Angiosperm mitochondrial genomes and mutations

Flowering plants harbor the largest mitochondrial genomes reported so far. At present, the nucleotide sequences of 15 mitochondrial genomes from seven angiosperm species are available, making detailed comparative analysis feasible. The gene content is variable among the species, but the most striking feature is the fluidity of intergenic regions, where species-specific sequences predominate. Additionally, angiosperm mitochondrial genomes, even within a species, show a remarkable amount of rearrangement. We also review mitochondrial mutants in angiosperms from a genomic viewpoint, and discuss how they have arisen. The involvement of nuclear genes in mitochondrial genome stability and organization is currently being revealed through the analysis of mutants.     

In silico analysis of SSRs in mitochondrial genomes of plants

Simple sequence repeats (SSRs) or microsatellites constitute a countable portion of genomes. However, the significance of SSRs in organelle genomes has not been completely understood. The availability of organelle genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. In the current study we surveyed the patterns of SSRs in mitochondrial genomes of different taxa of plants. A total of 16 mitochondrial genomes, from algae to angiosperms, have been considered to analyze the pattern of simple sequence repeats present in them. Based on study, the mononucleotide repeats of A/T were found to be more prevalent in mitochondrial genomes over other repeat types. The dinucleotides repeats, TA/AT, were the second most numerous, whereas tri-, tetra-, and pentanucleotide repeats were in less number and present in intronic or intergenic portions only. Mononucleotide repeats prevailed in protein-coding exonic portions of all organisms. These results indicates that microsatellite pattern in mitochondrial genomes is different from nuclear genomes and also focuses on organization and diversity at SSR locuses in mitochondrial genomes. This is the novel report of microsatellite polymorphism in plant mitochondrion on whole genome level.     

Simple sequence repeats in organellar genomes of rice: frequency and distribution in genic and intergenic regions

MOTIVATION: Simple sequence repeats (SSRs) are abundant across genomes. However, the significance of SSRs in organellar genomes of rice has not been completely understood. The availability of organellar genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. RESULTS: We have analyzed SSRs in mitochondrial and chloroplast genomes of rice. We identified 2528 SSRs in the mitochondrial genome and average 870 SSRs in the chloroplast genomes. About 8.7% of the mitochondrial and 27.5% of the chloroplast SSRs were observed in the genic region. Dinucleotides were the most abundant repeats in genic and intergenic regions of the mitochondrial genome while mononucleotides were predominant in the chloroplast genomes. The rps and nad gene clusters of mitochondria had the maximum repeats, while the rpo and ndh gene clusters of chloroplast had the maximum repeats. We identified SSRs in both organellar genomes and validated in different cultivars and species.     

Sequence organization of the mitochondrial genome of yeast--a review

We have compiled the available primary structural data for the mitochondrial genome of Saccharomyces cerevisiae and have estimated the size of the remaining gaps, which represent 12-13% of the genome. The lengths of sequenced regions and of gaps lead to a new assessment of genome sizes; these range (in round figures) from 85 000 bp for the long genomes, to 78 000 bp for the short genomes, to 74 000 bp for the supershort genome of Saccharomyces carlsbergensis. These values are 8-11% higher than those previously estimated from restriction fragments. Interstrain differences concern not only facultative intervening sequences (introns) and mini-inserts, but also insertions/deletions in intergenic sequences. The primary structure appears to be extremely conserved in genes and ori sequences, and highly conserved in intergenic sequences. Since coding sequences represent at most 33-35% of the genome, at least two thirds of the genome are formed by noncoding and yet highly conserved sequences. The G + C level of genes or exon is 25%, and that of intronic open reading frames (ORFs) 22%; increasingly lower values are shown by intronic closed reading frames (CRFs), 20%, ori sequences, 19%, intergenic ORFs, 17.5% and intergenic sequences, 15%.     

