THE LOCALIZATION OF BASIC PROTEINS IN THE NUCLEI OF LARVAL Drosophila SALIVARY GLANDS.

The identification and specific localization of chemical constituents within the cell nucleus have lagged considerably behind the qualitative and quantitative characterization of these classes of substances, with the possible exception of one component, the deoxyribonucleic acid (DNA). In addition to the latter, the nucleus contains ribonucleic acid1 2 (RNA), histone or basic protamine3 (BP), and at least one other type of protein, the chromosomin of Stedman and Stedman,4 the tryptophane-containing protein (Tr.Pr.) of Mirsky and Pollister,5 or the nonhistone protein fraction which Hamer6 has subjected to comparative amino acid analysis. The localization of these substances in the live nucleus is dependent almost wholly on inference from a variety of analyses and observations made on fixed preparations. The much disputed and maligned Feulgen reaction points to the DNA as a major constituent of the chromosomes. Studies with ultraviolet absorption spectroscopy,' enzymatic attack, and pyronin staining2 indicate that the RNA of the nucleus is predominantly nucleolar. The distribution of the BP seems to be much less precisely delineated. For example, Caspersson7 has reported its presence in the chromosomes and nucleolus of Drosophila salivary gland nuclei from his ultraviolet absorption studies. Vincent,8 from his chemical studies of the isolated nucleoli of starfish odcytes and of maize, could find no basic protein. Bloch and Godman9 have reported a proportional quantitative relationship between DNA and histone synthesis per rat fibroblast or liver cell. They have also shown cytochemically that, in a general manner, DNA and histone have the same nuclear distribution. In the present study the Feulgen reaction for DNA localization, combined with the fast-green-staining method of Alfert and Geschwind'0 for basic protein, has been employed with Drosophila salivary gland material to determine more precisely the localizations of BP with respect to DNA. The method employed is essentially that of Bloch and Godman :9 salivary glands of D. virilis fixed with trichloroacetic acid (TCA) were partially crushed under a cover slip; these preparations provided some whole nuclei. After the slide was frozen on a block of "dry ice," the cover slip was flicked off and the preparation stained directly by a modified Feulgen technique following 24 hours' lipid extraction in 95 per cent alcohol. Other salivary squashes were prepared similarly, using the conventional acetic acid fixative that permits rupture of the nuclei, yielding fully extended chromosomes. After a Feulgen treatment in which TCA was substituted for HCl, photographs were taken of a number of chromosomes or of whole nuclei; the position of each was recorded on a calibrated mechanical stage. The preparations were then re-exposed, and, after TCA extraction (15 minutes at 900 C.) to remove the DNA, were stained by the fast-green technique of Alfert and Geschwind. The recorded fields were then rephotographed.