Abstract
We have determined the complete nucleotide sequence of the avian tRNATrp which serves as primer for avian retrovirus DNA synthesis by the viral polymerase. The sequence is identical to that reported for tRNATrp present in uninfected avian cells (Harada, F., Sawyer, R. C., and Dahlberg, J. E. (1975) J. Biol. Chem. 250, 3487-3497). Although there appears to be only a single species of tRNATrp in avian cells, two functionally different forms within the population can be distinguished. We show that the tRNATrp isolated from virions can act in vitro as an efficient suppressor for UGA. The suppressor activity is roughly 3-fold greater with viral tRNATrp than with cellular tRNATrp. In addition, it has been reported (Panet, A., Haseltine, W. A., Baltimore, D., Peters, G., Harada, F., and Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2535-2539) that the viral polymerase can bind 100% of viral tRNATrp, but only 30% of cellular tRNATrp. Hence, avian retroviruses seem to selectively incorporate and utilize only one of these forms. Since the nucleotide sequence and nucleoside modifications are identical between viral and cellular tRNATrp, two conformations of avian tRNATrp may exist which can influence several biological activities of the molecule.
Highlights
We have determined the complete nucleotide sequence of the avian tRNATv which serves as primer for the intiation of DNA synthesis by the viral polymerase
Our attention was focused on possible differences in base modification since the avian sarcoma virus (ASV) polymerase can distinguish the tRNATw isolated from virions and tRNATwderived from the uninfected avian cell, even though there appears to boenly one isoacceptor species of tryptophan tRNA [5, 6]
Panet et al [7] found that theavian myeloblastosis virus tRNAT"is completely bound by the polymerase in solution, whereas only 30%of the cellular tRNATrpis bound
Summary
Materiah-The sources of most materials have been described previously [8]. [32P]Orthophosphatewas purchased from International Chemical and Nuclear Corp. ['HIMethylmethionine (specific activity, 10.5 Ci/mmol) was purchased from New England Nuclear. Reactions of 0.1 ml containing 1to 10 pg of tRNA, 10 mM Tris-HC1, pH 7.3, 10 mM MgCIZ, 10mM KC1.6 mM 2-mercaptoethanol, 6 mM CTP, 10 mM ATP, 5 pCi of r3H]amino acid at a specific activity -10 ci/mmol (New England Nuclear), and 10 pl of a crude chicken liver or chick embryo synthetases were prepared according to Nishimura [17]. Coli cell-free translation as described [27].The preparation of 9 S of DNase (Worthington) (at an enzyme to nucleic acid ratio of 1:lOO) rabbit globin mRNA wasby the method previously described [28],as for 30 min at 23°C followed by phenol extraction and precipitation was QB bacteriophage RNA [29]. The RNA was resuspended in 1 M NaCl and stirred at 4°C for 3 h after which the rRNA was removed by pelleting at 2000
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