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PubMed2005

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations.

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.

Adolescent↗
PubMed1992

Doublet translocation at GGA is mediated directly by mutant tRNA(2Gly).

Members of the sufS class of -1 frameshift suppressors have alterations of the GGA/G-decoding tRNA(2Gly). Suppressor-promoted frameshifting at GGA was shown in this study to be directly mediated by the mutant tRNA(2Gly). We disproved the possibility that, in the presence of the compromised mutant tRNA(2Gly), either wild-type tRNA(1Gly), wild-type tRNA(3Gly), a GGA-reading mutant form of tRNA(3Gly), or any other agent suppresses the frameshift mutation trpE91.

Escherichia coli↗
PubMed1998

Congenital adrenal hypoplasia: clinical spectrum, experience with hormonal diagnosis, and report on new point mutations of the DAX-1 gene.

X-linked congenital adrenal hypoplasia (AHC) is a rare developmental disorder of the human adrenal cortex and is caused by deletion or mutation of the DAX-1 gene, a recently discovered member of the nuclear hormone receptor superfamily. Hypogonadotropic hypogonadism is frequently associated with AHC. AHC occurs as part of a contiguous gene syndrome together with glycerol kinase deficiency (GKD) and Duchenne's muscular dystrophy. The present series, collected over the past 2 decades, includes 18 AHC boys from 16 families: 4 with AHC, GKD, and Duchenne's muscular dystrophy; 2 with AHC and GKD; and 12 with AHC (5 young adults with hypogonadotropic hypogonadism). Most of the boys presented with salt wasting and hyperpigmentation during the neonatal period. Plasma steroid determinations performed in the first weeks of life often showed confusing results, probably caused by steroids produced in the neonates' persisting fetocortex. Aldosterone deficiency usually preceded cortisol deficiency, which explains why the patients more often presented with salt-wasting rather than with hypoglycemic symptoms. An ACTH test was often necessary to detect cortisol deficiency in the very young infants. In some patients, serial testing was necessary to establish the correct diagnosis. In 4 boys studied during the first 3 months after birth, we found pubertal LH, FSH, and testosterone plasma levels indicating postnatal transient activation of the hypothalamic-pituitary-gonadal axis as in normal boys. Previous studies have shown that the DAX-1 gene is deleted in the AHC patients with a contiguous gene syndrome and is mutated in nondeletion patients. Most of the point mutations identified in AHC patients were frameshift mutations and stop mutations. In the 15 patients available for molecular analysis of the DAX-1 gene, there were large deletions in 6 patients and point mutations in another 7 patients. All of the point mutations identified in the present study resulted in a nonfunctional truncated DAX-1 protein. Two brothers with primary adrenal insufficiency and a medical history that strongly suggested AHC had no mutation in the DAX-1 gene. Thus, additional, as yet unknown genes must play a part in normal adrenal cortical development.

Adrenal Insufficiency↗
PubMed1999

Germline hMSH2 and differential somatic mutations in patients with Turcot's syndrome.

Turcot's syndrome is characterized clinically by the occurrence of primary brain tumor and colorectal tumor and has in previous reports been shown to be associated with germline mutations in the genes APC, hMLH1, and hPMS2. Here we describe three patients with Turcot's syndrome, each having colorectal adenocarcinoma and malignant glioma. All the colorectal and brain tumors from these patients showed replication errors in most of the microsatellite loci investigated. Search for underlying germline mutations in the nucleotide mismatch repair genes revealed three different hMSH2 mutations. All colorectal tumors showed a frameshift in the A(10) tract in the coding sequence of the transforming growth factor beta type II receptor (TGFBRII) gene, but no such change was detected in any of the brain tumors. Frameshift mutation in the BAX gene was found in one colon carcinoma and mutations in insulin-like growth factor type II receptor (IGFIIR) gene in one glioma. Our data have broadened the possible mutation spectrum of patients with Turcot's syndrome. The difference in the mutation spectrum of TGFBRII, BAX, and IGFIIR between brain and colorectal tumors in these individuals suggests that the mutator phenotype may target different pathogenic pathways in the oncogenic process of the two organs.

Adenomatous Polyposis Coli↗
PubMed1999

Von Hippel-Lindau syndrome. A pleomorphic condition.

