Mitochondrial 12S Ribosomal RNA Gene Research Articles

Takifugu obscurus is a euryhaline fugu species very close to Takifugu rubripes and suitable for studying osmoregulation

BackgroundThe genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family.ResultsThe following six Takifugu species were used for the analyses: T. obscurus, T. rubripes, T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus. When transferred to FW, only T. obscurus could survive while the others could not survive more than ten days in FW. During this course of FW adaptation, serum Na+ concentration of T. obscurus decreased only slightly, but a rapid and large decrease occurred even in the case of T. niphobles, a peripheral fresh water species that is often seen in brackish river mouths. Phylogenetic analysis using nucleotide sequences of the mitochondrial 16S ribosomal RNA gene of each species indicated that the six Takifugu species are very closely related with each other.ConclusionT. obscurus is capable of adapting to both FW and SW. Its genomic sequence shares a very high homology with those of the other Takifugu species such that the existing Takifugu genomic information resources can be utilized. These properties make "mefugu", which has drawn little attention from animal physiologists until this study, a useful model animal for studying the molecular mechanism of maintaining body fluid homeostasis.

Phylogeny of Japanese Stag Beetles (Coleoptera: Lucanidae) Inferred from 16S mtrRNA Gene Sequences, with Reference to the Evolution of Sexual Dimorphism of Mandibles

As a first step in reconstructing the phylogeny of world stag beetles (Coleoptera: Lucanidae), phylogenetic relationships among the major members of Japanese stag beetles were explored by analyzing a sequence of 1030 nucleotides from the mitochondrial 16S ribosomal RNA (16S rRNA) gene. A total of 20 species and three additional subspecies representing 13 genera were examined to provide basic information on the phylogeny of world Lucanidae. The resultant phylogenetic tree indicates that the family Lucanidae is monophyletic, and contains two major lineages: one consists of the genera Platycerus, Aesalus, Ceruchus, and Nicagus, and the other includes Dorcus, Rhaetulus, Prosopocoilus, Aegus, Neolucanus, Prismognathus, Lucanus, Figulus, and Nigidius. Generic members of the latter lineage are further divided into the following four sublineages: i) Figulus and Nigidius; ii) Prismognathus and Lucanus; iii) Aegus and Neolucanus; and iv) Dorcus, Rhaetulus, and Prosopocoilus. These molecular phylogenetic relationships are used as a basis for a preliminary exploration of the evolution of sexual dimorphism in the shape of the mandible. The results of this investigation suggest that strong sexual dimorphism with well-developed mandibles in males evolved independently at least twice, once in the genus Aegus and once in the ancestor of the Lucanus-Prismognathus and Dorcus-Rhaetulus-Prosopocoilus clades. Alternatively, it is possible that sexual dimorphism of mandibles has undergone secondary loss in the genera Figulus and Nigidius.

Application of Polymerase Chain Reaction to Detect Adulteration of Sheep's Milk with Goats’ Milk

The polymerase chain reaction has been applied for the specific detection of goats’ milk in sheep's milk using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of goat-specific primers yielded a 122-bp fragment from goats’ milk DNA, whereas no amplification signal was obtained in sheep's, cows’, and water buffaloes’ milk DNA. Polymerase chain reaction analysis of raw and heat-treated milk binary mixtures of sheep/goat enabled the specific detection of goats’ milk with a sensitivity threshold of 0.1%. This study demonstrates the usefulness of the proposed polymerase chain reaction assay for authentication of milk products in routine analysis.

Nucleotide diversity and Pleistocene population expansion in Atlantic and Mediterranean scallops ( Pecten maximus and P. jacobaeus) as revealed by the mitochondrial 16S ribosomal RNA gene

