Exp. Eye Hes. (1991)
53, 135-138
LETTER
One Member
TO THE EDITORS
of the y-Crystallin Gene is Expressed in Birds
Crystallins are the structural proteins in the vertebrate lens. The unique properties of this organ, like transparency and refractive index, are the result of the distribution and abundance of a limited number of these proteins (Bloemendal, 1981; Lubsen, Aarts and Schoenmakers, 1988). The fact that crystallins and also their mRNAs are very abundant in lenses of vertebrates has made it relatively easy to isolate the corresponding cDNAs of these proteins and their genes. At the moment much is known about the crystallins and the organization of their genes. This organization is characteristic for each of the gene families ; for instance ys, formerly called ps, is encoded by a gene which has the characteristics of the y-gene family. Therefore it must be a member, albeit a distant one, of this family (Van Rens et al., 1989). The crystallins can be divided into four major classes: c1-, p-, y- and S-crystallin. The first three classes are expressed in most of the vertebrates, whereas S-crystallin is only found in birds and reptiles. In addition, several species contain taxon-specific crystallins, like ,%in rabbit and hare, r in lamprey, some fishes, birds and reptiles, p in frogs of the genus Rana and E in many birds and crocodiles (Wistow and Piatigorsky, 1988). The diversity of the crystallins is the result of a long and complex evolutionary process. In different vertebrate species representatives of the different gene families are found. In general the /3-crystallins are the most abundant crystallins present except for rodent and fish lenses which contain predominantly ycrystallins (Lubsen et al., 1988). Intensive searches for y-crystallin sequences in an avian genomic library were without any positive result. The hybridization signals found were due to a dG.dC-tail in the probe (T&on, et al., 1984). On the other hand, McDevitt and Croft (19 77) found a monomeric protein fraction in pigeon lenses and concluded from the amino acid composition that it was more similar to ys (ps) than to the other y-crystallins. To investigate the presence of genomic y-crystallin sequences in several vertebrate species we screened Southern blots loaded with genomic DNA (Xba I restriction fragments) from six different classes of vertebrates. One of these blots was screened with a probe for rat yC which hybridizes with all paralogous rat y sequences (Moorman et al.. 1982). The other one was screened with a probe for bovine ys (Van Rens et al., 1989). In Fig. 1 the results of these screenings are shown. In Fig. l(B), in the lanes of man, ape, calf and rat, positive hybridization signals are found for yC. 0014-4835/91/070135+04
$03.00/O
Family,
ys,
Rat yC is capable of hybridizing to mammalian, but not to avian, reptilian, amphibian or fish genomic sequences ; the signals in the trout and lamprey lane do not represent discrete bands. However, it is known that amphibians like Rana temporuriu express at least four non-identical y-crystallin genes of which cDNA sequences have been published (Tomarev et al., 1984). Comparison of these sequences with the rat and bovine sequences showed that the rate of molecular evolution is higher in y-crystallins than in a-crystallins. In caiman (Cuimun crocodylus) two ycrystallins have been N-terminally sequenced (Chiou et al., 1987) and a y-like crystallin fraction was also found in river turtle (Amydu sinensis)but not in a snake (Trimeresurusmucrosquamutis)in fractions of a gel permeation column analysed on SDS-gels by Chiou, Chang and Lo (1988). Also, from carp two y-crystallin sequences are known, They have a homology of 75 %, similar to the 69% homology among the frog ycrystallins. In mammals the homology between the paralogous y-crystallins is always higher (Chang et al., 1988). It is therefore likely that the members of the fish and frog (and perhaps also the reptilian) ygene families arose independently from those of the mammalian y-crystallin family and followed a different evolutionary pathway (Tomarev et al., 1984; Chang et al., 1988). In the y-crystallin family the first duplication gave rise to the ancestors of the ys gene and those of the other y-crystallins. Further duplications gave rise to the present-day gene families of y-crystallins. The duplications in the mammalian ycrystallm family occurred before the mammalian radiation but after divergence of the fish and amphibian lineages (Lubsen et al., 1988). The homology between the y-crystallins of the frog and the yC of the rat is 68%. For carp it is 63-64x (Tomarev et al., 1984; Chang et al., 1988). These levels of homology and the nature of the homologous sequences are apparently not capable of revealing hybridization signals in the genomic DNA of lower vertebrates. Hybridization signals indicative of ys-like sequences are observed in lamprey, trout, monitor lizard, pigeon, duck, rat, calf, ape and man [Fig. l(A)]. In all lanes, except that of the amphibian species Xenopus laevis, hybridizing bands were found. This is the first time that positive signals are found with sequences of a ycrystallin cDNA (ys) in birds. As ys is a very ancient offshoot and a well-conserved crystallin, signals are found in species as distant from calf as lamprey. From an evolutionary tree constructed by Quax-Jeuken et al. (1985) it was concluded that the separation of the 0 199 1 Academic Press Limited
G. L. M. VAN
136
RENS
ET AL.