Von Hippel-Lindau (VHL) syndrome (OMIM 193300) is an autosomal dominant disorder caused by deletions or mutations in a tumor suppressor gene mapped to human chromosome 3p25. It is characterized clinically by vascular tumors, including retinal and central nervous system hemangioblastomas (cerebellar, spinal, and brain stem). Hemangioblastomas are benign and do not metastasize. Other features include cysts of the kidneys, liver, and pancreas. Clear cell renal cell carcinoma occurs in up to 70% of patients with VHL and is a frequent cause of death. Pheochromocytomas occur in association with specific alleles of the VHL gene; therefore, a family history of pheochromocytoma in association with VHL is an indication for thorough surveillance for pheochromocytoma in affected family members. Recently, it has been appreciated that patients with VHL may develop endolymphatic sac tumors, which can cause tinnitus or deafness. The diagnosis of VHL may be made in a patient with a family history of VHL based on a single retinal or cerebellar hemangioblastoma, renal cell carcinoma or pheochromocytoma, and, possibly, multiple pancreatic cysts. Renal and epididymal cysts are not sufficient to make the diagnosis of VHL. In the absence of a family history of VHL the presence of two or more retinal or cerebellar hemangioblastomas, or one hemangioblastoma with one visceral tumor, is required for diagnosis. Studies of the natural history of VHL showed a life expectancy less than 50 years before surveillance protocols were developed. Annual assessments (physical and ophthalmologic examinations) should begin in infancy. Imaging of abdominal organs and the brain and spine should be added in teenagers and adults. Renal cysts and tumors should be monitored by computed tomography every 6 months. Mutation analysis has allowed presymptomatic identification of affected family members; those found not to have inherited the gene do not need to be monitored. The VHL gene coding sequence contains three exons, and two isoforms of mRNA exist, reflecting the presence or absence of exon 2. Tumors arise after the loss or inactivation of the wild type allele in a cell. About 20% of patients have large germline mutations detectable by Southern blot analysis, 27% have missense mutations, and 27% have nonsense or frameshift mutations. In about 20% of VHL families no deletion or mutation can be detected. Families may be characterized by the presence (type 2; 7% to 20% of families) or absence (type 1) of pheochromocytomas. Most type 2 families have missense mutations, whereas most type 1 families are affected by deletions or premature termination mutations. Prognostic counseling regarding the lifetime risk of pheochromocytoma can be aided by determination of the underlying mutation in patients without family histories of VHL.

DNA Mutational Analysis↗
PubMed2000

Specificity of mutations induced by the food-associated heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine in colon cancer cell lines defective in mismatch repair.

Recently, we have shown a hypermutable response to the food-associated heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP) in human cells defective in mismatch repair (MMR). These findings suggest that exogenous compounds such as PhIP may play an important role in the generation of tumors in MMR-defective individuals. The specificity of mutations induced by PhIP exposure at the endogenous HPRT locus was determined in cell lines defective in MMR to better understand the mutagenic effects of PhIP in MMR-defective individuals and to gain insight into the molecular mechanism of carcinogenesis induced by PhIP. Eighty-six induced HPRT mutants from two different cell lines were isolated and sequenced after exposure to 10 microM PhIP. Nineteen (22%) of these mutants contained G:C to T:A transversion mutations, consistent with the promutagenic adduct of PhIP at the C8 position of guanine miscoding with adenine. This level of PhIP-induced G:C to T:A transversions was approximately 4.5-fold higher than spontaneous G:C to T:A frequencies. Additionally, a hotspot for mutation was observed in a run of six guanines in HPRT exon 3, where a total of 23 (27%) of all PhIP-induced mutations occurred. These mutations consisted of transversions, transitions, and frameshift mutations. The increase in mutant frequency at this run of guanines corresponded to a 24-fold elevation above the spontaneous frequency in one cell line and a 3.3-fold increase in the other. These data suggest that PhIP may increase the risk of human carcinogenesis mediated by MMR by increasing mutations at runs of guanine residues. PhIP may thereby promote tumorigenesis by mutating growth-regulating genes that contain runs of guanines in their coding sequences, such as BAX, the insulin-like growth factor II receptor IGFIIR, and even the mismatch repair gene hMSH6.

Base Pair Mismatch↗
PubMed2005

Mutations of the PKD2 gene in Taiwanese patients with autosomal dominant polycystic kidney disease.

