Abstract
Previous studies from this laboratory have provided a high resolution map for 16 tRNA genes located on the continuous heavy DNA strand of bacteriophage T5 DNA (Chen, M.-J., Locker, J., and Weiss, S.B. (1976) J. Biol. Chem. 251, 536-547). All of the T5 tRNA genes were located in three clusters within the DNA C segment, except for tRNAArg, which mapped on the left end of the DNA D segment (Desai, S.M., Hunt, C., Locker, J., and Weiss, S.B. (1978) J. Biol. Chem. 253, 6544-6550). In this report, we present evidence for the presence of eight additional T5 tRNA species, five of which are located in two new loci within the DNA C segment. We also describe a two-dimensional gel electrophoresis system for the separation and isolation of T5 tRNA species from crude infected RNA preparations. The gel electrophoresis system separates tRNA isoacceptors specific for different amino acids; evidence is presented that the isoacceptors for isoleucine, histidine, and serine are coded by different T5 genes.
Highlights
DNA (Chen, M.-J., LockerJ,., and Weiss,S.B. (1976) J
Map of Fig. 1 shows that all of the genes for the T5 tRNAspecies examined far are present in the C segment of the heavy T5 DNA strand except for tRNAArK, which is located at theleft end of the DNA D segment (8).In this report, we present 1) evidence for the presence of five additional T5 tRNAs anfdor the isoacceptors of tRNAH'" and tRNASe', and 2) a description of the two-dimensional gel electrophoresis and chromatographic procedures used for the isolation and identification of individual T5 tRNAspecies
Panel D shows the separation of the T5 tRNA species glutamineg, lycine, and arginine when the RNA extract of Spot 1-2 was subjected to athud dimension electrophoresis in 16%polyacrylamide containing 7 M urea
Summary
Growth a n d Purification of T5 Phages and Isolation of Phage DNA-Wild type T5' and all the T5 deletion mutants were grown and purified, and phage DNA was isolated, as described by Chen et al (5). In Procedure A, the first dimension was a 10% gel polymerized in 0.5 X T E B buffer (1 X T E B contained 21.6 g of Tris base, 1.86 g of Na'EDTA, and 11 g of boric acid/liter, adjusted to a final pH of 8.3) containing 0.4% 3-dimethylaminopropionitrile and 0.1% ammonium persulfate This gel was formed in a vertical electrophoresis apparatus (10 x 20 cm) (E-C Apparatus Corp., St. Petersburg, Fla.), cooled to 13°C by a circulating water bath, and run a4t00 V in 0.5 X T E B buffer until the bromphenol blue marker dye had migrated about 18 cm. E , C , D,and E designate the DNA segments produced by the major nicks in tRNA Gene Mop the light strand; the approximatemolecular weights
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