Diversity of Telomere Palindromic Sequences and Replication Genes among Streptomyces Linear Plasmids

Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.     

NAR Breakthrough Article: Telomere-associated proteins add deoxynucleotides to terminal proteins during replication of the telomeres of linear chromosomes and plasmids in Streptomyces

Typical telomeres of linear chromosomes and plasmids of soil bacteria Streptomyces consist of tightly packed palindromic sequences with a terminal protein (‘TP’) covalently attached to the 5′ end of the DNA. Replication of these linear replicons is initiated internally and proceeds bidirectionally toward the telomeres, which leaves single-strand overhangs at the 3′ ends. These overhangs are filled by DNA synthesis using the TPs as the primers (‘end patching’). The gene encoding for typical TP, tpg, forms an operon with tap, encoding an essential telomere-associated protein, which binds TP and the secondary structures formed by the 3′ overhangs. Previously one of the two translesion synthesis DNA polymerases, DinB1 or DinB2, was proposed to catalyze the protein-primed synthesis. However, using an in vitro end-patching system, we discovered that Tpg and Tap alone could carry out the protein-primed synthesis to a length of 13 nt. Similarly, an ‘atypical’ terminal protein, Tpc, and its cognate telomere-associated protein, Tac, of SCP1 plasmid, were sufficient to achieve protein-primed synthesis in the absence of additional polymerase. These results indicate that these two telomere-associated proteins possess polymerase activities alone or in complex with the cognate TPs.     

Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5' ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3' ends during replication. Most ('archetypal') Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA.     

How Many Processed Pseudogenes Are Accumulated in a Gene Family?

A simple kinetic model is developed that describes the accumulation of processed pseudogenes in a functional gene family. Insertion of new pseudogenes occurs at rate ν per gene and is countered by spontaneous deletion (at rate δ per DNA segment) of segments containing processed pseudogenes. If there are k functional genes in a gene family, the equilibrium number of processed pseudogenes is k(ν/δ), and the percentage of functional genes in the gene family at equilibrium is 1/[1 + (ν/δ)]. ν/δ values estimated for five gene families ranged from 1.7 to 15. This fairly narrow range suggests that the rates of formation and deletion of processed pseudogenes may be positively correlated for these families. If δ is sufficiently large relative to the per nucleotide mutation rate µ (δ > 20µ), processed pseudogenes will show high homology with each other, even in the absence of gene conversion between pseudogenes. We argue that formation of processed pseudogenes may share common pathways with transposable elements and retroviruses, creating the potential for correlated responses in the evolution of processed pseudogenes due to direct selection for control of transposable elements and/or retroviruses. Finally, we discuss the nature of the selective forces that may act directly or indirectly to influence the evolution of processed pseudogenes.

Anything produced by evolution is bound to be a bit of a mess—S. Brenner

     

Association of the I264T Variant in the Sulfide Quinone Reductase-Like (SQRDL) Gene with Osteoporosis in Korean Postmenopausal Women

To identify novel susceptibility variants for osteoporosis in Korean postmenopausal women, we performed a genome-wide association analysis of 1180 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 405 individuals with osteoporosis and 722 normal controls of the Korean Association Resource cohort. A logistic regression analysis revealed 72 nsSNPs that showed a significant association with osteoporosis (p<0.05). The top 10 nsSNPs showing the lowest p-values (p = 5.2×10^-4–8.5×10^-3) were further studied to investigate their effects at the protein level. Based on the results of an in silico prediction of the protein’s functional effect based on amino acid alterations and a sequence conservation evaluation of the amino acid residues at the positions of the nsSNPs among orthologues, we selected one nsSNP in the SQRDL gene (rs1044032, SQRDL I264T) as a meaningful genetic variant associated with postmenopausal osteoporosis. To assess whether the SQRDL I264T variant played a functional role in the pathogenesis of osteoporosis, we examined the in vitro effect of the nsSNP on bone remodeling. Overexpression of the SQRDL I264T variant in the preosteoblast MC3T3-E1 cells significantly increased alkaline phosphatase activity, mineralization, and the mRNA expression of osteoblastogenesis markers, Runx2, Sp7, and Bglap genes, whereas the SQRDL wild type had no effect or a negative effect on osteoblast differentiation. Overexpression of the SQRDL I264T variant did not affect osteoclast differentiation of the primary-cultured monocytes. The known effects of hydrogen sulfide (H₂S) on bone remodeling may explain the findings of the current study, which demonstrated the functional role of the H₂S-catalyzing enzyme SQRDL I264T variant in osteoblast differentiation. In conclusion, the results of the statistical and experimental analyses indicate that the SQRDL I264T nsSNP may be a significant susceptibility variant for osteoporosis in Korean postmenopausal women that is involved in osteoblast differentiation.     

