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Sample records for ii gene cosegregation

  1. Lack of cosegregation of the subgroup II antigens on genes 2 and 6 in porcine rotaviruses.

    PubMed Central

    Svensson, L; Padilla-Noriega, L; Taniguchi, K; Greenberg, H B

    1990-01-01

    The rotavirus subgroup I and II specificities associated with gene 2 and 6 products (vp2 and vp6, respectively) were shown not to cosegregate in a number of porcine rotavirus strains. The porcine OSU rotavirus strain and OSU-vp7-like strains were all found to possess a subgroup II-specific region on vp2 and a subgroup I-specific region on vp6. Of interest is the observation that the subgroup II-specific epitope on vp2 appears to be present only in human and porcine rotavirus strains, suggesting a possible human-pig ancestral lineage for gene 2. Images PMID:1688386

  2. A novel point mutation in the translation initiation codon of the pre-pro-vasopressin-neurophysin II gene: Cosegregation with morphological abnormalities and clinical symptoms in autosomal dominant neurohypophyseal diabetes insipidus

    SciTech Connect

    Rutishauser, J.; Boeni-Schnetzler, M.; Froesch, E.R.; Wichmann, W.; Huisman, T.

    1996-01-01

    Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare variant of idiopathic central diabetes insipidus. Several different mutations in the human vasopressin-neurophysin II (AVP-NP II) gene have been described. We studied nine family members from three generations of an ADNDI pedigree at the clinical, morphological, and molecular levels. AVP concentrations were measured during diagnostic fluid restriction tests. Coronal and sagittal high resolution T1-weighted images of the pituitary were obtained from affected and healthy family members. PCR was used to amplify the AVP-NP II precursor gene, and PCR products were directly sequenced. Under maximal osmotic stimulation, AVP serum levels were close to or below the detection limit in affected individuals. Magnetic resonance imaging studies revealed the characteristic hyperintense ({open_quotes}bright spot{close_quotes}) appearance of the posterior pituitary in two healthy family members. This signal was absent in all four ADNDI patients examined. The coding sequences of AVP and its carrier protein, neurophysin II, were normal in all family members examined. Affected individuals showed a novel single base deletion (G 227) in the translation initiation codon of the AVP-NP II signal peptide on one allele. The mutation in the AVP-NP II leader sequence appears to be responsible for the disease in this kindred, possibly by interfering with protein translocation. The absence of the hyperintense posterior pituitary signal in affected individuals could reflect deficient posterior pituitary function. 56 refs., 4 figs., 3 tabs.

  3. A Unique Mutation in a MYB Gene Cosegregates with the Nectarine Phenotype in Peach

    PubMed Central

    Dondini, Luca; Pacheco, Igor; Dettori, Maria Teresa; Gazza, Laura; Scalabrin, Simone; Strozzi, Francesco; Tartarini, Stefano; Bassi, Daniele; Verde, Ignazio; Rossini, Laura

    2014-01-01

    Nectarines play a key role in peach industry; the fuzzless skin has implications for consumer acceptance. The peach/nectarine (G/g) trait was described as monogenic and previously mapped on chromosome 5. Here, the position of the G locus was delimited within a 1.1 cM interval (635 kb) based on linkage analysis of an F2 progeny from the cross ‘Contender’ (C, peach) x ‘Ambra’ (A, nectarine). Careful inspection of the genes annotated in the corresponding genomic sequence (Peach v1.0), coupled with variant discovery, led to the identification of MYB gene PpeMYB25 as a candidate for trichome formation on fruit skin. Analysis of genomic re-sequencing data from five peach/nectarine accessions pointed to the insertion of a LTR retroelement in exon 3 of the PpeMYB25 gene as the cause of the recessive glabrous phenotype. A functional marker (indelG) developed on the LTR insertion cosegregated with the trait in the CxA F2 progeny and was validated on a broad panel of genotypes, including all known putative donors of the nectarine trait. This marker was shown to efficiently discriminate between peach and nectarine plants, indicating that a unique mutational event gave rise to the nectarine trait and providing a useful diagnostic tool for early seedling selection in peach breeding programs. PMID:24595269

