Cloning of the yeast methionyl-tRNA synthetase gene.

F Fasiolo,J Bonnet,F Lacroute

doi:10.1016/s0021-9258(19)69782-6

Abstract

A pool of random wild type yeast DNA fragments obtained by partial Sau IIIA restriction enzyme digestion and inserted in the Bam HI site of the hybrid yeast Escherichia coli plasmid ((pFL1) has been used to transform to prototrophy a methionyl-tRNA synthetase-impaired mutant requiring methionine. In the numerous prototroph strains recovered at least two independent clones have been obtained which show nonchromosomic inheritance character and an approximately 30-fold increase in methionyl-tRNA synthetase activity as compared to the wild type. Measurement of the Km for methionine in the transformed yeast cells indicates that the activity has been restored by decreasing the Km for methionine to the same level as found for the wild type methionyl-tRNA synthetase. Southern blotting experiments show that the yeast DNA's fragments inserted in the two independent plasmids share a common sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase. They also suggest that the methionyl-tRNA synthetase gene is differently orientated in the two plasmids

Highlights

Obtained by partial Sau IIIA restriction enzyme diges- Yeast and Bacteria”F.F.1 sp 1, used as recipient for transformation and inserted in the B a n HI site of the hybrid yeast tion has a rnesl,ura3 genotype and was constructed by recombining Escherichia coli plasmidhas been used to trans- the H.19.3.4 mutant provided by McLaughlin and Hartwell ( 2 ) with form to prototrophy a methionyl-tRNA synthetase-im- ura3 triple mutantisogenic to FL 100.This strain requiresuracil and paired mutant requiring methionine
The frequency of transformation was measured by plating an aliquot of the hybrid plasmid carrying the yeast DNA pool on a minimal plate supplemented with methionine (5 mg), selecting only for the Ura' phenotype born by each plasmid
Determination of Methionyl-tRNA Synthetase Aminoacylation Activity-Aliquots of the cultures(50ml) harvested in log phase were washed in extraction buffer and stored at -25°C

Summary

Introduction

Obtained by partial Sau IIIA restriction enzyme diges- Yeast and Bacteria”F.F.1 sp 1, used as recipient for transformation and inserted in the B a n HI site of the hybrid yeast tion has a rnesl,ura3 genotype and was constructed by recombining Escherichia coli plasmid (pFL1)has been used to trans- the H.19.3.4 mutant provided by McLaughlin and Hartwell ( 2 ) with form to prototrophy a methionyl-tRNA synthetase-im- ura3 triple mutantisogenic to FL 100.This strain requiresuracil and paired mutant requiring methionine. Fragment of the yeast 5 plasmid [7],a 1.1kilobase NindlII fragment methionyl-tRNA synthetase. Ments show that the yeast DNA’s fragments inserted in the two independent plasmids share acommon sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase.

Results

Conclusion