Transmission of the yeast mitochondrial genome to progeny: The impact of intergenic sequences

Summary In a previous publication it was shown that the output of yeast mitochondrial loci lacking nearby intergenic sequences (encompassing ori/rep elements) was reduced in crosses to strains with wild-type mtDNAs. In the present work, mitochondrial genomes carrying the intergenic deletions were marked at unlinked, loci by introducing specific antibiotic resistance mutations against erythromycin, oligomycin and paromomycin. These marked genomes were used to follow the output of unlinked regions of the genome from crosses between the intergenic deletion mutants and wild-type strains. Transmission of genetically unlinked markers in coding regions was substantially reduced when an intergenic deletion was present on the same genome. In general the transmission of the antibiotic markers was the same as or slightly higher than the corresponding intergenic marker. These results indicate that the presence of an intergenic deletion in the regions studied impairs the transmission to progeny of a mitochondrial genome as a whole. More specifically, the results suggest that ori/rep sequences, present in the regions that have been deleted, confer a competitive advantage over genomes lacking a full complement of such sequences. These results support the hypothesis that intergenic sequences, and specifically ori/rep elements, have a biological role in the mitochondrial genome. However, because of the exclusive presence of ori/rep sequences in the genus Saccharomyces, it may be that these sequences evolved in (or invaded) the mitochondrial genome relatively late in the evolution of the yeasts. Therefore, in a more general sense, variations in the amount and structure of intergenic sequences in various yeasts may reflect processes that have been of selective advantage in the metabolism of individual mitochondrial DNA in a particular environment and that have not drastically interrupted the respiratory phenotype.     

Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes

Despite the agricultural importance of both potato and tomato, very little is known about their chloroplast genomes. Analysis of the complete sequences of tomato, potato, tobacco, and Atropa chloroplast genomes reveals significant insertions and deletions within certain coding regions or regulatory sequences (e.g., deletion of repeated sequences within 16S rRNA, ycf2 or ribosomal binding sites in ycf2). RNA, photosynthesis, and atp synthase genes are the least divergent and the most divergent genes are clpP, cemA, ccsA, and matK. Repeat analyses identified 33–45 direct and inverted repeats ≥30 bp with a sequence identity of at least 90%; all but five of the repeats shared by all four Solanaceae genomes are located in the same genes or intergenic regions, suggesting a functional role. A comprehensive genome-wide analysis of all coding sequences and intergenic spacer regions was done for the first time in chloroplast genomes. Only four spacer regions are fully conserved (100% sequence identity) among all genomes; deletions or insertions within some intergenic spacer regions result in less than 25% sequence identity, underscoring the importance of choosing appropriate intergenic spacers for plastid transformation and providing valuable new information for phylogenetic utility of the chloroplast intergenic spacer regions. Comparison of coding sequences with expressed sequence tags showed considerable amount of variation, resulting in amino acid changes; none of the C-to-U conversions observed in potato and tomato were conserved in tobacco and Atropa. It is possible that there has been a loss of conserved editing sites in potato and tomato.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Microsatellite analysis in organelle genomes of Chlorophyta

Simple Sequence Repeats (SSRs) or microsatellites constitute a significant portion of genomes however; their significance in organellar genomes has not been completely understood. The availability of organelle genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. In the present work, SSRs were identified and categorized in 14 mitochondrial and 22 chloroplast genomes of algal species belonging to Chlorophyta. Based on the study, it was observed that number of SSRs in non-coding region were more as compared to coding region and frequency of mononucleotides repeats were highest followed by dinucleotides in both mitochondrial and chloroplast genomes. It was also observed that maximum number of SSRs was found in genes encoding for beta subunit of RNA polymerase in chloroplast genomes and NADH dehydrogenase in mitochondrial genomes. This is the first and original report on whole genomes sequence analysis of organellar genomes of green algae.     

Excision sequences in the mitochondrial genome of yeast

We report an analysis of the sequences used in the excision of the mitochondrial genomes of 22 spontaneous and ten ethidium bromide (EtBr)-induced Saccharomyces cerevisiae petite mutants. In all cases, excision sequences were found to be perfect direct repeats, often flanked on one or both sides by regions of patchy homology. Sequences used in the excision of the genomes of spontaneous petites were always located in the AT spacers and GC clusters of intergenic regions of the genome; the GC clusters corresponded to ori and ori^s sequences, namely to canonical and surrogate origins of DNA replication, respectively. In the case of the ethidium bromide-induced petites, excision sequences were found not only in intergenic sequences, but also in the introns and exons of mitochondrial genes.     

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

ABSTRACT: BACKGROUND: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. RESULTS: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. CONCLUSIONS: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.     

