Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution

Restriction–modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.     

Differential transmission of the Cucumis organellar genomes

 Although plants generally show maternal transmission of the organellar genomes, previous research has demonstrated that the mitochondrial (mt) genome of cucumber is paternally transmitted. In this study, we identified RFLPs in the organellar genomes of melon, squash, and watermelon to establish organellar DNA transmission. Serial dilutions of DNA demonstrated that our hybridizations revealed the presence of a polymorphic cytoplasm when it represented at least 1% of the DNA sample. At this level of sensitivity, the chloroplast genomes of melon, squash, and watermelon were maternally transmitted. The mitochondrial genomes of squash and watermelon were maternally transmitted; however, melon, like cucumber, showed paternal transmission of the mitochondrial genome. Because most angiosperms and the related genera Cucurbita and Citrullus show maternal transmission of the mtDNA, paternal transmission in Cucumis is likely the derived state. The Cucumis mitochondrial genomes are several-fold larger than those of other cucurbits. Based on 55 probe-enzyme combinations, mtDNA size differences could not be explained by duplication of the entire genome or partial duplication of regions hybridizing with the mitochondrial probes. Because the chloroplast, mitochondrial, and nuclear genomes of Cucumis are differentially transmitted, this genus is an excellent system to study the role of intergenomic transfer in the evolution of extremely large mitochondrial genomes. Received: 20 November 1997 / Accepted: 30 December 1997     

What tangled web: barriers to rampant horizontal gene transfer

Dawkins in his The Selfish Gene(1) quite aptly applies the term "selfish" to parasitic repetitive DNA sequences endemic to eukaryotic genomes, especially vertebrates. Doolittle and Sapienza(2) as well as Orgel and Crick(3) enlivened this notion of selfish DNA with the identification of such repetitive sequences as remnants of mobile elements such as transposons. In addition, Orgel and Crick(3) associated parasitic DNA with a potential to outgrow their host genomes by propagating both vertically via conventional genome replication as well as infectiously by horizontal gene transfer (HGT) to other genomes. Still later, Doolittle(4) speculated that unchecked HGT between unrelated genomes so complicates phylogeny that the conventional representation of a tree of life would have to be replaced by a thicket or a web of life.(4) In contrast, considerable data now show that reconstructions based on whole genome sequences are consistent with the conventional "tree of life".(5-10) Here, we identify natural barriers that protect modern genome populations from the inroads of rampant HGT.     

Genome structure and gene content in protist mitochondrial DNAs.

Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

Evolution of organellar genomes

Accumulating molecular data, particularly complete organellar genome sequences, continue to advance our understanding of the evolution of mitochondrial and chloroplast DNAs. Although the notion of a single primary origin for each organelle has been reinforced, new models have been proposed that tie the acquisition of mitochondria more closely to the origin of the eukaryotic cell per se than is implied by classic endosymbiont theory. The form and content of the ancestral proto-mitochondrial and proto-chloroplast genomes are becoming clearer but unusual patterns of organellar genome structure and organization continue to be discovered. The 'single-gene circle' arrangement recently reported for dinoflagellate chloroplast genomes is a notable example of a highly derived organellar genome.     

Multiplication of a restriction-modification gene complex

Previous works have suggested that some gene complexes encoding a restriction (R) enzyme and a cognate modification (M) enzyme may behave as selfish mobile genetic elements. RM gene complexes, which destroy 'non-self' elements marked by the absence of proper methylation, are often associated with mobile genetic elements and are involved in various genome rearrangements. Here, we found amplification of a restriction-modification gene complex. BamHI gene complex inserted into the Bacillus chromosome showed resistance to replacement by a homologous stretch of DNA. Some cells became transformed with the donor without losing BamHI. In most of these transformants, multiple copies of BamHI and the donor allele were arranged as tandem repeats. When a clone carrying one copy of each allele was propagated, extensive amplification of BamHI and the donor unit was observed in a manner dependent on restriction enzyme gene. This suggests that restriction cutting of the genome participates in the amplification. Visualization by fluorescent in situ hybridization revealed that the amplification occurred in single cells in a burst-like fashion that is reminiscent of induction of provirus replication. The multiplication ability in a bacterium with natural capacity for DNA release, uptake and transformation will be discussed in relation to spreading of RM gene -complexes.     

