Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation.     

Drosophila duplication hotspots are associated with late-replicating regions of the genome

Duplications play a significant role in both extremes of the phenotypic spectrum of newly arising mutations: they can have severe deleterious effects (e.g. duplications underlie a variety of diseases) but can also be highly advantageous. The phenotypic potential of newly arisen duplications has stimulated wide interest in both the mutational and selective processes shaping these variants in the genome. Here we take advantage of the Drosophila simulans-Drosophila melanogaster genetic system to further our understanding of both processes. Regarding mutational processes, the study of two closely related species allows investigation of the potential existence of shared duplication hotspots, and the similarities and differences between the two genomes can be used to dissect its underlying causes. Regarding selection, the difference in the effective population size between the two species can be leveraged to ask questions about the strength of selection acting on different classes of duplications. In this study, we conducted a survey of duplication polymorphisms in 14 different lines of D. simulans using tiling microarrays and combined it with an analogous survey for the D. melanogaster genome. By integrating the two datasets, we identified duplication hotspots conserved between the two species. However, unlike the duplication hotspots identified in mammalian genomes, Drosophila duplication hotspots are not associated with sequences of high sequence identity capable of mediating non-allelic homologous recombination. Instead, Drosophila duplication hotspots are associated with late-replicating regions of the genome, suggesting a link between DNA replication and duplication rates. We also found evidence supporting a higher effectiveness of selection on duplications in D. simulans than in D. melanogaster. This is also true for duplications segregating at high frequency, where we find evidence in D. simulans that a sizeable fraction of these mutations is being driven to fixation by positive selection.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.     

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.     

High Frequency Repeat-Induced Point Mutation (Rip) Is Not Associated with Efficient Recombination in Neurospora

Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.     

Multiple origins and rapid evolution of duplicated mitochondrial genes in parthenogenetic geckos (Heteronotia binoei; Squamata, Gekkonidae)

Accumulating evidence for alternative gene orders demonstrates that vertebrate mitochondrial genomes are more evolutionarily dynamic than previously thought. Several lineages of parthenogenetic lizards contain large, tandem duplications that include rRNA, tRNA, and protein-coding genes, as well as the control region. Such duplications are hypothesized as intermediate stages in gene rearrangement, but the early stages of their evolution have not been previously studied. To better understand the evolutionary dynamics of duplicated segments of mitochondrial DNA, we sequenced 10 mitochondrial genomes from recently formed ( approximately 300,000 years ago) hybrid parthenogenetic geckos of the Heteronotia binoei complex and 1 from a sexual form. These genomes included some with an arrangement typical of vertebrates and others with tandem duplications varying in size from 5.7 to 9.4 kb, each with different gene contents and duplication endpoints. These results, together with phylogenetic analyses, indicate independent and frequent origins of the duplications. Small, direct repeats at the duplication endpoints imply slipped-strand error as a mechanism generating the duplications as opposed to a false initiation/termination of DNA replication mechanism that has been invoked to explain duplications in other lizard mitochondrial systems. Despite their recent origin, there is evidence for nonfunctionalization of genes due primarily to deletions, and the observed pattern of gene disruption supports the duplication-deletion model for rearrangement of mtDNA gene order. Conversely, the accumulation of mutations between these recent duplicates provides no evidence for gene conversion, as has been reported in some other systems. These results demonstrate that, despite their long-term stasis in gene content and arrangement in some lineages, vertebrate mitochondrial genomes can be evolutionary dynamic even at short timescales.     

Molecular structure and genetic stability of human hypoxanthine phosphoribosyltransferase (HPRT) gene duplications.

We have determined the genetic stability of three independent intragenic human HPRT gene duplications and the structure of each duplication at the nucleotide sequence level. Two of the duplications were isolated as spontaneous mutations from the HL60 human myeloid leukemia cell line, while the third was originally identified in a Lesch-Nyhan patient. All three duplications are genetically unstable and have a reversion rate approximately 100-fold higher than the rate of duplication formation. The molecular structures of these duplications are similar, with direct duplication of HPRT exons 2 and 3 and of 6.8 kb (HL60 duplications) or 13.7 kb (Lesch-Nyhan duplication) of surrounding HPRT sequence. Nucleotide sequence analyses of duplication junctions revealed that the HL60-derived duplications were generated by unequal homologous recombination between clusters of Alu repeats contained in HPRT introns 1 and 3, while the Lesch-Nyhan duplication was generated by the nonhomologous insertion of duplicated HPRT DNA into HPRT intron 1. These results suggest that duplication substrates of different lengths can be generated from the human HPRT exon 2-3 region and can undergo either homologous or nonhomologous recombination with the HPRT locus to form gene duplications.     

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.     

Tandem genetic duplications in phage lambda. IV. The locations of spontaneously arising tandem duplications.

