The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene

Summary In the ascomycete Podospora anserina strain ageing (senescence) is caused by a mitochondrial plasmid. In juvenile mycelia it is an integral part of the mtDNA and becomes liberated during ageing. The nucleotide sequence of this plasmid and of its flanking regions was determined. It consists of 2,539 by and contains an un identified reading frame (URF) originating in the adjacent mtDNA upstream of excision point 1. Within the URF a putative 48 by autonomously replicating sequence (ars) was identified. At both excision sites of the plasmid there are two short nonidentical interrupted palindromes and a few base pairs apart from these palindromes, both upstream and downstream, two short inverted repeats are localised. The experimental data make it evident that the mt plasmid is an intron of the cytochrome c oxidase gene (subunit I) which may be excised at the DNA level and thus become the mobile infective agent causing senescence. The concept of this mobile intron and current hypotheses concerning the relationship between introns and transposons are stressed.     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.     

Genome organization in Fusarium oxysporum: clusters of class II transposons

Several families of transposable elements (TEs) are present in the genome of the phytopathogenic fungus Fusarium oxysporum. They are present in copy numbers ranging from just a few elements to tens or hundreds per genome. Sequence analysis of contiguous stretches of genomic DNA surrounding insertion sites of one family revealed that they are packed with repeated sequences. We have carried out a detailed study of the composition and arrangement of these repeats in three chromosomal regions. We found that they are essentially mixtures of several types of TEs, most of them being DNA transposons, different from those previously characterized. Some repeats are frequently reiterated and many of them are inserted into other elements. Parts of these regions are also duplications. These regions appear prone to rearrangement and transposition and are subject to rapid reorganization. Received: 22 December 1999 / 17 February 2000     

Structural and functional analysis of the origin of replication of mitochondrial DNA from Paramecium aurelia

Summary Replication of mitochondrial DNA in Paramecium aurelia involves the formation of a covalent crosslink at one end of this linear molecule and proceeds unidirectionally, producing a dimer consisting of two head to head monomers. The initiation regions within the dimer molecules have been sequenced and shown to be palindromic except for a central nonpalindromic A+T rich sequence, arranged in direct tandem repeats. This nonpalindromic region (see accompanying paper) has been identified as the cross-link which converts the initiation terminus into a continuous sequence. In this study, yeast transformation was used to assay the dimer initiation regions of P. aurelia mtDNA for the presence of autonomously replicating sequences. P. aurelia mtDNA fragments from species 1 and 4 were cloned into the yeast vector YIP5 and the hybrid plasmids (YPaM) were used to transform yeast. The dimer initiation regions from both species promoted high frequency transformation and extrachromosomal maintenance of YPaM plasmids. Subcloning analysis of the ARS-containing mtDNA fragments indicates, specifically, that the nonpalindrome, repetitive sequences are responsible for the autonomously replicating properties of YPaM plasmids.     

Structure of nuclear-localized <Emphasis Type="Italic">cox3</Emphasis> genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and in its colorless close relative <Emphasis Type="Italic">Polytomella</Emphasis> sp.

Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.     

Small interspersed sequences that serve as recombination sites at the cox2 and atp6 loci in the mitochondrial genome of soybean are widely distributed in higher plants

Mitochondrial DNA (mtDNA) fragments that contain cox2 and atp6 were cloned from a wild soybean (Glycine soja, accession `B09002') and from a cultivated soybean (G. max, `Harosoy'). Comparison of these DNAs revealed that two sets of repeated sequences, namely, 299 bp and 23 bp, were present in the 5′ regions of cox2 and atp6. The 299-bp and 23-bp repeats were present close to each other on the 5′ flanking region and the 5′ part of the coding region of cox2 in both `Harosoy' and `B09002', as well as on the 5′ flanking region of atp6 in `Harosoy', while these two repeats were separated by a 706-bp nucleotide sequence that contained a truncated sequence of nad3 at the 5′ flanking region of atp6 in `B09002'. The mtDNA configurations upstream from atp6 and cox2 found in `Harosoy' appeared to have been generated from configurations of cox2 and atp6 found in `B09002' via recombination across the 299-bp or 23-bp repeated sequences, or vice versa, in the mitochondrial genome of the hypothetical progenitor of these plants. The 299-bp sequence was found to be interspersed in the mitochondrial genome. Eight loci were identified that include mtDNA configurations that are inter-convertible with each other via recombination across this sequence in `B09002'. Various loci on the mitochondrial genomes of higher plants that harbor segments of the 299-bp repeats in Glycine were identified. Received: 16 August / 15 December 1997     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

Characterization of a variant Neurospora crassa mitochondrial DNA which contains tandem reiterations of a 1.9 kb sequence

Summary We have identified a second variant of Neurospora mtDNA which contains tandem, head-to-tail repeats of sequences at the boundary of Eco RI-4 and 6. This region may contain a. major replication origin of Neurospora mtDNA.     

