POLYNUCLEOTIDE SYNTHESIS IN NUCLEOLUS AND CHROMOSOMES I77 



cell ol the salivary gland of Diosophila. In cells fixed in acid fixatives the nucle- 

 olus shrinks somewhat and separates from the chromosomes. In such material 

 the only part of the chromosome attached to or includeti in the nucleolus is the 

 small region around the nucleolus organizer which is very small in proportion 

 to the total amount of chromatin in the nucleus. Therefore, the DNA associated 

 with the nucleolus is insignificant compared to the total in the nucleus. 



Plan of Experiment. Taylor ct a/.i^j) and McMaster-Kayc and Taylor (4) 

 have described the methods and plan ol similar experiments using P''- as the 

 label. Adenine-8-C^* was used in the experiment to be described, which was per- 

 formed by one of us (Dr. Taylor). After extraction of cold acid-soluble nucleo- 

 tides, nearly all of the remaining label from C'^-adenine is in RNA and DNA. 

 Since there are periods in early third instar when very little incorporation into 

 DNA occurs, the label is an excellent one for the study of RNA metabolism. 



Drosophila repleta larvae moult two times during the first 8 days when grown 

 at 20°C. After the second moult these third instar larvae grow during a period 

 of about 5 days before forming pupae. During this period the salivary glands 

 are presumably very active in secretion of proteins, and in addition the gland 

 cells grow tremendously. The chromosomes are polytene giant structures at the 

 beginning of third instar, but at one or more intervals during third instar they 

 synthesize DNA, presumably duplicating, and eventually reach the large size 

 typically studied by cytogeneticists. 



The RNA concentration of the salivary gland cells is relatively high during 

 the first half of third instar. Relative concentrations in the structures to be studied 

 have been estimated by McMaster-Kaye and Taylor (4) by two quite dififerent 

 techniques which gave the same answer. The amounts of the basic dye. Azure B, 

 bound by nucleolus and cytoplasm were compared by a mircophotometric tech- 

 nique. During the first half of third instar, while the cytoplasmic volume c]uad- 

 rupled, the ratio of RNA concentration in nucleolus and cytoplasm did not change 

 appreciably. The ratio is very near i all during this period. 



The other method is based on grain counts in autoradiographs. Larvae were 

 fed continuously on a food containing P"'--labeled phosphate. Larvae were fixed 

 at intervals until the label in RNA of all structures reached an equilibrium. At 

 this time the number of grains over any area of the cell should reveal the con- 

 centration of RNA, since all RNA would be expected to have the same specific 

 activity. Of course, all non-nucleic acid P"^'- was first removed from the cells and 

 replicate slides were prepared with RNA removed from one by ribonuclease. 

 The difference in number of grains over the cells on the two slides gave relative 

 concentrations. The concentrations in nucleolus and cytoplasm were again shown 

 to be ec]ual (4). The chromosomes, i.e., the nuclear region without a nucleolus, 

 had a little less than one-half the concentration of the nucleolus and cytoplasm. 



With this information, grain counts from autoradiographs of cells in larvae 



