60 120 180 240 300 360 

 ml 



120 180 240 300 360 



Fig. 14. 



The fractionation of phenol extracted nucleic acids from 



calf lens epithelial cells ( ) and calf cortex fiber 



cells ( ) on methylated albumin columns (MAK). 



A total of 35.4 O.D. 2^0 units from epithelial cells were 

 placed on the column; 45.0 O.D. 250 units from the fiber 

 cells were placed on the column. The nucleic acids were 

 eluted with a linear salt gradient ranging from 0.2 M to 

 1.4 M NaCl in 0.05 M sodium-phosphate pH 6.8. (Fig. 12, 

 J. Papaconstantlnou, Science, in press; copyright 1966 by 

 the American Association for the Advancement of 

 Science. 1 



and now the epithelial and fiber cell patterns 

 are almost alike except for the quantitative 

 differences between peaks A and C (Fig. 15). 

 Our next step was to determine whether 

 the RNA of peak A is t-RNA or a mixture of 

 t-RNA and a ribosomal RNA breakdown product. 

 (This mixture will be referred to below as 

 total soluble RNA). In his studies of the RNA 

 fractions from E. coli Midgely showed that 

 t-RNA and ribosomal RNA could be separated 

 on DEAE columns using 0.05 M tris pH 7.4 with 

 an increasing NaCl gradient (44). We carried 

 out a similar fractionation to determine whether 

 peak A is a mixture of t-RNA and ribosomal 

 RNA. We used this procedure to determine 

 whether the total soluble RNA from lens 

 epithelial and fiber cells could be resolved 

 into two fractions. In one experiment, phenol- 

 extracted RNA was first placed on a sucrose 

 gradient to eliminate the ribosomal RNA. The 

 material remaining at the top of the gradient 

 was dialyzed against tris buffer and was then 

 fractionated on a DEAE column. The elution 

 diagrams in Figs. 16A and 16B show a fiber 



Fig. 15. 



The fractionation of DNase-treated phenol-extracted 



nucleic acids from calf lens epithelial cells ( ) and 



calf cortex fiber cells ( ). The conditions of 



fractionation are exactly as described in Fig. 14. (Fig. 13, 

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

 American Association for the Advancement of Science.) 



cell and epithelial cell pattern respectively. 

 Firstly, it can be seen from the OD26O readings 

 that there is a small amount of RNA eluted by 

 0.5 M NaCl, which corresponds to the region 

 where bacterial t-RNA is eluted. Another larger 

 fraction is eluted by 0.7-0.8 M NaCl, which 

 corresponds to the region where bacterial 

 ribosomal RNA is eluted. Secondly, it can be 

 seen that there is a significant increase in the 

 ribosomal RNA fraction in the fiber cell pattern. 

 In another experiment the total soluble RNA 

 from calf cortex fiber cells was separated from 

 ribosomal RNA by fractionation on a MAK 

 column. The soluble RNA fractions (peak A) 

 were pooled, dialyzed against tris buffer and 

 fractionated on DEAE-cellulose. This fractiona- 

 tion is shown in Fig. 16C. It can be seen that 

 this elution pattern is identical to that obtained 

 for the total soluble RNA from the sucrose 

 gradient (Fig. 16A). These preliminary data 

 indicate that the increase in total soluble RNA 

 (peak A) of the fiber cell (Figs. 14, 15) is due 

 to the accumulation of a nondialyzable ribosomal 

 breakdown product which has chromatographic 

 (MAK) properties similar to t-RNA. Further 

 evidence that this may be a ribosomal break- 

 down product was obtained by a base ratio 



61 