Molecular mechanisms of extensive mitochondrial gene rearrangement in plethodontid salamanders

Extensive gene rearrangement is reported in the mitochondrial genomes of lungless salamanders (Plethodontidae). In each genome with a novel gene order, there is evidence that the rearrangement was mediated by duplication of part of the mitochondrial genome, including the presence of both pseudogenes and additional, presumably functional, copies of duplicated genes. All rearrangement-mediating duplications include either the origin of light-strand replication and the nearby tRNA genes or the regions flanking the origin of heavy-strand replication. The latter regions comprise nad6, trnE, cob, trnT, an intergenic spacer between trnT and trnP and, in some genomes, trnP, the control region, trnF, rrnS, trnV, rrnL, trnL1, and nad1. In some cases, two copies of duplicated genes, presumptive regulatory regions, and/or sequences with no assignable function have been retained in the genome following the initial duplication; in other genomes, only one of the duplicated copies has been retained. Both tandem and nontandem duplications are present in these genomes, suggesting different duplication mechanisms. In some of these mitochondrial DNAs, up to 25% of the total length is composed of tandem duplications of noncoding sequence that includes putative regulatory regions and/or pseudogenes of tRNAs and protein-coding genes along with the otherwise unassignable sequences. These data indicate that imprecise initiation and termination of replication, slipped-strand mispairing, and intramolecular recombination may all have played a role in generating repeats during the evolutionary history of plethodontid mitochondrial genomes.     

A Genome-Wide Analysis of Genetic Diversity in Trypanosoma cruzi Intergenic Regions

Background

Trypanosoma cruzi is the causal agent of Chagas Disease. Recently, the genomes of representative strains from two major evolutionary lineages were sequenced, allowing the construction of a detailed genetic diversity map for this important parasite. However this map is focused on coding regions of the genome, leaving a vast space of regulatory regions uncharacterized in terms of their evolutionary conservation and/or divergence.

Methodology

Using data from the hybrid CL Brener and Sylvio X10 genomes (from the TcVI and TcI Discrete Typing Units, respectively), we identified intergenic regions that share a common evolutionary ancestry, and are present in both CL Brener haplotypes (TcII-like and TcIII-like) and in the TcI genome; as well as intergenic regions that were conserved in only two of the three genomes/haplotypes analyzed. The genetic diversity in these regions was characterized in terms of the accumulation of indels and nucleotide changes.

Principal Findings

Based on this analysis we have identified i) a core of highly conserved intergenic regions, which remained essentially unchanged in independently evolving lineages; ii) intergenic regions that show high diversity in spite of still retaining their corresponding upstream and downstream coding sequences; iii) a number of defined sequence motifs that are shared by a number of unrelated intergenic regions. A fraction of indels explains the diversification of some intergenic regions by the expansion/contraction of microsatellite-like repeats.     

A novel gene family NBPF: intricate structure generated by gene duplications during primate evolution

Partial and complete genome duplications occurred during evolution and resulted in the creation of new genes and gene families. We identified a novel and intricate human gene family located primarily in regions of segmental duplications on human chromosome 1. We named it NBPF, for neuroblastoma breakpoint family, because one of its members is disrupted by a chromosomal translocation in a neuroblastoma patient. The NBPF genes have a repetitive structure with high intragenic and intergenic sequence similarity in both coding and noncoding regions. These similarities might expose these genomic regions to illegitimate recombination, resulting in structural variation in the NBPF genes. The encoded proteins contain a highly conserved domain of unknown function, which we have named the NBPF repeat. In silico analysis combined with the isolation of multiple full-length cDNA clones showed that several members of this gene family are abundantly expressed in a large variety of tissues and cell lines. Strikingly, no discernable orthologues could be identified in the completed genomes of fruit fly, nematode, mouse, or rat, but sequences with low homology could be isolated from the draft canine and bovine genomes. Interestingly, this gene family shows primate-specific duplications that result in species-specific arrays of NBPF homologous sequences. Overall, this novel NBPF family reflects the continuous evolution of primate genomes that resulted in large physiological differences, and its potential role in this process is discussed.     

Regions flanking ori sequences affect the replication efficiency of the mitochondrial genome of ori+ petite mutants from yeast

The mitochondrial genomes of progenies from 26 crosses between 17 cytoplasmic, spontaneous, suppressive, ori+ petite mutants of Saccharomyces cerevisiae have been studied by electrophoresis of restriction fragments. Only parental genomes (or occasionally, genomes derived from them by secondary excisions) were found in the progenies of the almost 500 diploids investigated; no evidence for illegitimate, site-specific mitochondrial recombination was detected. One of the parental genomes was always found to be predominate over the other one, although to different extents in different crosses. This predominance appears to be due to a higher replication efficiency, which is correlated with a greater density of ori sequences on the mitochondrial genome (and with a shorter repeat unit size of the latter). Exceptions to the 'repeat-unit-size rule' were found, however, even when the parental mitochondrial genomes carried the same ori sequence. This indicates that noncoding, intergenic sequences outside ori sequences also play a role in modulating replication efficiency. Since in different petites such sequences differ in primary structure, size, and position relative to ori sequences, this modulation is likely to take place through an indirect effect on DNA and nucleoid structure.     