The Leishmania major maxicircle divergent region is variable in different isolates and cell types

The maxicircle divergent region (DR) was partially sequenced in several isolates of Leishmania major. The sequence contains various repeated elements: two types of long GC-rich repeats alternating with clusters of short AT-rich repeats. The arrangement of repeats appears to be similar in the studied Leishmania species and their relative Leptomonas seymouri. Furthermore, a conserved sequence containing putative promoters within a palindrome was revealed in the DRs of these species. Unexpectedly, the DR sequence proved to be dissimilar in promastigotes and amastigotes of the same isolate perhaps through selection of parasites with particular maxicircle variants in the course of the promastigote-amastigote differentiation. Different number of repeats and numerous single nucleotide polymorphisms are observed in the compared sequences. We have also investigated the DR structure in 21 L. major isolates by PCR and demonstrated its great variability. We suppose, however, that different variants of the DR structure are generated by combination of several highly conserved domains.     

Evolutionary dynamics of transposable elements in the short-tailed opossum Monodelphis domestica

The genome of the gray short-tailed opossum Monodelphis domestica is notable for its large size ( approximately 3.6 Gb). We characterized nearly 500 families of interspersed repeats from the Monodelphis. They cover approximately 52% of the genome, higher than in any other amniotic lineage studied to date, and may account for the unusually large genome size. In comparison to other mammals, Monodelphis is significantly rich in non-LTR retrotransposons from the LINE-1, CR1, and RTE families, with >29% of the genome sequence comprised of copies of these elements. Monodelphis has at least four families of RTE, and we report support for horizontal transfer of this non-LTR retrotransposon. In addition to short interspersed elements (SINEs) mobilized by L1, we found several families of SINEs that appear to use RTE elements for mobilization. In contrast to L1-mobilized SINEs, the RTE-mobilized SINEs in Monodelphis appear to shift from G+C-rich to G+C-low regions with time. Endogenous retroviruses have colonized approximately 10% of the opossum genome. We found that their density is enhanced in centromeric and/or telomeric regions of most Monodelphis chromosomes. We identified 83 new families of ancient repeats that are highly conserved across amniotic lineages, including 14 LINE-derived repeats; and a novel SINE element, MER131, that may have been exapted as a highly conserved functional noncoding RNA, and whose emergence dates back to approximately 300 million years ago. Many of these conserved repeats are also present in human, and are highly over-represented in predicted cis-regulatory modules. Seventy-six of the 83 families are present in chicken in addition to mammals.     

Further characterization of a large inverted repeat in the mitochondrial genomes of Agaricus bisporus (=A. brunnescens) and related species

The mitochondrial (mt) genome of Agaricus bisporus Ag50 (a heterokaryon) is a 136-kilobase (kb) circular molecule which contains a pair of large inverted repeats (IRs). Two large BAMHI fragments (B1 and B2) which contain the IR regions were further mapped. The repeated regions were determined to be approximately 7.7 kb in length. The mt small ribosomal RNA (S rRNA) gene is located adjacent to one of the repeated regions. Orientational isomers, generated by homologous recombination between the repeated regions, were not observed in mtDNA extractions from Ag50 mycelium (liquid culture) or from Ag50 fruit bodies. We also did not observe any orientational isomers in Ag50HA or Ag50HB, two homokaryons somatically isolated from Ag50. DNA homologous to the Ag50 mt repeated regions was observed in ten other isolates of Agaricus including four isolates of A. bisporus, two isolates of A. subperonatus, two isolates of A. subfloccosus, one isolate of A. bitorquis, and one isolate of A. pattersonae. The repeated regions and the small unique regions in two other heterokaryotic strains of A. bisporus, Ag2 and Ag85, were physically mapped. The repeated regions in these two strains are also in the inverted forms. Restriction endonuclease mapping indicated that the two copies of the IR in Ag85 were not identical.     

Involvement of a large inverted repeated sequence

Summary Southern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two singlecopy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.     

In-frame length mutations associated with short tandem repeats are located in unassigned open reading frames of Oenothera chloroplast DNA

Chloroplast DNAs were compared between two closely related species in the subsection Munzia of the genus Oenothera. A restriction fragment length dimorphism (273 bp) within the large inverted repeats was localized to an unassigned open reading frame that is homologous to ORF 2280 of tobacco chloroplast DNA. This dimorphism is due to different copy numbers of various short tandem repeated sequences, with each repeat unit specifying an in-frame addition or deletion. Other small length mutations were detected within an unassigned reading frame that appears to be homologous to the tobacco ORF 1244, and in the non-coding sequence upstream of that frame. These insertions and/or deletions are all associated with short direct repeats that lie in tandem.     

