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Genetics, Vol 119, 805-814, Copyright © 1988
INVESTIGATIONS |
Cytoplasmic Leucyl-tRNA Synthetase of Neurospora crassa Is Not Specified by the leu-5 Locus
R. Benarous, C. M. Chow and U. L. RajBhandary
Present address: Institut de Pathologie Moleculaire, Faculte de Medecine Cochin Port-Royal, Paris, France.
We generated a {lambda}gt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, {lambda}NCLRSC1 and {lambda}NCLRSC2, were obtained which have inserts of ~2 kbp and ~1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that {lambda}NCLRSC1 and {lambda}NCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by {lambda}NCLRSC1 or {lambda}NCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an ~115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is ~3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located.