quite low. However, only a small fraction of 

 the hybridizable radioactivity in preparations 

 like this is ribosomal RNA. From competition 

 experiments, we get a crude estimate of the 

 fraction of the genome that's involved in the 

 synthesis of ribosomal RNA and that appears 

 to be about 0.2%. Most of the counts that do 

 hybridize appear to be attached to sites on the 

 DNA for which ribosomal RNA does not com- 

 pete. I might say finally that unless one is 

 familiar with this field, one might tend to be 

 impressed with the result just described. How- 

 ever, there are, in principle, much better ways 

 of doing it. The best, by far, seems at the 

 moment to be an experiment showing that in 

 the unfertilized egg there is a kind of RNA 

 capable of supporting protein synthesis in vitro, 

 an RNA other than degraded ribosomal or 

 transfer RNA, and if one obtains an in vitro 

 system that demonstrates this in a reliable 

 way, then the problem has really been solved 

 properly. There is a result from Monroy's 

 laboratory (10) that appeared about a year ago, 

 showing this to be the case, although the total 

 incorporated activities were quite low. Never- 

 theless, their claim, and it seems to be a 

 justified one, was that there is as much tem- 

 plate RNA in an unfertilized egg as there is 

 in an early blastula. That is certainly in accord 

 with the indirect evidence described earlier. 

 We come now to the final point, which 

 concerns proteins. First, there is some reason 

 to suspect that among the proteins made at the 

 beginning of development, are some that must 

 be important for mitosis. Inhibitors of proteins 

 synthesis, such as puromycin, also inhibit 

 cleavage (11). They inhibit all of development, 

 of course, but they do stop an ongoing cleavage 

 if applied before metaphase. These inhibitors 

 therefore stop division at a characteristic 

 cytologic stage - a stage just before the mitotic 

 spindle is formed and the nuclear membrane 

 breaks down. All of this suggests that there 

 are among the early proteins some that have 

 something to do with mitosis. Autoradiograms 

 of eggs labeled with amino acids make this 

 suggestion in another way. We make such 

 autoradiograms as a control whenever we label 

 sea urchin eggs. The reason for this is that 

 when dealing with animal cells, in a medium 

 like sea water, the problem of bacterial con- 

 tamination is ever present. One way that one 

 can be reasonably sure that the radioactivity 

 being studied is really inside the cells is to 

 make autoradiograms and, hence, we do so 

 routinely. 



Fig. 12. 



(Fig. 7, Gross, Malkln and Hubbard, /. Mol. Biol. 13, 463, 

 1965; reproduced with permission of Academic Press.) 



Examining autoradiograms of cells that 

 have been labeled with amino acids, during 

 the first division cycle, we observe the sort 

 of thing shown in Fig. 12. In cells that were 

 at metaphase or early anaphase, there was a 

 heavy concentration and, indeed, an almost ex- 

 clusive localization of radioactivity in the mitotic 

 spindle. Shown in the figure is an early anaphase 

 mitotic apparatus. Now there are two possible 

 interpretations of this result, and the one you 

 accept depends on your hypothesis of the or- 

 ganization of the mitotic apparatus. If you believe 

 that the mitotic spindle as seen in situ is a 

 simple structure, most or all of whose protein 

 is uniquely characteristic of it, then an auto- 

 radiogram of the type shown proves that most 

 of the radioactivity goes into one protein, i.e., 

 that all protein synthesis at the beginning of 

 development has to do with the mitotic apparatus. 

 The alternative arises if you don't believe that 

 the spindle has in it only spindle proteins, but 

 that it may have others as well. Then you have 

 to decide whether the localization may mean 

 something else. Figure 13, which is an elec- 

 tronmicrograph, shows why it is our conviction 

 that the second alternative has to be accepted. 

 This is a section through an early anaphase 

 spindle at moderate magnification, and it is 



11 



