Nucleotide Positions (nucleotide + position)

Distribution by Scientific Domains

Selected Abstracts

Maternal transmission of diabetes

J. C. Alcolado
Abstract Type 2 diabetes mellitus represents a heterogeneous group of conditions characterized by impaired glucose homeostasis. The disorder runs in families but the mechanism underlying this is unknown. Many, but not all, studies have suggested that mothers are excessively implicated in the transmission of the disorder. A number of possible genetic phenomena could explain this observation, including the exclusively maternal transmission of mitochondrial DNA (mtDNA). It is now apparent that mutations in mtDNA can indeed result in maternally inherited diabetes. Although several mutations have been implicated, the strongest evidence relates to a point substitution at nucleotide position 3243 (A to G) in the mitochondrial tRNAleu(UUR) gene. Mitochondrial diabetes is commonly associated with nerve deafness and often presents with progressive non-autoimmune ,-cell failure. Specific treatment with Coenzyme Q10 or L-carnitine may be beneficial. Several rodent models of mitochondrial diabetes have been developed, including one in which mtDNA is specifically depleted in the pancreatic islets. Apart from severe, pathogenic mtDNA mutations, common polymorphisms in mtDNA may contribute to variations of insulin secretory capacity in normal individuals. Mitochondrial diabetes accounts for less than 1% of all diabetes and other mechanisms must underlie the maternal transmission of Type 2 diabetes. Possibilities include the role of maternally controlled environments, imprinted genes and epigenetic phenomena. [source]

Identification of a potential "hotspot" DNA region in the RUNX1 gene targeted by mitoxantrone in therapy-related acute myeloid leukemia with t(16;21) translocation

Tiziana Ottone
The translocation t(16;21) involving RUNX1 (AML1) and resulting in the RUNX1-CBFA2T3 fusion is a rare but recurrent abnormality mostly found in therapy-related acute myeloid leukemia (t-AML) associated with agents targeting topoisomerase II (topo II). We characterized, at the genomic level, the t(16;21) translocation in a patient who developed t-AML after treatment of multiple sclerosis with mitoxantrone (MTZ). Long template nested PCR of genomic DNA followed by direct sequencing enabled the localization of RUNX1 and CBFA2T3 (ETO2) breakpoints in introns 5 and 3, respectively. Sequencing of the cDNA with specific primers showed the presence of the expected RUNX1-CBFA2T3 fusion transcript in leukemic cells. The RUNX1 intron 5 breakpoint was located at nucleotide position 24,785. This region contained an ATGCCCCAG nucleotide sequence showing ,90% homology to a "hotspot" DNA region ATGCCCTAG present in intron 6 of PML previously identified in therapy-related acute promyelocytic leukemia cases arising following treatment with MTZ. This study suggests a wider distribution in the human genome, and particularly at genes involved in chromosome translocations observed in t-AML, of DNA regions (hotspot) targeted by specific topo II drugs. © 2008 Wiley-Liss, Inc. [source]

Ethnic variation in AMD-associated complement factor H polymorphism p.Tyr402His,

HUMAN MUTATION, Issue 9 2006
Michael A. Grassi
Abstract Age-related macular degeneration (AMD) is the most common cause of irreversible visual loss in the developed world. Previous studies have demonstrated that the c.1204T>C, p.Tyr402His allelic variant in the complement factor H (CFH) gene is associated with an approximately three-fold increased risk for AMD in Caucasians of predominantly European descent. Both the prevalence as well as the phenotypic spectrum of AMD varies widely among persons of different ethnicities. We hypothesized that populations with a lower prevalence of AMD might also have a lower prevalence of the CFH risk allele. In this study we sought to determine the frequency of this sequence variant in control populations of Caucasians, African Americans, Hispanics, Somalis, and Japanese. Normal control populations were assembled for each ethnic group: Caucasian (n=148), Somali (n=128), African American (n=75), Hispanic (n=81), and Japanese (n=82). Individuals were genotyped using a restriction digest assay and the frequency of the C allele at nucleotide position 1204 of the CFH gene was determined. A bioinformatic approach was used to identify SNPs in linkage disequilibrium with rs1061170 (c.1204T>C, p.Tyr402His) from the human haplotype map project database (HapMap) in order to validate the findings. We found widely discordant frequencies of the risk allele between some of the different ethnic groups: Japanese 0.07±0.02, Hispanics 0.17±0.03, African-Americans 0.35±0.04, Caucasians 0.34±0.03, and Somalis 0.34±0.03. Allele frequencies generated by analysis of the HapMap database were consistent with these findings. This study suggests that there are other yet unidentified genetic factors important in the pathogenesis of AMD that may mitigate the effects of c.1204T>C, p.Tyr402His variant. Hum Mutat 27(9), 921,925, 2006. © 2006 Wiley-Liss, Inc. [source]

Genetic variation at the alpha-1-fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds

