The localization of spermidine in salivary gland cells of Drosophila melanogaster and its effect on H3-uridine incorporation.

Although the polyamines putrescine, spermidine, and spermine-are ubiquitous biological compounds, their specific distribution and function at the subcellular level remain uncertain.As cellular cations they probably serve an important function as part of the ionic milieu in the mediation of some cellular processes.Stabilization of cellular membranes,1 2 mitochondria,2 ribosomes, 4 and nuclei5 by polyamines has been demonstrated.Stabilization of polynucleotide by the polyamines6' I is reflected in the stimulation of RNA synthesis,8 9 DNA synthesis,1 and, in the case of ribosomes, in the enhancement of amino acid incorporation.1The problem of polyamine redistribution,12' 13 inherent in cell fractionation, has discouraged and cast doubt on investigations of polyamine localization at the cellular level.The present paper describes a reliable method for the radioautographic localization of spermidine, and the effect of polyamines on RNA synthesis in situ in polytene chromosomes.Materials and Methods.-Polyamine and RNA analyses: Stocks of Drosophila melanogaster were maintained at 23.0 +4 0.50C on a medium described by Pearl et al.,"4 modified by the addition of propionic acid (0.90 ml/liter) for the control of mold.Various stages of development were col- lected, washed in Drosophila saline, and frozen at -20'C.The polyamines were concentrated from TCA extracts of the developmental stages according to the method of Raina.15The separation and quantitation of the polyamines were effected by the thin-layer chromatographic method of Hammond and Herbst.16The hydrochloride salts of the polyamines (Calbiochem) were used in all experiments.RNA analyses'7 were performed on the TCA-insoluble precipitates, using yeast RNA as the standard.Radioautography.-Localization of H3-uridine: Salivary glands of late third-instar larvae of D. melanogaster were cleanly dissected in a drop of Medium 199 (Difco Laboratories, Detroit, Michigan), washed briefly in a fresh drop of the same medium, and preincubated for 1 hr in sealed depression slides containing Medium 199 (50 Al) in the presence and absence of spermidine.At the end of the preincubation period the glands were rapidly rinsed in Medium 199, transferred to Medium 199 containing H3-uridine (30 tic/ml; 4 c/mmole), and incubated for 30 min.The preincubation and incubation procedures were carried out with shaking in a Dubnoff water bath at 27-280C.The incubations were stopped by adding an equal volume of 10%7 TCA (w/v), and