THEJOURNAL OF BIOLOGICAL

CHEMISTRY Vol. 259,No. 15,Issue of August 10,pp. 9821-9825,1984

Printed in U.S.A.

The Chicken &Crystallin Gene Family

TWO GENES OF SIMILAR STRUCTURE IN CLOSE CHROMOSOMAL APPROXIMATION’

(Received for publication, February 13, 1984)

James W. Hawkins, JohnM. Nickerson, MargeryA. Sullivan$, and Joram Piatigorsky

From the Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health,
Bethesda, Maryland 20205 and SGenex Corporation, Rockville, Maryland 20852

&Crystallinis a major protein product of the differ- 13-15 intervening sequences (17, 19, 20). A brief report has
entiated chicken lens. We have isolatedtwo, non-allelic indicated a close linkage of the two &crystallin genes (21).
&crystallingenes using a recombinant bacteriophage/ In thisreport, we provide evidence for the existence of only
chicken genomicDNA library. There appear be to only one &crystallin gene locus for the chicken. This locus contains
thesetwo &crystallin genes inthehaploidchicken two non-allelic b-crystallin genes oriented with a similar 5’-
genome. Southern hybridization and R-loop analyses 3’ polarity on the chromosome and separated by approxi-
indicate that the two genes are oriented on the chro- mately 4.2 kb2of DNA. Electron microscropic analysis reveals
mosome with similar5‘-3’polarity. 6 1 , arbitrarily des- at least 17 exons and 16 introns in each &crystallin gene.
ignated as the directionally5’ of the twogenes, is 6.7 We have isolated the chicken &crystallin locus from three
kilobases in length,while 6 2 is 9.2 kilobases. The two recombinant bacteriophage clones, screened from a chicken
b-crystallin genes are about 4.2 kilobases apart. Struc- genomic library. The bacteriophages gXbCr61,gXSCr65, and
turally, bothgenes are arranged in a similar and char-
acteristic pattern of 17 exons/l6 introns, as judged by gXSCr54 are overlapping. gX6Cr61 contains one gene desig-
electron microscopy. The &crystallin gene locus rep- nated 61. gX6Cr65 contains the 3’ end of 61 and the 5’ end of
resents a simple model for the study of structural co- a second gene, designated 62. gX6Cr54 contains 62. We have
evolution and/or functional co-expression of two re- also cloned several kilobases of chromosomal DNA flanking
lated geneswithin a developmentally modulated regionthe 6 1 and 62 genes.
of the genome. Our isolation and initial characterization of the d-crystallin
gene locus will facilitate detailed structural and functional
investigations pertaining to the specialization (6, 14) and
specificity (22) of b-crystallin synthesis in the chicken lens.
The crystallins comprise the major group of structural
proteins found in the vertebrate lens (1-3). There are four EXPERIMENTALPROCEDURES
immunologically distinct crystallin families ( a , p, y, and 6 ) ,
each having a particular spatial and temporal expression in Hybridization Probes-Probes used in all experiments have been
previously described in detail. The full-length &crystallin cDNA clone
the developing tissue (4-6). p8Cr17 (13) was used to screen the chicken genomic library, and 8-
&Crystallin is found only in birds and reptiles (3, 7). In the crystallin clones P1181 (16), P1188 (16), and p6Crl7 were employed
chicken, it comprises 60-80% of the protein in the embryonic for DNA blot hybridizations. Heteroduplex experiments were carried
lens and about 50% of the protein in the adult lens (8-10). out with pbCrl7.
Chicken &crystallin is a noncovalently associated poly- Screening of Phage Genomic Library-The chicken genomic library
meric protein. Typically, at least two very similar polypeptide employed for this study was generously provided by Dr. J. D. Engel
subunits combine in various assortments to compose tetra- and colleagues (23). This library was constructed from the erythrocyte
DNA of a single White Leghorn chicken. Purified DNA was partially
mers (11).Early studies, employing sodium dodecyl sulfate- digested with H a d 1 and AluI and cloned into the bacteriophage X
polyacrylamide gel electrophoresis, determined the 6-crystal- Charon 4A using synthetic EcoRI linkers. The library was screened
lin polypeptide molecular weights to be about 48,000 and at a density of 1.0 X lo4 plaques/plate by the procedure of Benton
50,000 (12). Recent cDNA sequence analysis is consistent and Davis (24).
with the presence of a b-crystallin polypeptide with a molec- DNA Blot Hybridization-Phage DNA (1.0 pg) or genomic DNA
ular weight near 48,000 (13).’ (10 pg) was digested with EcoRI. Digested DNAs were size fraction-
ated in 0.9% agarose gels using 40 mM Tris (pH 8.2), 20 mM sodium
A considerable amount of work has been directed toward acetate, 18 mM NaCl, and 2 mM EDTA as a buffer or in 5% polyac-
the study of d-crystallin nucleic acids (14). A number of 6- rylamide gels using 90 mM Tris (pH 8.4), 90 mM boric acid, and 2.4
crystallin cDNAs have been constructed for analysis (15-18), mM EDTA as a buffer. The size-fractionated DNAs were denatured,
including a full-length cDNA (13). transferred to nitrocellulose filters (25), and hybridized to nick-
Efforts to isolate &crystallin genes have yielded positive translated (26) 3ZP-labeledprobes (1.0 X lo8 cpm/pg) at 68 “C in
results. Several fragments of two non-allelic d-crystallin genes Denhardt’s solution and 6 X SSC. After hybridization, filters were
washed three times in 2 X SSC at 68 “C for a total of 3 h.
have been obtained (19, 20), and an intact b-crystallin gene Source of Chicken Genomic DNA-DNA referred to as SPAFAS
has been isolated (17). Cross-hybridization studies indicate was obtained from the pooled erythrocytes of adult inbred gs- White
that these genes are very similar (19). Both chicken 6-crys- Leghorn chickens supplied by SPAFAS Inc., Lancaster, PA. DNA
tallin genes are highly interrupted with initial estimates of referred to as USDA 6 3 was obtained from pooled, 6-12-day-old,
White Leghorn headless embryos of the extensively inbred chickens
* The costs of publication of this article were defrayed in part by supplied by the United States Department of Agriculture, East Lan-
the payment of page charges. This article must therefore be hereby sing, MI. DNA referred to as Truslow was obtained from pooled, 15-
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. * The abbreviations used are: kb, kilobase; Tricine, N-[2-hydroxy-
’ K. Yasuda and T. S. Okada, personal communication. 1,l-bis(hydroxymethyl)ethyl]-glycine.