In silico analysis of microsatellites in organellar genomes of major cereals for understanding their phylogenetic relationships

Microsatellites are abundant across prokaryotic and eukaryotic genomes. However, comparative analysis of microsatellites in the organellar genomes of plants and their utility in understanding phylogeny has not been reported. The purpose of this study was to understand the organization of microsatellites in the coding and non-coding regions of organellar genomes of major cereals viz., rice, wheat, maize and sorghum. About 5.8-14.3% of mitochondrial and 30.5-43.2% of chloroplast microsatellites were observed in the coding regions. About 83.8-86.8% of known mitochondrial genes had at least one microsatellite while this value ranged from 78.6-82.9% among the chloroplast genomes. Dinucleotide repeats were the most abundant in the coding and non-coding regions of the mitochondrial genome while mononucleotides were predominant in chloroplast genomes. Maize harbored more repeats in the mitochondrial genome, which could be due to the larger size of genome. A phylogenetic analysis based on mitochondrial and chloroplast genomic microsatellites revealed that rice and sorghum were closer to each other, while wheat was the farthest and this corroborated with the earlier reported phylogenies based on nuclear genome co-linearity and chloroplast gene-based analysis.     

Inheritance and recombination of mitochondrial genomes in plants, fungi and animals

It is generally assumed that mitochondrial genomes are uniparentally transmitted, homoplasmic and nonrecombining. However, these assumptions draw largely from early studies on animal mitochondrial DNA (mtDNA). In this review, we show that plants, animals and fungi are all characterized by episodes of biparental inheritance, recombination among genetically distinct partners, and selfish elements within the mitochondrial genome, but that the extent of these phenomena may vary substantially across taxa. We argue that occasional biparental mitochondrial transmission may allow organisms to achieve the best of both worlds by facilitating mutational clearance but continuing to restrict the spread of selfish genetic elements. We also show that methodological biases and disproportionately allocated study effort are likely to have influenced current estimates of the extent of biparental inheritance, heteroplasmy and recombination in mitochondrial genomes from different taxa. Despite these complications, there do seem to be discernible similarities and differences in transmission dynamics and likelihood of recombination of mtDNA in plant, animal and fungal taxa that should provide an excellent opportunity for comparative investigation of the evolution of mitochondrial genome dynamics.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

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.     

Have malaria parasites three genomes?

Recent efforts to define the mitochondrial genome of malaria parasites have uncovered an unexpected complexity: there are two almost totally dissimilar organellar DNA molecules. lain Wilson, Malcolm Gardner, Jean Feagin and Donald Williamson discuss the surprising possibility that Plasmodium may have, in addition to the nuclear genome, two unrelated organellar genomes, one evidently mitochondrial and the other of unknown function.     

Selfish Little Circles: Transmission Bias and Evolution of Large Deletion-Bearing Mitochondrial DNA in Caenorhabditis briggsae Nematodes

Selfish DNA poses a significant challenge to genome stability and organismal fitness in diverse eukaryotic lineages. Although selfish mitochondrial DNA (mtDNA) has known associations with cytoplasmic male sterility in numerous gynodioecious plant species and is manifested as petite mutants in experimental yeast lab populations, examples of selfish mtDNA in animals are less common. We analyzed the inheritance and evolution of mitochondrial DNA bearing large heteroplasmic deletions including nad5 gene sequences (nad5Δ mtDNA), in the nematode Caenorhabditis briggsae. The deletion is widespread in C. briggsae natural populations and is associated with deleterious organismal effects. We studied the inheritance patterns of nad5Δ mtDNA using eight sets of C. briggsae mutation-accumulation (MA) lines, each initiated from a different natural strain progenitor and bottlenecked as single hermaphrodites across generations. We observed a consistent and strong drive toward higher levels of deletion-bearing molecules in the heteroplasmic pool of mtDNA after ten generations of bottlenecking. Our results demonstrate a uniform transmission bias whereby nad5Δ mtDNA accumulates to higher levels relative to intact mtDNA in multiple genetically diverse natural strains of C. briggsae. We calculated an average 1% per-generation transmission bias for deletion-bearing mtDNA relative to intact genomes. Our study, coupled with known deleterious phenotypes associated with high deletion levels, shows that nad5Δ mtDNA are selfish genetic elements that have evolved in natural populations of C. briggsae, offering a powerful new system to study selfish mtDNA dynamics in metazoans.     

