Selected species of 4S RNA of chick embryo cells will hybridize in vitro with 35S RNA of avian myeloblastosis virus. A major tRNA component of the hybridizable 4S RNA is tryptophan tRNA. A hybrid prepared from purified tryptophan tRNA and 35S RNA of avian myeloblastosis virus in vitro is an efficient templateprimer for DNA synthesis catalyzed by reverse transcriptase (RNA-dependent DNA polymerase).Initiation sites or primers are required for reverse transcription of 35S RNA of RNA tumor viruses in vitro. It has been reported that in avian myeloblastosis virus (AMV) (1) and in Rous sarcoma virus (2, 3) the primer is a low-molecularweight RNA. In AMV, 4S RNA with structural (4) and functional (5) properties of transfer RNA has been shown to be closely associated with the viral 70S RNA. A 4S RNA capable of priming DNA synthesis in Rous sarcoma virus has been isolated from the virus and has been shown to be a single species (6, 7). A tRNA with similar properties has been demonstrated in both avian and mammalian cells and was reported to be specifically aminoacylatable with tryptophan (8).We have recently reported that distinct species of tRNA from a variety of mammalian cells in culture will hybridize with 35S RNA of AKR murine leukemia virus and of AMV in vitro (9). We now report that distinct species of 4S RNA of chick embryo cells will hybridize in vitro with AMV 35S RNA and that a major component of this RNA is tryptophan tRNA. Furthermore, this tRNA, when hybridized to AMV 35S RNA, will prime the synthesis of DNA in the presence of purified AMV RNA-dependent DNA polymerase. MATERIALS AND METHODSThe 35S RNA was prepared from AMV-containing chicken plasma (kindly provided by Dr. J. W. Beard of Life Sciences, Inc., Gulfport, Fla.) as described (9). 4S RNA of chick embryo cells, both radioactively labeled and nonlabeled, was prepared from primary cultures. The cells were grown in medium 199 in either petri plates or in roller bottles. 32p labeling was in medium 199 (containing 5% fetal calf serum) with 10% of the usual phosphate content, and RNA extractions were the same as reported (9). RPC-5 column packing was prepared as described (10). Chromatographic conditions were optimized for the separation of eukaryotic tRNAs by C. K. solution of 10 mM Tris-HCl (pH 7.6); 0.1 M NaCl; 0.1% sodium dodecyl sulfate; and 1 mM EDTA. The total mixture, 385 JAl, was contained in 1-ml ampules, which were flushed with nitrogen before sealing. The contents were heated to 800 for 5 min and quickly chilled in ice water. The sample was then heated at 650 for 30 min, and the temperature of the water bath reset and maintained at 550 for an additional 14-16 hr (approximately 75 min were required for the temperature to reach 550). After hybridization the samples were quickly chilled in ice water, and the hybridized 4S RNA was separated from the free 4S RNA by sucrose density gradient centrifugation at 50,000 rpm in an SW 56 rotor through a 10-30% sucrose gradient in a solution of 10 mM Tris-HCl (pH 7.6); 10 mM NaCl; 0.1% sodium dod...

A variety of some of the more commonly used metabolic inhibitors and several substances previously found to influence protein incorporation by other types of cells were tested for their effects on protein incorporation by growing oocytes in vitro. With the exception of the uncouplers and the sulfhydryl reagent iodoacetate, the metabolic inhibitors at concentrations of 10 -3 M or less had little influence on sequestering activity. Cycloheximide, at concentrations which essentially abolished all protein synthesis, reduced protein incorporation by only 4&50%. The most pronounced inhibition of protein uptake without apparent cytotoxic side effects was provided by vinblastine and sulfhydryl reagents (especially trivalent arsenicals). The sulfhydryl reagents appeared to exert their effect primarily on the oocyte rather than the proteins of the medium. The results are evaluated both in terms of their relevance to previous studies with other cell systems and their significance towards understanding the mechanisms involved in the incorporation of macromolecules by I cells. Finally, a preliminary model of presented for further testing. ' Research sponsored by the U. S. Atomic Energy Commission under contract w i t h Union Carbide Corporation.

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