NADH Dehydrogenase Genes Research Articles

A single nucleotide polymorphism map of the mitochondrial genome of the parasitic nematode Cooperia oncophora.

The 13,636 bp mitochondrial (mt) genome sequence of the trichostrongylid nematode Cooperia oncophora was determined. Like the mt genomes of other nematodes it is AT rich (76.75%) and cytidine is the least common nucleoside in the coding strand. There are 2 ribosomal RNA (rrn) genes, 22 transfer RNA (trn) genes and 12 protein coding genes. The relatively short AT-rich region (304 bp) and the lack of a non-coding region between two of the NADH dehydrogenase genes, nad3 and nad5, makes the mt genome of C. oncophora one of the smallest known to date, having only 525 bp of non-coding regions in total. The majority of the C. oncophora protein encoded genes are predicted to end in an abbreviated stop codon like T or TA. In total, 426 single nucleotide polymorphisms (SNP) were mapped on the mt genome of C. oncophora, which is an average of 1 polymorphism per 32 bp. The most common SNPs in the mt genome of C. oncophora were G/A (59.2%) and C/T (28.4%) transitions. Synonymous substitutions (86.4%) were favoured over non-synonymous substitutions. However, the degree of sequence conservation between individual protein genes of different parasitic nematode species did not always correspond to the relative number of non-synonymous SNPs. The mt genome sequence of C. oncophora presents the first mt genome of a member of the Trichostrongyloidea and will be of importance in refining phylogenetic relationships between nematodes. The, still limited, SNP map presented here provides a basis for obtaining insight into the genetic diversity present in the different protein coding genes, trn, rrn and non-coding regions. A more detailed study of the more variable regions will be of use in determining the population genetic structure of C. oncophora. Ultimately this knowledge will add to the understanding of the host-parasite relationship.

The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria

A DNA sequence homologous to non-proton-pumping NADH dehydrogenase genes was found in the genome of Neurospora crassa encoding a polypeptide of 577 amino acid residues, molecular mass of 64,656 Da, with a putative transmembrane domain. Analysis of fungal mitochondria fractionated with digitonin indicates that the protein is located at the outer face of the inner membrane of the organelle (external enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. Mitochondria from the resulting null-mutant nde2 are highly deficient in the oxidation of cytosolic NADH and NADPH. A triple mutant nde1/nde2/ndi1, lacking mitochondrial alternative NAD(P)H dehydrogenases, was obtained, indicating that these proteins are not essential in N. crassa. However, crosses between the nde2 mutant strain and complex I-deficient mutants yielded no viable double mutants. Transcription of the nde-2 gene, as well as of ndi-1 (internal enzyme), is repressed in the late exponential phase of fungal growth.

PCR-RFLP identification of endemic Chilean species of Rhagoletis (Diptera: Tephritidae) attacking Solanaceae.

Four species of the genus Rhagoletis are native to Chile: R. nova (Schiner), R. conversa, (Brèthes), R. penela Foote and R. tomatis Foote. Currently, identification of these species is based on morphological criteria, but their strong similarity makes precise recognition difficult. To clarify species separation for quarantine purposes, a reliable method based on a PCR-RFLP procedure is reported. A DNA region containing mitochondrial NADH dehydrogenase genes was selected as a target sequence for the analysis. The amplification products (c. 1 kb) were digested with either SspI or DdeI, yielding specific patterns that differentiated each of the endemic species. Complete nucleotide sequences were determined, confirming empirical restriction maps. This report updates information on the geographical distribution of Rhagoletis species in Chile.

Phylogenetics and differentiation among the western taxa of the Erebia tyndarus group (Lepidoptera: Nymphalidae)

The phylogenetic relationships among six members of the ‘tyndarus’ group in the Erebia genus of Satyrid butterflies, i.e. E. tyndarus, E. cassioides, E. nivalis, E. calcaria, E. hispania and E. ottomana were analysed using data from 19 presumptive enzyme loci, as well as 440 and 429 bp, respectively, from the mitochondrial large ribosomal subunit (16S) and subunit 1 from the NADH dehydrogenase (ND1) genes. The two types of molecular data (allozymes and mtDNA) yielded largely congruent tree topologies. The two basal, independent lineages formed by E. ottomana and E. hispania are contrasted by a group of Genetically closely related taxa, suggestive of a recent radiation associated with allopatric speciation, and competitive exclusion. The time of divergence for the radiation event is similar for both allozymes and mtDNA with an estimation of 440000 years ago. The lineages involved in this radiation do not comply with all the criteria necessary to assign to each of them full species rank, but they can no more be included in one single species unit. Such situations involving more than two alio- or parapatric lineages may explain why polytomies are so often met in phylogenetic reconstructions, after the lineages have reached full species rank. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 319–332.