9821

This is an Open Access article under the CC BY license.

9822 &Crystallin Genes
day-old, White Leghorn headless embryos of commercially available These data indicate the presence of a chicken 6-crystallin
chickens supplied by TNS~OW Farms, Chestertown, MD. Truslowstructural gene locus contained within a 31.5-kb stretch of
chickens are maintained as sequestered flocks for approximately 5
genomic DNA.This region maybe characterized as containing
years.
Heteroduplex Analysis-Highly purified recombinant phage DNA contiguous EcoRI fragments ordered as follows: >9.8,0.7,3.8,
5.4, 3.1, and >8.7 kb (Fig. 2). The possibility of very small
and cDNA (2 pg/ml) were mixed in a 1:l ratio in a solution of 100
mM Tricine (Boehringer Mannheim) buffer (pH 8.0),500 mM NaCI, (>20 bases) EcoRI fragments existing within these recombi-
nant genomic DNAs is unlikely, as judged by electrophoresis
and 50% formamide. This solution was heated to 85 "C for 5 min and
hybridized a t 52 "C for 6 h before analysis (19). Photographs of the
in a 5%polyacrylamide gel.
hybridized nucleic acids were measured with a Numonics digitizer. Orientation of 6-Crystallin-coding Sequences-In order to
R-loop Analysis-Highly purified recombinant phage DNA was
establish the minimum number of &crystallin genes and their
hybridized to wild-type X Charon 4A phage DNA in a 1:l ratio under
transcriptional polarity upon the EcoRI fragments of the 6-
conditions described for heteroduplex analysis (19). Incubation a t
52 "C was carried out for 2 h, and then poly(A) mRNA from lensescrystallin locus, blot hybridization analysis was undertaken.
Recombinant phage EcoRI fragments were hybridized sepa-
of 15-day-old chick embryos (13) was introduced in a 200-fold excess.
This solution was brought to a 70% formamide concentration and rately with the following three 6-crystallin cDNAprobes:
allowed to continue hybridization a t 37 "C for 6 h before analysis
P1181, containing a 3' sequence extending from the poly(A)
(19). tail; P1188, a clone contiguous to P1181 and extending farther
RESULTS in a 5' direction; and p6Cr17, a full-length cDNA containing
all elements of P1181 and P1188, with a complete 5' sequence.
Isolation of &-Crystallin GenomicClones-Approximately Hybridization results depicted in Fig. 1are consistent with
1.2 X IO6 bacteriophage plaques were screened from the ge- the interpretation that there aretwo &crystallin genes within
nomic library using the full-length 6-crystallin cDNA probe this locus with the polarities depicted in Fig. 2.
p6Cr17. This screening yielded 13 positive clones. Restriction Blot Hybridization of Chicken Genomic DNA-In order to
mapping identified six clones with a unique genomic structure. investigate the organization of the 6-crystallin locus within
Three clones representative of the 6-crystallin locus (gX6Cr61, native chicken chromosomal DNA, blot hybridization was
gX6Cr65, and gX6Cr54) were analyzed in detail as follows. carried out on the genomic DNAof three different strains of
Size and Ordering of Genomic EcoRI Fragments-In order chicken. The full-length 6-crystallin cDNA probe p6Cr17 was
to determine the size, order, and relative overlap of genomic hybridized to theEcoRI-digested genomic DNA of SPAFAS,
inserts, EcoRI digests were performed on the three recombi- USDA 63, and Truslow chickens (Fig. 3). (Unfortunately,
nant bacteriophages and genomic fragments were analyzed by DNA of the specific chicken from which this genomic library
gel electrophoresis. Clone gX6Cr61 (Fig. 1, lane 3, and Fig. 2) was constructed is notavailable for analysis.) We expected to
contains 0.7- and 3.8-kb fragments, identical in size with two observe all EcoRI fragments corresponding to observed re-
fragments from gX6Cr65 (Fig. 1, lane 4, and Fig. 2). By the combinant bacteriophage fragments from the internal region
same size criterion, gX6Cr65 and gX6Cr54 (Fig. 1, lane 5, and of the b-crystallin locus, namely, the 0.7- 3.8-, 5.4-, and 3.1-
Fig. 2) contain common 5.4- and 3.1-kb fragments. Ifwe kb fragments. The 3.8-, 5.4-, and 3.1-kb fragments were read-
assume that similar fragment size implies fragment identity, ily distinguishable (Fig. 3). The 0.7-kb fragment hybridized
then the three genomic clones must overlap in the order, weakly.
gXbCr61, gXdCr65, and gX6Cr54. One would also predict the presence of two additional

