Truncated Gene Product Research Articles

Interactome Analysis Identifies MSMEI_3879 as a Substrate of Mycolicibacterium smegmatis ClpC1.

The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coimmunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfoldase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.

Novel WNT10A variant in a Japanese case of nonsyndromic oligodontia

Congenital tooth agenesis is one of the most common anomalies in humans. Many genetic factors are involved in tooth development, including MSX1, PAX9, WNT10A, and LRP6. Thus, mutations in these genes can cause congenital tooth agenesis in humans. In this study, we identified a novel nonsense WNT10A variant, NM_025216.3(WNT10A_v001):c.1090A > T, which produces a C-terminal truncated gene product, p.(Lys364*), in a sporadic form of congenital tooth agenesis. The variant was not found in the healthy parents and thus was considered to cause congenital tooth agenesis in the case.

An IL17RA frameshift variant in a Holstein cattle family with psoriasis-like skin alterations and immunodeficiency

BackgroundSkin lesions and dermatoses in cattle are often associated with infections due to bacteria, fungi or environmental risk factors. Dermatoses with genetic etiology have been described in cattle. Among these rare disorders, there are primary congenital dermatoses that are associated with inherited nutritional deficiencies, such as bovine hereditary zinc deficiency or zinc deficiency-like syndrome. This study presents three cases of Holstein cattle with congenital skin lesions observed on a single farm that resemble zinc deficiency-like syndrome. Close clinical and pathological examinations took place in two cases. Pedigree analysis indicated autosomal recessive inheritance and whole-genome sequencing of both affected calves was performed.ResultsThe two calves showed retarded growth and suffered from severe ulcerative dermatitis with hyperkeratosis, alopecia furunculosis and subcutaneous abscess formation. Blood analysis showed correspondent leukocytosis with neutrophilia whereas minerals, macro- and micronutrients were within the reference ranges. Variant calling and filtering against the 1000 Bull Genomes variant catalogue resulted in the detection of a single homozygous protein-changing variant exclusively present in both sequenced genomes. This single-nucleotide deletion in exon 3 of IL17RA on bovine chromosome 5 was predicted to have a deleterious impact on the encoded protein due to a frameshift leading to a truncated gene product. Genotyping of the affected cattle family confirmed recessive inheritance.ConclusionsA loss-of-function mutation of the IL17RA transmembrane protein could be identified as most likely pathogenic variant for the psoriasis-like skin alterations observed in the two affected Holstein calves. In man, rare recessive diseases associated with IL17RA include immunodeficiency 51 and chronic mucocutaneous candidiasis. This supports the observed immunodeficiency of the presented cases. This study reports the first naturally occurring IL17RA-associated animal model.

Clinical manifestations and neurofibromatosis type 1 gene mutations of 25 patients with neurofibromatosis type 1 from 10 Chinese pedigrees

This study enrolled 25 patients with neurofibromatosis type 1 (NF1) from 10 Chinese pedigrees. Sanger sequencing analysis and multiplex ligation-dependent probe amplification analysis were used to detect mutations and large fragment losses of the NF1 gene. This study identified 10 NF1 mutations, which comprised six novel and four recurrent mutations. Majority of the mutations can lead to termination codon production, which results in the synthesis of the truncated gene product neurofibromin.

Parallel Evolution in Streptococcus pneumoniae Biofilms.

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.

Abstract A48: Role of premature mRNA cleavage and alternative polyadenylation in driving breast cancer

