the ys gene is located on a single chromosomal fragment approx. 10 kb long. The BumHI fraction of about 10 kb was electro-eluted from an LMT gel (Maniatis et al., 1982). This fraction was ligated in X47 vector arms and packaged as described under MATERIALS AND

METHODS, section d. Screening of 8 x lo5 recombi- nants was done with our l/s cDNA clone ys 11. Three positive clones were isolated, one of which carried an approx. lo-kb insert. The latter was subcloned in pUC18 and used for further characterization.

The insert of clone ysll, and a 5’ and 3’ P.stI fragment of pBL@ were used to identify the restric- tion enzyme fragments containing the coding se- quences of the #% gene which appeared to be 5.6 kb long (Fig. 3).

(d) Sequence analysis of the bovine ys gene

To answer the question whether the structure of the ys gene is more y- than ~-c~st~lin-Ike, its intron/exon organization was determined. In @ystallin-coding genes separate exons encode

H Sa X

6 EX H x St 7 Sm HSa St X 6 -

L__J 05 kb

Fig. 3. Physical map of the ys gene. The gene clone, of which the sites of some restriction enzymes are shown in the upper line, was ligated into pUC18 (wavy lines). Restriction endonuclease sites are abbreviated: B, BumHI; E,EcoRI; H, HindHI; Sa, MI; Sm, SmaI; St, &I; X, XM. Open bars in the lower line represent the sequenced noncodmg regions while blackened bars represent the coding sequences (see Fig. 4) which are numbered with Roman numerals.

229

TATA-box A tcctatctcatRaactaagcaaatgtttgatttgdaacca8ccca~tataaatgtctct~tECCtgtaactctctA~CA~CCT~ATTTCA~ACT~~AAACCA~CTAT~ACCA

10 20 30 40 50 60 70 80 90 100 110 120

fMiKAGTZ_ IRTRON 1

AAC~TCTAAACCTGCAACCAAAgtaagtaaatggtaaatggtcaCgct~atttctagcctacc~~tttcttttcacgtaatgatacattgaaagaaacattttgcttctttacct8~a~gtga~

130 140 150 160 170 180 190 200 210 220 230 240

ctcatagttagaaggctggatggttgaagaaaagatgaaggaattc------------------------------ _"__3.,5kb_-____--___________-----________-_

250 260 270 280

B tCCttggCCttgaaagaagtctcggtgggagagagaCa8ataaaaaagtgaaaa~tgtattaCdtdatgtggtaa5agaatg~ggaagCaga8aaggCaatggCaCCCCaCtCCa~a~tC

10 20 30 40 50 60 70 80 90 100 110 120

tt8CrtggaaaatCct8tg8aC8gagcctggtaggcttaagagtt88CtaC8aCtgagtgaCttCtttCaCttttCaCtttCat8Catttggagaa88aa

130 1 a0 150 160 170 180 190 200 210 220 230 240

atggcaacccactccagtgttcttgcctggagaatccagg8atgggggagcctggtggctgccatctat8gg8tcg~acggggtcg~acagagtcg~aca~ct8aagcgacttagcagc 250 260 270 280 290 300 310 320 330 340 350 360

agcagcaga8t8ag8aagaagccatgaacctgagtctgaaaataatggCttata8taga8CaCtCtgtttggCttCatCttaagccctcataaaa8tCCtCattCCtat8CtgaCaaC~aa

370 380 390 400 410 420 430 440 k50 460 470 480

agtgtgccagaaggcagatgtgaggtacagtagaactatatattctctcctcaaaatg~agggaaaaggaattcttaaagtgggaaataatgc~cttaattttcatttttctRtaagcgc

490 500 510 520 530 540 550 560 570 580 590 600

INTRON 1 7 \ITFFEDKNFQGRHXDSDCDCADFHM

agcca~ctcacccctcagtcCttttcttcgtttg~cctgta~tc~agATTACTT?CTTTGAAGACAAAAACTTTCAAG~CGCCACTATGACA~~TT~GACTGTGCAGATTTCCACAT

610 620 630 640 650 660 670 680 690 700 710 720

YLSRCNSIRVEGCTWAVYERPNFAGXMYILPRGEYPEYQH

GTACCTCACCCGCTGCAACTCCATCACACTCCAACGAAGGAG~ACCT~~TGTGTATGAAA~CCAATTTT~TGGGTACATGTACATCCTACCCCGGG~GAGTATCCTGAGTACCA~A

730 740 750 760 770 780 790 800 810 820 830 840

87 INTRON 2 WYGLNDRLSSCRAVHL/----

CTGGATCGCCCTCAACGACCGCCYCAGCTCCT~AGG~TGTTCACCT~gt8~gt~tgg~Rgtggtgga~t~t~t~t~t~t~~tt~~~~tt=~8ag~=~t~tt~gg~tttt~tgtaaagg

