Alan C. Christensen

Plant Mitochondrial Recombination Surveillance Requires Unusual RecA and MutS Homologs

For >20 years, the enigmatic behavior of plant mitochondrial genomes has been well described but not well understood. Chimeric genes appear, and occasionally are differentially replicated or expressed, with significant effects on plant phenotype, most notably on male fertility, yet the mechanisms of DNA replication, chimera formation, and recombination have remained elusive. Using mutations in two important genes of mitochondrial DNA metabolism, we have observed reproducible asymmetric recombination events occurring at specific locations in the mitochondrial genome. Based on these experiments and existing models of double-strand break repair, we propose a model for plant mitochondrial DNA replication, chimeric gene formation, and the illegitimate recombination events that lead to stoichiometric changes. We also address the physiological and developmental effects of aberrant events in mitochondrial genome maintenance, showing that mitochondrial genome rearrangements, when controlled, influence plant reproduction, but when uncontrolled, lead to aberrant growth phenotypes and dramatic reduction of the cell cycle.

Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS

The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination. Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules. This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types. In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels. We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis. The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination. We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.

Dual-Domain, Dual-Targeting Organellar Protein Presequences in Arabidopsis Can Use Non-AUG Start Codons

The processes accompanying endosymbiosis have led to a complex network of interorganellar protein traffic that originates from nuclear genes encoding mitochondrial and plastid proteins. A significant proportion of nucleus-encoded organellar proteins are dual targeted, and the process by which a protein acquires the capacity for both mitochondrial and plastid targeting may involve intergenic DNA exchange coupled with the incorporation of sequences residing upstream of the gene. We evaluated targeting and sequence alignment features of two organellar DNA polymerase genes from Arabidopsis thaliana. Within one of these two loci, protein targeting appeared to be plastidic when the 5′ untranslated leader region (UTR) was deleted and translation could only initiate at the annotated ATG start codon but dual targeted when the 5′ UTR was included. Introduction of stop codons at various sites within the putative UTR demonstrated that this region is translated and influences protein targeting capacity. However, no ATG start codon was found within this upstream, translated region, suggesting that translation initiates at a non-ATG start. We identified a CTG codon that likely accounts for much of this initiation. Investigation of the 5′ region of other nucleus-encoded organellar genes suggests that several genes may incorporate upstream sequences to influence targeting capacity. We postulate that a combination of intergenic recombination and some relaxation of constraints on translation initiation has acted in the evolution of protein targeting specificity for those proteins capable of functioning in both plastids and mitochondria.

Diversity of the Arabidopsis Mitochondrial Genome Occurs via Nuclear-Controlled Recombination Activity

The plant mitochondrial genome is recombinogenic, with DNA exchange activity controlled to a large extent by nuclear gene products. One nuclear gene, MSH1, appears to participate in suppressing recombination in Arabidopsis at every repeated sequence ranging in size from 108 to 556 bp. Present in a wide range of plant species, these mitochondrial repeats display evidence of successful asymmetric DNA exchange in Arabidopsis when MSH1 is disrupted. Recombination frequency appears to be influenced by repeat sequence homology and size, with larger size repeats corresponding to increased DNA exchange activity. The extensive mitochondrial genomic reorganization of the msh1 mutant produced altered mitochondrial transcription patterns. Comparison of mitochondrial genomes from the Arabidopsis ecotypes C24, Col-0, and Ler suggests that MSH1 activity accounts for most or all of the polymorphisms distinguishing these genomes, producing ecotype-specific stoichiometric changes in each line. Our observations suggest that MSH1 participates in mitochondrial genome evolution by influencing the lineage-specific pattern of mitochondrial genetic variation in higher plants.

dELL is an essential RNA polymerase II elongation factor with a general role in development

Several eukaryotic proteins increase RNA polymerase II (Pol II) transcription rates in vitro. The relative contributions of these factors to gene expression in vivo is unknown. The ELL family of proteins promote Pol II elongation in vitro, and the Drosophila ELL homolog (dELL) is associated with Pol II at sites of transcription in vivo. The purpose of this study was to test whether an ELL family protein is required for gene expression in vivo. We show that dELL is encoded by the Suppressor of Triplo-lethal locus [Su(Tpl)]. We have characterized seven distinct mutant alleles of Su(Tpl) and show that a dELL transgene rescues recessive lethality of Su(Tpl). Su(Tpl) mutations cause abnormal embryonic segmentation and dominantly modify expression of diverse genes during development. These data show that an ELL family elongation factor is essential, acts broadly in development, and is not functionally redundant to other elongation factors in vivo.

Mitochondrial genome dynamics in plants and animals: convergent gene fusions of a MutS homologue.

