These observations cannot, however, explain 

 the persistence of LDH-1 in the lens fiber cells, 

 since the oxygen tension in the lens is lower 

 than that in the blood and the pathways of 

 oxidative metabolism are practically negligible. 

 Even under these conditions the highly glycolyz- 

 ing fiber cells retain LDH-1 thus showing that 

 within this tissue some other factor or factors 

 related to the replicative capacity must also be 

 considered in explaining the regulation of LDH 

 subunit synthesis. 



I have now come to the end of our observa- 

 tions on the regulation of synthesis of tissue 

 specific proteins associated with a specific 

 stage of cellular differentiation. Our data have 

 shown that the synthesis of y-crystallins is 

 specifically associated with the differentiation of 

 the epithelial cell to the fiber cell. Thus, the 

 a- and ^-crystallins are structural proteins of 

 the epithelial cell and the a-, p- and y-crystal- 

 lins are structural proteins of the fiber cell. 

 At the beginning of my talk I described some 

 cytological changes which occur in elongating 

 epithelial cells such as an enlargement of the 

 nucleus and nucleoli and an increase in the 

 ribosomal population. These observations are 

 indicative of an increase in protein synthesis 

 and may be associated with the initiation of 

 y-crystallin synthesis. 



The lactate dehydrogenases on the other 

 hand have shown us the simultaneous "turning 

 off" of a specific protein which is associated 

 not only with fiber cell formation, but also 

 with the aging and replicative activity of the 

 cell. Thus, the ability of the cell to regulate 

 LDH subunit synthesis in the absence of mor- 

 phological changes brings out a significant 

 difference between the regulation of y-crystallin 

 synthesis and LDH subunit synthesis. The y- 

 crystallins are highly tissue specific proteins 

 whose function may be essentially involved in 

 the structure of the lens whereas the LDHs are 

 widespread and are essential for metabolic 

 activity. In both cases, differential gene action 

 is required. Whether these regulatory mech- 

 anisms are similar must await further experi- 

 mentation. 



IV. The Role of Nucleic Acids in Lens Fiber 

 Cell Differentiation 



A. The status of m-RNA in differentiating 

 lens cells: the stabilization of m-RNA 



It has recently been shown that the synthe- 

 sis of specific proteins such as hemoglobins 



(32), feather keratins (33) and lens crystallins 

 (34-38) occurs on relatively long lived m-RNA 

 templates. These long lived messengers are 

 found in highly differentiated cells and are 

 involved in the synthesis of proteins specific 

 for these cells. Bacterial m-RNA for example, 

 which is considered to be short lived has a 

 half -life of 2 minutes (39), while the half-life of 

 m-RNA for feather keratin synthesis has been 

 reported to be longer than 24 hours (33). At 

 present, the only way to show stable m-RNA is 

 through the insensitivity of a protein-synthe- 

 sizing polysomal unit to actinomycin D, and all 

 the cases described so far are based on the 

 observation that protein synthesis continues 

 long after RNA synthesis has been halted by 

 actinomycin. On the basis of these preliminary 

 observations, it appears that the stability of 

 m-RNA is a very important feature of the 

 differentiated cell in which a large percentage 

 of the proteins synthesized are tissue specific 

 proteins. Although many tissue specific proteins 

 appear in the initial stages of tissue differen- 

 tiation, the basic question we would like to 

 consider is whether these proteins are synthe- 

 sized on "pre-existing" stable templates or 

 whether there is a progressive transition from 

 an actinomycin sensitive to an actinomycin 

 insensitive period of protein synthesis. 



In a series of experiments carried out by 

 Mr. James A. Stewart, Dr. Paul V. Koehn and 

 myself (36-38), we attempted to determine 

 whether (a) the lens crystallins of the epithelial 

 cells and fiber cells are synthesized on long 

 lived or short lived messenger templates and 

 (b) if there is some period of lens cell differen- 

 tiation in which the m-RNA passes from a stage 

 of actinomycin sensitivity to actinomycin insen- 

 sitivity, thus associating the stabilization of 

 m-RNA to a specific stage of cellular differen- 

 tiation. 



In these experiments, intact bovine calf 

 lenses were incubated in the presence of C^'*- 

 amino acids with and without actinomycin D 

 (10 fjg/ml). The epithelial and fiber cells were 

 separated and the crystallins from each cell 

 type were fractionated on DEAE-cellulose col- 

 umns. An elution diagram of the separation of 

 a-, ^- and y-crystallins of untreated and 

 actinomycin treated epithelial cells is shown 

 in Figs. 10 and 11. The incorporation of amino 

 acids into these proteins is also shown. It can 

 be seen that incorporation of amino acids into 

 epithelial cell crystallins could be extensively 

 inhibited by actinomycin. Both elution diagrams 

 show similar protein patterns. The specific 



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