Abstract There is increasing appreciation that alterations to epigenetic mechanisms contribute directly to breast cancer development. In cancer cells, one such mechanism, called alternative polyadenylation (APA), has been observed to cause a general shortening of mRNA through the usage and premature cleavage of upstream poly(A) signals (PAS). However, while it is hypothesized that APA may be linked to cancer pathogenesis, specific examples of causality between APA and cancer development have remained elusive. We report the identification of a truncated tumor suppressor, MAGI3, at both the mRNA and protein levels from the MDA-MB-231 breast cancer cell line. Using 3' rapid amplification of cDNA ends (RACE), we have determined that the truncated transcript arises due to premature cleavage and APA at a cryptic intronic PAS. We have previously demonstrated genetically and biochemically that MAGI3 functions as a mammary tumor suppressor by antagonizing the oncoprotein and Hippo pathway effector, YAP, via a physical sequestration mechanism. Importantly, here we find that the truncated gene product encoded by this aberrant MAGI3 transcript acts as a dominant-negative protein, preventing MAGI3-YAP physical association and thereby causing YAP nuclear activation and malignant transformation of cells. Since “epi-mutations” such as these play an important role in breast cancer development, yet cannot be detected by microarray or genome sequencing methodologies, we are currently undertaking analysis of large-scale, breast cancer RNA-Seq data to identify tumors that express the truncated driver mRNA and to determine the frequency of APA products in breast cancer. Knowledge of the clinically relevant proteins produced by APA that are unique to cancer cells may lead to new therapeutic opportunities for breast cancer. Citation Format: Thomas K. Ni, Charlotte Kuperwasser. Role of premature mRNA cleavage and alternative polyadenylation in driving breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A48.

Congenital erythropoietic porphyria: Insight into the molecular basis of the disease

Congenital Erythropoietic Porphyria (CEP) is a rare inborn error of metabolism charectorised by a deficiency of UROS III enzyme, an important enzyme in the heme biosythetic pathway. It is an autosomal recessive disease and only around 200 cases have been charectorised so far. The clinical presentation, genetic profile and the genotype-phenotype correlation of this disease is complex, and needs to understand completely for proper diagnose of the case and instituting specific therapy. Mutation analysis in the cases of CEP have revealed all types of mutations in the gene including additions, substitutions, insertion and deletions in the gene. Mutations have also been charectorised in the intron-exon junction as well as in the intron regions resulting in truncated gene product and hence a defective enzyme. Mutations in the promoter region too have been charectorised that affect the rate of gene expression. Trans-acting mutations resulting in a phenotype characteristic of CEP have also been recently charectorised. Various study in the molecular basis of the disease have demonstrated that the mutations result in the production of an unstable protein that gets destroyed rapidly resulting a critically low level of the enzyme in the biosystem. Targetting these factors which regulate the rapid degradation of the deformed proteins have been found to improve the clinical profile of the patient and offers potential for future therapy.

Rett syndrome like phenotypes in the R255X Mecp2 mutant mouse are rescued by MECP2 transgene.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that is usually caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). Four of the eight common disease causing mutations in MECP2 are nonsense mutations and are responsible for over 35% of all cases of RTT. A strategy to overcome disease-causing nonsense mutations is treatment with nonsense mutation suppressing drugs that allow expression of full-length proteins from mutated genes with premature in-frame stop codons. To determine if this strategy is useful in RTT, we characterized a new mouse model containing a knock-in nonsense mutation (p.R255X) in the Mecp2 locus (Mecp2(R255X)). To determine whether the truncated gene product acts as a dominant negative allele and if RTT-like phenotypes could be rescued by expression of wild-type protein, we genetically introduced an extra copy of MECP2 via an MECP2 transgene. The addition of MECP2 transgene to Mecp2(R255X) mice abolished the phenotypic abnormalities and resulted in near complete rescue. Expression of MECP2 transgene Mecp2(R255X) allele also rescued mTORC1 signaling abnormalities discovered in mice with loss of function and overexpression of Mecp2. Finally, we treated Mecp2(R255X) embryonic fibroblasts with the nonsense mutation suppressing drug gentamicin and we were able to induce expression of full-length MeCP2 from the mutant p.R255X allele. These data provide proof of concept that the p.R255X mutation of MECP2 is amenable to the nonsense suppression therapeutic strategy and provide guidelines for the extent of rescue that can be expected by re-expressing MeCP2 protein.

Editing CCR5: a novel approach to HIV gene therapy.

