Neurospora Crassa Mitochondrial DNA Research Articles

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

Development of an In Vitro Transcription System for Neurospora crassa Mitochondrial DNA and Identification of Transcription Initiation Sites

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

Duplication of the tRNA M Met and tRNACys genes and of fragments of a gene encoding a subunit of the NADH dehydrogenase complex in Neurospora grassa mitochondrial DNA

Neurospora crassa mitochondrial DNA (mtDNA) contains duplications of the tRNA(MMet) gene upstream of a gene (ND2) encoding a subunit of the NADH dehydrogenase complex and of the tRNA(Cys) gene which is found downstream of the apocytochrome b gene. Both duplicated genes are located upstream of the small rRNA gene. The duplications are extended to flanking sequences. In the case of the tRNA(MMet) duplication, two fragments of the ND2 gene are also duplicated. These two fragments, which are not contiguous in the ND2 gene, are connected to each other by a palindromic sequence of 37 bp and together they constitute an open reading frame. The possible involvement of this palindromic sequence in the processes of gene duplication and transfer is discussed. Two overlapping reading frames are present between the tRNA(MMet) and tRNA(Cys) copies. All information of the ND2 duplication and the two overlapping reading frames are present on a polycistronic transcript.

Some highlights of mitochondrial research based on analyses of Neurospora crassa mitochondrial DNA

Research on mitochondrial nucleic acids has produced major surprises. These include: (1) a novel mechanism for reading the genetic code, (2) the first examples of deviations from the ‘universal’ genetic code, and (3) the finding that protein genes can be located totally within introns of other genes. The first indication that sequences within introns are important in RNA splicing came from analyses of mitochondrial introns and recent studies have revealed a close relationship between the majority of mitochondrial introns on the one hand and one class of nuclear introns, the ‘self-splicing’ rRNA introns, on the other.

The Neurospora mitochondrial genome: the region coding for the polycistronic cytochrome oxidase subunit I transcript is preceded by a transfer RNA gene

We have sequenced a 682 bp fragment of Neurospora crassa mitochondrial DNA to complete the sequence between the gene for cytochrome b and the unassigned reading frame, URF U. The sequence contains a gene for a cysteine tRNA. The 5' end of the 6 kb polycistronic transcript of cytochrome c oxidase subunit 1 is immediately downstream from this tRNA. This shows that also in fungal mitochondria tRNAs can be used as processing signals, whereas palindromic sequences containing double Pst I sites, also present in this region, are not used for processing.

Cytochrome b gene of Neurospora crassa mitochondria. Partial sequence and location of introns at sites different from those in Saccharomyces cerevisiae and Aspergillus nidulans.

We have sequenced a 2614-base pair fragment of the Neurospora crassa mitochondrial DNA which contains part of the structural gene for apocytochrome b. This gene is split by at least two introns. The sequence reported here begins within one intron, extends through the next exon, another intron 1276 base pairs long, and the last exon which encodes the COOH terminus of cytochrome b. Within the 254 amino acids encoded by the two exons, there is a high degree of sequence conservation, 81%, with cytochrome b of Aspergillus nidulans. Surprisingly, both introns in the N. crassa cytochrome b gene are located at positions different from introns in the corresponding genes in Saccharomyces cerevisiae or A. nidulans. The upstream intron is located 22 nucleotides before the first intron in the long form of the S. cerevisiae cytochrome b gene. The downstream intron is located 16 nucleotides before the third intron in the long form of the S. cerevisiae gene and the only intron in the A. nidulans cytochrome b gene. The 1276-base pair downstream intron contains a 314 amino acid long open reading frame, which is in-phase with the preceding exon. The protein product of this reading frame has some resemblance to intron-encoded proteins, known as "mRNA maturases," which are thought to participate in RNA splicing in the mitochondria of S. cerevisiae. Another feature shared by the downstream intron and most other mitochondrial introns is the presence of the Box 9 and Box 2 consensus sequences, which may also be important for RNA splicing.

The structure of the gene for subunit I of cytochrome c oxidase in Neurospora crassa mitochondria

We have sequenced the gene for cytochrome c oxidase subunit 1 (CO I) in Neurospora crassa mitochondrial DNA. The gene is coded by the same strand as the rRNA and tRNA genes. The coding sequence predicts a protein of 557 amino acids, starting with methionine, and ending with asparagine. Comparison to the N-terminal amino acid sequence of the mature protein (Werner et al. 1980) reveals that the methionine is located at position -2. No other upstream AUG codons have been found in frame. The C-terminal part of the gene is about 70 basepairs longer than the corresponding parts of the Saccharomyces and mammalian genes. The homology between the Neurospora coding sequence and those of yeast and mammals is very high. As compared to Saccharomyces, the introns il through i5 are absent.