BACKGROUND: Mutation analysis in the context of clinical phenotypes helps clarify the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). Over 78 PKD2 gene mutations have been reported in the literature, but few have been described from an Asian population. This study attempted to characterize PKD2 mutations and their clinical implications among Taiwanese. METHODS: Twenty unrelated ADPKD patients with uncharacterized genotypes were screened for mutations in the PKD2 gene via single-strand conformation polymorphism (SSCP) of PCR products from genomic DNA, using previously reported PCR conditions and primers. RESULTS: This study identified two novel mutations (C681A and 2136-2137delG) and one mutation (C2407T) previously reported in a Cypriot family. Overall, we found PKD2 mutations in 15% (three out of 20) of the ADPKD patients screened. The mutations included two nonsense mutations (Y227X and R803X) and one frameshift mutation (712-715X) that could all lead to premature termination of translation. The locations of mutations in this study spanned the entire PKD2 gene on exons 2, 11, and 13 without clustering and did not influence the renal disease severity. CONCLUSIONS: The study identified two novel mutations and one recurrent mutation of the PKD2 gene in 20 Taiwanese patients. The characteristics of the mutations in this study resemble those reported among Western populations.

Adult↗
PubMed2006

Six novel mutations of the LDL receptor gene in FH kindred of Sicilian and Paraguayan descent.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.

Adult↗
PubMed1998

[The relationship between p53 mutation and esophageal cancer biological behavior].

OBJECTIVE: To investigate the relationship between p53 mutation and esophageal cancer biological behavior as well as its prognostic role in esophageal cancer. METHOD: Mutations in exon 5-8 of p53 were screened in 30 sporadic esophageal cancers by a combination of PCR-SSCP and PCR-direct DNA silver sequencing. RESULTS: Mutations were found in 11 cases (36.7%). There were 9 point mutations, including 4 missense mutations, 2 nonsense mutations, 3 silent mutations. The other 2 cases were frameshift mutations due to base insertion and deletion. With statistical analysis, the p53 mutation rate was 56.3% in moderately and poorly-differentiated esophageal cancers, 14.3% in well-differentiated cases, with a highly significant difference between the two groups (P=0.025). The mutation rate of the cancers with all layers invaded (52.6%) was remarkably higher than that of the cancers without all layers invaded (9.1%)(P=0.024). A significantly higher rate of p53 mutation was also seen in cases with lymph nodes metastasis (61.5%) than in cases with no lymph nodes metastasis (17.6%) (P=0.024). CONCLUSION: The data suggest that there is a strong correlation between the presence of p53 mutation and the biological behavior of human esophageal cancer, such as histologic differentiation, invasion stage, and regional lymph nodes metastasis. Detection of p53 mutation can be helpful in identifying more progressive esophageal cancer and assessing prognosis. The study also discussed the dominant negative effect of p53 and the genetic effect of silent mutation.

Esophageal Neoplasms↗
PubMed2001

MECP2 truncating mutations cause histone H4 hyperacetylation in Rett syndrome.

Rett syndrome (RTT) is a mostly sporadic disorder of developmental regression, with loss of speech and purposeful hand use, microcephaly and seizures. It affects 1 in 10 000-15 000 females. RTT is caused by mutations in the MECP2 gene, which is located in Xq28 and subject to X inactivation. MECP2 encodes a methyl-CpG-binding protein that binds to 5-methyl-cytosine in DNA through its methyl-binding domain. Recruitment of a transcriptional silencing complex through MeCP2's transcriptional repression domain results in histone deacetylation and chromatin condensation. To study the effects of two common truncating RTT mutations (R168X and 803delG), we examined mutant MeCP2 expression and global histone acetylation levels in clonal cell cultures from a female RTT patient with the mutant R168X allele on the active X chromosome, as well as in cells from a male hemizygous for the frameshift mutation 803delG (V288X). Both mutant alleles generated stable RNA transcripts, but no intact MeCP2 protein was detected with an antibody against the C-terminal region of MeCP2. Western blots with antibodies against acetylated histones H3 and H4 revealed that H4, but not H3, was hyperacetylated. By using antibodies against individual acetylated lysine residues, the observed H4 hyperacetylation was attributed to increased acetylation of lysine 16. Therefore, expression of endogenous truncating MECP2 alleles, in the absence of wild-type MeCP2 protein, is specifically associated with an increase in the mono-acetylated histone isoform H4K16. This observed effect may result in over-expression of MeCP2 target genes and, thus, play a role in the pathogenesis of RTT.

Acetylation↗
PubMed1995

Strain-dependent occurrence of functional GTP:AMP phosphotransferase (AK3) in Saccharomyces cerevisiae.

