Linda Bonen

The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize

We have determined the sequence of the wheat mitochondrial gene for cytochrome oxidase subunit II (COII) and find that its derived protein sequence differs from that of maize at only three amino acid positions. Unexpectedly, all three replacements are non‐conservative ones. The wheat COII gene has a highly‐conserved intron at the same position as in maize, but the wheat intron is 1.5 times longer because of an insert relative to its maize counterpart. Hybridization analysis of mitochondrial DNA from rye, pea, broad bean and cucumber indicates strong sequence conservation of COII coding sequences among all these higher plants. However, only rye and maize mitochondrial DNA show homology with wheat COII intron sequences and rye alone with intron‐insert sequences. We find that a sequence identical to the region of the 5′ exon corresponding to the transmembrane domain of the COII protein is present at a second genomic location in wheat mitochondria. These variations in COII gene structure and size, as well as the presence of repeated COII sequences, illustrate at the DNA sequence level, factors which contribute to higher plant mitochondrial DNA diversity and complexity.

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene☆

Abstract The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa . The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1- cox 1 intron in Podospora anserina . It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5′ region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox 1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.

Genes for tRNAAsp, tRNAPro, tRNATyr and two tRNAsSer in wheat mitochondrial DNA

We have begun a systematic search for potential tRNA genes in wheat mtDNA, and present here the sequences of regions of the wheat mitochondrial genome that encode genes for tRNAAsp (anticodon GUC), tRNAPro (UGG), tRNATyr (GUA), and two tRNAsSer (UGA and GCU). These genes are all solitary, not immediately adjacent to other tRNA or known protein coding genes. Each of the encoded tRNAs can assume a secondary structure that conforms to the standard cloverleaf model, and that displays none of the structural aberrations peculiar to some of the corresponding mitochondrial tRNAs from other eukaryotes. The wheat mitochondrial tRNA sequences are, on average, substantially more similar to their eubacterial and chloroplast counterparts than to their homologues in fungal and animal mitochondria. However, an analysis of regions ∼ 150 nucleotides upstream and ∼ 100 nucleotides downstream of the tRNA coding regions has revealed no obvious conserved sequences that resemble the promoter and terminator motifs that regulate the expression of eubacterial and some chloroplast tRNA genes. When restriction digests of wheat mtDNA are probed with 32P-labelled wheat mitochondrial tRNAs, <20 hybridizing bands are detected, whether enzymes with 4 bp or 6 bp recognition sites are used. This suggests that the wheat mitochondrial genome, despite its large size, may carry a relatively small number of tRNA genes.

Ribosomal RNA homologies and the evolution of the filamentous blue-green bacteria

SummaryRibosomal RNA (rRNA) sequence homology (as determined by comparisons of T1 oligonucleotide catalogs of32P-labeled 16S rRNAs) has been used to assess phylogenetic relationships within the filamentous and unicellular blue-green bacteria, and to identify regions of evolutionary conservatism within blue-green bacterial 16S rRNAs.Nostoc andFischerella, representatives of two morphologically distinct and highly differentiated orders, are shown to be as closely related (on the basis of RNA sequence homology) as typical members of the non-blue-green bacterial genusBacillus. They are further shown to be (on the same basis) indistinguishable from typical unicellular members of a subgroup of the unicellular blue-green bacterial order Chroococcales. These results have general implications for studies of the origin of differentiated prokaryotes and of evolutionary change in prokaryotic macromolecules. In particular, they provide indirect evidence that the divergences of contemporary major prokaryotic groups are truly ancient ones.

Can partial methylation explain the complex fragment patterns observed when plant mitochondrial DNA is cleaved with restriction endonucleases

It is widely accepted (e.g. [ 1,2]) that the mitochondrial DNA (mtDNA) of higher plants consists of a uniform population of 30 nm circular molecules, of mol. wt -70 X lo6 [3,4]. However, restriction endonuclease patterns of plant mtDNA are considerably more complex than expected for a homogeneous population of this size [5-71. In contrast, the simpler restriction patterns of chloroplast DNA [8] are compatible with the presence of a homogeneous population of molecules [9], even though studies [IO-121 of higher plant chloroplast DNA have suggested that it is physically larger and kinetically more complex than the mtDNA of the same organisms (cf. [4]). To rationalize this apparent discrepancy, it has been proposed [5] that plant mtDNA may actually be heterogeneous, consisting of several types of physically indistinguishable molecules having different sequence arrangements of the same genetic information. Alternatively, an unexpectedly complex restriction pattern could result from failure of a restriction endonuclease to cleave all potential sites in what appears to be a homogeneous DNA population. This could occur if each restriction site existed in either modified (e.g., methylated) or unmodified versions in different mtDNA molecules, with only the unmodified site being susceptible to cleavage by the endonuclease in question. To test this latter possibility, we have examined the distribution of 5-methyldeoxycytidine (m5C) in