Mitochondrial NADH Dehydrogenase gene (mtND2) Phylogeny of Egyptian Tilapia species

NADH dehydrogenase is a very important protein and is expressed by the mitochondrial NADH dehydrogenase gene (mtND2). Dehydrogenase enzyme is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP. Also, it reversibly catalyses the oxidation of NADH to NAD and reduced acceptor. The size of mtDN2 of Tilapia species and their hybrids is ~1050 base pairs and was detected by using the polymerase chain reaction technique. To identify the molecular phylogeny and the physical characteristics of mtND2 gene of Tilapia species were done by using the restriction fragment length polymorphisms (RFLPs) with some restriction endonucleases(AccI, AvaII, AvaI, StyI, Bg1I and EaeI). The PCR-RFLPs of NADH dehydrogenase gene of Tilapia species and their hybrids may prove that the gene is quite evolution phylogenetic difference from one species to another. At the same time, This study investigated the feasibility of mitochondrial DNA (mtDNA) based approaches in addressing problems of identification of Tilapia species and their hybrids, isolated from the River Nile by using the PCR-RFLPs analysis of mtND2 gene.

Spatial and temporal population structure of sea trout at the Island of Gotland, Sweden, delineated from mitochondrial DNA

In a study of the genetic relationships among 879 anadromous brown trout Salmo trutta from 13 streams at the Island of Gotland, Sweden, using RFLP analysis of a mitochondrial DNA segment (NADH dehydrogenase‐1 gene), six haplotypes were detected. Significant genetic divergence was observed among streams as well as between cohorts within streams. Approximately 8–9% of the total variation was due to differences between populations, and 4–5% was explained by differences between cohorts within populations. The female effective population size (Nef) was assessed from temporal haplotype frequency differences between consecutive cohorts; the estimated average Nef over all populations was just below 30, suggesting that these populations were effectively quite small. With such small effective sizes the populations are expected to loose genetic variability quickly, but the observed levels do not appear particularly low. This indicates that female migration between streams occurs. The observed level of differentiation does not support the presumption that a particular pre‐smolt migratory behaviour observed in Gotland streams, with large portions of fry leaving for the sea soon after hatching, results in a reduced homing ability. From a conservation management perspective the Gotland brown trout streams should be regarded as a population system where the vitality and survival of brown trout in one stream is dependent on the opportunity of contact and exchange of individuals from other streams.

Spatial and temporal population structure of sea trout at the Island of Gotland, Sweden, delineated from mitochondrial DNA

In a study of the genetic relationships among 879 anadromous brown trout Salmo trutta from 13 streams at the Island of Gotland, Sweden, using RFLP analysis of a mitochondrial DNA segment (NADH dehydrogenase-1 gene), six haplotypes were detected. Significant genetic divergence was observed among streams as well as between cohorts within streams. Approximately 8–9% of the total variation was due to differences between populations, and 4–5% was explained by differences between cohorts within populations. The female effective population size (Nef) was assessed from temporal haplotype frequency differences between consecutive cohorts; the estimated average Nef over all populations was just below 30, suggesting that these populations were effectively quite small. With such small effective sizes the populations are expected to loose genetic variability quickly, but the observed levels do not appear particularly low. This indicates that female migration between streams occurs. The observed level of differentiation does not support the presumption that a particular pre-smolt migratory behaviour observed in Gotland streams, with large portions of fry leaving for the sea soon after hatching, results in a reduced homing ability. From a conservation management perspective the Gotland brown trout streams should be regarded as a population system where the vitality and survival of brown trout in one stream is dependent on the opportunity of contact and exchange of individuals from other streams.

NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH.

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.

NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH–ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH–ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.

Assessment of concordance among genealogical reconstructions from various mtDNA segments in three species of Pacific salmon (genus Oncorhynchus).