Acquired immunodeficiency syndrome (AIDS) is a life-threatening disorder caused by infection of individuals with the human immunodeficiency virus (HIV). Entry of HIV-1 into target cells depends on the presence of two surface proteins on the cell membrane: CD4, which serves as the main receptor, and either CCR5 or CXCR4 as a co-receptor. A limited number of people harbor a genomic 32-bp deletion in the CCR5 gene (CCR5∆32), leading to expression of a truncated gene product that provides resistance to HIV-1 infection in individuals homozygous for this mutation. Moreover, allogeneic hematopoietic stem cell (HSC) transplantation with CCR5∆32 donor cells seems to confer HIV-1 resistance to the recipient as well. However, since Δ32 donors are scarce and allogeneic HSC transplantation is not exempt from risks, the development of gene editing tools to knockout CCR5 in the genome of autologous cells is highly warranted. Targeted gene editing can be accomplished with designer nucleases, which essentially are engineered restriction enzymes that can be designed to cleave DNA at specific sites. During repair of these breaks, the cellular repair pathway often introduces small mutations at the break site, which makes it possible to disrupt the ability of the targeted locus to express a functional protein, in this case CCR5. Here, we review the current promise and limitations of CCR5 gene editing with engineered nucleases, including factors affecting the efficiency of gene disruption and potential off-target effects.

Copy Number Variants in the Kallikrein Gene Cluster

The kallikrein gene family (KLK1-KLK15) is the largest contiguous group of protease genes within the human genome and is associated with both risk and outcome of cancer and other diseases. We searched for copy number variants in all KLK genes using quantitative PCR analysis and analysis of inheritance patterns of single nucleotide polymorphisms. Two deletions were identified: one 2235-bp deletion in KLK9 present in 1.2% of alleles, and one 3394-bp deletion in KLK15 present in 4.0% of alleles. Each deletion eliminated one complete exon and created out-of-frame coding that eliminated the catalytic triad of the resulting truncated gene product, which therefore likely is a non-functional protein. Deletion breakpoints identified by DNA sequencing located the KLK9 deletion breakpoint to a long interspersed element (LINE) repeated sequence, while the deletion in KLK15 is located in a single copy sequence. To search for an association between each deletion and risk of prostate cancer (PC), we analyzed a cohort of 667 biopsied men (266 PC cases and 401 men with no evidence of PC at biopsy) using short deletion-specific PCR assays. There was no association between evidence of PC in this cohort and the presence of either gene deletion. Haplotyping revealed a single origin of each deletion, with most recent common ancestor estimates of 3000-8000 and 6000-14 000 years for the deletions in KLK9 and KLK15, respectively. The presence of the deletions on the same haplotypes in 1000 Genomes data of both European and African populations indicate an early origin of both deletions. The old age in combination with homozygous presence of loss-of-function variants suggests that some kallikrein-related peptidases have non-essential functions.

Detection of a novel frameshift mutation and regions with homozygosis within ARHGEF28 gene in familial amyotrophic lateral sclerosis

Rho guanine nucleotide exchange factor (RGNEF) is a novel NFL mRNA destabilizing factor that forms neuronal cytoplasmic inclusions in spinal motor neurons in both sporadic (SALS) and familial (FALS) ALS patients. Given the observation of genetic mutations in a number of mRNA binding proteins associated with ALS, including TDP-43, FUS/TLS and mtSOD1, we analysed the ARHGEF28 gene (approx. 316 kb) that encodes for RGNEF in FALS cases to determine if mutations were present. We performed genomic sequencing, copy number variation analysis using TaqMan real-time PCR and spinal motor neuron immunohistochemistry using a novel RGNEF antibody. In this limited sample of FALS cases (n=7) we identified a heterozygous mutation that is predicted to generate a premature truncated gene product. We also observed extensive regions of homozygosity in the ARHGEF28 gene in two FALS patients. In conclusion, our findings of genetic alterations in the ARHGEF28 gene in cases of FALS suggest that a more comprehensive genetic analysis would be warranted.

Multiple and independent origins of short seeded alleles of GS3 in rice.

GRAIN SIZE 3 (GS3) is a cloned gene that is related to seed length. Here we report the discovery of new deletion alleles at the GS3 locus, each of which confer short seed. We selected ten short seeded cultivars from a collection of 282 diverse cultivars. Sequence analysis across the GS3 gene in these ten cultivars identified three novel alleles and a known allele that contain several independent deletion(s) in the fifth exon of GS. These independent deletion variants each resulted in a frameshift mutation that caused a premature stop codon, and they were functionally similar to one another. Each coded for a truncated gene product that behaved as an incomplete dominant allele and conferred a short seeded phenotype. Haplotype analysis of these sequence variants indicated that two of the variants were of japonica origin, and two were from indica. Transformation experiments demonstrated that one of the deletion alleles of GS3 decrease the cell number in the upper epidermis of the glume, resulting in a significant reduction in seed length. The multiple and independent origins of these short seeded alleles indicate that farmers and early breeders imposed artificial selection favoring short seeds.