  4. A Co-segregating Microduplication of Chromosome 15q11.2 Pinpoints Two Risk Genes for Autism Spectrum Disorder

    PubMed Central

    van der Zwaag, Bert; Staal, Wouter G; Hochstenbach, Ron; Poot, Martin; Spierenburg, Henk A; de Jonge, Maretha V; Verbeek, Nienke E; van ’t Slot, R.; van Es, Michael A; Staal, Frank J; Freitag, Christine M; Buizer-Voskamp, Jacobine E; Nelen, Marcel R; van den Berg, Leonard H; van Amstel, Hans K Ploos; van Engeland, Herman; Burbach, J Peter H

    2010-01-01

    High resolution genomic copy-number analysis has shown that inherited and de novo copy-number variations contribute significantly to autism pathology, and that identification of small chromosomal aberrations related to autism will expedite the discovery of risk genes involved. Here, we report a microduplication of chromosome 15q11.2, spanning only four genes, co-segregating with autism in a Dutch pedigree, identified by SNP microarray analysis, and independently confirmed by FISH and MLPA analysis. Quantitative RT-PCR analysis revealed over 70 % increase in peripheral blood mRNA levels for the four genes present in the duplicated region in patients, and RNA in situ hybridization on mouse embryonic and adult brain sections revealed that two of the four genes, CYFIP1 and NIPA1, were highly expressed in the developing mouse brain. These findings point towards a contribution of microduplications at chromosome 15q11.2 to autism, and highlight CYFIP1 and NIPA1 as autism risk genes functioning in axonogenesis and synaptogenesis. Thereby, these findings further implicate defects in dosage-sensitive molecular control of neuronal connectivity in autism. However, the prevalence of this microduplication in patient samples was statistically not significantly different from control samples (0.94% in patients vs 0.42% controls, p=0.247), which suggests that our findings should be interpreted with caution and indicates the need for studies that include large numbers of control subjects to ascertain the impact of these changes on a population scale. PMID:20029941

  5. A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass.

    PubMed

    Manzanares, Chloé; Barth, Susanne; Thorogood, Daniel; Byrne, Stephen L; Yates, Steven; Czaban, Adrian; Asp, Torben; Yang, Bicheng; Studer, Bruno

    2016-04-01

    The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic self-incompatibility (SI) mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. Here, we report a fine-mapping approach to identify the male component of the S-locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins. Our results represent a major step forward toward understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S-locus.

  6. The powdery mildew resistance gene REN1 co-segregates with an NBS-LRR gene cluster in two Central Asian grapevines

    PubMed Central

    2009-01-01

    Background Grape powdery mildew is caused by the North American native pathogen Erysiphe necator. Eurasian Vitis vinifera varieties were all believed to be susceptible. REN1 is the first resistance gene naturally found in cultivated plants of Vitis vinifera. Results REN1 is present in 'Kishmish vatkana' and 'Dzhandzhal kara', two grapevines documented in Central Asia since the 1920's. These cultivars have a second-degree relationship (half sibs, grandparent-grandchild, or avuncular), and share by descent the chromosome on which the resistance allele REN1 is located. The REN1 interval was restricted to 1.4 cM using 38 SSR markers distributed across the locus and the segregation of the resistance phenotype in two progenies of collectively 461 offspring, derived from either resistant parent. The boundary markers delimit a 1.4-Mbp sequence in the PN40024 reference genome, which contains 27 genes with known functions, 2 full-length coiled-coil NBS-LRR genes, and 9 NBS-LRR pseudogenes. In the REN1 locus of PN40024, NBS genes have proliferated through a mixture of segmental duplications, tandem gene duplications, and intragenic recombination between paralogues, indicating that the REN1 locus has been inherently prone to producing genetic variation. Three SSR markers co-segregate with REN1, the outer ones confining the 908-kb array of NBS-LRR genes. Kinship and clustering analyses based on genetic distances with susceptible cultivars representative of Central Asian Vitis vinifera indicated that 'Kishmish vatkana' and 'Dzhandzhal kara' fit well into local germplasm. 'Kishmish vatkana' also has a parent-offspring relationship with the seedless table grape 'Sultanina'. In addition, the distant genetic relatedness to rootstocks, some of which are derived from North American species resistant to powdery mildew and have been used worldwide to guard against phylloxera since the late 1800's, argues against REN1 being infused into Vitis vinifera from a recent interspecific