Seven segments of mitochondrial DNA (mtDNA), comprising 97% of the mitochondrial genome, were amplified by polymerase chain reaction (PCR) and examined for restriction site variation using 13 restriction endonucleases in three species of Pacific salmon: pink (Oncorhynchus gorbuscha), chum (O. keta) and sockeye (O. nerka) salmon. The distribution of variability across the seven mtDNA segments differed substantially among species. Little similarity in the distribution of variable restriction sites was found even between the mitochondrial genomes of the even- and odd-year broodlines of pink salmon. Significantly different levels of nucleotide diversity were detected among three groups of genes: six NADH-dehydrogenase genes had the highest; two rRNA genes had the lowest; and a group that included genes for ATPase and cytochrome oxidase subunits, the cytochrome b gene, and the control region had intermediate levels of nucleotide diversity. Genealogies of mtDNA haplotypes were reconstructed for each species, based on the variation in all mtDNA segments. The contributions of variation within different segments to resolution of the genealogical trees were compared within each species. With the exception of sockeye salmon, restriction site data from different genome segments tended to produce rather different trees (and hence rather different genealogies). In the majority of cases, genealogical information in different segments of mitochondrial genome was additive rather than congruent. This finding has a relevance to phylogeographic studies of other organisms and emphasizes the importance of not relying on a limited segment of the mtDNA genome to derive a phylogeographic structure.

Phylogeny of Limia (Teleostei: Poeciliidae) Based on NADH Dehydrogenase Subunit 2 Sequences

Complete sequences for the mitochondrial gene NADH Dehydrogenase 2 (ND2) and partial sequences for the tRNA-Met and tRNA-Trp genes were obtained for 11 populations of the poeciliid fish genus (or subgenus) Limia, including species from Hispaniola, Cuba, Jamaica, and Grand Cayman Islands. Additional sequences for Limia (2 species), Pamphorichthys (3), Poecilia (16), and Xiphophorus (1), all from the tribe Poeciliini, were extracted from GenBank, as was a sequence for Heterandria formosa, from the tribe Heterandriini. Phylogenetic analyses included parsimony, distance methods, and maximum-likelihood. Several Limia species groups that had been proposed based on morphological data were evaluated. The versicolor species group was strongly supported, as was the close relationship between the Cuban Limia vittata and the Grand Cayman Limia caymanensis. However, the proposed vittata species group was not upheld by the ND2 data. The phylogenetic position of Limia melanogaster, from Jamaica, was either sister to the versicolor species group or basal to all other included Limia species. Limia was found to be monophyletic; however, Limia species from the island of Hispaniola were not monophyletic. There was little support for any proposed sister group to Limia. The phylogeny was used to reconsider a previous comparative study of poeciliid courtship behavior and sexual dimorphism. The data indicated that there may have been two independent appearances of courtship display behavior in Limia; considering the tribe Poeciliini as a whole, there is evidence for as many as five appearances of display (including two within Limia), or a complex pattern of gains and losses of such behavior. The application of phylogenetic information to the comparative study did not refute the previously hypothesized correlation between the presence of a courtship display and the presence of sexual dimorphism in poeciliid fish.

Multiple lineages of the mitochondrial gene NADH dehydrogenase subunit 1 (ND1) in parasitic helminths: implications for molecular evolutionary studies of facultatively anaerobic eukaryotes.

The task of using partial ND1 sequences to infer a phylogeny for species of the genus Paragonimus (Trematoda: Digenea) was complicated by the discovery of at least two ND1 lineages within individual worms. The divergence of the ND1 lineages is shown by phylogenetic analysis not only to predate the divergence of the three Paragonimus species or species groups investigated but also the divergence of some trematode families. Some sequences are clearly pseudogenes as they contain single base deletions and/or premature termination codons. The presence of both pseudogenes and/or mitochondrial heteroplasmy are invoked to explain the presence of multiple and divergent ND1 lineages in these trematodes, which have two distinct cytochemical types of mitochondria. The implications for phylogenetic studies generally and of parasitic helminths specifically, using ND1 sequence data, are discussed. The ability of these organisms to adapt their metabolic processes to the variable availability of oxygen as an electron acceptor are proposed to explain some of the molecular diversity observed in parasitic helminths and possibly also in other anaerobically adapted eukaryotes.

Mitochondrial gene mutations in gestational diabetes mellitus

Mitochondrial DNA mutations have been implicated in many diseases including diabetes mellitus. Although gestational diabetes mellitus (GDM) has been suggested to have genetic determinant and to be etiologically indistinct with non-insulin-dependent diabetes mellitus (NIDDM), its association with mitochondrial gene mutations is still unknown. In this study, 137 patients with GDM and 292 non-diabetic pregnant controls were examined for mitochondrial DNA mutations from the nucleotide 3130–4260 encompassing tRNA-Leu gene and adjacent NADH dehydrogenase 1 gene by polymerase chain reaction, single-stranded conformation polymorphism, restriction fragment length polymorphism and DNA sequencing. One heteroplasmic mutation at the position of 3398 (T-C), which changed a highly conserved methionine to threonine in NADH dehydrogenase subunit 1, was identified in 2.9% GDM patients but not in the controls, indicating its association with GDM ( P=0.01). Two novel mutations, a heteroplasmic C3254A and a homoplasmic A3399T, were also found in GDM subjects, the functional meaning of which merits further investigation. G3316A and T3394C mutations implicated in NIDDM, were seen at higher frequencies in patients with GDM than the controls. Our results suggest that mitochondrial DNA mutations may contribute to the development of GDM in some patients.

