The use of cloned tRNA genes for the purification and measurement of specific tRNAs.

Abstract

We have previously reported the ability of a cloned tRNAMeti gene (pt145) to bind tRNAMeti specifically [5]. In this paper, we show that a pBR322 plasmid containing the tRNAAsn gene of Xenopus (pt38 - donated by Stuart Clarkson) will specifically bind to mouse tRNAAsn when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One-dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band and several small-molecular-weight minor bands. The hybridizing tRNA has been identified as tRNAAsn by partial RNA sequencing and the detection of both the Q base and t6A. The steady-state concentration of tRNAAsn in the uninduced Friend cell was determined by hybridizing tRNA labeled in vitro to pt38. 1% of the total tRNA hybridized, representing 0.017 pg tRNAAsn/cell. The fraction of newly synthesized tRNA representing tRNAAsn or tRNAMeti was also determined by hybridizing tRNA labeled in vivo to either pt38 or pt145, respectively. 0.96% and 0.85% of the tRNA hybridized to pt38 and pt145, respectively.

• Authors: L Kleiman, C Essagian, K Nicoghosian, R J Cedergren
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 782 Issue 3 Pg. 269-73 (Jul 18 1984) ISSN: 0006-3002 [Print] Netherlands
• PMID: 6733110 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Asparagine
• RNA, Transfer

Topics

• Animals
• Asparagine
• Cloning, Molecular
• Nucleic Acid Hybridization
• RNA, Transfer (biosynthesis, genetics, isolation & purification)
• Xenopus laevis (genetics)