The gene for yeast GTP:AMP phosphotransferase (PAK3) was found to encode a nonfunctional protein in 10 laboratory strains and one brewers' strain. The protein product showed high similarity to vertebrate AK3 and was located exclusively in the mitochondrial matrix. The deduced amino acid sequence revealed a protein that was shorter at the carboxyl terminus than all other known adenylate kinases. Introduction of a +1 frameshift into the 3'-terminal region of the gene extended homology of the deduced amino acid sequence to other members of the adenylate kinase family including vertebrate AK3. Frameshift mutations obtained after in vitro and in vivo mutagenesis were capable of complementing the adk1 temperature-conditional deficiency in Escherichia coli, indicating that the frameshift led to the expression of a protein that could phosphorylate AMP. Some yeasts, however, including strain D273-10B, two wine yeasts, and two more distantly related yeast genera, harbored an active allele, named AKY3, which contained a +1 frameshift close to the carboxyl terminus as compared with the laboratory strains. The encoded protein exhibited GTP:AMP and ITP:AMP phosphotransferase activities but did not accept ATP as phosphate donor. Although single copy in the haploid genome, disruption of the AKY3 allele displayed no phenotype, excluding the possibility that laboratory and brewers' strains had collected second site suppressors. It must be concluded that yeast mitochondria can completely dispense with GTP:AMP phosphotransferase activity.

Alleles↗
PubMed1998

Characterization of IS1515, a functional insertion sequence in Streptococcus pneumoniae.

We describe the characterization of a new insertion sequence, IS1515, identified in the genome of Streptococcus pneumoniae I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585-602, 1959). A copy of this element located in the cap1EI41R gene was sequenced. The 871-bp-long IS1515 element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS1381 from S. pneumoniae, ISL2 from Lactobacillus helveticus, IS702 from the cyanobacterium Calothrix sp. strain PCC 7601, and IS112 from Streptomyces albus G. IS1515 appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain 141R is the result of both the presence of an IS1515 copy and a frameshift mutation in the cap1EI41R gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS1515 behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS1515 were also able to excise and move to another locations in the chromosome of S. pneumoniae. To our knowledge, this is the first report of a functional IS in pneumococcus.

Amino Acid Sequence↗
PubMed2005

Interplay between GCN2 and GCN4 expression, translation elongation factor 1 mutations and translational fidelity in yeast.

Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs.

Alcohol Oxidoreductases↗
PubMed2004

Molecular basis of beta-thalassemia in the population of Tunisia.

The present study attempts to delineate the spectrum of beta-thalassemia (thal) mutations in Tunisia by studying a large population from different parts of the country. A total of 285 unrelated subjects, 190 of whom had beta-thal major, 72 with Hb S/beta-thal, one with Hb C/beta-thal, one with Hb O-Arab/beta-thal and 21 beta-thal carriers, were studied. The molecular defects were detected in 97.7% of the beta-thalassemic chromosomes (n=475). Nineteen different beta-thalassemic alleles were identified. Two mutations, namely codon 39 (C-->T) and IVS-I-110 (G-->A) accounted for 70.0% of the studied chromosomes, followed by IVS-I-1 (G-->A) (4.5%). Five other mutations, frameshift codon (FSC) 44 (-C), codon 30 (G-->C), IVS-I-2 (T-->G), IVS-II-745 (C-->G), and FSC 6 (-A), are not uncommon in this population, while the remaining 11 mutations, IVS-I-5 (G-->A), -30 (T-->A), codons 25/26 (+T), IVS-I-6 (T-->C), FSC 5 (-CT), IVS-II-848 (C-->A), FSC 8 (-AA), -87 (C-->G), IVS-I-5 (G-->C), IVS-II-1 (G-->A) and IVS-II-849 (A-->C) are quite rare; four of these have not been previously reported in the Tunisian population. Potential origin and spread of these mutations to Tunisia are also discussed.