  7. Co-segregation analysis and mapping of the anthracnose Co-10 and angular leaf spot Phg-ON disease resistance genes in common bean cultivar Ouro Negro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anthracnose (ANT) and angular leaf spot (ALS) are devastating diseases of common bean. Ouro Negro is a highly productive Mesoamerican black-seeded common bean cultivar possessing the dominant Co-10 and Phg-ON genes that confer resistance to ANT and ALS, respectively. In this study we elucidate the ...

  8. Schizophrenia and the androgen receptor gene: Report of a sibship showing co-segregation with Reifenstein Syndrome but no evidence for linkage in 23 multiply affected families

    SciTech Connect

    Arranz, M.; Sharma, T.; Sham, P.; Kerwin, R.

    1995-10-09

    Crow et al. have reported excess sharing of alleles by male sibling pairs with schizophrenia, at a triplet repeat marker within the androgen receptor gene, indicating that mutations at or near this gene may be a risk factor for males. In this report, we describe a pair of male siblings concordant for both schizophrenia and Reifenstein syndrome, which is caused by a mutation in this gene. This provides support for the hypothesis that the androgen receptor may contribute to liability to develop schizophrenia. Because of this, we have examined a collection of 23 pedigrees multiply affected by schizophrenia for linkage to the androgen receptor. We have found no evidence for linkage by both the LOD score and affected sibling-pair methods, under a range of genetic models with a broad and narrow definition of phenotype, and when families with male-to-male transmission are excluded. However, because of the small number of informative male-male pairs in our sample, we cannot confirm or refute the excess allele sharing for males reported by Crow. 35 refs., 1 fig., 2 tabs.

  9. Dupuytren's Contracture Cosegregation with Limb-Girdle Muscle Dystrophy

    PubMed Central

    Lace, Baiba; Inashkina, Inna; Micule, Ieva; Vasiljeva, Inta; Naudina, Maruta Solvita; Jankevics, Eriks

    2013-01-01

    Limb-girdle muscular dystrophies (LGMDs) is a heterogeneous group of muscular dystrophies that mostly affect the pelvic and shoulder girdle muscle groups. We report here a case of neuromuscular disease associated with Dupuytren's contracture, which has never been described before as cosegregating with an autosomal dominant type of inheritance. Dupuytren's contracture is a common disease, especially in Northern Europe. Comorbid conditions associated with Dupuytren's contracture are repetitive trauma to the hands, diabetes, and seizures, but it has never before been associated with neuromuscular disease. We hypothesize that patients may harbor mutations in genes with functions related to neuromuscular disease and Dupuytren's contracture development. PMID:24024053

  10. Cosegregation of Christmas disease and major affective disorder in a pedigree.

    PubMed

    Gill, M; Castle, D; Duggan, C

    1992-01-01

    Three males with factor-IX deficiency (Christmas disease) in one pedigree all had severe affective disorder. This apparent cosegregation, if true, would support the hypothesis that in some pedigrees, a gene for major affective disorder is located on the X chromosome.

  11. Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer.

    PubMed

    Torchard, D; Blanchet-Bardon, C; Serova, O; Langbein, L; Narod, S; Janin, N; Goguel, A F; Bernheim, A; Franke, W W; Lenoir, G M

    1994-01-01

    Epidermolytic palmoplantar keratosis (EPPK) cosegregates with breast and ovarian cancers in a large French pedigree, raising the possibility that a single genetic mutation might cause these conditions and offering a potential lead to the identification of a hereditary breast/ovarian cancer gene. We have performed linkage analysis and show that the EPPK locus lies on the long arm of chromosome 17 near the type I keratin gene cluster and the proposed breast cancer gene (BRCA1). The type I keratin 9 gene has been partially sequenced in four affected individuals. A single base mutation within the rod domain of the protein cosegregates with EPPK in all affected individuals tested. Although inheritance of this mutation is likely responsible for EPPK, it is unlikely to be the cause of the breast and ovarian cancer.