Highly conserved GC-rich palindromic DNA sequences flank tRNA genes in Neurospora crassa mitochondria

In sequencing a 2200 bp region of the Neurospora crassa mitochondrial DNA encoding the 3′ end of the large rRNA gene and a cluster of six tRNA genes, we have found that the tRNA genes are flanked by highly conserved GC-rich palindromic DNA sequences. An 18 bp long core sequence, 5′-CC CTGCAG TA CTGCAG GG-3′, containing two closely spaced Pst I sites, is common to all these palindromic sequences. Each of the eight Pst I sites mapped in the 2200 bp region consists of two closely spaced Pst I sites; thus this 2200 bp long segment actually contains 16 Pst I sites. Between 5–10% of the N. crassa DNA may consist of these GC-rich palindromic sequences that include the 18 base long core sequence. The same core sequence is present within both the 5′ and 3′ side of the intervening sequence of the large rRNA gene, close to, but not at, the intron-exon boundaries. We discuss probable roles for these sequences in N. crassa mitochondrial function, including their role as signals either in the synthesis or processing (or both) of RNA in the mitochondria.

Characterization of a variant Neurospora crassa mitochondrial DNA which contains tandem reiterations of a 1.9 kb sequence

We have identified a second variant of Neurospora mtDNA which contains tandem, head-to-tail repeats of sequences at the boundary of Eco RI-4 and 6. This region may contain a. major replication origin of Neurospora mtDNA.

Transcripts and processing patterns for the ribosomal RNA and transfer RNA region of Neurospora crassa mitochondrial DNA.

Transcripts and processing patterns for the ribosomal RNA and transfer RNA region of Neurospora crassa mitochondrial DNA.

Deletion mutants of Neurospora crassa mitochondrial DNA and their relationship to the "stop-start" growth phenotype.

"Stoppers" are a class of Neurospora crassa extranuclear mutants characterized by gross deficiencies of cytochromes b and aa3 and an unusual growth phenotype which involves irregular periods of growth andnongrowth. In the present work, mtDNAs from all four stopper mutants were found to contain deletions or insertions detectable by restriction enzyme analysis. [stp] mtDNA consists predominantly of defective molecules which retain a 16-megadalton segment (EcoRI-1, -4, and -6) of wild-type mtDNA (40 megadaltons). The other stopper mutants show smaller alterations: [stp A18t]-618, a 0.35-kilobase deletion in EcoRI-7b; [stp B2]-651, a 4-kilobase insertion in EcoRI-2; and [stp A]-574, a 5-kilobase deletion in EcoRI-2 and -10. Based on these results, we propose that "stop-start" growth results from competition between certain defective mtDNAs which have a tendency to predominate and low concentrations of less defective mtDNA species which must be retained to sustain growth. Three additional extranuclear mutants ("nonstoppers") have also been found to contain deletions in mtDNA. Remarkably, the defective mtDNA species in two of these mutants ([poky]H1-10 and [SG-3]-551) retain different sizes (18 and 13 megadlatons, respectively) of the same region retained in [stp] mtDNA (i.e., EcoRI-1, -4, and -6). The findings suggest that production of defective mtDNAs in Neurospora is nonrandom with a preferred mechanism leading to retention of this segment. It may be significant that the retained segment contains both mitochondrial rRNA genes and most mitochondrial tRNA genes. These deletion mutants may provide a tool for genetic mapping of Neurospora mtDNA.

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.

The anatomy of the tRNA-rRNA region of the Neurospora crassa mitochondrial DNA

To obtain more information about the arrangement of Hind III restriction fragments in the tRNA-rRNA region of the Neurospora crassa mitochondrial (mt) DNA we have cleaved the mtDNA with Hpa I and Hind II. We could construct additional cleavage maps for these enzymes. Hybridization of rRNAs to Hind II fragments confirmed the existence of an intervening region of about 2,300 basepairs in the 24S rRNA (Hahn et al., Cell, in press). About seven tRNA genes, among which the genes for tRNA1Ser and tRNAMetM, are located in a segment of about 5,000 bp separating the 24S and 17S rRNA genes. Another cluster of 14 tRNA genes is found adjacent to the other end of the 24S gene. The genes for tRNALeu1 and tRNAMetF are located in this cluster.

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 · 10 6, which is in good agreement with earlier measurements on intact DNA, using the electron microscope.

Physical map of Neurospora crassa mitochondrial DNA and its transcription unit for ribosomal RNA.

A circular denaturation and restriction map of mitochondrial DNA from Neurospora crassa is presented. The map shows the position of all twelve fragments produced by restriction endonuclease Eco R I and the position of the largest Hin III fragment along the previously established map of AT-rich sequences. The two wild type strains Em 5256 and 7A differ in the lengths of two Eco R I fragments. No difference was found between the mitochondrial mutant "poky" and its parent strain. The position of the DNA segment carrying the transcription unit for the two ribosomal RNA molecules has been determined by molecular hybridization.