30 90 150 210 

 ml 



Fig. 16. 



The fracnonatlon of total soluble RNA from epithelial 

 cells and fiber cells on DEAE-cellulose. (A) Total 

 soluble RNA from calf cortex fiber cells. The rlbosomal 

 RNA and soluble RNA were first separated by sucrose 

 gradient. The soluble RNA fractions from the top of 

 the gradient were combined and dialyzed against 0.01 M 

 Tris-HCl pH 7.3 + 0.01 M MgCl2 +5|/g/ml PVS before 

 being placed on the column. (B) Total soluble RNA from 

 calf epithelial cells. The experimental procedures were 

 the same as in A. (C) Total soluble RNA from calf 

 cortex fiber cells. The rlbosomal RNA and soluble RNA 

 were first separated by fractionation on a MAK column. 

 The soluble RNA (peak A) fractions were combined and 

 dialyzed as described in Fig. 16A before being placed on 

 the column. (Fig. 14, J. Papaconstantinou, Science, in 

 press; copyright by the American Association for the 

 Advancement of Science.) 



analysis of the rlbosomal RNA eluted from the 

 DEAE column. This fraction has a GC content 

 of 64%, which is typical of rlbosomal RNA. As 

 a further characterization of these fractions 

 we are presently studying the extent to which 

 they can be charged with C^"* -amino acids. 



C. Inactivation of DNA: the loss of nuclear 

 activity in lens fiber cell 



The MAK column patterns in Fig. 14 

 indicate that there is a significant decrease in 

 the DNA in the cortex fiber cells. These ob- 

 servations are in agreement with cytological 

 reports that the nucleus of the cortex fiber 

 cell decreases in size and is ultimately lost in 



180 240 300 360 



Fig. 17. 



The fractionation of phenol-extracted nucleic acids from 

 calf lens epithelial cells incubated in ^H-thymidine. 



CD. 260 ' ; counts per minute/ml . (Fig. 15, 



J. Papaconstantinou, Science, in press; copyright by the 

 American Association for the Advancement of Science. 



the older fiber cells. 



As I stated previously, the lens fiber cells 

 do not have the ability to replicate. Although 

 small amounts of DNA could be detected in the 

 nucleic acids extracted from the fiber cells it 

 is not known whether this DNA is metabolically 

 active. In view of this, we performed experi- 

 ments to determine whether ^H-thymidine is 

 incorporated into the DNA of the fiber cells. 

 After incubating calf lenses in ^H-thymidine, 

 the nucleic acids from epithelial cells and fiber 

 cells were phenol extracted and fractionated 

 on MAK columns (Fig. 17). The DNA (peak B) 

 fractions were counted and it was found that 

 the ^H-thymidine was incorporated into the 

 DNA of the epithelial cells. On the other hand, 

 the corresponding experiment with the fiber 

 cell DNA fraction showed that there is no 

 incorporation of ^H-thymidine into the fiber 

 cell DNA. We concluded from these experiments 

 that although the DNA of the epithelial cells is 

 metabolically active, this activity is lost in the 

 fiber cell. This observation, we feel, has an 

 important and obvious bearing on our observa- 

 tions that the m-RNA in the fiber cell is stable. 



V. Summary and Conclusions 



I would like to return to the beginning of 

 my lecture, where I listed the cytological events 

 marking the differentiation of the lens epithelial 

 cell to the fiber cell and now attempt to corre- 

 late these events with our biochemical observa- 

 tions. The morphological and biochemical alter- 

 ations characteristic of all stages of lens cell 



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