-
B
I2
3
4
5
6
7
8
9
IO
FIG. 1. Southern blotting of genomic DNA of several vertebrate species. Ten-microgram aliquots of genomic DNA were digested with Xba I for 2 hr. After digestion the samples were loaded on a 0.6% agarose gel and size-fractionated. The DNA was transferred to nitrocellulose with 10% SSC and hybridization experiments were carried out according to the method of Church and Gilbert (1984) with a randomly primed 32P-labeled probe (Feinberg and Vogelstein. 1983) after the addition of 100 ,ug of herring sperm DNA per ml of hybridization mixture. The probes used for screening the blots were cDNAs of ys and yC in Fig. l(A) and (B), respectively. The filters were washed with the prescribed buffers. In lane 1 chromosomal DNA was loaded from man (Homo sapiens); in lane 2, ape (Pun troglodytes); lane 3, calf (Bos taurus); lane 4, rat (Rattus norvegicus): lane 5, pigeon (Columbu liviu): lane 6. duck (Anasplatyrhynchos): lane 7, monitor lizard (Varunus exunthemuticus): lane 8. toad (Xenupus luevis): lane 9, trout (Sulmo truttu); lane 10, lamprey (Petromyzon murinus). The AHind III marker is indicated by bars. The arrow points to the slots where the digests of the chromosomal DNA were loaded.
y-CRYSTALLIN
GENE
FAMILY
IN
BIRDS
found for all species (see Fig. 2). The hybridizing signals were of similar size as could be concluded from a Northern blot which was loaded with 10 pg of glyoxyiated mRNA from bovine lenses (data not shown). All birds examined express a sequence homologous to ys crystallin. The same Northern blots were screened with the yC probe. In neither one of the lanes (chicken, pigeon or duck) could a positive signal be found. Even after prolonged exposure of the Northern blots no positive signal could be detected (data not shown). Therefore, one has to conclude that ys is expressed in birds. In view of our results on Southern blotting and the studies by Chiou et al. (198711, who found an N-terminally blocked y-fraction, it seems that ys exists also in reptiles. It appears that ys is evolutionarily well-conserved, and hybridizing sequences can be detected in species as distant from mammals as lamprey. The other members of the y-crystallin family are less conserved, and they cannot be detected in birds.
13531078872 603-
1353-
-
1078872603
137
-
Acknowledgements We would like to thank Dr J. A. M. Leunissen for assistance with the CAOS/CAMM computer programs, F. Ho1 for genomic digests for the Southern blots, and W. Vree Egberts for construction of the Northern blots.