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

Rickettsial evolution in the light of comparative genomics

Rickettsia are best known as strictly intracellular vector‐borne bacteria that cause mild to severe diseases in humans and other animals. Recent advances in molecular tools and biological experiments have unveiled a wide diversity of Rickettsia spp. that include species with a broad host range and some species that act as endosymbiotic associates. Molecular phylogenies of Rickettsia spp. contain some ambiguities, such as the position of R. canadensis and relationships within the spotted fever group. In the modern era of genomics, with an ever‐increasing number of sequenced genomes, there is enhanced interest in the use of whole‐genome sequences to understand pathogenesis and assess evolutionary relationships among rickettsial species. Rickettsia have small genomes (1.1–1.5 Mb) as a result of reductive evolution. These genomes contain split genes, gene remnants and pseudogenes that, owing to the colinearity of some rickettsial genomes, may represent different steps of the genome degradation process. Genomics reveal extreme genome reduction and massive gene loss in highly vertebrate‐pathogenic Rickettsia compared to less virulent or endosymbiotic species. Information gleaned from rickettsial genomics challenges traditional concepts of pathogenesis that focused primarily on the acquisition of virulence factors. Another intriguing phenomenon about the reduced rickettsial genomes concerns the large fraction of non‐coding DNA and possible functionality of these “non‐coding” sequences, because of the high conservation of these regions. Despite genome streamlining, Rickettsia spp. contain gene families, selfish DNA, repeat palindromic elements and genes encoding eukaryotic‐like motifs. These features participate in sequence and functional diversity and may play a crucial role in adaptation to the host cell and pathogenesis. Genome analyses have identified a large fraction of mobile genetic elements, including plasmids, suggesting the possibility of lateral gene transfer in these intracellular bacteria. Phylogenetic analyses have identified several candidates for horizontal gene acquisition among Rickettsia spp. including tra, pat2, and genes encoding for the type IV secretion system and ATP/ADP translocase that may have been acquired from bacteria living in amoebae. Gene loss, gene duplication, DNA repeats and lateral gene transfer all have shaped rickettsial genome evolution. A comprehensive analysis of the entire genome, including genes and non‐coding DNA, will help to unlock the mysteries of rickettsial evolution and pathogenesis.     

Cucumber: a model angiosperm for mitochondrial transformation?

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.     

Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae.

The mitochondrial DNA (mtDNA) of Porphyra purpurea, a circular-mapping genome of 36,753 bp, has been completely sequenced. A total of 57 densely packed genes has been identified, including the basic set typically found in animals and fungi, as well as seven genes characteristic of protist and plant mtDNAs and specifying ribosomal proteins and subunits of succinate:ubiquinone oxidoreductase. The mitochondrial large subunit rRNA gene contains two group II introns that are extraordinarily similar to those found in the cyanobacterium Calothrix sp, suggesting a recent lateral intron transfer between a bacterial and a mitochondrial genome. Notable features of P. purpurea mtDNA include the presence of two 291-bp inverted repeats that likely mediate homologous recombination, resulting in genome rearrangement, and of numerous sequence polymorphisms in the coding and intergenic regions. Comparative analysis of red algal mitochondrial genomes from five different, evolutionarily distant orders reveals that rhodophyte mtDNAs are unusually uniform in size and gene order. Finally, phylogenetic analyses provide strong evidence that red algae share a common ancestry with green algae and plants.