850 860 870 880 890 900 910 920 930 9'(0 950 960

~gttttcttaagattttcctaacctcttaatgggtggttggt~tcttgttggctgctgagacttcagt~aaetettaagatcagttcatcataagaccacacgatttatttccactttct

970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080

tta~c~~ttatatattttaaggaataaagttttccttactgaacgtcagctgttttagtcagtacacagttgegatgggagttcagatcatctcgcttteaagatggtgggtcctgaact

1090 1100 111s 1120 1130 1140 1150 1160 1170 1180 1190 1200

INTRON 2 88 -SSGGQYKL

g~t~t~ttctctgagttcctagcctgctijttcattttc~gt~~C~gttgct5tCttttC~~CttgttttCtgtgtttttttgtttttt~ttttC~~CTAGTGGAGGCCAGTATAA~T

1210 1220 1230 1240 1250 1?60 1270 1280 1290 1300 1310 1320

QIFEKGDFNGQHHETTEDCPSIMEQFHMREVHSCKVLEGA TCACATCTTTGACAAAGGCGATTTTAATGCTCAGATGCATCAGACCACCGAAGACTCCCCTTCCATCATCCAGCAGTTCCACAT~~A~TCCA~CCT~AA~T~TGGAGG~~

1330 13'io 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440

WIFYELPNYRC RQXLLDKKEYRKPVDUGAASPAVQSFRRI

CTGCATCTTCTATGACCTCCCCAACTACCCAGGCAGCCACTACCTCCTGCACAAGAACGAGTACCGGAACCCCGTCGACTCGCCTGCA~TCCCCAGCTCTCCAGTCT~CCCCCCCAT

1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560

177 V E stop-codon POly-A

TGTGGAG~TGATACAGATGCGGCCAAACGCTGCCTGGCCTTGTCATCCAAATAAGCATTATA~ACAATT~CAT~attccgctgtttactgtgacgcctctctttaaacacc

1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680

tt~aa~agggaccagtcaagagtgccctccacagcacgcagtcacataaagcaactccctcttcag5t~ccgttct~gtctttgtgccaaccaaaataggattgcattgaaaggata

1690 1700 1110 1720 1730 1740 1750 1160 1770 1780 1790 1800

g~c~~tcttact-gacatggtggcccttgttgcttaaggtctatgttaattcaccag5acagtcatcatagctRccg~tattgtgtgccttcattttcccatttctctt~caacagc

1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920

cctataagtgagggggtaggBg8C=gtgtctaattgc=tttc=c=a=t===t===ttg=8gg~g 1930 1940 1950 1960 1970 1980

Fig. 4. Complete coding sequences of the p gene and noncoding sequences flanking the exons. (A) Sequence of the first intron and adjacent nucleotides; (B) sequence of the second exon, second intron and the third intron together with flanking regions. Introns are marked by bent lines. Exonic regions of the gene starting at the cap site and ending at the poly(A) site, as well as the deduced amino acid sequence are given in capitals. The last digit of the numerals is aligned with the corresponding nt and given underneath the sequence. The numbering of aa is above the residues.

230

each of the four predicted motifs forming two domains in the protein, whereas in the y-crystallins the information for a domain is fused into one exon. The dideoxy chain-termination method, performed on recombinants of numerous random and site- directed restriction fragments (Messing and Vieira, 1982) of the y.r gene, enabled us to determine the sequence of both strands as depicted in Fig. 4. Com- parison of the gene sequences with the reported ys cDNA and amino acid sequence (Quax-Jeuken et al., 1985a) allowed us to locate the different exons, as shown in Figs. 3 and 4. It appears that the ys gene consists of three exons and two introns, of which the second one was sequenced to completion. For the first intron only the 5’ and 3’ sequences, flanking exons 1 and 2, were elucidated. The first exon

contains the start codon and the coding information for the lirst 6 aa of ys-crystallin as well as a 5’-non- coding sequence of 48 bp. The cap site was deduced from the primer extension experiment (see RESULTS AND DISCUSSION, section a). The second exon contains the information for aa residues 7-87. Finally, the third exon contains the coding informa- tion for aa 88-177 and the 3’-noncoding sequence of 73 bp. The coding sequences determined for the gene match for 100% with the cDNA sequence. Introns 1 and 2 are 4.25 kb and 0.4 kb long, respectively, and are exactly located at positions corresponding to those of the two introns in the y genes.