Atlantic ( Pecten maximus) and Mediterranean ( Pecten jacobaeus) scallops have been traditionally considered as distinct species, but recent genetic studies have shown that they are races or subspecies separated by the Almeria-Oran oceanographic front in SE Spain. We have scored the nucleotide polymorphism of a 511 base pairs long fragment of the mitochondrial 16S ribosomal RNA gene in 85 individuals from 4 populations of P. maximus and 3 of P. jacobaeus. The populations were characterized by sharing the 2 most common haplotypes. We found no significant differences in haplotype frequencies among populations or species. However, slight, significant differentiation between taxa appeared when haplotypes were pooled in two groups according to their phylogenetic relationships and after analysis of molecular variance, in agreement with previous allozyme studies. Levels of within-population nucleotide diversity were similar in all populations except in P. jacobaeus from the northern Adriatic Sea, suggesting a smaller effective population size in that area which could be due to variable recruitment. Finally, populations showed an excess of rare haplotypes. The mismatch distribution and several population genetic statistics indicate that the excess of rare variants is due to a population expansion which occurred in the second half of the Pleistocene period, less than 0.9 my before present, and probably well after the origin of the two scallop races.

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Abstract Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.

A simple method to distinguish two commercially valuable eel species in Japan Anguilla japonica and A. anguilla using polymerase chain reaction strategy with a species-specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.

Phylogenetic studies of Chinese labeonine fishes (Teleostei: Cyprinidae) based on the mitochondrial 16S rRNA gene

The mitochondrial 16S ribosomal RNA gene is sequenced from 24 ingroups taxa, including 18 species from Labeoninae grouped in 13 genera. Phylogenetic analyses are subjected to neighbor joining, maximum parsimony, maximum likelihood and Bayesian analyses. Phylogenetic analysis indicates that Labeoninae is basically a monophyletic assemblage and can be divided into 2 major clades: one comprising the genera Cirrhinus, Crossocheilus and Garra; and the other consisting of the genera Labeo, Sinilabeo, Osteochilus, Pseudoorossocheilus, Parasinilabeo. Ptychidio, Semilabeo, Pseudogyricheilus, Rectori and Discogobio. According to our present analysis, the features such as the presence of the adhesive disc on the chin and the pharyngeal teeth in 2 rows used in the traditional taxonomy of Labeoninae provide scarce information for phylogeny of labeonine fishes.

TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures

A rapid and highly specific real-time quantitative PCR, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA), has been developed for the quantitation of pork ( Sus scrofa) in binary pork/beef muscle mixtures. The method combines the use of pork-specific primers, that amplify a 411 bp fragment from pork DNA, and mammalian-specific primers amplifying a 425–428 bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan), that hybridizes in the “pork-specific” and also in the “mammalian” DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number ( C t) at which mammalian and pork-specific PCR products are first detected, in combination with the use of reference standards of known pork content, allows the determination of the percentage of pork in a mixed sample. Analysis of experimental pork/beef muscle binary mixtures demonstrated the specificity and sensitivity of the assay for detection and quantitation of pork in the range 0.5–5%.

Genetic Factors in Aminoglycoside Toxicity

Ototoxicity is the major irreversible toxicity of aminoglycosides, and it occurs both in a dose-dependent and idiosyncratic fashion. The idiosyncratic pathway is presumably due to genetic predispositions, and an inherited mutation in the mitochondrial 12S ribosomal RNA gene that predisposes carriers to aminoglycoside ototoxicity was identified in 1993. Up to a third of patients with aminoglycoside ototoxicity carry this mutation. Two other mutations in the same mitochondrial gene affect a small minority of additional patients. Thus, the prevention of aminoglycoside-induced ototoxicity through family history and molecular diagnosis is possible in many cases. It is the challenge of genomic medicine to translate this more than a decade-old knowledge into clinical practice.

A Quick Method for Species Identification of Japanese Eel (Anguilla japonica) Using Real-Time PCR: An Onboard Application for Use During Sampling Surveys

To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.

Deafness due to A1555G mitochondrial mutation without use of aminoglycoside.