FULL-LENGTH MID-cDNA 3"cDNA

PROBEPROBEPROBE cDNA
FIG. 1. Analysis of Recombinant
bacteriophage clones. Lanes 1-5, a
0.9% agarose gelof EcoRI-digested re-
combinant phage clones: lane 1 , DNA
size standards ( X DNA digested with

-.
HindIII and 6x174 DNA digested with
HaeIII); lane 2, pdCrl7 &-crystallin
cDNA in pBR322 digested with HindIII;

-
lane3, gXdCr61 digested with EcoRI; lane 1 2 3 4 5 kb 6 7 8 9 15 14
10 13
11 12
4, gX6Cr65 digested with EcoRI; lane 5, = - q
gX6Cr54 digested with EcoRI. Lanes 6- t 9 8 -
9,blot hybridization of lanes 2-5 using -8:7 -
32P-labeledpbCrl7 probe: lane 6 corre-
sponds to lane 2; lane 7 corresponds to 4 -5.4-
lane 3; lane 8 corresponds to lane 4; lane - - 3.8
9 corresponds to lane 5. Lunes 10-12,
blot hybridization of lanes 3-5 using 32P-
- -3.1-
labeled P1181 probe:lane 10 corresponds
to lane 3; lane 11 corresponds to lane 4;
lane 12 corresponds to lane 5. Lanes 13-
15, blot hybridization of lanes 3-5 using
3ZP-labeledP1188 probe: lane I3 corre- 0.7 -
sponds to lane 3; lane I4 corresponds to
lane 4; lane I5 corresponds to lane 5.
Sizes of principal genomic fragments are
indicated.
 &Crystallin Genes 9823

FIG.2. The chicken &crystallin

locus. The continuous stretch of chro-
mosomal DNA, upon which, the 6-crys-
tallin genes reside, is depicted. The po-
sitions occupied by 61 and 62 are repre-
sented by two rectangles. The dark areas
of the rectangles represent exons (desig- I
I 1
,,
,
nated by numbers), while the light areas I
I
1
,
represent introns (designated by capital dLOCUS 1 -I
letters). The position and size of EcoRI
fragments, characteristic of the chicken
genomic DNAs used in this study, are
indicated below the locus. Belowthis, the
genomic inserts of three recombinant
bacteriophages (gXdCr61,gX6Cr65, and gAdCr6l 1 tl
gX6Cr54) are illustrated with respect to gAdCr65 1 :: 1
their overlap and orientation within the gAdCr54
I
I
6-crystallin locus.

