Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications.

This paper reports the first comprehensive analysis of Displacement loop (D-loop) region sequences from ten different mammalian orders. It represents a systematic evolutionary study at the molecular level on regulatory homologous regions in organisms belonging to a well defined class, mammalia, which radiated about 150 million years ago (Mya). We have aligned and analyzed 26 complete D-loop region sequences available in the literature and the fat dormouse sequence, recently determined in our laboratory. The novelty of our alignment consists of the extensive manual revision of the preliminary output obtained by computer program to optimize sequence similarity, particularly for the two peripheral domains displaying heterogeneity in length and the presence of repeated sequences. The multialignment is available at the WWW site: http://www.ba.cnr.it/dloop.html. Our comparative study has allowed us to identify new conserved sequence blocks present in all the species under consideration and events of insertion/deletion which have important implications in both functional and evolutionary aspects. In particular we have detected two blocks, about 60 bp long, extended termination associated sequences (ETAS1 and ETAS2) conserved in all the organisms considered. Evaluation against experimental work suggests a possible functional role of ETAS1 and ETAS2 in the regulation of replication and transcription and targeted experimental approaches. The analyses on conserved sequence blocks (CSBs) clearly indicate that CSB1 is the only very essential element, common to all mammalian mt genomes, while CSB2 and CSB3 could be involved in different though related functions, probably species specific, and thus more linked to nuclear mitochondrial coevolutionary processes. Our hypothesis on the different functional implications of the conserved elements, CSBs and TASs, reported so far as main regulatory signals, would explain the different conservation of these elements in evolution. Moreover the intra-order comparison of the D-loop regions highlights peculiar features useful to define the evolutionary dynamics of this region in closely related species.

Transcription mapping of the Ori L region reveals novel precursors of mature RNA species and antisense RNAs in rat mitochondrial genome.

We have identified new transcripts in the region surrounding the L-strand replication origin (Ori L) of rat liver mitochondrial DNA. In particular, we have detected previously unidentified intermediates of RNA processing on both the heavy and the light strands, such as precursors of the ND2 mRNA plus the Trp-tRNA and precursors of the tRNAs clustered in the Ori L region. This indicates that the mechanism of RNA processing in mitochondria proceeds step-wise producing a variety of precursors of the mature forms. The other striking finding is the detection of antisense RNA species in the region of L-strand replication. Since a variety of antisense transcripts were also found in the D-loop region of rat mitochondrial DNA, we suggest that they might play a regulatory role in the replication and expression of the mitochondrial genome.

Detection of novel transcripts in the human mitochondrial DNA region coding for ATPase8-ATPase6 subunits.

We have analyzed the tRNA(Lys), ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNA(Lys), ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNA(Lys) plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.

Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature.

The complete and symmetric transcription of the main non coding region of rat mitochondrial genome: in vivo mapping of heavy and light transcripts.

The experiments here reported demonstrate that the main non-coding region of rat mitochondrial DNA is symmetrically transcribed. We have identified stable heavy and light transcripts, whose pattern is rather complex, in the D-loop region of rat mitochondrial DNA. Their relative concentrations have been determined. We detected heavy transcripts which encompass the whole D-loop and more abundant heavy RNA species which we interpreted as transcripts terminating downstream of the 3' end of the last coded gene (Thr-tRNA). The processed heavy RNA species contain polyA, suggesting a strict association between cleavage and polyadenylation. The pattern of light transcripts shows a long RNA, which, starting from the light strand promoter, covers the whole segment, and shorter RNA species which seems to be actively processed at the level of the conserved sequence boxes, probably acting as primers. The symmetric transcription of the D-loop containing region of rat mitochondrial DNA, and in particular the presence of stable transcripts complementary to the putative RNA primers, suggest that mechanisms mediated by interaction between complementary transcripts (antisense RNAs) might play a role in the regulation of mitochondrial DNA replication and expression.

Mitochondrial DNA variability of West New Guinea populations.

This paper reports human mitochondrial DNA variability in West New Guinea (the least known, western side of the island of New Guinea), not yet described from a molecular perspective. The study was carried out on 202 subjects from 12 ethnic groups, belonging to six different Papuan language families, representative of both mountain and coastal plain areas. Mitochondrial DNA hypervariable region 1 (HVS 1) and the presence of the 9-bp deletion (intergenic region COII-tRNA(Lys)) were investigated. HVS 1 sequencing identified 73 polymorphic sites defining 89 haplotypes; the 9-bp deletion, which is considered a marker of Austronesian migration in the Pacific, was found to be absent in the whole West New Guinea study sample. Statistical analysis applied to the resulting haplotypes reveal high heterogeneity and an intersecting distribution of genetic variability in these populations, despite their cultural and geographic diversity. The results of subsequent phylogenetic approaches subdivide mtDNA diversity in West New Guinea into three main clusters (groups I-III), defined by sets of polymorphisms which are also shared by some individuals from Papua New Guinea. Comparisons with worldwide HVS 1 sequences stored in the MitBASE database show the absence of these patterns outside Oceania and a few Indonesian subjects, who also lack the 9-bp deletion. This finding, which is consistent with the effects of genetic drift and prolonged isolation of West New Guinea populations, lead us to regard these patterns as New Guinea population markers, which may harbor the genetic memory of the earliest human migrations to the island.

MmtDB: a Metazoa mitochondrial DNA variants database.

The present paper describes the structure of MmtDB-a specialized database designed to collect Metazoa mitochondrial DNA variants. Priority in the data collection is given to the Metazoa species for which a large amount of variants is available, as it is the case for human variants. Starting from the sequences available in the Nucleotide Sequence Databases, the redundant sequences are removed and new sequences from other sources are added. Value-added information are associated to each variant sequence, e.g. analysed region, experimental method, tissue and cell lines, population data, sex, age, family code and information about the variation events (nucleotide position, involved gene, restriction site's gain or loss). Cross-references are introduced to the EMBL Data Library, as well as an internal cross-referencing among MmtDB entries according to their tissual, heteroplasmic, familiar and aplotypical correlation. MmtDB can be accessed through the World Wide Web at URL [see text].