W.B. Bao
Summary Escherichia coli F18 bacteria producing enterotoxins and/or shigatoxin (ETEC/STEC) are main pathogens that cause oedema disease and postweaning diarrhoea in piglets, and alpha-1-fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of ETEC F18 receptor. The genetic variations at nucleotide position 307 in open reading frame of FUT1 gene in one wild boar breed and 20 western commercial and Chinese native pig breeds were investigated by polymerase chain reaction,restriction fragment length polymorphism. The results showed that the genetic polymorphisms of the FUT1 locus were only detected in western pig breeds and the Chinese Taihu (including Meishan pig, Fengjing pig and Erhualian pig), Huai and Lingao pig breeds; only Duroc and Pietrain possessed the resistant AA genotype, while the wild boar and other Chinese pig breeds only presented the susceptible genotype GG. The results indicated that Chinese native pig breeds lack genetic factors providing resistance to ETEC F18 bacteria. The resistant allele to ETEC F18 might originate from European wild boar. It was inferred that oedema and postweaning diarrhoea caused by ETEC F18 have close relationship with the growth rate, which can explain why on the contrary Chinese native pig breeds have stronger resistance to oedema and postweaning diarrhoea in piglets compared with western pig breeds. [source]

De novo mutation in the mitochondrial tRNALeu(UUR) gene (A3243G) with rapid segregation resulting in MELAS in the offspring

Abstract: A 14-year-old Chinese boy with a normal perinatal and early developmental history presented at 5 years of age with migraine, intractable epilepsy, ataxia, supraventricular tachycardia, paralytic ileus and progressive mental deterioration. Computerized tomography revealed multiple cerebral infarcts in the parieto-occipital region without basal ganglial calcification. Magnetic resonance imaging showed increased signal intensity in T2 weighted images in the same regions. A cerebral digital subtraction angiogram was normal. Venous lactate, pyruvate, lactate to pyruvate ratio and cerebrospinal fluid lactate were elevated. Muscle biopsy did not reveal any ragged red fibres; dinucleotide,tetrazolium reductase activity was normal. Mitochondrial DNA analysis detected an adenine to guanine mutation at nucleotide position 3243 of tRNALeu(UUR). All four tissues analysed demonstrated heteroplasmy: leucocyte 56%, hair follicle 70%; buccal cell 64%; muscle 54%. The mother and brother of the proband, both asymptomatic, were also found to have a heteroplasmic A3243G mutation in the leucocytes, hair follicle and buccal cells. Other members of the maternal lineage, including the maternal grandmother, did not have the mutation. This report describes a patient with mitochondrial encephalopathy, lactic acidosis, stroke-like episodes, who presented with multisystem involvement. The absence of ragged red fibres in muscle biopsy did not preclude the diagnosis. Mutational analysis of mitochodrial DNA conveniently confirmed the diagnosis of the disorder. A de novo mutaton is demonstrated in this family. [source]

Novel MPZ Mutation In A Sporadic CMT Patient

E Bellone
Mutations in the gene for the major structural protein component of peripheral nerve myelin, myelin protein zero (MPZ), are associated with some forms of hereditary neuropathies such as Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelinating neuropathy (CHN). The common pathological characteristics of these allelic disorders are severe demyelination and remyelination of peripheral nerves. Recently, MPZ mutations were also found in patients with the axonal form of CMT neuropathy (CMT2). We studied a patient with negative familiar history and clinical and electrophysiological features of Charcot-Marie-Tooth disease: distal muscle weakness and atrophy, foot deformities (pes cavus), and severely reduced nerve conduction velocities in the motor and sensory nerves. The sural nerve biopsy showed marked loss of myelinated fibers, few onion bulbs, and a high percentage of fibers showing excessive myelin outfoldings. DNA analysis excluded CMT1A duplication by Southern blot and by pulsed field gel electrophoresis methods. SSCP analysis of all six exons of MPZ revealed a shift band in exon 2 in the patient's DNA. No such difference was detected in normal controls. Direct sequencing disclosed a G , A transition at nucleotide position 181. This base substitution predicts the replacement of aspartic acid with asparagine at codon 61. A mutation at the same codon (but different amino acid replacement) was recently identified in a family with the axonal type of CMT, in which the disease was autosomal dominantly inherited. This finding provides further confirmation of the role of MPZ gene in peripheral neuropathies and suggests that MPZ coding region mutations may account for a considerable number of CMT cases which do not involve DNA duplication on 17p11.2-p12. This research was partially supported by a MURST and an Ateneo grant to FA, by a Ministero della Sanità grant to PM. Our laboratory is a member of the European Charcot-Marie-Tooth Consortium co-ordinated by Prof. Christine Van Broeckhoven. [source]

A case of WHIM syndrome associated with diabetes and hypothyroidism

Junji Takaya
Abstract: The WHIM syndrome is a rare immunological disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. We hypothesized that immunological or genetic mechanisms may link WHIM syndrome and type 1 diabetes. We report that the young girl with WHIM syndrome developed diabetes and transient hypothyroidism. A nonsense mutation (C,T) truncating the CXC chemokine receptor 4 (CXCR4) C-terminal cytoplasmic tail domain occurred at nucleotide position 1000(R334X) of the CXCR4 gene in one allele of the patient was identified, and the person was diagnosed as having WHIM syndrome. Recent observation suggested that the CXCR4, a G-protein-coupled receptor with a unique ligand, CXCL12, might be involved in the pathogenesis for type 1 diabetes. Taken into consideration the concurrent prevalence of the two disorders and the speculated common pathogenesis associated with the CXCR4, our patient may enable us to understand the genetic damage related to accelerated apoptosis. [source]