binant DNA fragments contain one genomic Hue111 or AluI

site which was ligated to a synthetic EcoRI linker during
cloning. For the SPAFAS chicken (Fig. 4, lane I), we observed
two bands, one of about 13.0 kb and a second of 7.6 kb. The
7.6-kb fragment probably arises from a polymorphism within
the &crystallin locus. A fragment of similar size, correspond-
ing to the5' end of d l has been isolated in our (19) and other
IA
I
c
C (17)laboratories. It would appear then that the SPAFAS
chicken contains only one allele for the &crystallin locus
which is detectable by EcoRI digestion. Moreover, since the
7.6-kb EcoRI fragment is found at the5' end of the chromo-
somal locus in the SPAFAS chicken (17, 19), the 13.0-kb
fragment isfound at the 3' end of the locus. The 7.6-kb
J
fragment anda new fragment of about 11.0 kb are present in
EcoRI-digested DNAs of USDA 6 3 (Fig. 4, lane 2) and Trus-
62

dl I
kb 1 2 3
,, ., I
FIG.3. R-loop and heteroduplex analysis of recombinant 13.0-
bacteriophage clones. A, line drawing corresponding to the electron 11 .o- -c, I

micrograph aboue it which represents the hybridization of 6-crystallin

mRNA to a heteroduplex formed between gX6Cr61 and bacteriophage 7.6 - 1
X Charon 4A DNA. The 5' and 3' ends of 61 are marked. Introns of
61 are designated A-P. B, a line drawing corresponding to theelectron
micrograph above it which represents the hybridization of 6-crystallin
cDNA, p6Cr17, to a heteroduplex formed between gX6Cr61 and bac- 5.4 -
teriophage X Charon 4A DNA. The 5' and 3' ends of 61 are marked.
C,a line drawing corresponding to the electron micrograph above it 3.8 -
which represents the hybridization of 6-crystallin mRNA to a heter-
oduplex formed between gX6Cr54 and bacteriophage X Charon 4A
DNA. The 5' and 3' ends of 12 are marked. Introns of62 are 3.1 -
designated A'-P'. D,a line drawing corresponding to the electron
micrograph to the left of it which represents the hybridization of two
6-crystallin mRNAs to a heteroduplex formed between gX6Cr65 and
bacteriophage X Charon 4A DNA. The 5' and 3' ends of a partial 61
and partial62 gene are marked. The dotted line represents the stretch
0
.
of genomic spacer DNA found between the two genes. The dashed
lines represent bacteriophage X DNA.
FIG.4. Genomic blot analysis of the chicken &crystallin
locus. Lanes 1-3, chicken genomic DNA digested with EcoRl, elec-
fragments corresponding to EcoRI fragments from the ex- trophoresed in a 0.9% agarose gel, blotted, and hybridized with a 32P-
tremities of the &crystallin chromosomal locus. These two labeled p6Crl7 probe; lane 1 , DNA from SPAFAS chickens; lane 2,
fragments should be larger than the corresponding 9.8- and DNA from USDA 6 3 chickens; lane 3, DNA from Truslow chickens.
8.7-kb recombinant bacteriophage fragments since therecom- Sizes of principal genomic fragments are indicated.
9824 &Crystallin Genes
low (Fig. 4, lane 3 ) chickens. The 11.0-kb fragment is presum- We have arbitrarily designated the exons of 6 1 by number
ably from a second allele and corresponds tothe 9.8-kb and the intronsby capital letters beginning at the 5' end of
fragment which we isolated from the genomic library. the gene. The exons and introns of62 are designated in a
Analysis of 6-Crystallin Genes by Electron Microscopy-In similar fasion but with an additional prime character. Al-
order to obtaina more detailed view of the 6-crystallin genes, though it would appear that exons or introns of 6 1 or 62 with
electron microscopic experiments were carried out. The three a similar character designation may be homologous in struc-
recombinant bacteriophages were hybridized with &crystallin ture, thisremains to be proven by DNA sequence analysis.
mRNA or cDNA (Fig. 3), and the resultinghybrids were In summary, evidence derived from EcoRI genomic frag-
examined in theelectron microscope. ment comparison, blot hybridization of recombinant phage
Analysis of gX6Cr61(Fig. 3, A and B ) indicated the presence and of total genomic DNA, and R-loop analysis indicates that
of one highly interrupted 6-crystallin-coding region. This gene there is one 6-crystallin locus in the haploid chicken genome.
was designated 61. The measurement of 15complete molecules About 31.5 kb of this locus have been isolated and found to