MOLECULAR SEQUENCE DATA INDICATING AN ENDOSYMBIOTIC ORIGIN FOR PLASTIDS

Photosynthetic mechanisms in cyanobacteria (and presumably in prochlorophytes I ) are so similar to those in eukaryotic photosynthesizers that it would be foolish to suggest that they evolved independently.’ If eukaryotes did not now segregate photosynthetic functions and the DNA encoding some of these functions into discrete, membrane-bounded organelles (plastids) , then we would all accept what Margulis has called the “botanical myth;” the notion that plants arose from eukaryotic algae (specifically chlorophytes) and that these in turn arose in some straightforward way from oxygenic-photosynthetic prokaryotes, of which we now know only two kinds-the cyanobacteria and the prochlorophytes. Unfortunately, eukaryotic photosynthesizers do segregate photosynthetic functions and Ihe DNA responsible for some of these functions. There are really only two ways to explain this. One (the “botanical myth” or, less pejoratively, the direcl filiation,‘ autogenous origin,: or developmental hypothesis) assumes that nuclear and organellar genomes diverged, intracellularly, after or during the formation of the first “protoeukaryote,” which was itself capable of oxygenic photosynthesis. The other (the endosymbiont or xenogenous’ origin hypothess) assumes that the nuclear genome arose in a nonphotosynthetic protoeukaryote, while plastid genomes are of foreign origin, the descendants of the genomes of once free-living oxygenic-photosynthetic prokaryotic endosymbionts. The question then is one of phylogenetic relationships between genomes. If plastid genomes prove to be more closely related to those of the nucleus o€ the cells in which they reside then they are to those of surviving oxygenicphotosynthetic prokaryotes. then autogenous origins are likely. If, on the other hand, they show stronger affinities to free-living prokaryotes, then xenogenous origins are likely. All the genomes in question have diverged so markedly that meaningful measures of overall homology cannot be obtained. But we can look at conservative parts of these genomes. The ribosomal RNA (rRNA) genes are conservative parts of genomes, and have the further singular advantage of being present on all nuclear, organellar, and prokaryotic genomes.h We know of no proteins with identifiable homologs3 coded for by each of these sorts of genome, and the plastid proteins whose sequences have been used to bolster xenogenous explanations are in most i f not all cases actually the products of nuclear genes.@

Mitochondrial Ribosomal RNAs of Triticum aestivum (Wheat): Sequence Analysis and Gene Organization

The complicated restriction patterns of plant mtDNA suggest that it is unusually large and complex and possesses a type of sequence heterogeneity not found in the mtDNAs of other eukaryotes (Leaver and Gray 1982). As a result, our knowledge of the genetic function and organization of this important organelle genome is much less advanced than in the case of animal and fungal mtDNAs. As one approach to improving this situation, we are identifying and characterizing large restriction fragments encoding specific plant mitochondrial genes. Our work has centered on the mtDNA of wheat, Triticum aestivum, and has focused particularly on the rRNA genes. DISCUSSION Novel Arrangement of rRNA Genes in Wheat mtDNA The mitochondrial 26S and 18S rRNAs of wheat differ from their cytosol counterparts in physicochemical properties (Cunningham and Gray 1977), T1 oligonucleotide fingerprints (Cunningham et al. 1976), and (in the case of the 18S rRNAs) T1 oligonucleotide catalogs (Bonen et al. 1977). Wheat mitochondrial 5S rRNA is also a distinct molecular species, as demonstrated by oligonucleotide cataloging (Cunningham et al. 1976) and, more recently, by determination of its complete primary sequence (Spencer et al. 1981). Using Southern hybridization, we found that bulk mtRNA from wheat is specifically encoded by wheat mtDNA and shares no detectable sequence homology with wheat cytosol RNA (Bonen and Gray 1980). We also identified restriction fragments encoding the individual 26S, 18S, and 5S wheat mitochondrial rRNAs, as well as the mitochondrial tRNAs (Fig. 1). These represent the first documented plant mitochondrial genes. In view of...