One novel and one recurrent mutation in the PROS1 gene cause type I protein S deficiency in patients with pulmonary embolism associated with deep vein thrombosis

Kazuhiro Mizukami
Abstract We investigated the molecular basis of type I protein S (PS) deficiency in two unrelated Japanese families, in which both probands developed pulmonary embolism associated with deep vein thrombosis. Nucleotide sequencing of amplified DNA revealed distinct point mutations in the PROS1 gene of the probands, which were designated protein S Sapporo 1 and protein S Sapporo 2. Additional mutations in the PROS1 gene were excluded by DNA sequencing of all exons and intron/exon boundaries. In the 25-year-old Japanese male patient who carried protein S Sapporo 1, we identified a heterozygous A-to-T change in the invariant ag dinucleotide of the acceptor splice site of intron f of the PROS1 gene. This mutation is a novel splice site mutation that impairs normal mRNA splicing, leading to exon 7 skipping, which was confirmed by platelet mRNA analysis. Translation of this mutant transcript would result in a truncated protein that lacks the entire epidermal growth factor-like domain 3 of the PS molecule. In a 31-year-old Japanese male and his younger brother who each carried protein S Sapporo 2, we detected a previously described heterozygous T-to-C transition at nucleotide position 1147 in exon 10 of the PROS1 gene, which predicts an amino acid substitution of tryptophan by arginine at residue 342 in the laminin G1 domain of the PS molecule. Both mutations would cause misfolding of the PS protein, resulting in the impairment of secretion, which is consistent with the type I PS deficiency phenotype. Am. J. Hematol., 2006. © 2006 Wiley-Liss, Inc. [source]

Endothelin receptor B2 (EDNRB2) is associated with the panda plumage colour mutation in Japanese quail

M. Miwa
Summary The panda mutant in Japanese quail (Coturnix japonica) displays spots of wild-type plumage on a white background and is controlled by an autosomal recessive allele (s). The dotted white is controlled by a third allele (sdw) of the s locus with sdw/sdw quail having less pigmentation than s/s quail. We mapped the s locus to the Japanese quail chromosome 4 (CJA04) in a previous study. The orthologous region of the chicken (Gallus gallus) genome includes endothelin receptor B2 (EDNRB2), an avian-specific paralog of endothelin receptor B (EDNRB). EDNRB mutations in mammals retard the migration of neural crest cells (NCCs), which results in a spotted coat colour and an enteric nervous defect. In the present study, we investigated the association between the s locus and EDNRB2 in Japanese quail. Sequence comparison among transcripts from livers of wild-type, panda and dotted white quail revealed a nucleotide substitution (c.995G>A) leading to a p.R332H amino acid change that was specific to panda, whereas no amino acid substitution was found in dotted white birds. The amino acid position 332 is located in the sixth transmembrane domain and is highly conserved in both avian and mammalian endothelin receptors. The A allele at nucleotide position 995 was specific to panda (s/s) birds among 10 strains, and was mapped to the same chromosomal region as the s locus. Quantitative RT-PCR revealed that EDNRB2 transcripts were reduced in both panda and dotted white mutants compared with wild-type. However, there was no difference between the early embryos of wild-type and panda with respect to the migration of NCCs. The genetic association of EDNRB2 with plumage colour in birds was found for the first time in this study. [source]

Mitochondrial DNA variability in Poles and Russians

Mitochondrial DNA (mtDNA) sequence variation was examined in Poles (from the Pomerania-Kujawy region; n = 436) and Russians (from three different regions of the European part of Russia; n = 201), for which the two hypervariable segments (HVS I and HVS II) and haplogroup-specific coding region sites were analyzed. The use of mtDNA coding region RFLP analysis made it possible to distinguish parallel mutations that occurred at particular sites in the HVS I and II regions during mtDNA evolution. In total, parallel mutations were identified at 73 nucleotide sites in HVS I (17.8%) and 31 sites in HVS II (7.73%). The classification of mitochondrial haplotypes revealed the presence of all major European haplogroups, which were characterized by similar patterns of distribution in Poles and Russians. An analysis of the distribution of the control region haplotypes did not reveal any specific combinations of unique mtDNA haplotypes and their subclusters that clearly distinguish both Poles and Russians from the neighbouring European populations. The only exception is a novel subcluster U4a within subhaplogroup U4, defined by a diagnostic mutation at nucleotide position 310 in HVS II. This subcluster was found in common predominantly between Poles and Russians (at a frequency of 2.3% and 2.0%, respectively) and may therefore have a central-eastern European origin. [source]

Mutation screening of the macrophage migration inhibitory factor gene: Positive association of a functional polymorphism of macrophage migration inhibitory factor with juvenile idiopathic arthritis