FIG. 2. Northern blotting of avian lens mRNA. RNA was isolated from young chicken (G&s domesticus), duck (Anus platyrhynchos) and pigeon (Columba livia) lenses. On two different Northern blots in each lane 1Opg of glyoxylated total RNA of lenses of chicken, duck or pigeon (lane 2, 3, 5, respectively) was loaded. Glyoxylated hHind III and $xHue III DNA (lanes 1 and 4) was taken as a marker. Hybridization experiments were carried out, with a randomly primed 32Plabeled ys probe, according to Church and Gilbert (1984) with the addition of 100 ,ug of herring sperm DNA per ml hybridization buffer. Filters were washed as in the Southern blotting experiments.
ys gene indeed occurred before the divergence of the ancestors of frog and mammals. The only ys-crystallin sequenced from lower vertebrates is a carp (Cyprinus carpio) one of which has very similar sequence to that of the bovine ys (Chang and Chang, 1987). The homology at the nucleotide level in the coding region is 71%, just enough to detect sequences in lower vertebrates. To check whether or not the ys sequences are also expressed in birds we made Northern blots of pigeon, duck and chicken lens RNA. Positive signals were
G. L. M. VAN RENS W. W. DE JONG H. BLOEMENDAL Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
References
Bloemendal, H. ( 198 1). The lens proteins. In Molecular and Cellular Biology of the Eye Lens (Ed. Bloemendal, H.). Chapter 1. Pp. 147. John Wiley: New York. Chang, T. and Chang, W.-C. (1987). Cloning and sequencing of a carp ,&-crystallin cDNA. Biochem. Biophys. Acta 910, 89-92. Chang, T., Jiang, Y.-J., Chou, S.-H. and Chang, W.-C. (1988). Carp gamma-ctystallin with high methionine content: cloning and sequencing of the complementary DNA. Biochem. Biophys. Actu 951, 226-V. Chiou. S.-H., Chang, W.-P. and Lo, C.-H. (1988). Biochemical comparison of lens crystallins from three reptilian species. Biochem. Biophys. Actu 955, 1-V. Chiou. S.-H., Chang, W.-P., Lo, C.-H. and Chen, S.-W. ( 198 7). Sequence comparison of y-crystallins from the reptilian and other vertebrate species. FEBS Lett. 221. 134-8. Church. G. M. and Gilbert, W. (1984). Genomic sequencing. Proc. N&J. Acad. Sci. U.S.A. 81, 1991-5. Feinberg, A. P. and Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137, 266-7.
138
G. L. M. VAN
Lubsen. N. H., Aarts, H. J. M. and Schoenmakers, J. G. G. (1988). The evolution of lenticular proteins : the p- and y-crystallin gene superfamily. Progr. Biophys. Molec. Biol. 51, 47-76. McDevitt, D. S. and Croft, L. R. (1977). On the existenceof y-crystallin in bird lens. Exp. Eye Res. 25, 473-81. Moorman, R. J. M.. Den Dunnen,J. T., Bloemendal,H. and Schoenmakers,J. G. G. (1982). Extensive intragenic sequencehomologyin two distinct rat lensy-crystallin cDNAs suggestduplicationsof a primordial gene.Proc. Natl. Acad. Sci. U.S.A. 79, 6876-80.
Quax-Jeuken,Y., Driessen,H., Leunissen,J., Quax, W., De Jong,W. W. and Bloemendal,H. (1985). ,f%-Crystallin: structure and evolution of a distinct memberof the pr superfamily.EMBO 1. 4, 259 7-602.
RENS
ET AL.
Tomarev.S. I.. Zinovieva, R. D.. Chalovka,P., Krayev, A. S., Skryabin, K. G. andGause.G. G. (1984). Multiple genes coding for the frog eye lens y-crystallins. Gene27, 301-8. T&on, J. A., Jones,R. E., King, C. R. and Piatigorsky. J. (1984). Evidence against y-crystallin DNA or RNA sequences in the chicken. Exp. Eye Res. 39, 513-22. Van Rens,G. L. M., Raats,J. M. H.. Oldenburg.O., Wijnen. J. T.. Meera Khan, P., DeJong.W. W. and Bloemendal. H. (1989). Structure of the bovine eyelensys-crystallin gene(formerly /3s).Gene78. 225-33. Wistow, G. and Piatigorsky, J. (1988). Lenscrystallins: the evolution and expressionof proteins for a highly specializedtissue.Arm. Rev. Biochem. 57, 479-504.