  12. Regulation of major histocompatibility complex class II genes

    PubMed Central

    Choi, Nancy M.; Majumder, Parimal; Boss, Jeremy M.

    2010-01-01

    Summary The major histocompatibility complex class II (MHC-II) genes are regulated at the level of transcription. Recent studies have shown that chromatin modification is critical for efficient transcription of these genes, and a number of chromatin modifying complexes recruited to MHC-II genes have been described. The MHC-II genes are segregated from each other by a series of chromatin elements, termed MHC-II insulators. Interactions between MHC-insulators and the promoters of MHC-II genes are mediated by the insulator factor CCCTC-binding protein and are critical for efficient expression. This regulatory mechanism provides a novel view of how the entire MHC-II locus is assembled architecturally and can be coordinately controlled. PMID:20970972

  13. Mutation in CEP63 co-segregating with developmental dyslexia in a Swedish family.

    PubMed

    Einarsdottir, Elisabet; Svensson, Idor; Darki, Fahimeh; Peyrard-Janvid, Myriam; Lindvall, Jessica M; Ameur, Adam; Jacobsson, Christer; Klingberg, Torkel; Kere, Juha; Matsson, Hans

    2015-11-01

    Developmental dyslexia is the most common learning disorder in children. Problems in reading and writing are likely due to a complex interaction of genetic and environmental factors, resulting in reduced power of studies of the genetic factors underlying developmental dyslexia. Our approach in the current study was to perform exome sequencing of affected and unaffected individuals within an extended pedigree with a familial form of developmental dyslexia. We identified a two-base mutation, causing a p.R229L amino acid substitution in the centrosomal protein 63 kDa (CEP63), co-segregating with developmental dyslexia in this pedigree. This mutation is novel, and predicted to be highly damaging for the function of the protein. 3D modelling suggested a distinct conformational change caused by the mutation. CEP63 is localised to the centrosome in eukaryotic cells and is required for maintaining normal centriole duplication and control of cell cycle progression. We found that a common polymorphism in the CEP63 gene had a significant association with brain white matter volume. The brain regions were partly overlapping with the previously reported region influenced by polymorphisms in the dyslexia susceptibility genes DYX1C1 and KIAA0319. We hypothesise that CEP63 is particularly important for brain development and might control the proliferation and migration of cells when those two events need to be highly coordinated.

  14. The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

    PubMed Central

    Evers, R; Cornelissen, A W

    1990-01-01

    We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit. Images PMID:2169604

  15. [Osteochondrodysplasia determined genetically by a collagen type II gene mutation].

    PubMed

    Czarny-Ratajczak, M; Rogala, P; Wolnik-Brzozowska, D; Latos-Bieleńska, A

    2001-01-01

    Chondrodysplasias are a heterogenous group of skeletal dysplasias, affecting the growing cartilage. The main part of chondrodysplasias is caused by mutations in various types of collagen genes. The current classification within this group of disorder relies on clinical, histological and radiographic features. Type II collagenopathies comprise part of chondrodysplasias, consisting of hereditary disorders caused by defects in the type II collagen. Collagen type II is coded by a large gene--COL2A1. The chromosomal location for the human COL2A1 gene is 12q13.11-q13.12. Defects in collagen type II are caused by point mutations in the COL2A1 gene. Type II collagenopathies form a wide spectrum of clinical severity ranging from lethal achondrogenesis type II, hypochondrogenesis, through severe forms like spondyloepiphyseal dysplasia congenita, spondyloepimetaphyseal dysplasia congenita, Marshall syndrome, to the mild forms--Stickler syndrome and early osteoarthritis. The pathological changes in the patients are observed in the growth plate, nucleus pulposus and vitreous body, where the abnormal collagen type II is distributed. This article presents the genetic background of collagenopathies type II and the results of current molecular studies of the patients. Both the molecular and the clinical studies may promise a better understanding of the relationship between the genotype and the phenotype. We present the patients, who were diagnosed at the Department of Medical Genetics and in the Orthopaedic Department in Poznań. PMID:11481990

