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Nucleic Acids Research, 1983, Vol. 11, No. 9 2585-2598
© 1983

MOLECULAR BIOLOGY

The isolation and measurement of tRNAimet using RNA/DNA hybridization

L. Meiman*, T. Heron*, C. Essagian*, K. Nicoghosian+ and R.J. Cedergren+

*Lady Davis Institute for Medical Research of the Sir Mortimer B.Davis-Jewish General Hospital 3755 Cote St. Catherine Road, Montreal, Quebec +Département de Biochimie, Université de Montréal Case Postale 6128, Montréal, Québec, Canada

Received February 23, 1983. Revised April 6, 1983. Accepted April 6, 1983.

A pBR322 plasmid containing the initiator tRNAmet gene of Xenopus (ptl45 -donated by Stuart Clarkson) will specifically bind to mouse initiator tRNAmet (tRNAmet) when total mouse tRNA, extracted from uninduced Friend erythroleukenda cells, is hybridized to the gene probe. One dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band (95%) and a minor band. The hybridizing tRNA has been identified as initiator tRNAmet by RNA sequencing. Hybridization of tRNAtotal to another plasmid containing the Xenopus gene for tRNAasn results in two bound species with different electro-phoretic mobilities than the tRNA bound to the initiator tRNAmet gene. ptl45 has been used to measure the steady state concentration of initiator tRANmet in the uninduced and erythroid Friend cell, and in the unfertilized egg and 21 h blastula of the sea urchin. Initiator tRNAmet represents 0.91% and 0.52% of the tRNA populations extracted from uninduced and erythroid Friend cells, respectively. Based upon the total tRNA content per cell, there is a 3.8 fold decrease in initiator tRNAmet per cell during erythroid differentiation. tRNA extracted from unfertilized eggs and 21 h blastula of the sea urchin both have 0.5% of total tRNA as initiator tRNAmet (approximately 1.5 pg).


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