(Received 17 December 1990 and accepted 3 January 1991)
53, 135-138
LETTER
One Member
TO THE EDITORS
of the y-Crystallin Gene is Expressed in Birds
Crystallins are the structural proteins in the vertebrate lens. The unique properties of this organ, like transparency and refractive index, are the result of the distribution and abundance of a limited number of these proteins (Bloemendal, 1981; Lubsen, Aarts and Schoenmakers, 1988). The fact that crystallins and also their mRNAs are very abundant in lenses of vertebrates has made it relatively easy to isolate the corresponding cDNAs of these proteins and their genes. At the moment much is known about the crystallins and the organization of their genes. This organization is characteristic for each of the gene families ; for instance ys, formerly called ps, is encoded by a gene which has the characteristics of the y-gene family. Therefore it must be a member, albeit a distant one, of this family (Van Rens et al., 1989). The crystallins can be divided into four major classes: c1-, p-, y- and S-crystallin. The first three classes are expressed in most of the vertebrates, whereas S-crystallin is only found in birds and reptiles. In addition, several species contain taxon-specific crystallins, like ,%in rabbit and hare, r in lamprey, some fishes, birds and reptiles, p in frogs of the genus Rana and E in many birds and crocodiles (Wistow and Piatigorsky, 1988). The diversity of the crystallins is the result of a long and complex evolutionary process. In different vertebrate species representatives of the different gene families are found. In general the /3-crystallins are the most abundant crystallins present except for rodent and fish lenses which contain predominantly ycrystallins (Lubsen et al., 1988). Intensive searches for y-crystallin sequences in an avian genomic library were without any positive result. The hybridization signals found were due to a dG.dC-tail in the probe (T&on, et al., 1984). On the other hand, McDevitt and Croft (19 77) found a monomeric protein fraction in pigeon lenses and concluded from the amino acid composition that it was more similar to ys (ps) than to the other y-crystallins. To investigate the presence of genomic y-crystallin sequences in several vertebrate species we screened Southern blots loaded with genomic DNA (Xba I restriction fragments) from six different classes of vertebrates. One of these blots was screened with a probe for rat yC which hybridizes with all paralogous rat y sequences (Moorman et al.. 1982). The other one was screened with a probe for bovine ys (Van Rens et al., 1989). In Fig. 1 the results of these screenings are shown. In Fig. l(B), in the lanes of man, ape, calf and rat, positive hybridization signals are found for yC. 0014-4835/91/070135+04
$03.00/O
Family,
ys,
Rat yC is capable of hybridizing to mammalian, but not to avian, reptilian, amphibian or fish genomic sequences ; the signals in the trout and lamprey lane do not represent discrete bands. However, it is known that amphibians like Rana temporuriu express at least four non-identical y-crystallin genes of which cDNA sequences have been published (Tomarev et al., 1984). Comparison of these sequences with the rat and bovine sequences showed that the rate of molecular evolution is higher in y-crystallins than in a-crystallins. In caiman (Cuimun crocodylus) two ycrystallins have been N-terminally sequenced (Chiou et al., 1987) and a y-like crystallin fraction was also found in river turtle (Amydu sinensis)but not in a snake (Trimeresurusmucrosquamutis)in fractions of a gel permeation column analysed on SDS-gels by Chiou, Chang and Lo (1988). Also, from carp two y-crystallin sequences are known, They have a homology of 75 %, similar to the 69% homology among the frog ycrystallins. In mammals the homology between the paralogous y-crystallins is always higher (Chang et al., 1988). It is therefore likely that the members of the fish and frog (and perhaps also the reptilian) ygene families arose independently from those of the mammalian y-crystallin family and followed a different evolutionary pathway (Tomarev et al., 1984; Chang et al., 1988). In the y-crystallin family the first duplication gave rise to the ancestors of the ys gene and those of the other y-crystallins. Further duplications gave rise to the present-day gene families of y-crystallins. The duplications in the mammalian ycrystallm family occurred before the mammalian radiation but after divergence of the fish and amphibian lineages (Lubsen et al., 1988). The homology between the y-crystallins of the frog and the yC of the rat is 68%. For carp it is 63-64x (Tomarev et al., 1984; Chang et al., 1988). These levels of homology and the nature of the homologous sequences are apparently not capable of revealing hybridization signals in the genomic DNA of lower vertebrates. Hybridization signals indicative of ys-like sequences are observed in lamprey, trout, monitor lizard, pigeon, duck, rat, calf, ape and man [Fig. l(A)]. In all lanes, except that of the amphibian species Xenopus laevis, hybridizing bands were found. This is the first time that positive signals are found with sequences of a ycrystallin cDNA (ys) in birds. As ys is a very ancient offshoot and a well-conserved crystallin, signals are found in species as distant from calf as lamprey. From an evolutionary tree constructed by Quax-Jeuken et al. (1985) it was concluded that the separation of the 0 199 1 Academic Press Limited
G. L. M. VAN
136
RENS
ET AL.