indicates that 6 1 contains at least 17 exons and 16 introns. contain two 6-crystallin genes designated 6 1 and 62. 6 1 and 62
Their sizes are listed in Table IA. have a similar transcriptionalpolarity, with 61 in the most 5'
When gXdCr65 was analyzed (Fig. 3 0 ) , two regions of position. 61 is approximately 6.7 kb inlength, and 62 is
hybridization were observed. The first, at one end of the approximately 9.2 kb in length. 6 1 and 62 are about 4.2 kb
genomic region, was homologous to the putative 3' region of apart. Both genes contain at least 17 exons and 16 introns.
61, observed in gX6Cr61. A spacer of some 4.2 kb, estimated These data have been used to derive a structural map of the
from five measurements, separates this portion of 6 1 from a &crystallin locus (Fig. 2).
second hybridizing figure which extends to the other end of
the genomic region. This second figure represents the 5' end DISCUSSION
of a second haystallin gene, designated 62.
Complete figures of 62 were obtained when the third over- Structure of the &Crystallin Locus-The studies presented
lapping recombinant phage, gX6Cr54, was examined (Fig. 4C). here reveal the presence of a single chicken 6-crystallin locus
The 5' region of 62 observed in gX6Cr65 was homologous to comprised of contiguous EcoRI fragments ordered from 5' to
the 5' portion of the &crystallin gene in gX6Cr54. Once again, 3' as follows: >9.8, 0.7, 3.8, 5.4, 3.1, and X . 7 kb. Sequence
a total of 17 exons and 16 introns were found in 62, the sizes analysis to be presented elsewhere3established unequivocally
of which are reported in Table IB (averaged from five com-
that these DNA fragments contain two 6-crystallin genes. In
plete molecules). From the analysis of all three clones, the an earlier description of the 6-crystallin locus, Jones et al.
total lengths of 61 and 62 were determined to be approximately (19) defined three chicken genomic EcoRI fragments (g6Cr3,
6.7 and 9.2 kb, respectively. g6Cr4, and g6Cr2) which appear by size and hybridization to
be analogous to the3.8-, 3.1-, and >8.7-kb fragments, respec-
tively. Studies by Yasuda et al. (17) describe genomic EcoRI
TABLEI fragments of 0.8 and 4.0 kb from the recombinant bacterio-
Exon and intron size of the 61 and 62 genes
phage XC6106 and of3.0 and 11.0 kb from XC6109 which
A. 61 B. 62 correspond to the 0.7-,3.8-, 3.1-, and >8.7-kb fragments,
Exon Intron ~~~~&~
S.D. Exon Intron
Estimated
no. bases
S,D, respectively. A fragment of 5.4 kb, from the clone Ch618, has
been reported by Yasuda et al. (21) and is probably the same
1 95 34 1' 138 81 as the 5.4-kb fragmentreported here. Conspicuous in the
A 977 200 A' 1695 394 literature is the presence of a 7.6-kb fragment reportedin the
2 164 2' 48 213 37 clone XC6106 by Yasuda et al. (17) and by Jones et al. (19) as
B 92 1 101 B' 1332 274 the fragment g6Crl. Blot hybridization experiments reported
3 86 19 3' 103 20
C 333 46 C' 302 90 here indicate that this 7.6-kb fragment is contained within
4 71 33 4' 114 31 the >9.8-kb fragment and arises as a resultof a polymorphism
D 193 80 D' 735 181 within the 6-crystallin locus. This conclusion is supported by
5 80 26 5' 115 9 the fact that both the 7.6- and the >9.8-kb fragments have
E 264 94 E' 357 210 been demonstrated to contain 5' coding sequence of a 6-
6 82 23 6' 107 31 crystallin gene adjacent to the0.7-kb fragment characteristic
F 234 76 F' 152 67
of 61. It should also be noted that anEcoRI fragment of 10.0
7 77 34 7' 114 41
G 128 108 G' 476 176 kb has been reported in separate studiesby Yasuda et al. (21)
8 75 24 8' 89 20 which is directly analogous to the >9.8-kb fragment. If one
H 196 80 H' 465 176 accepts the likelihood of polymorphism within the chicken
9 72 17 9' 135 62 genome, then allstudies todate areconsistent with the
I 190 119 I' 86 38 existence of just one 6-crystallin locus, one allele of which has
10 97 31 10' 140 62
J 588 96 J' 79 231 been described in thisstudy.
11 97 55 11' 113 25 With regard to the number, linkage, and fine structure of
K 114 44 K' 109 58 6-crystallin genes, the present results agree closely with pre-
12 70 26 12' 80 22 vious reports. Jones et al. (19) and Yasuda et al. (17) have
