cell formation in the regenerating salamander 

 lens (10). 



In my second proposal, I stated that the 

 variation in DEAE column properties between 

 adult cortex and adult nucleus r-crystallins 

 imply distinct differences exist between the 

 y-crystallins of the nucleus region and cortex 

 region of the adult lens. Since the adult nucleus 

 fibers are cells which were formed during the 

 earlier period of lens growth, these regional 

 differences in r-crystallins of the adult lens 

 (Figs. 3B and 3C) may be due to amino acid 

 differences in the r-crystallins formed by fiber 

 cell differentiation at differentiation at different 

 ages. Thus, the y-crystallins from lenses of 

 younger animals (embryos and young calves) 

 should have the same chromatographic prop- 

 erties as y-crystallins from the nucleus fibers 

 of an adult lens. Evidence for this is presented 

 by the elution patterns for proteins from calf 

 cortex (Fig. 4B), calf nucleus (Fig. 4C) and 

 embryonic lenses (Fig. 5), which show that the 

 y-crystallins in the fiber cells of these younger 

 lenses are chromatographically similar to the 



o 



Cl 



e 



200 400 600 800 1000 

 ml effluent 



Fig. 5. 



Fractionation of the soluble proteins from the combined 

 lenses of U5 day and 130 day embryos, 426.24 mg pro- 

 tein in a volume of 7.4 ml were added to a DEAE-cellu- 

 lose column (2 cm x 10 cm). 302.9 mg protein were 

 recovered at the end of the experiment. The elution se- 

 quence and volume of buffers used are as follows: 

 I. 100 ml 0.05 M sodium-phosphate pH 7; II. 200 ml 

 0.0075 M sodium-phosphate pH 6.5; III. 150 ml 0.01 M 

 sodium-phosphate pH 6; IV. 200 ml 0.02 M sodium- 

 phosphate pH 5.7; v. 500 ml 0.02 M sodium-phosphate 

 pH 5.7 + 0.1 M NaCl; VI. 150 ml 0.1 M sodium-phos- 

 phate pH 5.7 + 0.1 NaCl; VII. 150 ml 0.1 M sodium- 

 phosphate pH 5.7 + 0.3 NaCl. The fractions were col- 

 lected in 10 ml aUquots. (Fig. 3, J. Papaconstantinou, 

 Biochim. Biophys. Acta 107,81, 1965; reproduced with per- 

 mission of Elsevier Publishing Company.) 



y-crystallins of the adult nucleus fibers 

 (Fig. 3C). Furthermore, it can be seen that only 

 in the elution pattern of the calf cortex fibers 

 (Fig. 4B), where the predominating y-crystallins 

 are of the "embryonic type", are there indica- 

 tions of the appearance of the types of y-crys- 

 tallins observed in the adult cortex fiber cells, 

 i.e., peaks a 2 and b (Fig. 3B). The patterns for 

 calf nucleus fiber cell proteins and embryonic 

 lens proteins show a complete absence of adult 

 cortex fiber type y-crystallins. 



In view of the differences in chromato- 

 graphic properties of the y-crystallins attempts 

 were made to obtain further evidence for more 

 distinct differences between the embryonic and 

 adult y-crystallins. Further purification of adult 

 cortex, adult nucleus and embryonic y-crystal- 

 lins was achieved by DEAE-cellulose fractiona- 

 tion using tris buffer ranging in pH from 10 to 

 7 (Fig. 6). In each case the y-crystallins were 

 resolved into 4 major proteins. Our observations 

 are similar to those of Bjork (11) who, through 

 the use of alternative procedures of fractiona- 

 tion, was able to resolve the y-crystallins into 4 

 distinct fractions. The purified y-crystallins 

 from each of these fractions were concentrated 

 and their relative mobilities were determined 

 by paper electrophoresis. The electrophoretic 

 patterns (Figs. 7, 8) show that the y-crystallins 

 from embryonic and adult nucleus fibers have 

 the same mobility, whereas the y-crystallins of 

 the adult cortex have a different mobility. 

 These data are in agreement with the prelimi- 

 nary observations on DEAE-columns. 



From these observations it can be con- 

 cluded that (a) y-crystallin synthesis is initiated 

 during fiber cell formation and is associated 

 with this specific stage of lens cell differentia- 

 tion and (b) that the y-crystallins synthesized 

 during embryonic and early post-natal fiber 

 cell differentiation are electrophoretically dis- 

 tinct from those synthesized in the fibers of the 

 adult lens. Thus, the type of y-crystallin syn- 

 thesized depends on the age of the animal or 

 possibly the rate at which fiber cells are laid 

 down (5, 6). 



With respect to the initiation of y-crystallin 

 synthesis we have an example of gene activation 

 at the molecular level, and one question which 

 concerns us now is whether the activation of 

 y-crystallin synthesis is intimately associated 

 with the genetic regulation of the morphological 

 changes in the cell. Furthermore, since fiber 

 cell formation involves the transition of a 

 replicative cell to a non-replicative cell, is 

 y-crystallin in any way associated with this 

 aspect of lens cell differentiation? We are 



52 