The cell wall binding domain of Listeria bacteriophage endolysin PlyP35 recognizes terminal GlcNAc residues in cell wall teichoic acid

The cell wall binding domains (CBD) of bacteriophage endolysins target the enzymes to their substrate in the bacterial peptidoglycan with extraordinary specificity. Despite strong interest in these enzymes as novel antimicrobials, little is known regarding their interaction with the bacterial wall and their binding ligands. We investigated the interaction of Listeria phage endolysin PlyP35 with carbohydrate residues present in the teichoic acid polymers on the peptidoglycan. Biochemical and genetic analyses revealed that CBD of PlyP35 specifically recognizes the N-acetylglucosamine (GlcNAc) residue at position C4 of the polyribitol-phosphate subunits. Binding of CBDP35 could be prevented by removal of wall teichoic acid (WTA) polymers from cell walls, and inhibited by addition of purified WTAs or acetylated saccharides. We show that Listeria monocytogenes genes lmo2549 and lmo2550 are required for decoration of WTAs with GlcNAc. Inactivation of either gene resulted in a lack of GlcNAc glycosylation, and the mutants failed to bind CBDP35. We also report that the GlcNAc-deficient phenotype of L. monocytogenes strain WSLC 1442 is due to a small deletion in lmo2550, resulting in synthesis of a truncated gene product responsible for the glycosylation defect. Complementation with lmo2550 completely restored display of characteristic serovar 1/2 specific WTA and the wild-type phenotype.

CMV promoter is inadequate for expression of mutant human RyR2 in transgenic rabbits

Introduction Fundamental differences in Ca 2+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome. Methods The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2. Results Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct > 30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3′ end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart. Discussion Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits.

Novel GH10 Xylanase, with a Fibronectin Type 3 Domain, from Cellulosimicrobium sp. Strain HY-13, a Bacterium in the Gut of Eisenia fetida

The gene encoding a novel modular xylanase from Cellulosimicrobium sp. strain HY-13 was identified and expressed in Escherichia coli, and its truncated gene product was characterized. The enzyme consisted of three distinct functional domains, an N-terminal catalytic GH10 domain, a fibronectin type 3 domain, and C-terminal carbohydrate-binding module 2.

Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei : a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30

We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIalpha) of the industrially important fungus Trichoderma reesei. This subunit is the catalytic part of the glucosidase II heterodimeric enzyme involved in the structural modification within the endoplasmic reticulum (ER) of N-linked oligosaccharides present on glycoproteins. The gene encoding GIIalpha (gls2alpha) in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product. Based on the peculiar monoglucosylated N-glycan pattern on proteins produced by the strain, we concluded that the truncated protein can still hydrolyze the first alpha-1,3-linked glucose residue but not the innermost alpha-1,3-linked glucose residue from the Glc2Man9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2alpha gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIalpha protein. Since glucosidase II is also involved in the ER quality control of nascent polypeptide chains, its transcriptional regulation was studied in a strain producing recombinant tissue plasminogen activator (tPA) and in cultures treated with the stress agents dithiothreitol (DTT) and brefeldin A (BFA), which are known to block protein transport and to induce the unfolded protein response. While the mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement was observed after DTT addition.

Mutational analysis of theTCOF1gene in 11 Japanese patients with Treacher Collins Syndrome and mechanism of mutagenesis

Treacher Collins Syndrome (TCS) (OMIM 154500) is a congenital, craniofacial disorder inherited as an autosomal dominant trait. The responsible gene for TCS, TCOF1, was mapped to 5q32-33.1 and identified in 1996. Since then, TCOF1 mutations in patients with TCS have been reported from Europe, North and South America, however, no TCS cases from an Asian country have been molecularly characterized. Here we report mutational analysis for 11 Japanese patients with TCS for the first time, and have identified TCOF1 mutations in 9 of them. The mutations detected were various, but most likely all the mutations are predicted to result in a truncated gene product, known as treacle. One mutation frequently reported was included in our cases, but no missense mutations were detected. These findings are similar to those for the previous studies for TCS in other races. We have speculated about the molecular mechanisms of the mutations in most cases. Collectively, we have defined some of the characteristic molecular features commonly observed in TCS patients, irrespective of racial difference.

Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans.

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.

Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain.

Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the alpha-ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimer's disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH-SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD-deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse-label experiment revealed that the loss of the subunits is a post-translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes.

Genetic susceptibility in type 1 diabetes and its associated autoimmune disorders.

Animal models of type 1 diabetes There are several animal models of type 1A diabetes (immune mediated). The first inbred animal model evolved from the discovery of diabetic animals in an outbred colony of Wistar rats maintained at the Bio Breeding Research Laboratories in Ottawa, Canada. The Bio Breeding (BB) rat develops spontaneous immune-mediated diabetes with at least four loci contributing to disease: the major histocompatibility complex (MHC, iddm2) on rat chromosome 20; the lymphopenic locus iddm1 on rat chromosome 4 [1], iddm3 on rat chromosome 2 [2], and a fourth locus on rat chromosome 15. The lymphopenic phenotype (iddm1 or lyp) locus has been mapped to a syntenic region of rat chromosome 4 [1,3]. Mutation at this locus in a homozygous state produces a severe T cell lymphopenia. Hornum and co-workers have mapped the gene to a 0.3-cM region between markers D4Got59 and Abp1 and constructed a 550-kb P1-derived artificial chromosome (PAC) contig covering the region [4]. They found a frameshift mutation in Immune-Associated Nucleotide4 (Ian4) gene which encodes a GTP-binding protein localized in the outer mitochondrial membrane [5]. This mutation results in a truncated gene product that lacks twothirds of the total protein. They hypothesized that Ian 4 is involved in the regulation of T-cell apoptosis, making it an excellent candidate for iddm1. The Ian4 gene product is anchored to the outer mitochondrial membrane, and this organelle is an important site for the regulation of cell death [6]. The same mutation was found by MacMurray and co-workers but they termed the gene Ian5 [7]. Some Ian genes are expressed in mature T cells and switched on during thymic T-cell development [8–10]. The Komeda diabetes-prone (KDP) rat [11], a diabetesprone substrain of the Long-Evans Tokushima lean (LETL) rat is characterized by autoimmune destruction of pancreatic β cells [12], rapid onset of overt diabetes with no sex difference, and no significant T cell lymphopenia. The gene Cblb (Casitus B-lineage lymphoma b, also known as Cas-Br-M murine ectropic retroviral transforming sequence b) was a potential candidate given its influence on immune function. Yokoi et al. [13] confirmed a C → T homozygous nonsense mutation (arginine to a stop at codon 455, Arg455X) that removes 484 amino acids, including the proline-rich region and leucine zipper domain. The mutation is specific to the KDP rat and its original LETL strain. Approximately 80% of the Iddm/kdp1 homozygous mutant animals develop diabetes with severe insulitis, and most of the remainder showed mild to moderate insulitis. Neither heterozygous nor wildtype animals developed diabetes. They demonstrated that Cblb is a important regulator of autoimmunity and Cblb is a major susceptibility gene for rat type 1 diabetes [14]. The non-obese diabetic mouse is the best studied spontaneous animal model of type 1 diabetes. Diabetes susceptibility in the NOD is polygenic, with a major influence of genes within the MHC. Linkage analyses have identified at least 19 susceptibility loci (Idd1-Idd19) that contribute to disease pathogenesis [15,16]. Lymphocytes mediate the specific destruction of insulin-producing β cells. These loci can either confer susceptibility or resistance to expression of insulin autoantibodies, insulitis and or disease development. Idd1 has been mapped to the MHC locus on mouse chromosome 17 [17,18]. Idd1 mapped to class II I-A and I-E regions as a gene complex [19], but the incidence of the disease is strongly affected by a gene or genes outside of this segment (Idd16). Variants of the beta-2 microglobulin gene is one of the first non-MHC genes to by clearly identified. Though neither the insulin 2 gene on chromosome 7 nor the insulin 1 gene on chromsome 19 show linkage to diabetes, in crosses with normal mouse strains, knockouts of either of these genes greatly influences the development of diabetes. Several new mouse models of human type 1 diabetes have been developed using transgenic mice. We found