180 240 

 ml 



Fig. 13. 



The fractionation of calf lens cortex fiber cell proteins 

 Incubated with l^C-algal hydrolysate and lO^g/mlac- 

 tinomycin D. The experimental conditions are the same 

 as described for Fig. 10. (Fig. 11, J. Papaconstantlnou, 

 Science, in press, copyright 1966 by the American Asso- 

 ciation for the Advancement of Science.) 



GROSS: Well, whether its GC or not, they're 

 not as sensitive. 



PAPACONSTANTINOU: Right. I'm just 

 presenting all these ideas which have come out 

 in the literature. They're not my ideas, and I'm 

 just trying to fit some of our data into any one 

 or all of these as we go along. However, I think 

 the evidence does seem to indicate that we can 

 pinpoint the stage at which the messenger RNA 

 becomes stabilized, and that is at the time when 

 the epithelial cells are starting to elongate. 

 The y-crystallin is still sensitive to actino- 

 mycin at that stage, but when they've finally 

 elongated, it's no longer sensitive. This holds 

 true, also, for the a- and ^-crystallins. The 

 period of stabilization seems to fall in con- 

 currently with the breakdown of ribosomes and 

 the decrease in the size of the nucleus and 

 nucleoli. 



Secondly, this stimulation might be attrib- 

 uted to inhibition of the synthesis of a repressor 

 protein by actinomycin (41). It has been shown 

 that the actinomycin D stimulation of tryptophan 

 pyrrolase and tyrosine transaminase occurs 

 after these enzymes have been induced by 

 hydrocortisone, when their m-RNA is relatively 

 stable. By inhibiting the m-RNA responsible 

 for the synthesis of repressor protein the level 

 of this repressor is decreased and a stimula- 



tion of these enzymes results. Finally, the 

 lens epithelial cells are essential for the active 

 transport of nutrients into the lens fiber cells. 

 It is, therefore, possible that actinomycin alters 

 these properties such that there is an increase 

 in the transport of amino acids into the fiber 

 cell layer, resulting in a stimulation of protein 

 synthesis on stable RNA templates. 



B. Ribosomal breakdown in lens fiber cells 



In this final phase of my talk I would like 

 to describe a phenomenon, again associated with 

 fiber cell differentiation which may explain the 

 reduced rate of protein synthesis observed in 

 the final cell. I shall start by reiterating that 

 Eguchi and Karasaki (3, 4) showed by electron 

 microscope studies that during fiber cell forma- 

 tion, in the elongating epithelial cell there is an 

 increase in ribosomes, whereas in the completed 

 fiber cell the ribosomal population is decreased. 

 We feel that we have been able to show essen- 

 tially the same thing in our chemical analyses of 

 the ribosomal RNA in the fiber cell. In these 

 studies we have used methylated albumin col- 

 umns (42) to fractionate nucleic acids from lens 

 epithelial cells and fiber cells and to detect any 

 qualitative or quantitative differences that may 

 occur in the nucleic acids of these cells. The 

 fractionation procedure involved the use of a 

 linear NaCl gradient in 0.05 M Na-phosphate 

 buffer pH 6.8. An elution patternof the epithelial 

 cell nucleic acids is shown in Fig. 14. The t-RNA 

 (peak A) is eluted between 0.4 M - 0.6 M NaCl; 

 DNA (peak B) is eluted between 0.6 M - 0.8 M 

 NaCl; and ribosomal RNA (peak C) is eluted 

 between 0.8 M - 1.0 M NaCl. This sequence of 

 elutions compares well with a similar elution 

 system used to fractionate t-RNA, DNA and 

 ribosomal RNA from E. coli (43). A pattern 

 for the RNA extracted from fiber cells is super- 

 imposed over the epithelial cell pattern to 

 facilitate comparisons between them. It can be 

 seen that there are striking differences between 

 the two patterns: (a) the pattern for fiber cell 

 RNA shows a significantly larger amount of 

 material eluted in peak A (t-RNA) with respect 

 to the amount of material in the ribosomal RNA 

 (peak C); (b) in addition, there is a sharp 

 decrease in the DNA (peak B) of the fiber cell 

 pattern. The quantitative differences between 

 t-RNA and R-RNA of epithelial and fiber cells 

 are better seen in the patterns of Fig. 15. Phenol 

 extracted nucleic acids from epithelial and 

 fiber cells were DNase treated to remove DNA, 

 prior to fractionation on methylated albumin 

 columns. The DNA (peak B) is completely lost, 



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