268 VINCENT G. ALLFREY 



AMINO ACID INCORPORATION INTO NUCLEAR RIBOSOMES 



Much of the protein synthesis in the intact nucleus occurs in its 

 ribonucleoprotein components [5, 37]. These are readily extractable from 

 the nucleus in dilute salt solutions or neutral buffers. Recent fractionation 

 studies of such nuclear extracts has revealed a heterogeneous population of 

 ribonucleoprotein particles which can be separated into classes of different 

 chemical composition and biosynthetic activity [37]. 



When the different fractions are examined under the electron micro- 

 scope, one observes the presence of large mmibers of dense particles of 

 about 100 A diameter. Particles of this size are clearly discernible in 

 electron micrographs of sections of intact thymus nuclei, and in tissue 

 sections. Similar particles have also been reported in nucleoli [38, 40], 

 chromosomal loops [41], in the Balbiani rings of certain chromosomes [42], 

 and throughout the nuclear sap in other tissues [43]. Dr. J. H. Frenster 

 has made some attempts at further morphological characterization of these 

 nuclear ribonucleoprotein particles (ribosomes), but problems of resolution 

 make this very difficult [37]. 



Tracer studies have shown that the nuclear ribonucleoprotein particles, 

 like those of the cytoplasm, are actively engaged in protein synthesis. Their 

 activity within the thymus nucleus has been followed by incubating isolated 

 nuclei in the presence of [^^C]-labelled amino acids and subsequently isolat- 

 ing the various RNP fractions [37]. The results of such experiments indicate 

 that the different particle fractions, separated on the basis of differing 

 sedimentation properties, also differ greatly in their metabolic activity [37]. 



There is convincing evidence that amino acid uptake takes place in 

 ribosomal particles within the nucleus, (i) The incorporation of amino 

 acids requires the presence of sodium ions in the nuclear suspension. (As 

 will be shown later, this requirement for sodium, rather than potassium 

 ions, is a sure indication of nuclear localization.) (2) Preincubation of the 

 nuclei with deoxyribonuclease effectively inhibits the subsequent uptake of 

 [^^C]-labelled amino acids into the proteins of the RNP particles. This 

 evidence for DNA dependency, apart from establishing nuclear localization, 

 also reflects the need for the nuclear phosphorylating system as a source 

 of ATP [13]. 



The synthetic activity of the nuclear ribosomes can also be studied 

 outside of the cell nucleus because, if they are properly supplemented, free 

 RNP particles prepared from thymus nuclei can incorporate [^"^C-Jamino 

 acids in vitro [37, 44]. The process requires, in addition to the particles, 

 ATP and an ATP-regenerating system, the nuclear pH 5 enzymes, and 

 guanosine triphosphate (GTP) [37, 44]. The time course of incorporation 

 of [i-^'*C]-leucine into the proteins of isolated nuclear ribosomes is 

 depicted in Fig. 3. It is of special interest that amino acid incorporation 

 can take place only in particles which were prepared under isotonic con- 