Chromosomes↗
PubMed1998

The influence of 3TC resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

A common target for therapies against human immuno-deficiency virus type 1 (HIV-1) is the viral reverse transcriptase (RT). Treatment with the widely used nucleoside analog (-)-2', 3'-deoxy-3'-thiacytidine (3TC) leads to the development of resistance-conferring mutations at residue M184 within the YMDD motif of RT. First, variants of HIV with the M184I substitution appear transiently, followed by viruses containing the M184V substitution, which persist and become the dominant variant for the duration of therapy. In the three-dimensional crystal structure of HIV-1 RT complexed with double-stranded DNA, the M184 residue lies in the vicinity of the primer terminus, near the incoming dNTP substrate. Recent studies have shown that 3TC resistance mutations, including M184I, increase the nucleotide insertion and mispair extension fidelity. Therefore, we have examined the effects of the M184I mutation on the overall polymerase fidelity of HIV-1 RT via an M13-based forward mutation assay. We found the overall error rate of the M184I variant of HIV-1 RT to be 1.7 x 10(-5) per nucleotide. This represents a 4-fold increase in fidelity over wild-type HIV-1Hxb2RT (7.0 x 10(-5) per nucleotide) and a 2.5-fold increase in fidelity over the M184V variant (4.3 x 10(-5) per nucleotide). Of the nucleoside analog resistance mutations studied using the forward assay, the M184I variant has shown the greatest increase in fidelity observed to date. Interestingly, the M184I variant RT displays significantly altered error specificity, both in terms of error rate at specific sites and in the overall ratio of substitution to frameshift mutations in the entire target.

Bacteriophage M13↗
PubMed1998

A gene-targeted mouse model for familial hypobetalipoproteinemia. Low levels of apolipoprotein B mRNA in association with a nonsense mutation in exon 26 of the apolipoprotein B gene.

Familial hypobetalipoproteinemia, a syndrome characterized by abnormally low plasma levels of low density lipoprotein cholesterol, is caused by mutations in the apolipoprotein (apo) B gene that interfere with the synthesis of a full-length apoB100. In many cases of familial hypobetalipoproteinemia, nonsense or frameshift mutations result in the synthesis of a truncated apoB protein. To understand why these mutations result in low plasma cholesterol levels, we used gene targeting in mouse embryonic stem cells to introduce a nonsense mutation (N1785Stop) into exon 26 of the mouse Apob gene. The sole product of this mutant Apob allele was a truncated apoB, apoB39. Mice homozygous for this "apoB39-only" (Apob39) allele had low plasma levels of apoB39 and markedly reduced plasma levels of very low density lipoprotein and low density lipoprotein cholesterol when fed a high fat diet. Analysis of liver and intestinal RNA from heterozygous apoB39-only mice revealed that the Apob39 mRNA levels were 60-70% lower than those from the wild-type allele. Interestingly, apoB39 was not cleared as rapidly from the plasma as apoB48. The apoB39-only mice provide new insights into the mechanisms of familial hypobetalipoproteinemia and the structural features of apoB that are important for lipoprotein metabolism.

Animals↗
PubMed1998

The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme.

Seven of the approximately 40 subunits of the mammalian respiratory NADH dehydrogenase (Complex I) are encoded in mitochondrial DNA (mtDNA). Their function is almost completely unknown. In this work, a novel selection scheme has led to the isolation of a mouse A9 cell derivative defective in NADH dehydrogenase activity. This cell line carries a near-homoplasmic frameshift mutation in the mtDNA gene for the ND6 subunit resulting in an almost complete absence of this polypeptide, while lacking any mutation in the other mtDNA-encoded subunits of the enzyme complex. Both the functional defect and the mutation were transferred with the mutant mitochondria into mtDNA-less (rho0) mouse LL/2-m21 cells, pointing to the pure mitochondrial genetic origin of the defect. A detailed biosynthetic and functional analysis of the original mutant and of the rho0 cell transformants revealed that the mutation causes a loss of assembly of the mtDNA-encoded subunits of the enzyme and, correspondingly, a reduction in malate/glutamate-dependent respiration in digitonin-permeabilized cells by approximately 90% and a decrease in NADH:Q1 oxidoreductase activity in mitochondrial extracts by approximately 99%. Furthermore, the ND6(-) cells, in contrast to the parental cells, completely fail to grow in a medium containing galactose instead of glucose, indicating a serious impairment in oxidative phosphorylation function. These observations provide the first evidence of the essential role of the ND6 subunit in the respiratory function of Complex I and give some insights into the pathogenic mechanism of the known disease-causing ND6 gene mutations.

Animals↗
PubMed2000

Inactivation of the glutamate decarboxylase gene in Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. lactis strains show glutamate decarboxylase activity, whereas L. lactis subsp. cremoris strains do not. The gadB gene encoding glutamate decarboxylase was detected in the L. lactis subsp. cremoris genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.

Base Sequence↗