Inhibition of Mitochondrial Function in HL60 Cells Is Associated with an Increased Apoptosis and Expression of CD14

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1α,25-dihydroxyvitamin D3 or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.

Consensus multiplex PCR–restriction fragment length polymorphism (RFLP) for rapid detection of plant mitochondrial DNA polymorphism

A fast, simple, and efficient approach, termed consensus multiplex PCR–RFLP, was developed and employed to detect mitochondrial (mt)DNA variation in three orchid species, Spiranthes hongkongensis, S. sinensis, and S. spiralis. Using multiplex PCR, three pairs of consensus mitchondrial primers were added simultaneously into each reaction tube to amplify three nonoverlapping introns located in the NADH dehydrogenase genes. Fragment length differences in the multiplex PCR amplicons were directly detectable between S. spiralis and the other two species. Further restriction analysis of the multiplex PCR amplicons revealed sufficient mtDNA polymorphism, suitable for phylogenetic studies at the interspecific level. This approach is well suited for large‐scale population surveys of mitochondrial genome diversity in plants. Additionally, the maternal mode of inheritance of organelle genomes renders this approach valuable for rapid identification of the origin and specific parentage of hybrid or allopolyploid species.

Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.

The role, if any, that mitochondrial (mt) DNA alterations play in the carcinogenic process remains unclear. To determine whether mtDNA instability occurs in cancers, nine microsatellite sequences in the mtDNA were examined in 45 sporadic colorectal carcinomas. Alteration in a polycytidine (C)n tract within a non-coding displacement-loop (D-loop) region was detected in 20 carcinomas (44%), three of which also exhibited frameshift mutations in a polyadenosine (A)8 or polycytidine (C)6 tract within NADH dehydrogenase (ND) genes. Interestingly, all three mutant genes were predicted to encode truncated ND proteins, which lacked a large portion of the C-terminus. These results suggested that certain repair systems, like the mismatch repair systems in the nuclear genome, are required for mtDNA maintenance and that defects in these systems can lead to target mitochondrial gene mutations in colorectal carcinomas.

Reassessment of Dalbulus Leafhopper (Homoptera: Cicadellidae) Phylogeny Based on Mitochondrial Dna Sequences

Conflicting hypotheses of phylogenetic relationships among leafhopper species in the genus Dalbulus DeLong have resulted from analyses of morphological and allozyme data. To improve the phylogenetic estimate, nucleotide sequence data from 340 bp of the mitochondrial 12S rDNA gene and 942 bp of the NADH dehydrogenase 1 (ND1) gene were compiled for 10 species of Dalbulus , 1 species of its putative sister group, Baldulus , and 2 outgroups. Intraspecific branch lengths were estimated for Dalbulus gelbus DeLong, Dalbulus maidis (DeLong and Wolcott), and Baldulus tripsaci Kramer and Whitcomb. Sequences were aligned based on secondary structure models of the 12S gene and along codons in the ND1 gene. Phylogenetic analyses of the sequence data using maximum parsimony, genetic distance-neighbor joining and maximum likelihood methods yielded nearly identical results. A test for incongruence indicated that the phylogenetic estimate based on sequence data was not significantly different from that derived based on morphological characters. Allozyme data yielded a topology that was significantly incongruent with both morphological and sequence data. Therefore, the morphological and sequence datasets were combined into a single analysis, which yielded a well-resolved phylogenetic estimate for Dalbulus . The resulting tree supports the hypothesis that the ancestral hosts of Dalbulus were gamagrasses ( Tripsacum spp.) and that the acquisition of a preference for maize ( Zea mays ) arose independently in 2 Dalbulus lineages.

Searching for the closest living relative(s) of tetrapods through evolutionary analyses of mitochondrial and nuclear data.

