tau-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey tau-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck tau-crystallin was obtained and sequenced, confirming the close relationship with alpha-enolase. Southern blot analysis showed that the duck genome contains a single alpha-enolase gene, while Northern blot analysis showed that the message for tau-crystallin/alpha-enolase is present in embryonic duck lens at 25 times the abundance found in liver. tau-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. tau-Crystallin/alpha-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene.

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We have utilized [3H]cDNA . DNA annealing (using S1 endonuclease resistance as an assay) to detect the presence of sequences complementary to chick 6-crystallin mRNA in the genome of fish (salmon, herring), amphibians (frog, newt), reptiles (python, gekko, caiman), birds (quail, turkey, duck), and mammals (mouse, calf), and have compared several physical parameters of d-crystallin of the reptiles and birds. The rate and extent of chick d-crystallin [3H]cDNA . DNA annealing were comparable among the birds, and indicated that the d-crystallin sequences are in the unique fraction of the genome. On the average, about one-fifth as much annealing took place with the reptiles as with the birds, and no annealing was observed with the fish, amphibians and mammals. The avian and reptilian d-crystallins had subunit molecular weights near 50 000 and native molecular weights near 200 000, as judged by sodium dodecylsulfate/urea/polyacrylamide gel electrophoresis and agarose gel chromatography, respectively, indicating that the subunit structure of d-crystallin has been largely conserved. Electrophoresis of the peptides generated by digestion with Staphylococcus aureus V8 protease indicated that the primary structures of the different reptilian and avian d-crystallins were similar but not identical. The isoelectric points of the &crystallins ranged between pH 5 and 7, depending on the organism. In general, the reptilian d-crystallins had the higher isoelectric points.Immunological studies have shown that lenses of birds and reptiles possess crystallins which are different from those of fish, amphibians and mammals [l-71. Originally, these avian and reptilian proteins were considered as members of the P-crystallins. Rabaey [4] was the first to suggest that the predominant crystallin unique to birds and reptiles be considered separately from the /I-crystallins ; he called it FISC, for 'first important soluble crystallin', due to its prevalence in embryonic lenses. Subsequently this protein was called d-crystallin [8].Much of our knowledge of d-crystallin comes from studies of chickens, where it comprises 60-80% of the protein in the embryonic lens and at least 50 % of the protein in the adult lens [4,9,10] In the present study we have taken advantage of our ability to isolate and reverse-transcribe embryonic chick d-crystallin mRNA [15] in order to test by CDNA . DNA annealing for the presence of d-crystallin DNA sequences in the genome of mammals, birds, reptiles, amphibians and fish. In addition, we have compared the subunit structure and peptides of d-crystallins of four species of birds and three species of reptiles. The results provide evidence that the &crystallin DNA sequences arose with the reptiles and that the subunit structure of &crystallin has been largely conserved. MATERIALS AND METHODS rganismsThe following organisms were used : 15-day-old white Leghorn chick embryos (Gallus gallus, order galliformes), 15 -18-day-old Japanese quail embryos (Coturnix coturnix, order galliformes), 14-18-dayold turkey embryos (M...

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