Rachelle Donn
Objective To determine if polymorphisms of the macrophage migration inhibitory factor (MIF) gene are associated with juvenile idiopathic arthritis (JIA). Methods Denaturing high-performance liquid chromatography was used to screen the MIF gene in 32 UK Caucasian controls and 88 UK Caucasian JIA patients. Ninety-two healthy UK Caucasian controls were then genotyped for each of the polymorphic positions identified. A panel of 526 UK Caucasian JIA patients and 259 UK Caucasian controls were subsequently genotyped for a single-nucleotide polymorphism (SNP) identified in the 5,-flanking region of the gene, using SNaPshot ddNTP primer extension and capillary electrophoresis. The functional significance of this polymorphism was also studied using luciferase-based reporter gene assays in human T lymphoblast and epithelial cell lines. Results A tetranucleotide repeat CATT(5,7) beginning at nucleotide position ,794 and 3 SNPs at positions ,173 (G to C), +254 (T to C), and +656 (C to G) of the MIF gene were identified. No JIA-specific mutations were found. Allele and genotype frequencies differed significantly between the controls and the JIA patients for the MIF-173 polymorphism. Individuals possessing a MIF-173*C allele had an increased risk of JIA (34.8% versus 21.6%) (odds ratio 1.9, 95% confidence interval 1.4,2.7; P = 0.0002). Furthermore, the MIF-173* G and C variants resulted in altered expression of MIF in a cell type,specific manner. Serum levels of MIF were also significantly higher in individuals who carried a MIF-173*C allele (P = 0.04). Conclusion The ,173-MIF*C allele confers increased risk of susceptibility to JIA. Our data suggest a cell type,specific regulation of MIF, which may be central to understanding its role in inflammation. [source]

Identification of four novel mutations in five unrelated Korean families with Fabry disease

J-K Lee
Fabry disease is a X-linked recessively inherited metabolic disorder, which results from the deficient activity of the lysosomal hydrolase ,-galactosidase A leading to the systemic deposition of glycosphingolipids with terminal ,-galactosyl moieties. Single-strand conformation polymorphism (SSCP) analysis was performed, followed by DNA sequencing of PCR amplified exons of the human ,-galactosidase A gene in 5 unrelated Korean patients with classic Fabry disease. Five different mutations were identified; two nonsense mutations (Y86X and R342X), one missense mutation (D266N), and two small deletions (296del2 and 802del4). Except for R342X mutation, four were novel mutations (Y86X, D266N, 296del2, 802del4). A T to G transversion at nucleotide position 5157 in exon 2 caused a tyrosine-to-stop substitution at codon 86. A G to A transition at position 10 287 in exon 5 substituted an asparagine for an aspartate at codon 266. Mutation 296del2 in exon 2 resulted in a frame shift with a stop signal at the 22th codon downstream from the mutation, whereas mutation 802del4 resulted in a stop codon at the site of 4 bp deletion. In addition, the 802del4 was found to be a de novo mutation. This is the first report on mutation analysis of the human ,-galactosidase A gene in Korean patients with Fabry disease. [source]

Ectopic activation of the transcription promoter for the testis-specific mouse Pgk-2 gene on elimination of a cis -acting upstream DNA region

Hiroshi Ando
Transgenic mice carrying the coding sequence of ,-galactosidase, for which expression was driven by various upstream regions including the transcription promoter of the testis-specific mouse Pgk-2 gene, were generated. Expression of ,-galactosidase mRNA driven by the region between nucleotide positions , 1404 and + 61, with respect to the transcription initiation site numbered + 1, was examined by reverse transcription-mediated polymerase chain reaction, blot hybridization and in situ hybridization, and compared with that of endogenous Pgk-2 mRNA. The results revealed that the 1.4 kb DNA region is sufficient for determining the organ-specific, developmental stage-specific and spermatogenic stage-specific transcription of the mouse Pgk-2 gene. When the region between , 684 and + 61 was used to generate transgenic mice, ,-galactosidase mRNA was detectable not only in the testis, but also in other organs such as brain and lung. However, the timing and cell-type specificity of testicular expression of ,-galactosidase mRNA were retained in these mice. Because the region between , 1404 and , 685 repressed the Pgk-2 promoter in somatic cell-derived cell lines, it is suggested that the organ specificity of Pgk-2 transcription is achieved at least partly by negative regulation. [source]

DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression

FEBS JOURNAL, Issue 6 2003
Anke Homann
We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba). In functional analyses using both promoter and factor mutants, the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directed muta- genesis of SASIG1 identified critical residues within the conserved regions 2.4 and 4.2 necessary for binding of the ,10 and ,35 promoter elements, respectively. SASIG1 and 2, but not SASIG3, each have a typical region 2.5 for binding of the extended ,10 promoter element. SASIG3 has a pro-sequence reminiscent of ,K from Bacillus subtilis, suggesting that proteolytic cleavage from an inactive precursor is involved in the regulation of plastid transcription. In addition, SASIG2 was found to be more abundant in light-grown as compared to dark-grown mustard seedlings, while the converse was true for SASIG3. [source]

Genetic diversity of the hyperparasite Sphaerellopsis filum on Melampsora willow rusts

