The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis.
The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.
Kubelik
A R
AR
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Turcq
B
B
Lambowitz
A M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003360:Cosmids; D003600:Cytosol / Q000201:enzymology; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014637:Valine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
D003062:Codon; D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D014637:Valine-tRNA Ligase