The role of insertions/deletions in the evolution of the intergenic region betweenpsbA andtrnH in the chloroplast genome

Summary TrnH and the intergenic region betweentrnH andpsbA of the chloroplast genomes of alfalfa (Medicago sativa), Fabaceae, and petunia (Petunia hybrida), Solanaceae, were sequenced and compared to published sequences of that region from other members of those families. A striking feature of these comparisons is the occurrence of insertions/deletions between short, nearly perfect AT-rich direct repeats. The directionality of these mutations in the petunia, tobacco andNicotiana debneyi lineages within the Solanaceae cannot be discerned. However, we present several alternative hypotheses that are consistent with Goodspeed's 1954 evolutionary treatment of the genusNicotiana and family Solanaceae. Within the Fabaceae, the major size differences in the intergenic region between alfalfa, pea and soybean are due to insertions/ deletions between direct repeats. The alfalfa intergenic region has an inverted repeat stem-loop structure of 210 bases directly 5 totrnH. This structure is an insert relative to the liverwort.Marchantia polymorpha. Portions of the insert are found also in pea and soybean as well as in published sequences from other dicots representing diverse orders: petunia, tobacco,N. debneyi (Scrophulariales), spinach (Caryophyllales), and Brassica napus (Capparales). Some of the regions of the insert that are missing in these plants appear to have resulted from deletions of sequences between different imperfect direct repeats within, or 5 to and within the insert. Other deletions are not flanked by repeated sequences. A shrot insert flanked by imperfect direct repeats inB. napus occurs just witin the longer alfalfa insert suggesting that both alfalfa andB. napus have remnants of an even longer insert relative toM. polymorpha. From these analyses we hypothesize the insertion of a stem-loop structure into anM. polymorpha-like ancestral land plant, followd by deletions of sequences, often between different imperfect direct repeats within and upstream of the insert, leading to thepsbA-trnH intergenic sequences represented by the present-day plants examined.     

Comparative analysis of human and mouse 3' Igh regulatory regions identifies distinctive structural features

Immunoglobulin heavy chain (Igh) locus rearrangements are controlled in part by an approximately 30 b complex 3' regulatory region located 3' of C alpha: this region contains several enhancers. We report here the comparison of the genomic sequences of the 3' regulatory region and further downstream sequences from mouse, rat, human and chimpanzee. Only short segments of homology were detected in the 3' regulatory region, and these were located in the vicinity of the known 3' enhancers. The nearest highly conserved segment is the nearest non-Igh gene, hole, which is located approximately 62 kb downstream of mouse C alpha. Analysis of murine 3' Igh sequences by single nucleotide polymorphism (SNP) and restriction fragment length polymorphism (RFLP) detected a transition region (high to low SNP or RFLP density) approximately 120 kb downstream of mouse C alpha. Although there is only limited sequence identity between rodent and primate 3' Igh regulatory regions, all of these regulatory regions contain a palindrome and locally repetitive elements. Locally repetitive elements in primates comprise blocks of "switch-like" sequences that differ from the families of inverted and tandem repeats that are present in rodents. We propose that together with enhancers, these "conserved" structural features are essential for the activity of the 3' Igh regulatory region in vivo.     

Evolution of heteroplasmy at a mitochondrial tandem repeat locus in cultured rabbit cells

Surveys of animal mitochondrial DNA (mtDNA) polymorphism reveal that mtDNA length variation is common. Much of this variation involves non-coding tandem repeat arrays in the main control region of the molecule. Despite a high mutation rate, generating systematic individual mtDNA length heteroplasmy, the number of repeats in a tandem array is maintained within a narrow range in lagomorphs. To investigate the basis for this apparent paradox, we studied the evolution of mtDNA length polymorphism in several rabbit cell clones containing different proportions of mtDNA, with four or five 153-bp repeats. Our data show that equivalent amounts of two mtDNA molecular types are not stable (evolution towards a predominant type being the rule) and that other types remain represented, maintaining the length polymorphism. The data suggest that mtDNA molecules with a longer array of repeats have a replicative advantage that could depend on the nuclear background.     

Sequence and organization of the genomic termini of equine herpesvirus type 1

The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DR1-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the 'a' sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV 'a' sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DR1-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DR1-Uc (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/packaging elements of other herpesviruses are discussed.     

Chromosomal distribution of reverse transcriptase-containing retroelements in two Triticeae species

A large portion of plant and particularly cereal genomes consist of repetitive DNA families, many of which are likely to be or to have evolved from retroelements. Molecular evidence suggests that repeated DNA sequences, although perhaps originating as innocuous or 'selfish' elements, can have dramatic effects on genome organization and function. Knowledge of chromosomal distribution of retroelements is important for understanding plant chromosome structure/functional organization, and could shed light on the dynamics of retroelements and their role in the evolutionary process. In the present study we aim to find a possible correlation between physical location of the regions with species-specific sequences and the distribution of conserved RT domains of the Ty1-copia, Ty3-gypsy and LINE groups of retroelements on the chromosomes of two diploid species that belong to the different branches of the tribe Triticeae, namely Aegilops speltoides Tausch (2n=2x=14) and Hordeum spontaneum L (2n=2x=14). All three groups of retroelements were found in large quantities in the genomes of the tested species. They are cluster-distributed, and the important role of these elements in the formation of terminal heterochromatin is shown. We found that there was a predominance of Ty1-copia and LINE elements in the chromosome regions with preferential content of species-specific sequences.