The objective was to clarify the characteristics of deafness associated with the A1555G mutation within mitochondrial 12S ribosomal RNA gene in the absence of aminoglycoside exposure. Clinical and genetic studies in family members with the A1555G mitochondrial mutation were performed. The subjects were 123 maternally related members of a large Japanese family with the A1555G mutation. All subjects had no previous history of exposure to aminoglycosides. Hearing disability and handicap, tinnitus, and medical histories were analyzed by interviews in all of the subjects, genetic testing was performed in 41 subjects, and pure-tone audiometry was conducted in 26 subjects with hearing disability and handicap. The A1555G mutation was detected in a homoplasmic form (meaning that all the mitochondrial DNA carries the mutation) in all 41 subjects who were screened. The risk for developing postlingual hearing loss was likely to be much higher in the present subjects than in the general population. Both the severity and age at onset of the phenotype were similar in affected subjects within the same sibling group. Pure-tone averages were significantly worse in subjects who developed hearing loss before age 10 years than in those who developed hearing loss later. The present study demonstrated that the prevalence of deafness in individuals with the A1555G mitochondrial mutation was likely to be high even in the absence of aminoglycoside exposure and clearly showed the association of severe to profound hearing loss with the onset of hearing loss before age 10 years.

Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction.

A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.

PCR Identification of Beef, Sheep, Goat, and Pork in Raw and Heat-Treated Meat Mixtures

A PCR assay has been developed for the specific and qualitative detection of pork (Sus scrofa domesticus), beef (Bos taurus), sheep (Ovis aries), and goat (Capra hircus) in raw and heat-treated meat mixtures. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear species identification. Analysis of experimental meat mixtures demonstrated that the detection limit of the assay was 1% (wt/wt) for each species analyzed. This assay can be useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in meat mixtures.

Phylogenetic relationships of commercial European and Australasian king scallops ( Pecten spp.) based on partial 16S ribosomal RNA gene sequences

King scallops ( Pecten sp.) are commercially important in regions as distant as Europe, Australia, and Japan, and some species are cultured. The taxonomy of king scallops has been traditionally based in characteristics of the shell. Recently, the analysis of protein and DNA polymorphism has shown that the two forms of king scallops known from Europe, Pecten maximus and Pecten jacobaeus, are the same species in spite of showing clear morphological differences. This casts doubts on the distinction of other king scallop species based on morphology alone. We have studied the phylogenetic relationships of the two European species with two other Pecten taxa which show a striking morphological similarity with them in spite of living in the opposite side of the world: Pecten fumatus and Pecten novaezelandiae, from the southern Indo-Pacific around the coasts of Australia and New Zealand. We scored the nucleotide variability of a 501-base-pair-long fragment of the mitochondrial 16S ribosomal RNA gene in a total of 27 individuals and performed a phylogenetic analysis. Atlantic and Australasian taxa constituted two clearly separated lineages, which rules out an explanation for morphological similarity based on conspecificity due to human transfer, as has been proposed for other bivalve species-pairs in the studied regions. However, we found that the amount of differentiation observed between the two oceanic lineages was modest when compared with that expected under the generally accepted paleobiogeographical model of origin of Atlantic and Indo-Pacific king scallops, based on the closure of the Tethys Sea at the beginning of the Miocene, ca. 14 million years ago (average Kimura 2-parameter distance=0.0154±0.0053). We present an alternative model based on Beu and Darragh's [Proc. R. Soc. Victoria 113 (2001) 1] hypothesis of South Africa to Australia Pleistocene dispersal and Vermeij's [Mar. Biol. 112 (1992) 343] hypothesis of pre-Pleistocene transequatorial dispersal from Europe to South Africa. Finally, our phylogenetic analysis did not separate P. fumatus and P. novaezelandiae as different species, contrary to previous studies based on allozymes.

Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction.

A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.

How Much Should We Worry About Aminoglycoside Ototoxicity

Two mutations in the mitochondrial 12S ribosomal RNA gene that predispose individuals to aminoglycoside ototoxicity -- a major and potentially preventable form of acquired hearing loss -- have been previously described. These researchers used molecular techniques to …

Otzi's last meals: DNA analysis of the intestinal content of the Neolithic glacier mummy from the Alps.

