Mitochondrial 12S RNA Research Articles

Romanian cyprinids phylogeny based on 16S ARN mitochondrial genes

The vertebrate mitochondrial genome has been an important model system for studying molecular evolution, organism phylogeny, and genome structure. Phylogenetic relatioships were inferred from analysis of 570 base pairs (bp) of mithocondrial DNA (mtDNA), representing a conserved region of 16S rRNA. We sequenced 13 cyprinids species and one putative outgroup (Misgurnus fossilis) from Romania. Based upon nucleotide sequence comparisons of cyprinid mitochondrial 16SRNA genes, we established the phylogenetic relationships between analyzed species. The phylogenetic trees obtained by two different methods (neighbor-joining and maximum parsimony) have the same topology and show that most species examined have supported the traditional division of the Cyprinidae into two subfamilies: Cyprininae and Leuciscinae.

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss.

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .

A putative RNA editing from U to C in a mouse mitochondrial transcript.

Recently, we isolated and characterized a new mouse mitochondrial RNA molecule containing the mitochondrial 16S RNA plus 121 nt joined to the 5' end of the RNA. This fragment arises from the L strand of the same gene and we have named this transcript chimeric RNA. At position 121 of the RNA there is a C, which, according to the sequence of the mitochondrial 16S RNA gene, should be a U. We hypothesized that this RNA is synthesized having a U at position 121, which is later substituted to a C by a putative editing reaction. Based on the presence of sites for the restriction endonucleases RsaI and Fnu4HI around position 121, both forms of the RNA were detected in mouse tissues. To confirm the presence of the non-edited and putative edited RNA, a fragment containing the first 154 nt of the RNA was amplified by RT-PCR and cloned. The substitution of U for C was demonstrated by sequencing these clones. In vitro transcription experiments demonstrated that the substitution of U for C is not due to artifact of amplification or cloning. Moreover, in mitochondria from testis only the non-edited form was found. This, together with other experimental evidence, demonstrated that the base substitution was not due to polymorphism of the mitochondrial 16S RNA gene. This is the first demonstration of a substitution reaction from U to C in a mammalian mitochondrial transcript.

Venomous cone snails: molecular phylogeny and the generation of toxin diversity

In order to investigate the generation of conotoxin diversity, δ-conotoxin sequences from nine Conus species were analyzed in the context of their phylogeny. Using a standard molecular marker, mitochondrial 16S RNA, we determined that the δ-conotoxins were derived from three distinct species clades based on the phylogenetic reconstruction of a large set (>80) of Conus species and other toxoglossate molluscs. Four different mechanisms appear to have contributed to the diversity of the δ-conotoxins analyzed: (1) Speciation: δ-Conotoxins in different species diverge from each other (the prepro regions of orthologous genes somewhat more slowly than the reference rRNA rate, the mature toxin regions significantly faster). (2) Duplication: Intraspecific δ-conotoxin divergence is initiated by gene duplication events, some of which may have predated the species itself. (3) Recombination: A novel δ-conotoxin may arise through recombination of two parental δ-contoxin genes. (4) ‘ Focal hypermutation’: This sudden, almost saltatory change in sequence is always restricted to the mature toxin region. The first three have been recognized previously as mechanisms important for the evolution of gene families in other phylogenetic systems; the last is a remarkable, mechanistically unexplained and specialized feature of Conus peptide diversification.

Phylogeny of higher taxa of hexapoda according to 12sRNA sequences

The phylogenetic relationship of Hexapoda has been debated for a long time, which will be resolved mainly depending on the settlement of monophyly, affinities and interrelationships among Protura, Collembola and Diplura. Mitochondrial 12sRNA gene about 355 bp fragments from one proturan species, two collembolan species, two dipluran species and one oribatid species were sequenced. The Kimura 2-parameter distances were calculated and a series of molecular phylogenetic trees were reconstructed by using the N-J method, from which the following points were drawn: (i) Protura and Collembola compose a monophyletic group representing absent-cerci; (ii) Diplura is not a monophyletic group, in which Campodeoid with filiform cerci belongs to a clade and Japygoid with pincer cerci and Ectognatha compose another clade, that is, Insecta s.str. stemmed from Japygoid. So it would be suggested that the phylogenetic relationship of Hexapoda is [Parainsecta (Collembola + Protura) +Campodeoid +Insecta (Japygoid + Ectognatha)].