Twenty-four spontaneously arising, long DNA addition derivatives of phage lambda have been isolated by two methods (one physical, one genetic) based on phage DNA content. All are shown to contain a tandem duplication of phage DNA by a number of criteria. The location of the duplicated segment in each has been determined by electron microscopy of DNA hereroduplexes. The duplications are found to lie at random throughout the chromosome, with no preferential locations for endpoints. This rules out the possibility that duplications are formed by crossing-over at regions of homology on the phage chromosome.     

Structural conservation and variation in the D-loop-containing region of vertebrate mitochondrial DNA

The nucleotide sequences of the D-loop-containing regions of three rat mitochondrial DNAs (mtDNAs), two from the species Rattus norvegicus and one from R. rattus, were determined. Comparisons made among these sequences and with the mouse sequence showed that, on the basis of both base composition and frequency of nucleotide alterations, three domains could be defined within the D-loop-containing region: a central conserved segment, poor in L-strand adenine, flanked by two divergent, adenine-rich regions. Deletions and insertions were found to occur at an unexpectedly high frequency in these sequences and the conserved sequence block called CSB-1 was found not to be intact in the R. rattus sequence. Although in comparisons of more distantly related mtDNAs the D-loop region is the most divergent on the molecule, it does not diverge more than typical protein genes between R. norvegicus and R. rattus, and its central conserved domain appears to be one of the molecule's most conserved regions. The most variable domain borders the tRNAPhe gene and contains the L and H-strand promoters and the 5' terminus for H-strand DNA synthesis. Within this region we have found sequences in all the mtDNAs we have examined, including those of human, two artiodactyls and Xenopus, that are capable of folding into cloverleaf structures. In the other divergent domain of the same mtDNAs, we find sequences capable of assuming similar secondary structural configurations at or near the sites for the termination of D-loop DNA synthesis. The evolutionary preservation of the potential to form such structures despite the high primary-structural divergence of the regions they occur in, suggests the structures are of principal importance for some processes occurring in the D-loop-containing region.     

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

Elevated Levels of Petite Formation in Strains of SACCHAROMYCES CEREVISIAE Restored to Respiratory Competence. I. Association of Both High and Moderate Frequencies of Petite Mutant Formation with the Presence of Aberrant Mitochondrial DNA

When recently arisen spontaneous petite mutants of Saccharomyces cerevisiae are crossed, respiratory competent diploids can be recovered. Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation. On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5–16%) and those with a high frequency (>60%) of petite formation. Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules. Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern. Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations. In addition, sectored colony siblings can have different aberrant mtDNAs. The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome. Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency.     

Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication]

Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deo operon of E. coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

Unequal crossing over is the principal pathway of homologous recombination in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.     

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.     

Comparative analysis of Fusarium mitochondrial genomes reveals a highly variable region that encodes an exceptionally large open reading frame

The mitochondrial (mt) genomes of Fusarium verticillioides, Fusarium solani and Fusarium graminearum were annotated and found to be 53.7, 63.0 and 95.7 kb in length, respectively. The genomes encode all genes typically associated with mtDNAs of filamentous fungi yet are considerably larger than the mt genome of F. oxysporum. Size differences are largely due to the number of group I introns. Surprisingly, the genomes contain a highly variable region of 7-9 kb that encodes an exceptionally large, unidentified open reading frame (uORF). The region has the hallmarks of a horizontally transmitted DNA and was likely acquired prior to the divergence of Fusarium species. Two additional uORFs were detected that are also under positive selection. DNA repeats associated with the uORFs suggest that 3' gene duplication may be an adaptive mechanism to modify coding regions or generate new ORFs. The acquisition of these new genes contrasts to the wide-scale size reduction experienced by fungal mt genomes.     

MITOCHONDRIAL-DNA ANALYSES AND THE ORIGIN AND RELATIVE AGE OF PARTHENOGENETIC LIZARDS (GENUS CNEMIDOPHORUS). IV. NINE SEXLINEATUS-GROUP UNISEXUALS

Mitochondrial DNAs (mtDNAs) from nine morphologically distinct unisexual species and five bisexual species of lizards, all from the sexlineatus species-group of Cnemidophorus, were compared using restriction endonucleases. The unisexual lizards have mtDNAs that are identical at all or nearly all of the 128 sites cleaved. Although differing little in sequence, some mtDNAs differed in size due to the presence of tandem sequence duplications. Phylogenetic analysis of cleavage maps indicates that the mtDNAs of the unisexuals are most similar to that of the bisexual species C. inornatus. Considerable mtDNA diversity exists among C. inornatus populations, and one geographically restricted subspecies, C. i. arizonae, was identified as the most probable maternal ancestor of all nine unisexuals. All but one of these are triploid, and all have at least one C. inornatus gene complement. This, together with the homogeneity of their mtDNAs, suggests that all stem from one or a small number of allodiploid females (presumably parthenogenetic) that originated in a restricted geographic area in the recent past. These data, when combined with those from allozyme studies, preclude the possibility that most of the triploid unisexuals could have arisen via fertilization of an unreduced diploid ovum from one species by a haploid sperm from a different species.