Biallelic SUN5 Mutations Cause Autosomal-Recessive Acephalic Spermatozoa Syndrome

Acephalic spermatozoa syndrome is a rare and severe form of teratozoospermia characterized by a predominance of headless spermatozoa in the ejaculate. Family clustering and consanguinity suggest a genetic origin; however, causative mutations have yet to be identified. We performed whole-exome sequencing in two unrelated infertile men and subsequent variant filtering identified one homozygous (c.824C>T [p.Thr275Met]) and one compound heterozygous (c.1006C>T [p.Arg356Cys] and c.485T>A [p.Met162Lys]) SUN5 (also named TSARG4) variants. Sanger sequencing of SUN5 in 15 additional unrelated infertile men revealed four compound heterozygous (c.381delA [p.Val128Serfs^∗7] and c.824C>T [p.Thr275Met]; c.381delA [p.Val128Serfs^∗7] and c.781G>A [p.Val261Met]; c.216G>A [p.Trp72^∗] and c.1043A>T [p.Asn348Ile]; c.425+1G>A/c.1043A>T [p.Asn348Ile]) and two homozygous (c.851C>G [p.Ser284^∗]; c.350G>A [p.Gly114Arg]) variants in six individuals. These 10 SUN5 variants were found in 8 of 17 unrelated men, explaining the genetic defect in 47.06% of the affected individuals in our cohort. These variants were absent in 100 fertile population-matched control individuals. SUN5 variants lead to absent, significantly reduced, or truncated SUN5, and certain variants altered SUN5 distribution in the head-tail junction of the sperm. In summary, these results demonstrate that biallelic SUN5 mutations cause male infertility due to autosomal-recessive acephalic spermatozoa syndrome.     

Antigen Diversity in the Parasitic Bacterium Anaplasma phagocytophilum Arises from Selectively-Represented,Spatially Clustered Functional Pseudogenes

Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and other animals, and is an obligate intracellular parasite. Throughout the course of infection, hosts acquire temporary resistance to granulocytic anaplasmosis as they develop immunity specific for the major antigen, major surface protein 2 (Msp2). However, the bacterium then utilizes a novel recombination mechanism shuffling functional pseudogenes sequentially into an expression cassette with conserved 5′ and 3′ ends, bypassing host immunity. Approximately 100 pseudogenes are present in the only fully sequenced human-origin HZ genome, representing the possibility for almost unlimited antigenic diversity. In the present study, we identified a select group of 20% of the A. phagocytophilum HZ msp2 pseudogenes that have matched preferentially to human, canine, and equine expression cassettes. Pseudogenes cluster predominantly in one spatial run limited to a single genomic island in less than 50% of the genome but phylogenetically related pseudogenes are neither necessarily located in close proximity on the genome nor share similar percent identity with expression cassettes. Pseudogenes near the expression cassette (and the origin) are more likely to be expressed than those farther away. Taken together, these findings suggest that there may be natural selection pressure to retain pseudogenes in one cluster near the putative origin of replication, even though global recombination shuffles pseudogenes around the genome, separating pseudogenes that share genetic origins as well as those with similar identities.     

Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present in Mycobacterium fortuitum and Mycobacterium tuberculosis

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N′-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.     

A Functional Polymorphism C-509T in TGFβ-1 Promoter Contributes to Susceptibility and Prognosis of Lone Atrial Fibrillation in Chinese Population

Transforming growth factor-β1 (TGF-β1) is an important mediator of atrial fibrosis and atrial fibrillation (AF). But the involved genetic mechanism is unknown. Herein, the TGF-β1 C-509T polymorphism (rs1800469) was genotyped in a case-control study of 840 patients and 845 controls in Chinese population to explore the association between the polymorphism and susceptibility and prognosis of lone AF. As a result, the CT and/or TT genotypes had an increased lone AF risk [adjusted odds ratio (OR) = 1.50 for CT, OR = 3.72 for TT, and OR = 2.15 for CT/TT], compared with the TGF-β1CC genotype. Moreover, patients carrying CT/TT genotypes showed a higher possibility of AF recurrence after catheter ablation, compared with patients carrying CC genotype. In a genotype-phenotype correlation analysis using 24 normal left atrial appendage samples, increasing gradients of atrial TGF-β1 expression levels positively correlated with atrial collagen volume fraction were identified in samples with CC, CT and TT genotypes. The in vitro luciferase assays also showed a higher luciferase activity of the -509T allele than that of the -509C allele. In conclusion, the TGF-β1 C-509T polymorphism is involved in the etiology of lone AF and thus may be a marker for genetic susceptibility to lone AF and predicting prognosis after catheter ablation in Chinese populations. Therefore, we provide new information about treatment strategies and our understanding of TGF-β1 in AF.     

Recognizing the pseudogenes in bacterial genomes

Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1–8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as ‘hypothetical’ or ‘unknown’; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes.     