(e) Intron/exon distribution and junctions

From the exon/intron distribution one may con- clude that the gene coding for ys actually has the structure of a y-crystallin-coding gene. However, the length of the coding part of the first exon of ys differs from that of the y-crystallins. In the 7 genes this exon codes for 2 aa residues only, whereas the first exon of ys encodes 6 aa. Comparison of the cDNA se- quence of ps from carp (Chang and Chang, 1987) with the bovine p gene reveals that homology stops at the splice junction between the first and second exon. If the y.r gene of the carp has the same con- served intron/exon structure, its first exon encodes only 2 aa and is therefore more y-like in this respect as well.

There is a marked difference in length of the first intron of ys and those in the currently known ~-cryst~lin-~od~g genes (Lok et al., 1984; 1985;

Den Dunnen et al., 1985a; 1986; Meaking et al., 1987). All these y genes have a first intron of 85-l 10 bp, whereas the first intron of ys has the extreme length of 4.25 kb. As far as we know the intron contains at least one BDF repeat (Skowronski et al., 1984) from nt 89-368 (Fig. 4B). In ~~~stallins the first intron is more variable, ranging in length from 0.4 to 2 kb (Lubsen et al., 1988). All intron/exon junctions in the ;‘s gene obey the GT/AG consensus (Breathnach and Chambon, 1981; Mount, 1982; Padgett et al., 1986).

(f) Flanking regions of ys gene

In the 5’-flanking region at nt position 47 (Fig. 4A), a TATA box was found whose sequence is in accord with the published rule for TATA boxes (Breathnach and Chambon, 1981). Like in hamster and mouse orA-crystallin (King and Piatigorsky, 1983; Van den Heuvel et al., 1985) and the chicken 62-crystallin (Wistow and Piatigorsky, 1988) no plausible CAAT box (Dierks et al., 1983) could be found in the calf 7s gene. The conserved regions directly upstream from the TATA-box as described in tl-, p-, y- and b-crystallins (Thompson et al., 1987) were not found in the short 5’-flanking sequence of the ys gene. Important upstream sequences as observed in y gene promoter studies (Den Dunnen et al., 1986; Meaking et al., 1987) could not be com- pared because our clone lacked these sequences.

The transcription start point was determined by counting the number of nt derived from the primer extension experiment and was assigned to nt position 76 (Fig. 4A), 29 bp downstream from the TATA box. The polyadenylation signal AATAAA is situ- ated at position 1625 in Fig. 4B, 29 bp upstream from the poly(A) addition site.

(g) Chromosomal localization of the human ys gene

The rationale for the use of interspe~i~c somatic cell hybrids for genetic analysis of cloned gene sequences and chromosomal localization of the cor- responding human gene has adequately been exemplified earlier by Quax et al. (1985). Chinese hamster-human hybrids were found to be suitable for the assignment of the ys gene. Hybridization experi- ments were carried out with a 230-bp PstI fragment of pBL/Is or with a control probe for chromosome 3:

231

TABLE I

Distribution of human chromosome-specific DNA markers and CRYGS gene in Chinese hamster-human somatic cell hybrids a

Hybrids’ Chromosomes’ CRYGS”

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

MaYl + + + + + + + + + + + + MaY2 + + + + + + + + f + MaY5 + + + + + + + + + + + + MaY6 + + + + + + f + MaY7 + + + + + + + + + MeAl + + + + + + + + + + + MeBl + + + + + + + + + + + + + + + MaYll + + + + + + + + ++ + + + MaYl3.1 + + + + + NT + + + MaYl4 + f + + + + + + + + + + MaGl + + + + +r + + + MaG4 + + + + + + + + + + MaG14 + + + + + + + + + + + + + + + + + f MaR20 + + + + + + + + +++ + + + +

Chr./CRYGSd

+l-

-I+

% Discordantse 50 50 0 21 43 64 57 29 64 11 50 29 57 36 57 64 57 64 50 50 43 50 36

4 00 122222 2432243 3 123532

3 70 247627 83163465 854143

a CRYGS is the locus symbol proposed for the human gene coding for ys-crystallin, following the international system for gene

nomenclature (Shows et al, 1987). We use also an abbreviated p symbol.

