0.25 0.50 1.0 2.0 0.25 0.50 1.0 2.0 0.25 0.50 1.0 



SODIUM PiRCHLORATE COtiC.CM) 



Fig. 4. 



Relation between protein dissociated from rat liver chromatin and template activity (AMP 

 Incorporated). (Fig. 4, Marushlga and Bonner, ] . Mol. Biol. 15, 160, 1966; reproduced with 

 permission of Academic Press.) 



TABLE III 



Synthesis of Pea-Seed Globulin by Messenger RNA 

 Dependent Ribosomal System in Response to Messenger 

 RNA Generated by Two Different Kinds of Pea-Plant 

 Chromatin 



Template for RNA 

 synthesist 



C"-leucine incorporated 

 into protein 



Total soluble 



protein Globulin 



(cpm) (cpm) 



Globulin/total 



protein 



(°0 



t The reaction mixture contains all materials required for both RNA and protein 

 synthesis. Incubation for 30 minutes at 37^ All particulate material was then 

 centrifuged off at 105000 ■ g and pea-seed globulin content of soluble protein 

 synthesized determined by immunochemical assay. 



five-fold difference when comparing the coty- 

 ledon chromatin and the apical bud chromatin. 

 Do the preparations vary that much? 



CHALKLEY: Well, 1 suspect that in these 

 cotyledons the overall in vivo synthesis of RNA 

 may be considerably below the overall RNA 

 synthesis in apical buds and this may be mirrored 

 in the capacity of the chromatin to make RNA. 



The results also reflect different scales of ex- 

 periments. 



PAPACONSTANTINOU: Did you treat with 

 perchlorate? 



CHALKLEY: No, that's exactly as it was 

 isolated. They haven't been treated at all. 



In order to identify the protein as precisely 

 as possible they applied the following ap- 

 proaches. The in vitro synthesized protein was 

 purified using the procedure applicable to pea 

 seed globulin. If this material is synthesized 

 from C^* -labeled amino acids and then diluted 

 with cold globulin isolated from pea cotyledon, 

 it has been shown to have exactly the same 

 Svedberg constant as native globulin when studied 

 in the ultracentrifuge. In addition, radioactive 

 globulin was digested with trypsin and the re- 

 sulting peptide fragments separated by two- 

 directional paper chromatography, and com- 

 pared with the tryptic digestion pattern from 

 native pea seed globulin. Radioactivity was found 

 in positions corresponding to every peptide spot 

 (from native globulin) and no trace of radio- 

 activity was located in other regions of the 

 chromatogram. So it appears that it is really 

 genuine globulin. 



POLLARD: Have you done anything with 

 antibodies? Does it precipitate? 



134 