M. Liesebach
Summary The non-specific rust hyperparasite Sphaerellopsis filum occurs naturally on Melampsora rusts of many species of the genus Salix as well as on a large range of other rust genera worldwide. To study the genetic diversity of the hyperparasitic fungus 77 S. filum isolates collected from rusts on willow clones from plantations, clone collections and natural habitats of different sites were investigated using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis of the rDNA internal transcribed spacer regions including 5.8S rDNA and sequence analysis. Additionally, strains from Melampsora poplar rusts (4) and strains of Puccinia abrupta from Parthenium hysterophorus (5) and of P. obscura from Bellis perennis (1) were used for comparisons. Results of genetic analysis demonstrated distinct variation within the S. filum isolates. Two main groups with more than 32% difference between their nucleotide sequences were distinguished, indicating two taxa within S. filum. Within the first main group three profiles (I, II, III) were detected. The differences between these profiles were about 12%. The variation within each profile was very low (less than 2%). The second main group comprised two profiles (IV, V), which differed in 12 to 16% of their nucleotide positions. The isolates of group IV possessed a higher variation (up to 5%) within the group than those of the first main group (I, II, III). Group V was only represented by a single isolate. Neither interrelations between the S. filum profiles and the Melampsora genotypes nor a spatial distribution could be detected. It is remarkable that the six strains of S. filum from Puccinia rusts belong to one subgroup. Résumé Sphaerellopsis filum est un hyperparasite non spécifique des rouilles qui se rencontre naturellement sur les Melampsora affectant le genre Salix ainsi que sur une large gamme d'autres genres de rouille dans le monde entier. Pour caractériser la diversité de ce champignon hyperparasite, 77 isolats de S. filum, récoltés à partir de lésions de rouille sur des clones de plantation, des collections de clones et dans des sites naturels, ont étéétudiés par analyse PCR-RFLP des régions ITS de l'ADNr, incluant le 5.8S rDNA, et par analyse de séquences. Des souches provenant de Melampsora des peupliers (4), de Puccinia abrupta sur Parthenium hysterophorus (5) et de P. obscura sur Bellis perennis (1) ont également été utilisées pour comparaison. L'analyse génétique démontre une variation entre isolats de S. filum. Deux groupes principaux, avec plus de 32% de différence dans leur séquence nucléotidique, se distinguent, indiquant l'existence de deux taxons au sein de S. filum. A l'intérieur du premier groupe, trois profils (I, II, III) sont mis en évidence, avec une différence d'environ 12% entre profils mais très peu de variation (moins de 2%) à l'intérieur d'un profil. Le deuxième groupe comprend deux profils (IV, V) qui différent de 12 à 16% pour leur séquence nucléotidique. Les isolats du groupe IV présentent une variation intra-groupe plus importante (jusqu'à 5%) que ceux des groupes I, II et III. Le groupe V n'est représenté que par un isolat. Aucune relation n'a pu être établié entre les profils de S. filum et les génotypes de Melampsora ou la distribution spatiale. Il est à noter que les six isolats de S. filum provenant de rouilles àPuccinia appartiennent à un seul sous-groupe. Zusammenfassung Der Hyperparasit Sphaerellopsis filum kommt natürlich auf Melampsora -Rostpilzen bei Salicaceae und auf zahlreichen anderen Rostgattungen weltweit vor. Um die genetische Vielfalt dieses hyperparasitischen Pilzes zu untersuchen, wurden 77 S. filum -Proben, die von Weidenrosten in Plantagen, Klonsammlungen und von verschiedenen natürlichen Standorten stammen, isoliert. Die 5.8S-ITS-Abschnitte der rDNA wurden mit Hilfe der PCR-RFLP untersucht und Sequenzen analysiert. Zum Vergleich wurden vier Isolate von Pappelrosten sowie fünf Isolate von Puccinia abrupta von Parthenium hysterophorus und ein Isolat von Puccinia obscura von Bellis perennis herangezogen. Die Ergebnisse der genetischen Untersuchungen zeigten eine deutliche Variation zwischen den S. filum -Isolaten. Zwei Gruppen mit über 32% Unterschied in der Nukleotid-Sequenz ließen sich unterscheiden. Dies deutet auf zwei taxonomische Einheiten von Sphaerellopsis hin. Die erste Gruppe ließ sich in drei Untergruppen (I, II, III) einteilen, deren 5.8S-ITS-Profile im Mittel 12% Unterschied aufwiesen. Die Variation innerhalb dieser drei Profile war sehr gering (,2%). Die zweite Gruppe umfasste zwei Profile (IV, V), die sich an 12 bis 16% Positionen ihrer Nukleotid-Abfolge unterschieden. Die Variation innerhalb von Profil IV war höher (bis 5%) als die der Untergruppen I - III, Profil V war nur durch ein Isolat vertreten. Eine Beziehung des S. filum -Genotyps zum Melampsora -Genotyp der Weide oder der Pappel ließ sich bei dem untersuchten Material nicht nachweisen, ebenso konnte keine geographische Differenzierung gefunden werden. Auffällig ist, dass alle sechs Puccinia -Isolate einer Untergruppe angehörten. [source]

Three novel thiopurine S-methyltransferase allelic variants (TPMT*20, *21, *22) , association with decreased enzyme function,,

