Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase.
The cyt-2-1 mutant of Neurospora crassa is deficient in cytochromes aa3 and c and in cytochrome c heme lyase activity (Mitchell, M.B., Mitchell, H.K., and Tissieres, A. (1953) Proc. Natl. Acad. Sci. U.S.A. 39, 606-613; Nargang, F.E., Drygas, M.E., Kwong, P.L., Nicholson, D.W., and Neupert, W. (1988) J. Biol. Chem. 263, 9388-9394). By rescue of the slow growth character of the cyt-2-1 mutant, we have cloned the cyt-2+ gene from a N. crassa genomic library using sib selection. Analysis of the DNA sequence of the cyt-2+ gene revealed an open reading frame of 346 amino acids that has homology to the yeast cytochrome c heme lyase. The open reading frame is interrupted by two short introns. Codon usage and Northern hybridization analysis suggest that the cyt-2 gene is expressed at low levels. The cyt-2-1 mutant allele was cloned from a partial cyt-2-1 gene bank using the wild-type gene as a probe. Sequence analysis of the mutant gene revealed a 2-base (CT) deletion that alters the reading frame for 21 codons before generating an early stop codon in the protein-coding sequence. It was previously suggested that the cyt-2-1 mutation inactivates one of two regulatory circuits controlling the production of cytochrome aa3. The finding that the cyt-2-1 mutation affects the coding sequence for cytochrome c heme lyase provides a direct explanation for the deficiency of cytochrome c in the mutant and suggests that the lack of cytochrome aa3 is a regulatory response to the deficiency of cytochrome c.
Drygas
M E
ME
Department of Genetics, University of Alberta, Edmonton, Canada.
Lambowitz
A M
AM
Nargang
F E
FE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D008190:Lyases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009491:Neurospora / Q000235:genetics*; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D012326:RNA Splicing; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D008190:Lyases; C031581:cytochrome C synthetase