-
B
I2
3
4
5
6
7
8
9
IO
FIG. 1. Southern blotting of genomic DNA of several vertebrate species. Ten-microgram aliquots of genomic DNA were digested with Xba I for 2 hr. After digestion the samples were loaded on a 0.6% agarose gel and size-fractionated. The DNA was transferred to nitrocellulose with 10% SSC and hybridization experiments were carried out according to the method of Church and Gilbert (1984) with a randomly primed 32P-labeled probe (Feinberg and Vogelstein. 1983) after the addition of 100 ,ug of herring sperm DNA per ml of hybridization mixture. The probes used for screening the blots were cDNAs of ys and yC in Fig. l(A) and (B), respectively. The filters were washed with the prescribed buffers. In lane 1 chromosomal DNA was loaded from man (Homo sapiens); in lane 2, ape (Pun troglodytes); lane 3, calf (Bos taurus); lane 4, rat (Rattus norvegicus): lane 5, pigeon (Columbu liviu): lane 6. duck (Anasplatyrhynchos): lane 7, monitor lizard (Varunus exunthemuticus): lane 8. toad (Xenupus luevis): lane 9, trout (Sulmo truttu); lane 10, lamprey (Petromyzon murinus). The AHind III marker is indicated by bars. The arrow points to the slots where the digests of the chromosomal DNA were loaded.
y-CRYSTALLIN
GENE
FAMILY
IN
BIRDS
found for all species (see Fig. 2). The hybridizing signals were of similar size as could be concluded from a Northern blot which was loaded with 10 pg of glyoxyiated mRNA from bovine lenses (data not shown). All birds examined express a sequence homologous to ys crystallin. The same Northern blots were screened with the yC probe. In neither one of the lanes (chicken, pigeon or duck) could a positive signal be found. Even after prolonged exposure of the Northern blots no positive signal could be detected (data not shown). Therefore, one has to conclude that ys is expressed in birds. In view of our results on Southern blotting and the studies by Chiou et al. (198711, who found an N-terminally blocked y-fraction, it seems that ys exists also in reptiles. It appears that ys is evolutionarily well-conserved, and hybridizing sequences can be detected in species as distant from mammals as lamprey. The other members of the y-crystallin family are less conserved, and they cannot be detected in birds.
13531078872 603-
1353-
-
1078872603
137
-
Acknowledgements We would like to thank Dr J. A. M. Leunissen for assistance with the CAOS/CAMM computer programs, F. Ho1 for genomic digests for the Southern blots, and W. Vree Egberts for construction of the Northern blots.