A novel chimeric mitochondrial RNA localized in the nucleus of mouse sperm.

Six identical cDNA clones corresponding to an RNA of 1685 nucleotides that is enriched in mouse sperm compared with testis were isolated from a mouse testis cDNA library. The sequence of these clones corresponds to the 16S mitochondrial RNA plus an inverted repeat of 120 bp covalently joined to the 5' end of the RNA. By RT-PCR, it was demonstrated that this transcript, referred to as chimeric RNA, was present in mouse sperm, testis, liver, kidney, brain, and spleen. The absence of an equivalent sequence in mitochondrial DNA or as a mitochondrial pseudogene in total DNA extracted from sperm, testis, and somatic tissues suggests that the chimeric RNA is a post-transcriptional product, maybe resulting from a trans splicing reaction. The chimeric RNA was found by RT-PCR in total RNA extracted from purified sperm heads. This result was confirmed by in situ hybridization, which showed clear staining of the sperm nucleus with probes corresponding to sequences of the mitochondrial 16S RNA and the inverted repeat.

Rapid identification of an A1555G mutation in human mitochondrial DNA implicated in aminoglycoside-induced ototoxicity.

This article describes a multiplex allele-specific PCR (AS-PCR) approach for detection of an A to G mutation occurring in the human mitochondrial 12s RNA gene at nucleotide 1555. Possession of this mutation has been shown to be associated with irreversible hearing loss following administration of aminoglycoside antibiotics, and in some families is associated with profound sensorineural deafness in the absence of aminoglycoside antibiotics. We screened 206 unrelated individuals from the province of Otago, New Zealand, and found one who possessed the mitochondrial 1555 A to G mutation (0.48%; 95% confidence interval, 0.01-2.75).

Specific binding of aminoglycosides to a human rRNA construct based on a DNA polymorphism which causes aminoglycoside-induced deafness.

RNA constructs prepared from wild-type and mutant (1555(G)) human mitochondrial 12S RNA were studied with respect to their abilities to specifically bind aminoglycoside antibiotics. The 1555(G) point mutation had previously been found to be associated with hereditary deafness induced by aminoglycosides. It is shown here that the 1555(G) RNA analog stoichiometrically binds aminoglycosides with high affinities, while the wild-type construct does not bind aminoglycosides at all. Analogous mutations in a 16S bacterial rRNA construct show the opposite behavior. Bacterial 16S rRNA constitutes the functional target for aminoglycoside antibiotics. While the wild-type 16S rRNA decoding region construct binds aminoglycosides stoichiometrically with binding affinities in the micromolar range, the mutant is unable to specifically bind aminoglycosides. These results demonstrate the importance of a specific GC base pair in aminoglycoside binding in both the human and the bacterial rRNA constructs. These studies also provide quantitative evidence in support of the hypothesis that the 1555(G) point mutation in human mitochondrial 12S RNA causes aminoglycoside induced deafness.

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae.

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.

Phylogenetic relationships within the class mammalia: A study using mitochondrial 12S RNA sequences

The interrelationships of the three mammalian groups, Monotremata, Marsupialia, and Eutheria, have been studied using DNA sequences from the mitochondrial 12S ribosomal RNA gene. The results suggest that the monotremes diverged from the living therians only shortly before eutherians and marsupials separated from each other, although there is some evidence for a slowdown in rate of base change in the monotreme lineage. Whtin the Monotremata, the two extant species of tachyglossids show a very close genetic relationship and the data suggest a very recent divergence. We have also confirmed that the Patagonian Monito del Monte,Dromiciops australis, is more closely related to the australidephian marsupials than it is to other South American species.