b These 14 hybrid clones were derived from fusions involving two Chinese hamster (a3 and E36) cell lines and peripheral blood leukocyte

populations from five human individuals, Y, A, B, G and R, respectively. They resulted in five series designated as May, MeA, MeB,

MaG and MaR. The procedures used for their production, propagation, characterization and maintenance were described earlier

(Westerveld et al., 1971; Meera Khan, 1971). They were also employed in various other studies (Meera Khan et al., 1978; Quax-Jeuken

et al., 1985b). Molecular probes recognizing the following loci were used as chromosome-specific markers: 1 (DNFlSSl); 2 (APOB);

3 (D3S3); 4 (D4S.10); 5 (D5S37); 6 (C4); 7 (EGFR); 8 (PLAT); 9 (ABL); 10 (DlOZl); 11 (CALCl); 12 (Dl2Sl7; FIVWF); 13 (D13S7;

MYH2; Dl7S5); 14 (Dl4Sl); 15 (Dl5S2); 16 (Dl6S85); 17 (Dl7Sl; MYH2; Dl7S5); 18 (Dl8S6; Dl8S5); 19 (APOC2); 20 (ADA);

21 (D21S25); 22 (IGLV; D22Sl); X (DXS84; F9). We thank Drs. B. Carritt, SE. Humphries, R. White, J.F. Gusella, D.M. Kurnitt,

R. Fodde, A. Ulrich, J. Verheyen, G. Grosveld, P. Devilee, J.W.M. Hoppener, Y. Nakamura, F. Bernardi, T. Dryja, D.C. Page, D.R.

Higgs, C. Schwartz, P.L. Pearson, H. Tateishi, A.J. van der Eb, J.C. Kaplan and J.-L. Mandel for the relevant probes (see Pearson et al.,

1987).

L_ Only chromosome-specific DNA markers, were used to screen the hybrids. The symbol + means that the marker is present; NT,

not tested.

d The categories of discordants ( + / - indicates that the chromosome-specific DNA marker is present, CRYGS is absent; - / + indicates

the opposite) were counted separately.

e %D= Number of discordants (D) x loo

Number of hybrids scored (H)

Example (column for chromosome 1):

H = Total number of hybrids scored for chromosome 1 and CRYGS = 14

D = number of hybrids in which chromosome 1 and CRYGS were discordant is 4 + 3 = 7.

Thus, %D = i x 100 = h x 100 = 50 (see the equation above).

232

D3S3 (not shown). The data in Table I show an

absolute cosegregation of the human ?/‘s gene with

human chromosome 3 in a panel of 14 independent

clones of hybrids derived from four different fusion

experiments.

(h) Reconsideration of the /3s nomenclature and con-

clusions

We have previously proposed to rename Bs to yF

(Quax-Jeuken et al., 1985a) in view of its behavior on

a gel-filtration column, where it migrates in front of

the y crystallins (Van Dam, 1966). The correspond-

ing gene has three exons and two introns like all the

y-crystahins (Den Dunnen et al., 1985a,b; 1986;

Lok et al., 1984; Meaking et al., 1987). The de-

scribed mapping of the orthologous gene on the

human chromosome provided a difference with the

y-crystallins. The human y-crystallin-encoding genes

are assigned to chromosome 2 (Den Dunnen et al.,

1985b). Like mouse and rat y genes, they are

clustered on a single locus on the chromosome. As

the designation yF has recently been assigned to a

gene of the rat y-crystallin group (Lubsen et al.,

1988) it has been decided at the 8th International Eye

Congress (San Francisco, 1988) to choose the new

name ys. We have to agree with this decision.

Our present studies revealed that y.s had to be

assigned to chromosome 3. Hence it is not clustered

with the rest of the y genes on the same chromosome.

This confirms the data predicted by the phylogenetic

tree (Quax-Jeuken et al., 1985a) that p is an ancient

offshoot of the y-crystallin-coding genes. It diverged

after two duplications of a common ancestral gene.

After this event several duplications gave rise to the

present-day y gene family. In conclusion we propose

that ys is a product of a gene which became evolu-

tionarily separated from the y gene family, the latter

remaining closely linked.

ACKNOWLEDGEMENTS

We thank Rick Wansink for his contributions to

the sequence analysis, John Mulders for cooperation

in cDNA library construction and Jack Leunissen

for computer search assistance.

REFERENCES

Berbers, G.A.M., Hoekman, W.A., Bloemendal, H., De Jong,

W.W., Kleinschmidt, T. and Braunitzer, G.: Homology

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