FIG. 2. Northern blotting of avian lens mRNA. RNA was isolated from young chicken (G&s domesticus), duck (Anus platyrhynchos) and pigeon (Columba livia) lenses. On two different Northern blots in each lane 1Opg of glyoxylated total RNA of lenses of chicken, duck or pigeon (lane 2, 3, 5, respectively) was loaded. Glyoxylated hHind III and $xHue III DNA (lanes 1 and 4) was taken as a marker. Hybridization experiments were carried out, with a randomly primed 32Plabeled ys probe, according to Church and Gilbert (1984) with the addition of 100 ,ug of herring sperm DNA per ml hybridization buffer. Filters were washed as in the Southern blotting experiments.
ys gene indeed occurred before the divergence of the ancestors of frog and mammals. The only ys-crystallin sequenced from lower vertebrates is a carp (Cyprinus carpio) one of which has very similar sequence to that of the bovine ys (Chang and Chang, 1987). The homology at the nucleotide level in the coding region is 71%, just enough to detect sequences in lower vertebrates. To check whether or not the ys sequences are also expressed in birds we made Northern blots of pigeon, duck and chicken lens RNA. Positive signals were
G. L. M. VAN RENS W. W. DE JONG H. BLOEMENDAL Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
References
Bloemendal, H. ( 198 1). The lens proteins. In Molecular and Cellular Biology of the Eye Lens (Ed. Bloemendal, H.). Chapter 1. Pp. 147. John Wiley: New York. Chang, T. and Chang, W.-C. (1987). Cloning and sequencing of a carp ,&-crystallin cDNA. Biochem. Biophys. Acta 910, 89-92. Chang, T., Jiang, Y.-J., Chou, S.-H. and Chang, W.-C. (1988). Carp gamma-ctystallin with high methionine content: cloning and sequencing of the complementary DNA. Biochem. Biophys. Actu 951, 226-V. Chiou. S.-H., Chang, W.-P. and Lo, C.-H. (1988). Biochemical comparison of lens crystallins from three reptilian species. Biochem. Biophys. Actu 955, 1-V. Chiou. S.-H., Chang, W.-P., Lo, C.-H. and Chen, S.-W. ( 198 7). Sequence comparison of y-crystallins from the reptilian and other vertebrate species. FEBS Lett. 221. 134-8. Church. G. M. and Gilbert, W. (1984). Genomic sequencing. Proc. N&J. Acad. Sci. U.S.A. 81, 1991-5. Feinberg, A. P. and Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137, 266-7.
138
G. L. M. VAN
Lubsen. N. H., Aarts, H. J. M. and Schoenmakers, J. G. G. (1988). The evolution of lenticular proteins : the p- and y-crystallin gene superfamily. Progr. Biophys. Molec. Biol. 51, 47-76. McDevitt, D. S. and Croft, L. R. (1977). On the existenceof y-crystallin in bird lens. Exp. Eye Res. 25, 473-81. Moorman, R. J. M.. Den Dunnen,J. T., Bloemendal,H. and Schoenmakers,J. G. G. (1982). Extensive intragenic sequencehomologyin two distinct rat lensy-crystallin cDNAs suggestduplicationsof a primordial gene.Proc. Natl. Acad. Sci. U.S.A. 79, 6876-80.
Quax-Jeuken,Y., Driessen,H., Leunissen,J., Quax, W., De Jong,W. W. and Bloemendal,H. (1985). ,f%-Crystallin: structure and evolution of a distinct memberof the pr superfamily.EMBO 1. 4, 259 7-602.
RENS
ET AL.
Tomarev.S. I.. Zinovieva, R. D.. Chalovka,P., Krayev, A. S., Skryabin, K. G. andGause.G. G. (1984). Multiple genes coding for the frog eye lens y-crystallins. Gene27, 301-8. T&on, J. A., Jones,R. E., King, C. R. and Piatigorsky. J. (1984). Evidence against y-crystallin DNA or RNA sequences in the chicken. Exp. Eye Res. 39, 513-22. Van Rens,G. L. M., Raats,J. M. H.. Oldenburg.O., Wijnen. J. T.. Meera Khan, P., DeJong.W. W. and Bloemendal. H. (1989). Structure of the bovine eyelensys-crystallin gene(formerly /3s).Gene78. 225-33. Wistow, G. and Piatigorsky, J. (1988). Lenscrystallins: the evolution and expressionof proteins for a highly specializedtissue.Arm. Rev. Biochem. 57, 479-504.
(Received 17 December 1990 and accepted 3 January 1991)