The phylogenetic relationships of the African lungfish (Protopterus dolloi) and the coelacanth (Latimeria chalumnae) with respect to tetrapods were analyzed using complete mitochondrial genome DNA sequences. A lungfish + coelancanth clade was favored by maximum parsimony (although this result is dependent on which transition:transversion weights are applied), and a lungfish + tetrapod clade was supported by neighbor-joining and maximum-likelihood analyses. These two hypotheses received the strongest statistical and bootstrap support to the exclusion of the third alternative, the coelacanth + tetrapod sister group relationship. All mitochondrial protein coding genes combined favor a lungfish + tetrapod grouping. We can confidently reject the hypothesis that the coelacanth is the closest living relative of tetrapods. When the complete mitochondrial sequence data were combined with nuclear 28S rRNA gene data, a lungfish + coelacanth clade was supported by maximum parsimony and maximum likelihood, but a lungfish + tetrapod clade was favored by neighbor-joining. The seeming conflicting results based on different data sets and phylogenetic methods were typically not statistically strongly supported based on Kishino-Hasegawa and Templeton tests, although they were often supported by strong bootstrap values. Differences in rate of evolution of the different mitochondrial genes (slowly evolving genes such as the cytochrome oxidase and tRNA genes favored a lungfish + coelacanth clade, whereas genes of relatively faster substitution rate, such as several NADH dehydrogenase genes, supported a lungfish + tetrapod grouping), as well as the rapid radiation of the lineages back in the Devonian, rather than base compositional biases among taxa seem to be directly responsible for the remaining uncertainty in accepting one of the two alternate hypotheses.

Three mitochondrial DNA restriction polymorphisms in elite endurance athletes and sedentary controls.

This study examined the associations between elite endurance athlete (EEA) status and three mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) in the subunit 5 of the NADH dehydrogenase (MTND5) locus and one in the D-loop region. A group of 125 Caucasian male EEA well endowed with the phenotypic expression of VO2max (78.9 +/- 3.8 mL x kg(-1) x min(-1), mean +/- SD) and 65 sedentary controls (SCON: VO2max = 39.8 +/- 8.2 mL x kg(-1) x min(-1)) participated in the study. VO2max was determined during an incremental exercise test on a cycle ergometer or a motor-driven treadmill. mtDNA was extracted from white blood cells or lymphoblastoid cell lines and specific regions were amplified by the polymerase chain reaction. The Pearson Chi-square statistic test and Fisher exact test revealed no significant association (P > 0.05) between any of the three mtDNA RFLPs and EEA status. The MTND5-BamHI RFLP at bp 13,470 (morph 3) was found in 12.8% of the EEA and 12.3% of the SCON (chi2 = 0.009, P = 0.92). The prevalence of the MTND5-Ncil RFLP at bp 13,364 (morph 2) was 12.9% and 14% for the EEA and SCON, respectively (chi2 = 0.043, P = 0.83). The D-loop-KpnI RFLP at bp 16,133 (morph 1) was found in 5.8% of the EEA and in 1.6% of the SCON (Fisher exact test = 1.80, P = 0.18). The MTND5-HincII RFLP at bp 12,406 (morph 1) was not present in this study sample. These results indicate no evidence for a difference in the frequency of two polymorphic restriction sites in the subunit 5 of the NADH dehydrogenase gene of mtDNA and one in the D-loop region between elite endurance athletes and sedentary controls.

A new point mutation (3426, A to G) in mitochondrial NADH dehydrogenase gene in Korean diabetic patients which mimics 3243 mutation by restriction fragment length polymorphism pattern.

Mitochondrial tRNA(Leu)(UUR) gene mutation is one of the candidates in the pathogenesis of NIDDM. Especially the 3243 (A-->G) mutation is associated with the maternally-inherited diabetes and deafness. To evaluate the prevalence and characteristics of the 3243 point mutation in Koreans, we screened 433 Korean diabetic patients (220 men and 213 women). Genomic DNA was extracted from peripheral white blood cells and PCR was carried out with mitochondrial DNA primers (3130-3149, 3558-3539) encompassing the 3243 position. After digestion with Apa-1, five subjects showed polymorphism suggesting 3243 point mutation but when we directly sequenced the amplified DNA with an automatic sequencer, only 2 of the 5 patients were shown to have 3243 (A-->G) mutation and the other 3 subjects had 3426 (A-->G) mutation rather than 3243 mutation. Two diabetic patients with 3243 mutation were lean (BMI = 14.4, 17.0 kg/m2), had relatively lower fasting C-peptide concentrations (0.9 ng/ml each), and required insulin for management. In contrast, those with 3426 point mutation were not lean (BMI = 22.6-28.0 kg/m2), had relatively higher C-peptide levels (3.9-5.4 ng/ml), and could be managed with oral hypoglycemic agents. None of the 5 patients had deafness. In conclusion, the prevalence of 3243 point mutation in Korean diabetic patients was approximately 0.5% and we found a new mutation mimicking 3243 mutation by PCR-RFLP (restriction fragment length polymorphism) pattern. We suggest that sequencing of the PCR product or designing smaller PCR fragment size to enhance the specificity may help to identify the exact location of the point mutation.