Mitochondrial processes influence a broad spectrum of physiological and developmental events in higher eukaryotes, and their aberrant function can lead to several familiar disease phenotypes in mammals. In plants, mitochondrial genes directly influence pollen development and the occurrence of male sterility in natural plant populations. Likewise, in animal systems evidence accumulates to suggest important mitochondrial functions in spermatogenesis and reproduction. Here we present evidence for a convergent gene fusion involving a MutS-homologous gene functioning within the mitochondrion and designated Msh1. In only plants and soft corals, the MutS homologue has fused with a homing endonuclease sequence at the carboxy terminus of the protein. However, the endonuclease domains in the plants and the soft corals are members of different groups. In plants, Msh1 can influence mitochondrial genome organization and male sterility expression. Based on parallels in Msh1 gene structure shared by plants and corals, and their similarities in reproductive behavior, we postulate that this convergent gene fusion might have occurred in response to coincident adaptive pressures on reproduction.

A peritrophin-like protein expressed in the embryonic tracheae of Drosophila melanogaster

We have cloned and sequenced a cDNA from Drosophila melanogaster that encodes a protein homologous to the peritrophins, a family of chitin-binding proteins from the peritrophic matrix of insects. Unexpectedly, the gene, Gasp, is expressed in the embryonic tracheae. We suggest that this family of proteins may be present in other tissues than the peritrophic matrix, particularly where nutrient or gas exchange are important, and/or where invasion by parasites or viruses is possible. We have also mapped two similar genes that had been sequenced by the Berkeley Drosophila Genome Project, and find that these three very similar genes are not clustered, but are located on three different chromosomes.

Plant Mitochondrial Genome Evolution Can Be Explained by DNA Repair Mechanisms

Plant mitochondrial genomes are notorious for their large and variable size, nonconserved open reading frames of unknown function, and high rates of rearrangement. Paradoxically, the mutation rates are very low. However, mutation rates can only be measured in sequences that can be aligned—a very small part of plant mitochondrial genomes. Comparison of the complete mitochondrial genome sequences of two ecotypes of Arabidopsis thaliana allows the alignment of noncoding as well as coding DNA and estimation of the mutation rates in both. A recent chimeric duplication is also analyzed. A hypothesis is proposed that the mechanisms of plant mitochondrial DNA repair account for these features and includes different mechanisms in transcribed and nontranscribed regions. Within genes, a bias toward gene conversion would keep measured mutation rates low, whereas in noncoding regions, break-induced replication (BIR) explains the expansion and rearrangements. Both processes are types of double-strand break repair, but enhanced second-strand capture in transcribed regions versus BIR in nontranscribed regions can explain the two seemingly contradictory features of plant mitochondrial genome evolution—the low mutation rates in genes and the striking expansions of noncoding sequences.

Genes and Junk in Plant Mitochondria—Repair Mechanisms and Selection

Plant mitochondrial genomes have very low mutation rates. In contrast, they also rearrange and expand frequently. This is easily understood if DNA repair in genes is accomplished by accurate mechanisms, whereas less accurate mechanisms including nonhomologous end joining or break-induced replication are used in nongenes. An important question is how different mechanisms of repair predominate in coding and noncoding DNA, although one possible mechanism is transcription-coupled repair (TCR). This work tests the predictions of TCR and finds no support for it. Examination of the mutation spectra and rates in genes and junk reveals what DNA repair mechanisms are available to plant mitochondria, and what selective forces act on the repair products. A model is proposed that mismatches and other DNA damages are repaired by converting them into double-strand breaks (DSBs). These can then be repaired by any of the DSB repair mechanisms, both accurate and inaccurate. Natural selection will eliminate coding regions repaired by inaccurate mechanisms, accounting for the low mutation rates in genes, whereas mutations, rearrangements, and expansions generated by inaccurate repair in noncoding regions will persist. Support for this model includes the structure of the mitochondrial mutS homolog in plants, which is fused to a double-strand endonuclease. The model proposes that plant mitochondria do not distinguish a damaged or mismatched DNA strand from the undamaged strand, they simply cut both strands and perform homology-based DSB repair. This plant-specific strategy for protecting future generations from mitochondrial DNA damage has the side effect of genome expansions and rearrangements.

Evolution of a large, conserved, and syntenic gene family in insects.

The Osiris gene family, first described in Drosophila melanogaster, is clustered in the genomes of all Drosophila species sequenced to date. In D. melanogaster, it explains the enigmatic phenomenon of the triplo-lethal and haploinsufficient locus Tpl. The synteny of Osiris genes in flies is well conserved, and it is one of the largest syntenic blocks in the Drosophila group. By examining the genome sequences of other insects in a wide range of taxonomic orders, we show here that the gene family is well-conserved and syntenic not only in the diptera but across the holometabolous and hemimetabolous insects. Osiris gene homologs have also been found in the expressed sequence tag sequences of various other insects but are absent from all groups that are not insects, including crustacea and arachnids. It is clear that the gene family evolved by gene duplication and neofunctionalization very soon after the divergence of the insects from other arthropods but before the divergence of the insects from one another and that the sequences and synteny have been maintained by selection ever since.