HUMAN MUTATION, Issue 9 2006
Elke Schaeffeler
Abstract The genetic polymorphism of the thiopurine S-methyltransferase, TPMT, comprises at least 21 alleles causing three distinct drug metabolism phenotypes termed normal/high, intermediate, and deficient methylators. In consequence, adverse drug reactions may occur if standard doses of thiopurines are applied routinely. Genetic prediction of the methylator phenotype as a basis for dose selection requires the extensive knowledge of single nucleotide polymorphisms occurring naturally in the population. Here we describe three novel missense variants in the TPMT gene which were associated with an intermediate red blood cell TPMT activity in three Caucasians. The following alleles were designated: TPMT*20 (c.712A>G), *21 (c.205C>G), and *22 (c.488G>C). No further genetic variations in remaining coding regions as well as the 5,flanking region of TPMT were identified. These sequence variants are present in highly conserved nucleotide positions of the TPMT gene throughout various mammalian species and in zebra fish, and are predicted to be intolerant when the functional consequences of variations were analyzed using SIFT (Sorting Intolerant From Tolerant) algorithm. In Caucasians the occurrence of these genetic variants appears to be extremely rare since none of these alleles were identified in a randomly selected control population of 1048 individuals. © 2006 Wiley-Liss, Inc. [source]

Masking selected sequence variation by incorporating mismatches into melting analysis probes,

HUMAN MUTATION, Issue 3 2006
Rebecca L. Margraf
Abstract Hybridization probe melting analysis can be complicated by the presence of sequence variation (benign polymorphisms or other mutations) near the targeted mutation. We investigated the use of "masking" probes to differentiate alleles with similar probe melting temperatures. Selected sequence variation was masked by incorporating mismatches (deletion, unmatched nucleotide, or universal base) into hybridization probes at the polymorphic location. Such masking probes create a probe/target mismatch with all possible alleles at the selected polymorphic location. Any allele with additional variation at another site is identified by a lower probe melting temperature than alleles that vary only at the masked position. This "masking technique" was applied to RET protooncogene and HPA6 mutation detection using unlabeled hybridization probes, a saturating dsDNA dye, and high-resolution melting analysis. Masking probes clearly distinguished all targeted mutations from polymorphisms when at least 1 base pair (bp) separated the mutation from the masked variation. We were able to mask polymorphisms immediately adjacent to mutations, except in certain cases, such as those involving single-base deletion probes when both adjacent positions had the same polymorphic nucleotides. The masking probes can also localize mutations to specific codons or nucleotide positions. Masking probes can simplify melting analysis of complex regions and eliminate the need for sequencing. Hum Mutat 27(3), 269,278, 2006. © 2006 Wiley-Liss, Inc. [source]

Microelectronic DNA chip for hereditary hyperferritinemia cataract syndrome, a model for large-scale analysis of disorders of iron metabolism,

HUMAN MUTATION, Issue 2 2006
Francesca Ferrari
Abstract Hereditary hyperferritinemia cataract syndrome (HHCS) is caused by mutations in the regulatory iron responsive element (IRE) in the 5,UTR of the L-ferritin transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to a constitutive upregulation of the protein in tissue and serum. Twenty-nine mutations have been reported within the L-ferritin (FTL) IRE sequence, 21 of which were available to us. In addition, we included in this study three new mutations. Thus, we analyzed 24 mutations spanning over a DNA stretch of 48 nucleotides, including four deletions 2,29 nucleotides long and 20 substitutions, seven of which were conservative transversions. With this unique experimental model we developed a microchip diagnostic platform for identifying known molecular defects in the L-ferritin IRE structure with a microelectronic array approach, which we optimized after studying the effects of various parameters. The system enables electronic deposition of biotinylated amplicons to selected pads. Under optimized conditions, no cross-hybridization was found, even for mutations that affected the same or adjacent nucleotide positions. The same cartridge could be serially hybridized with all the 24 reporter probe sets, which allowed correct genotyping right up until the end of the analysis. Extensive validation on 200 samples in a blinded fashion gave total concordance of results. This pilot study represents a first step toward developing a diagnostic microchip for large-scale analyses for epidemiological studies and screening of mutations associated with iron disorders. Hum Mutat 27(2), 201,208, 2006. © 2006 Wiley-Liss, Inc. [source]

Minisequencing-Based Genotyping of Duffy and ABO Blood Groups for Forensic Purposes

Gianmarco Ferri Ph.D.
ABSTRACT: Duffy and ABO blood group genetic polymorphisms were studied by minisequencing analysis of single-nucleotide polymorphisms (SNPs) at nucleotide positions,33, 125, 265, and 298 of the Duffy gene and at nucleotide positions,261, 297, 467, 646, and 703 of the ABO gene. In an Italian population sample, we found four alleles and seven genotypes for the Duffy and six alleles and 16 genotypes for the ABO systems. The lower limit for reproducible results was 200 pg DNA, with a range of up to 10 ng and an optimum at 1 ng. All of the 16 analyzed inclusive paternity tests were also consistent with parentage and two out of four inconsistencies with parentage cases were excluded by one or more SNPs. Although Duffy and ABO SNP typing show lower informativeness than most current forensic tests, their robustness, the limited population distribution of FY*Fy type, and the sensitivity of the minisequencing technology suggest that these markers can be useful in selected forensic applications. [source]

The genus Adriohydrobia (Hydrobiidae: Gastropoda): polytypic species or polymorphic populations?