L 102 47 L' 229 95 demonstrated the existence of two non-allelic genes. Yasuda
13 97 20 13' 115 11 et al. (21) have also reported gene linkage. The estimate of
M 142 63 " 192 60
exon and intronnumber for the 6-crystallin genes has varied
14 87 27 14' 91 11
N 338 60 N' 183 69 with an upward trend. This is presumably due to theanalysis
15 112 31 15' 146 43 of partial gene fragments and to inherent limitations of the
0 432 87 0' 571 178 electron microscopic analysis techniques used by all groups.
16 70 16' 159 24
P 70 P' 79 16 T. Borras, J. M. Nickerson, J. W. Hawkins, and J. Piatigorsky,
17 70 17' 182 53
- - unpublished observations.
 Genes &Crystallin
A definitive assignment of intron and exon number awaits
nucleotide sequence analysis. Our results indicate that the
present estimates will be revised upward again. For example,
exon 1 and 1' are each divided by a small i n t r ~ n . ~
The remote possibility exists that there are multiple 6-
crystallin gene loci in the chicken, identical in structure with
respect to therestriction enzymes used in all genomic blotting.
It is, of course, also possible that distantly related genes or
pseudogenes could exist in the chicken genome.
Evolution of the &-CrystallinLocus-Recent analyses have
correlated exon number with polypeptide size in many eukar-
yotic genes (27). Typically there are two to three exons/100
amino acids of protein. The 6-crystallin genes are consistent
with this finding. The 61 gene encodes a 48-kDa polypeptide
containing 447 amino acids (including the initiating methio-
nine residue), as judged by cDNA sequencing (13). Thus, one
might expect 9-14 exons of coding sequence. Our data indicate
that exons 1-15 (as defined by electron microscopy and dia-
grammed in Fig. 2) encode the &crystallin polypeptide. Fur-
thermore, inspection of Table I indicates that the exons are
quite similar in size, ranging from 70 to 164 base pairs in 61
and from 79 to 213 base pairs in 62. Perhaps the present
structures of the chicken &crystallin genes have resulted from
repeated duplicationsof an ancestral exon encoding a protein
segment of stable structure (29). Intragenic duplicationsof an
ancestral exon encoding a stable structural motif appear to
be the mechanism of evolution for the y (28, 29)- and /3 (30)-
crystallin polypeptides which form a superfamily of related
lens proteins.
In is interesting from both structural and functional view-
points to compare the &-crystallin locus with that of the
albuminla-fetoprotein gene system. Theintronand exon
number are conserved in the albumin/a-fetoprotein system
(31) as in the &crystallin locus. In fact, the albumin and a -
fetoprotein genes share exact exon boundaries which correlate
exquisitely with native protein domains. A scheme for the
evolution of the albuminla-fetoprotein genes has been pro-
posed (32-34). This involves initial intragenic duplication of
an exonic subdomain to form a domain, followed by a second
set of intragenic duplications of this domain, resulting inthe
formation of a modern gene. Both genes of the 6-crystallin
and the albumin/a-fetoprotein systems are separated by sev-
eral kilobases of spacer DNA and have similar transcriptional
polarities (34). The similarity appears to end at this point,
however. Albumin and a-fetoprotein are expressed at high
rates at differentstages of development. By contrast, the
present evidence suggests that the two &crystallin genes are
expressed at similar times in development and at unequal
rates, with 61 being considerably more active than 62 (see Ref.
35). It is particularly interesting thatthese two gene systems
with such similar structure could be expressed so differently.
We may anticipate that further detailed analysisof the struc-
ture andexpression of the cloned &crystallin genes described
in the presentstudy will provide new insights into themodes
of their function.
Acknowledgments-We thank Dawn Sickles for expert secretarial
assistance and Drs. Teresa Borras, Ana B. Chepelinsky, Gokul Das,
and David McDevitt for critical reading of the manuscript. We also
thank Drs. Bruce Paterson and Tom Sargent for their advice and
help.
9825
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