Molecular Characterization of the Mitochondrial DNA of a New Stopper Mutant Er-3 of Neurospora Crassa

An ethidium bromide-induced stopper mutant of Neurospora crassa is characterized at the molecular level. The mutant has two populations of mitochondrial DNA: a defective predominant mutant molecule and a basal level of the wild-type molecule. The aberrant DNA resulted after a 25-kbp deletion from the wild-type mitochondrial chromosome, which included major genes such as cytb, co1 and oli2. The deletion endpoints are located in the second intron of the ND5 gene, and in a sequence 250 nucleotides upstream of the co2 gene. The recombination has taken place between two nine nucleotide repeats CCCCGCCCC, one of which is close to a PstI palindrome at its 5' end. Thus the mutant ER-3 differs from all the other stopper mutants described previously in the extent and location of the deletions in the mtDNA.     

Pet127 governs a 5' -> 3'-exonuclease important in maturation of apocytochrome b mRNA in Saccharomyces cerevisiae

The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3'-dodecamer sequence, but the 5'-ends have seven different sequences. To investigate whether apocytochrome b (COB) mRNA is processed at the 5'-end from a longer precursor by an endonuclease or an exonuclease, a 64-nucleotide sequence, which is required for the protection of COB mRNA by the Cbp1 protein and is found at the 5'-end of the processed COB mRNA, was duplicated in tandem. The wild-type 64-nucleotide element functioned in either the upstream or downstream position when paired with a mutant element. In the tandem wild-type strain, the 5'-end of the mRNA was at the 5'-end of the upstream unit, demonstrating that the mRNA is processed by an exonuclease. Accumulation of precursor COB RNA in single and double element strains with a deletion of PET127 demonstrated that the encoded protein governs the 5'-exonuclease responsible for processing the precursor to the mature form.     

A nuclear mutation prevents processing of a mitochondrially encoded membrane protein in Saccharomyces cerevisiae.

Subunit II of cytochrome oxidase is encoded by the mitochondrial OXI1 gene in Saccharomyces cerevisiae. The temperature-sensitive nuclear pet mutant ts2858 has an apparent higher mol. wt. subunit II when analyzed on lithium dodecylsulfate (LiDS) polyacrylamide gels. However, on LiDS-6M urea gels the apparent mol. wt. of the wild-type protein exceeds that of the mutant. Partial revertants of mutant ts2858 that produce both the wild-type and mutant form of subunit II were isolated. The two forms of subunit II differ at the N-terminal part of the molecule as shown by constructing and analyzing nuclear ts2858 and mitochondrial chain termination double mutants. The presence of the primary translation product in the mutant and of the processed form in the wild-type lacking 15 amino-terminal residues was demonstrated by radiolabel protein sequencing. Comparison of the known DNA sequence with the partial protein sequence obtained reveals that six of the 15 residues are hydrophilic and, unlike most signal sequences, this transient sequence does not contain extended hydrophobic parts. The nuclear mutation ts2858 preventing post-translational processing of cytochrome oxidase subunit II lies either in the gene for a protease or an enzyme regulating a protease.     

Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation.

To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing.     

Assembly of the mitochondrial membrane system: isolation of mitochondrial transfer ribonucleic acid mutants and characterization of transfer ribonucleic acid genes of Saccharomyces cerevisiae

A method is described for isolating cytoplasmic mutants of Saccharomyces cerevisiae with lesions in mitochondrial transfer ribonucleic acids (tRNA's). The mutants were selected for slow growth on glycerol and for restoration of wild-type growth by cytoplasmic "petite" testers that contain regions of mitochondrial deoxyribonucleic acid (DNA) with tRNA genes. The aminoacylated mitochondrial tRNA's of several presumptive tRNA mutants were analyzed by reverse-phase chromatography on RPC-5. Two mutant strains, G76-26 and G76-35, were determined to carry mutations in the cysteine and histidine tRNA genes, respectively. The cysteine tRNA mutant was used to isolate cytoplasmic petite mutants whose retained segments of mitochondrial DNA contain the cysteine tRNA gene. The segment of one such mutant (DS504) was sequenced and shown to have the cysteine, histidine, and threonine tRNA genes. The structures of the three mitochondrial tRNA's were deduced from the DNA sequence.