T. Wilke
In molluscs, the shell characters have historically played an important role in discrimination among species. However, because of the paucity, variability and degree of homoplasy of shell characters, their sole use for taxonomic and systematic studies is controversial in many groups. In the present paper the genus AdriohydrobiaRadoman, 1973 is used as a paradigm to test relationships of taxa that were considered to be species, mainly on the basis of the shell size variations. We tested whether the genus consists of several sympatric and polytypic species or a single species with polymorphic populations and whether the reported shell size differences, on which the description of three putative species is mainly based, are intrinsic or extrinsic. A fragment of the mitochondrial cytochrome oxidase I (COI) gene was used as an independent genetic marker. We found very little genetic variability in 40 specimens from four populations studied. The nucleotide-sequence diversity (,) within populations ranges from 0.0017 to 0.0056 and the nucleotide-sequence divergence (Dxy) between populations from 0.0018 to 0.0051. The phylogenetic network is very compact with two ,groups' of haplotypes that are separated by only two nucleotide positions. A plot of pairwise nucleotide differences against pairwise shell size differences did not reveal any distinct clusters and a Mantel test did not show any significant associations between the two matrices. Based on the very low genetic diversity, the lack of distinct clusters in the phylogenetic network and the lack of concordance between morphological and genetic differentiation it is concluded that only one species is involved, Adriohydrobia gagatinella. The previously reported morphogroups within Adriohydrobia are probably due to a discrete age structure in these population and/or due to the effect of trematode-induced gigantism. The observed genetic patterns in Adriohydrobia indicate a rapid population growth from an ancestral population of small evolutionary-effective size. The present study stresses the importance of testing species-level hypotheses based on shell characters using one or more independent markers. Die Gattung Adriohydrobia (Hydrobiidae: Gastropoda): polytypische Arten oder polymorphe Populationen? Schalenmerkmale spielen historisch eine wichtige Rolle bei der Bestimmung von Molluskenarten. Die alleinige Nutzung von Schalenmerkmalen für systematische und taxonomische Arbeiten ist jedoch in vielen Gruppen umstritten, da die relativ wenigen Schalenmerkmale oft sehr variabel und durch einen hohen Grad von Homoplasie gekennzeichnet sind. In der vorliegenden Arbeit wurde die Gattung AdriohydrobiaRadoman, 1973 als Fallbeispiel genutzt, um Beziehungen von Arten innerhalb einer Gattung zu untersuchen, die hauptsächlich anhand ihrer Schalengröße unterschieden werden. Es wurde getestet, ob die Gattung mehrere sympatrische und polytypische Arten oder nur eine Art mit polymorphen Populationen umfasst. Darüber hinaus wurde untersucht, ob die dokumentierten Unterschiede in der Schalenhöhe, auf welchen die Beschreibung der drei potentiellen Arten der Gattung hauptsächlich beruhte, intrinsisch oder extrinsisch sind. Als unabhängiger genetischer Marker wurde ein Fragment des mitochondrialen Gens für Cytochromoxidase I (COI) verwendet. Die untersuchten 40 Individuen von vier Populationen zeichneten sich durch eine nur sehr geringe genetische Variabilität aus. Die Nukleotidsequenz-Diversität (,) innerhalb der Populationen variiert zwischen 0.0017 und 0.0056; die Nukleotidsequenz-Divergenz (Dxy) zwischen den Populationen reicht von 0.0018 bi 0.0051. Das phylogenetische Netzwerk ist sehr kompakt und umfasst zwei ,Gruppen' von Haplotypen, welche durch nur zwei Nukleotidpositionen getrennt sind. Die graphische Darstellung von paarweisen Nukleotid-Differenzen gegen paarweise Gehäusegröße-Differenzen lässt keine diskreten Gruppen erkennen und ein Mantel-Test zeigt keine signifikanten Beziehungen zwischen den Matrices. Aufgrund der geringen genetischen Differenzierung, des Fehlens von diskreten Gruppen im phylogenetischen Netzwerk und des nicht-signifikanten Zusammenhanges von morphologischer and genetischer Differenzierung wird geschlussfolgert, dass nur eine Art involviert ist, Adriohydrobia gagatinella. Die in der Literatur dokumentierten Morpho-Gruppen beruhen vermutlich auf einer diskreten Altersstruktur in diesen Populationen und/oder auf den Auswirkungen von trematoden-induziertem Gigantismus. Die festgestellten genetischen Muster in Adriohydrobia lassen das schnelle Wachstum einer Stammpopulation von geringer evolutionär-effektiver Größe vermuten. Die vorliegende Studie ist ein Beispiel dafür, wie wichtig es sein kann, auf Schalenmerkmale beruhende Arthypothesen mit unabhängigen Markern zu verifizieren. [source]

DNA barcodes for globally threatened marine turtles: a registry approach to documenting biodiversity