Gene Replacement Analysis of the Streptomyces virginiae barA Gene Encoding the Butyrolactone Autoregulator Receptor Reveals that BarA Acts as a Repressor in Virginiamycin Biosynthesis

Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of Streptomyces species, act as a primary signal in Streptomyces virginiae to trigger virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesis, two barA mutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type S. virginiae on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in S. virginiae. In contrast, virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of virginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strain NH2, an internal EcoNI-SmaI fragment of barA was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strain NH2 and in a plasmid-integrated strain containing both intact and mutated barA genes, virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.     

Importance of the conserved nucleotides around the tRNA-like structure of Escherichia coli transfer-messenger RNA for protein tagging

A bacterial RNA functioning as both tRNA and mRNA, transfer-messenger RNA (tmRNA) rescues stalled ribosomes and clears the cell of incomplete polypeptides. For function, Escherichia coli tmRNA requires an elaborate interplay between a tRNA-like structure and an internal mRNA domain that are connected by a 295 nt long compact secondary structure. The tRNA-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserved among the known 140 tmRNA sequences. All these residues were mutated to define their putative role(s) in trans-translation. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all variants. As anticipated from the low sequence conservation, mutating positions 8–12 and position 15 affects neither aminoacylation nor protein tagging. Mutating a set of two conserved positions 13 and 14 abolishes both functions. Probing the solution conformation indicates that this defective mutant adopts an alternate conformation of its acceptor stem that is no more aminoacylatable, and thus inactive in protein tagging. Selected point mutations at the conserved nucleotide stretches 16–20 and 333–335 seriously impair protein tagging with only minor changes in their solution conformations and aminoacylation. Point mutations at conserved positions 19 and 334 abolish trans-translation and 70S ribosome binding, although retaining nearly normal aminoacylation capacities. Two proteins that are known to interact with tmRNA were purified, and their interactions with the defective RNA variants were examined in vitro. Based on phylogenetic and functional data, an additional structural motif consisting of a quartet composed of non-Watson–Crick base pairs 5′-YGAC-3′:5′-GGAC-3′ involving some of the conserved nucleotides next to the tRNA-like portion is proposed. Overall, the highly conserved nucleotides around the tRNA-like portion are maintained for both structural and functional requirements during evolution.     

Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut

In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae'' and ‘Candidatus Nucleococcus kirbyi''. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer.     

Fine Mapping of a GWAS-Derived Obesity Candidate Region on Chromosome 16p11.2

IntroductionLarge-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r²>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls.MethodsThe coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants.ResultsExcept for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function.ConclusionVariants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted.     

Interspecific Transfer of Streptomyces Giant Linear Plasmids in Sterile Amended Soil Microcosms

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30°C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 × 10⁻⁴ and 3.6 × 10⁻⁵ CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.     

Mitochondrial DNA associations with East Asian metabolic syndrome

Mitochondrial dysfunction has repeatedly been reported associated with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS), as have mitochondrial DNA (mtDNA) tRNA and duplication mutations and mtDNA haplogroup lineages. We identified 19 Taiwanese T2DM and MS pedigrees from Taiwan, with putative matrilineal transmission, one of which harbored the pathogenic mtDNA tRNA^Leu(UUR) nucleotide (nt) 3243A>G mutation on the N9a3 haplogroup background. We then recruited three independent Taiwanese cohorts, two from Taipei (N?=?498, mean age 52 and N?=?1002, mean age 44) and one from a non-urban environment (N?=?501, mean age 57). All three cohorts were assessed for an array of metabolic parameters, their mtDNA haplogroups determined, and the haplogroups correlated with T2DM/MS phenotypes. Logistic regression analysis revealed that mtDNA haplogroups D5, F4, and N9a conferred T2DM protection, while haplogroups F4 and N9a were risk factors for hypertension (HTN), and F4 was a risk factor for obesity (OB). Additionally, the 5263C>T (ND2 A165V) variant commonly associated with F4 was associated with hypertension (HTN). Cybrids were prepared with macro-haplogroup N (defined by variants m.ND3 10398A (114T) and m.ATP6 8701A (59T)) haplogroups B4 and F1 mtDNAs and from macro-haplogroup M (variants m.ND3 10398G (114A) and m.ATP6 8701G (59A)) haplogroup M9 mtDNAs. Additionally, haplogroup B4 and F1 cybrids were prepared with and without the mtDNA variant in ND1 3394T>C (Y30H) reported to be associated with T2DM. Assay of mitochondria complex I in these cybrids revealed that macro-haplogroup N cybrids had lower activity than M cybrids, that haplogroup F cybrids had lower activity than B4 cybrids, and that the ND1 3394T>C (Y30H) variant reduced complex I on both the B4 and F1 background but with very different cumulative effects. These data support the hypothesis that functional mtDNA variants may contribute to the risk of developing T2DM and MS.