  16. Cohesin regulates MHC class II genes through interactions with MHC class II insulators.

    PubMed

    Majumder, Parimal; Boss, Jeremy M

    2011-10-15

    Cohesin is a multiprotein, ringed complex that is most well-known for its role in stabilizing the association of sister chromatids between S phase and M. More recently, cohesin was found to be associated with transcriptional insulators, elements that are associated with the organization of chromatin into regulatory domains. The human MHC class II (MHC-II) locus contains 10 intergenic elements, termed MHC-II insulators, which bind the transcriptional insulator protein CCCTC-binding factor. MHC-II insulators interact with each other, forming a base architecture of discrete loops and potential regulatory domains. When MHC-II genes are expressed, their proximal promoter regulatory regions reorganize to the foci established by the interacting MHC-II insulators. MHC-II insulators also bind cohesin, but the functional role of cohesin in regulating this system is not known. In this article, we show that the binding of cohesin to MHC-II insulators occurred irrespective of MHC-II expression but was required for optimal expression of the HLA-DR and HLA-DQ genes. In a DNA-dependent manner, cohesin subunits interacted with CCCTC-binding factor and the MHC-II-specific transcription factors regulatory factor X and CIITA. Intriguingly, cohesin subunits were important for DNA looping interactions between the HLA-DRA promoter region and a 5' MHC-II insulator but were not required for interactions between the MHC-II insulators themselves. This latter observation introduces cohesin as a regulator of MHC-II expression by initiating or stabilizing MHC-II promoter regulatory element interactions with the MHC-II insulator elements, events that are required for maximal MHC-II transcription.

  17. Phylogenetic relationships of class II fumarase genes from trichomonad species.

    PubMed

    Gerbod, D; Edgcomb, V P; Noël, C; Vanácová, S; Wintjens, R; Tachezy, J; Sogin, M L; Viscogliosi, E

    2001-08-01

    Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species. All residues known to be highly conserved in this enzyme were present. Nuclear run-on assays showed that one of the two genes identified in Tritrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis. These findings corroborate previous biochemical data. Fumarase genes were also expressed in Monocercomonas sp. and Tetratrichomonas gallinarum but not in Pentatrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used. Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes. The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria. Furthermore, they are basal to other eukaryotes in rRNA analyses. However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal. Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes. All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution. In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs. This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes. The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in

  18. Phylogenetic relationships of class II fumarase genes from trichomonad species.

    PubMed

    Gerbod, D; Edgcomb, V P; Noël, C; Vanácová, S; Wintjens, R; Tachezy, J; Sogin, M L; Viscogliosi, E

    2001-08-01

    Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species. All residues known to be highly conserved in this enzyme were present. Nuclear run-on assays showed that one of the two genes identified in Tritrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis. These findings corroborate previous biochemical data. Fumarase genes were also expressed in Monocercomonas sp. and Tetratrichomonas gallinarum but not in Pentatrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used. Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes. The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria. Furthermore, they are basal to other eukaryotes in rRNA analyses. However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal. Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes. All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution. In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs. This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes. The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in

  19. HLA class II genes: typing by DNA analysis.

    PubMed

    Bidwell, J L; Bidwell, E A; Bradley, B A

    1990-04-01

    A detailed understanding of the structure and function of the human major histocompatibility complex (MHC) has ensued from studies by molecular biologist during the last decade. Virtually all of the HLA genes have now been cloned, and the nucleotide sequences of their different allelic forms have been determined. Typing for these HLA alleles is a fundamental prerequisite for tissue matching in allogeneic organ transplantation. Until very recently, typing procedures have been dominated by serological and cellular methods. The availability of cloned DNA from HLA genes has now permitted the technique of restriction fragment length polymorphism (RFLP) analysis to be applied, with remarkable success and advantage, to phenotyping of both HLA Class I and Class II determinants. For the HLA Class II genes DR and DQ, a simple two-stage RFLP analysis permits the accurate identification of all specificities defined by serology, and of many which are defined by cellular typing. At the present time, however, RFLP typing of HLA Class I genes is not as practicable or as informative as that for HLA Class II genes. The present clinical applications of HLA-DR and DQ RFLP typing are predominantly in phenotyping of living donors, including selection of HLA-matched volunteer bone marrow donors, in allograft survival studies, and in studies of HLA Class II-associated diseases. However, the time taken to perform RFLP analysis precludes its use for the typing of cadaveric kidney donors. Nucleotide sequence data for the alleles of HLA Class II genes have now permitted the development of allele-specific oligonucleotide (ASO) typing, a second category of DNA analysis. This has been greatly facilitated by the ability to amplify specific HLA Class II DNA 'target' sequences using the polymerase chain reaction (PCR) technique. The accuracy of DNA typing techniques should ensure that this methodology will eventually replace conventional HLA phenotyping.