Compact intergenic regions of the pufferfish genome facilitate isolation of gene promoters: characterization of Fugu 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (fPapss2) gene promoter function in transgenic Xenopus

The highly compact nature of the pufferfish (Fugu rubripes) genome renders it a useful tool not only for annotating coding regions within vertebrate genomes, but also for the identification of sequences important to gene regulation. Indeed, owing to this compaction it will be feasible in many instances to initiate analyses using entire intergenic regions when mapping gene promoters; a strategy that is very rarely feasible with the expanded genomes of other species. Stemming from our interest in studying promoters expressed in chondrocytes, we selected for study the intergenic region upstream of Fugu 3'-phosphoadenosine 5'-phosphosulfate synthase 2, fPapss2, a gene required for the normal development of cartilage extracellular matrix. Functional characterization of the entire fPapss2 5' intergenic region was carried out by monitoring expression of the enhanced green fluorescent protein (EGFP) gene reporter in the developing cartilage of transgenic Xenopus laevis. By evaluating a series of 5' intergenic region deletions we defined a minimal fPapss2 sequence of approximately 300 bp that was essential for EGFP expression in tadpole cartilage. This functional analysis of an entire Fugu intergenic region, combined with the efficiency of Xenopus transgenesis, serves as a model for the rapid characterization of evolutionarily-conserved regulatory regions of other pufferfish genes.     

Evolution of noncoding and silent coding sites in the Plasmodium falciparum and Plasmodium reichenowi genomes

We compared levels of sequence divergence between fourfold synonymous coding sites and noncoding sites from the intergenic and intronic regions of the Plasmodium falciparum and Plasmodium reichenowi genomes. We observed significant differences in the level of divergence between these classes of silent sites. Fourfold synonymous coding sites exhibited the highest level of sequence divergence, followed by introns, and then intergenic sequences. This pattern of relative divergence rates has been observed in primate genomes but was unexpected in Plasmodium due to a paucity of variation at silent sites in P. falciparum and the corollary hypothesis that silent sites in this genome may be subject to atypical selective constraints. Exclusion of hypermutable CpG dinucleotides reduces the divergence level of synonymous coding sites to that of intergenic sites but does not diminish the significantly higher divergence level of introns relative to intergenic sites. A greater than expected incidence of CpG dinucleotides in intergenic regions less than 500 bp from genes may indicate selective maintenance of regulatory motifs containing CpGs. Divergence rates of different classes of silent sites in these Plasmodium genomes are determined by a combination of mutational and selective pressures.     

Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.)

Marine mussels of the genus Mytilus have an unusual mode of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Female mussels are homoplasmic for the F mitotype, which is inherited maternally, while males are usually heteroplasmic, carrying a mixture of the maternal F mitotype and the paternally inherited M genome. Two classes of M genomes have been observed: "standard" M genomes and "recently masculinized" M genomes. The latter are more similar to F genomes at the sequence level but are transmitted paternally like standard M genomes. In this study we report the complete sequences of two standard male M. edulis and one recently masculinized male M. trossulus mitochondrial genome. A comparative analysis, including the previously sequenced M. edulis F and M. galloprovincialis F and M mtDNAs, reveals that these genomes are identical in gene order, but highly divergent in nucleotide and amino acid sequence. The large amount (>20%) of nucleotide substitutions that fall in coding regions implies that there are several amino acid replacements between the F and M genomes, which likely have an impact on the structural and functional properties of the mitochondrial proteome. Correlation of the divergence rate of different protein-coding genes indicates that mtDNA-encoded proteins of the M genome are still under selective constraints, although less highly than genes of the F genome. The mosaic F/M control region of the masculinized F genome provides evidence for lineage-specific sequences that may be responsible for the different mode of transmission genetics. This analysis shows the value of comparative genomics to better understand the mechanisms of maintenance and segregation of mtDNA sequence variants in mytilid mussels.