Samples of the intestinal content were collected from the ileum and colon of the Neolithic glacier mummy popularly known as the Tyrolean Iceman, or Otzi. DNA was extracted from the samples and PCR amplified, using a variety of primer pairs designed to bind to different genes (mammal mitochondrial 12S ribosomal RNA gene, plant/fungal nuclear 18S ribosomal RNA gene, plant chloroplast ribulose bisphosphate carboxylase large subunit gene). This made it possible to distinguish between animal and plant food residues (macroremains) and pollen (microremains). According to the DNA reconstruction, the man's last meal was composed of red deer (Cervus elaphus) meat, and, possibly, cereals; this meal had been preceded by another one based on ibex (Capra ibex), different species of dicots, and cereals. The DNA spectrum corresponding to pollen residues in the colon, on the other hand, fits with the hypothesis that the last journey of the Neolithic hunter/warrior was made through a subalpine coniferous forest to the site at over 3,200 m above sea level, where his mummified body was to be discovered 5,000 years later.

Analysis of the mitochondrial 12S rRNA gene supports a two-clade hypothesis of the evolutionary history of scleractinian corals

Scleractinian corals have long been assumed to be a monophyletic group characterized by the possession of an aragonite skeleton. Analyses of skeletal morphology and molecular data have shown conflicting patterns of suborder and family relationships of scleractinian corals, because molecular data suggest that the scleractinian skeleton could have evolved as many as four times. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 12S ribosomal RNA gene from 28 species of scleractinian corals and use this gene to infer the evolutionary history of scleractinians. We show that the sequences obtained fall into two distinct clades, defined by PCR product length. Base composition among taxa did not differ significantly when the two clades were considered separately or as a single group. Overall, transition substitutions accumulated more quickly relative to transversion substitutions within both clades. Spatial patterns of substitutions along the 12S rRNA gene and likelihood ratio tests of divergence rates both indicate that the 12S rRNA gene of each clade evolved under different constraints. Phylogenetic analyses using mt 12S rRNA gene data do not support the current view of scleractinian phylogeny based upon skeletal morphology and fossil records. Rather, the two-clade hypothesis derived from the mt 16S ribosomal gene is supported.

The double origin of Iberian peninsular chameleons

There is considerable controversy concerning the origin of Iberian populations of the Mediterranean chameleon, Chamaeleo chamaeleon. Current opinion dictates that Spanish populations result from introductions during the 18th and 19th centuries, with subsequent translocations from the original populations to other parts of Spain. The Portugese population in the Algarve is believed to have been introduced from Africa or Spain during the 1920s. However, Holocene remains of chameleons suggest that the Malaga population at least could have a much older origin. Analysis of sequences from the mitochondrial 16S ribosomal RNA gene of samples from the Iberian Peninsula and North Africa revealed a double origin for the Iberian population. The Mediterranean Iberian (Malaga) population is closely related to Mediterranean North African populations, with Atlantic Iberian populations more closely related to populations of the Atlantic coast of North Africa. The overall genetic differentiation and diversity observed was very low, preventing precise dating of the colonization events. However this low level of differentiation is not consistent with Plio-Pleistocene colonization, the assumed timing for a natural colonization event and suggests that chameleons were probably introduced twice by man in the recent past.

The mitochondrial 12S gene is a suitable marker of populations of Sarcoptes scabiei from wombats, dogs and humans in Australia.

We sequenced part of the mitochondrial 12S ribosomal RNA gene of 23 specimens of Sarcoptes scabiei from eight wombats, one dog and three humans. Twelve of the 326 nucleotide positions varied among these mites and there were nine haplotypes (sequences) that differed by 1-8 nucleotides. Phylogenetic analyses indicated that these mites were from two lineages: (1) mites from wombats from Victoria, Australia, and mites from the humans and dog from the Northern Territory, Australia (haplotypes 1-4, 9); and (2) mites from the humans and dog from the Northern Territory (haplotypes 5-8). Mites from the three different hosts (wombats, a dog and humans) had not diverged phylogenetically; rather, these mites had similar 12S sequences. Thus, we conclude that these mites from wombats, humans and a dog are closely related, and that they diverged from a common ancestor relatively recently. This conclusion is consistent with the argument that people and/or their dogs introduced to Australia the S. scabiei mites that infect wombats in Australia . So, S. scabiei, which has been blamed for the extinction of populations of wombats in Australia, may be a parasitic mite that was introduced to Australia with people and/or their dogs. These data show that the mitochondrial 12S rRNA gene may be a suitable population marker of S. scabiei from wombats, dogs and humans in Australia.