Abstract DNA barcoding is a global initiative that provides a standardized and efficient tool to catalogue and inventory biodiversity, with significant conservation applications. Despite progress across taxonomic realms, globally threatened marine turtles remain underrepresented in this effort. To obtain DNA barcodes of marine turtles, we sequenced a segment of the cytochrome c oxidase subunit I (COI) gene from all seven species in the Atlantic and Pacific Ocean basins (815 bp; n = 80). To further investigate intraspecific variation, we sequenced green turtles (Chelonia mydas) from nine additional Atlantic/Mediterranean nesting areas (n = 164) and from the Eastern Pacific (n = 5). We established character-based DNA barcodes for each species using unique combinations of character states at 76 nucleotide positions. We found that no haplotypes were shared among species and the mean of interspecific variation ranged from 1.68% to 13.0%, and the mean of intraspecific variability was relatively low (0,0.90%). The Eastern Pacific green turtle sequence was identical to an Australian haplotype, suggesting that this marker is not appropriate for identifying these phenotypically distinguishable populations. Analysis of COI revealed a north,south gradient in green turtles of Western Atlantic/Mediterranean nesting areas, supporting a hypothesis of recent dispersal from near equatorial glacial refugia. DNA barcoding of marine turtles is a powerful tool for species identification and wildlife forensics, which also provides complementary data for conservation genetic research. [source]

amfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus

Haruka Yamazaki
Summary In Streptomyces griseus, A-factor (2-isocapryloyl-3R -hydroxymethyl-,-butyrolactone) acts as a chemical signalling molecule that triggers morphological differentiation and secondary metabolism. A transcriptional activator, AdpA, in the A-factor regulatory cascade switches on a number of genes required for both processes, thus forming an AdpA regulon. amfR encoding a regulatory protein similar to response regulators of bacterial two-component regulatory systems and essential for aerial mycelium formation was found to be a member of the AdpA regulon. AdpA bound two sites at nucleotide positions approximately ,200 (site 1) and ,60 (site 2), with respect to the major transcriptional start point of amfR, and accelerated the transcription of amfR by assisting RNA polymerase in forming an open complex at an appropriate region including the transcriptional start point. Site 2 contributed more to the transcriptional activation of amfR by AdpA than site 1, although AdpA showed a much lower affinity to site 2 than to site 1. The amfR transcription enhanced by AdpA subsequently ceased at day 2 when aerial hyphae began to be formed in the wild-type strain, whereas in an adsA null mutant amfR was continuously transcribed even until day 3. This implied that amfR was repressed growth dependently by a gene product under the control of ,-AdsA. Transcription of the promoter upstream of amfT depended on amfR, which is consistent with the idea that AmfR serves as an activator for amfTSBA in the amf operon. The observations that the amfR gene contains a TTA codon, a potential target for bldA -mediated regulation, and a conserved Asp-54 residue, which might be phosphorylated by a sensor kinase, suggest that the amf operon is under transcriptional, translational and post-translational control systems. [source]

Simulating evolution by gene duplication of protein features that require multiple amino acid residues

PROTEIN SCIENCE, Issue 10 2004
Michael J. Behe
MR, multiresidue Abstract Gene duplication is thought to be a major source of evolutionary innovation because it allows one copy of a gene to mutate and explore genetic space while the other copy continues to fulfill the original function. Models of the process often implicitly assume that a single mutation to the duplicated gene can confer a new selectable property. Yet some protein features, such as disulfide bonds or ligand binding sites, require the participation of two or more amino acid residues, which could require several mutations. Here we model the evolution of such protein features by what we consider to be the conceptually simplest route,point mutation in duplicated genes. We show that for very large population sizes N, where at steady state in the absence of selection the population would be expected to contain one or more duplicated alleles coding for the feature, the time to fixation in the population hovers near the inverse of the point mutation rate, and varies sluggishly with the ,th root of 1/N, where , is the number of nucleotide positions that must be mutated to produce the feature. At smaller population sizes, the time to fixation varies linearly with 1/N and exceeds the inverse of the point mutation rate. We conclude that, in general, to be fixed in 108 generations, the production of novel protein features that require the participation of two or more amino acid residues simply by multiple point mutations in duplicated genes would entail population sizes of no less than 109. [source]

Sequence diversity and rates of molecular evolution between sheep and cattle genes

J. W. Kijas
Summary Experiments that aim to identify genes of importance in sheep are currently inhibited by a paucity of genomic resources. One approach, therefore, is to exploit the wealth of data and associated capabilities becoming available for the bovine genome. Cross-species application of microarrays and comparative sequencing to identify single nucleotide polymorphisms are two possibilities; however, both are dependant on the level of nucleotide sequence similarity between the two species. This study used 120 gene orthologues consisting of over 60 kb of aligned sequence to estimate the gene diversity between cattle and sheep. Less than 3% of protein-coding nucleotide positions were found to be different, indicating that the prospect for successfully using cross-species strategies is high. Substitution at synonymous sites ranged between 6.9 and 7.7% (± 0.3%), and was higher than at non-synonymous sites (1.4,1.7 ± 0.1%). The relative rate test was used to determine whether the observed mutation rates were constant between the two lineages. While the rate at synonymous sites appeared constant, the rate at non-synonymous sites was significantly higher within the caprinae lineage (sheep) when compared with bovinae (cattle; ,2 = 10.03; d.f. = 1, P < 0.01). This is the first demonstration that variable rates of molecular evolution may be present within the family Bovidae. [source]

cDNA cloning and genetic polymorphism of the swine major histocompatibility complex (SLA) class II DMA gene

A. Ando
cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM , chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes , chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR,restriction fragment length polymorphism (PCR,RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma. [source]