NUCLEAR SYNTHESIS OF RNA 533 



In this pulse experiment the densely labelled nucleus in an unlabelled 

 cytoplasm found at 12 min. contrasts sharply with unlabelled nucleus 

 surrounded by heavily labelled cytoplasm at 100 min. (Figs. 2(6) and 2(d)). 

 The presence of an unlabelled nucleus surrounded by heavily labelled 

 cvtoplasm not onlv indicates a transfer of RXA from nucleus to cytoplasm 

 but suggests in addition that the transfer of RXA in the reverse direction, 

 from cvtoplasm to nucleus, does not take place. This distribution of 

 labelling also implies that if breakdown of cytoplasmic RXA does occur, 

 the products are not used by the nucleus for RXA synthesis. Similar 

 conclusions were suggested bv nuclear transplantation studies in amoeba 

 by Goldstein and Plant [S]. 



The experiment in Fig. 3 also demonstrates that the pool into which 

 [■^H]-cvtidine (or its derivatives) enters must be large since tritium becomes 

 incorporated into RXA long after [-^Hj-cytidine has been eliminated from 

 the medium. Because the pool cannot be washed out of living Tetra/iyinena 

 with non-labelled medium, it may be that the ['^Hj-cytidine has been 

 converted to a form which is bound (but still acid-soluble) or which does 

 not readilv pass through the cell membrane. The pool may be in the form 

 of mono-, di-, or triphosphates of cytidine. Whether the pool is localized 

 in the nucleus or cvtoplasm or is present in both places is not known. 



Bv removing the nucleus from a cell, it becomes possible to compare 

 the capacities of nucleated and enucleated cells to synthesize RXA. 

 Tetrahvniena were cut into nucleated and enucleated fragments with a 

 glass needle controlled bv a micromanipulator. All of the nucleated frag- 

 ments survive longer than 40 hr. and most of them regenerate and resume 

 proliferation. The enucleated fragments survive in the complete nutrient 

 medium for 10 to 40 hr. and move about by ciliary activity. 



Fnucleated and nucleated fragments of Tetrahymeua have been 

 incubated for 20 to 240 min. immediately after cutting, in complete 

 medium containing ['^HJ-cytidine. The incorporation of activity into 

 nucleated fragments is always intense (Fig. 4). With short incubation (up 

 to 90 min.) the nucleus is more densely labelled than the cytoplasm. After 

 that time, labelling is so heavy in both nucleus and cytoplasm that no 

 difference between the two sites is apparent. About thirty-five enucleated 

 fragments of cytoplasm have been studied, and none has been found to in- 

 corporate p^HJ-cytidine into RXA (Fig. 4). 



In sharp contrast, enucleated Tetraliymena are still capable oi in- 

 corporation of [^^C]-amino acids. This activity occurs at a lower rate than 

 in the nucleated fragments of comparable size. The [^*C]-amino acids are 

 presumably incorporated into protein since they are not removed by 

 10 min. extraction with 5",, TCA at 90' and ether-alcohol treatment for 

 10 min. This capacity of enucleated Tetvahymena to incorporate [^^C]- 

 amino acids drops very rapidlv after enucleation. By 6 hr. the incorporation 