  20. Characterization of Schizothorax prenanti cgnrhII gene: fasting affects cgnrhII expression.

    PubMed

    Wang, T; Yuan, D; Zhou, C; Lin, F; Chen, H; Wu, H; Wei, R; Xin, Z; Liu, J; Gao, Y; Chen, D; Yang, S; Pu, Y; Li, Z

    2014-08-01

    In this study, the role of chicken gonadotropin-releasing hormone II (cgnrhII) in feeding regulation was investigated in Schizothorax prenanti. First, the full-length S. prenanti cgnrhII cDNA consisted of 693 bp with an open reading frame of 261 bp encoding a protein of 86 amino acids. Next, cgnrhII was widely expressed in the central and peripheral tissues. Last, there were significant changes in cgnrhII mRNA expression in the fasted group compared to the fed group in the S. prenanti hypothalamus during 24 h fasting (P < 0.05). Furthermore, the cgnrhII gene expression presented a significant decrease in the fasted group compared with the fed group (P < 0.05) on days 3, 5 and 7, after re-feeding, there was no significant changes in cgnrhII mRNA expression level between refed and fed group on day 9 (P > 0.05). Thus, the results suggest that cGnRH II expression is influenced by fasting and the gene may be involved in feeding regulation in S. prenanti.

  1. Mice lacking all conventional MHC class II genes

    PubMed Central

    Madsen, Lars; Labrecque, Nathalie; Engberg, Jan; Dierich, Andrée; Svejgaard, Arne; Benoist, Christophe; Mathis, Diane; Fugger, Lars

    1999-01-01

    MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCIIΔ/Δ mice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of Aα and Aβ knockout animals, notably a dearth of CD4+ lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCIIΔ/Δ mice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering “humanized” mouse models of human MHC-II-associated autoimmune disorders. PMID:10468609

  2. Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

    PubMed Central

    Hillenmeyer, Maureen E.; Vandova, Gergana A.; Berlew, Erin E.; Charkoudian, Louise K.

    2015-01-01

    Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties. PMID:26499248

  3. Interactions between the Class II Transactivator and CREB Binding Protein Increase Transcription of Major Histocompatibility Complex Class II Genes

    PubMed Central

    Fontes, Joseph D.; Kanazawa, Satoshi; Jean, Dickson; Peterlin, B. Matija

    1999-01-01

    Class II major histocompatibility (class II) genes are regulated in a B-cell-specific and gamma interferon-inducible fashion. The master switch for the expression of these genes is the class II transactivator (CIITA). In this report, we demonstrate that one of the functions of CIITA is to recruit the CREB binding protein (CBP) to class II promoters. Not only functional but also specific binding interactions between CIITA and CBP were demonstrated. Moreover, a dominant negative form of CBP decreased the activity of class II promoters and levels of class II determinants on the surface of cells. Finally, the inhibition of class II gene expression by the glucocorticoid hormone could be attributed to the squelching of CBP by the glucocorticoid receptor. We conclude that CBP, a histone acetyltransferase, plays an important role in the transcription of class II genes. PMID:9858618

  4. Bacterial control of host gene expression through RNA polymerase II

    PubMed Central

    Lutay, Nataliya; Ambite, Ines; Hernandez, Jenny Grönberg; Rydström, Gustav; Ragnarsdóttir, Bryndís; Puthia, Manoj; Nadeem, Aftab; Zhang, Jingyao; Storm, Petter; Dobrindt, Ulrich; Wullt, Björn; Svanborg, Catharina

    2013-01-01

    The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coli develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II–dependent (Pol II–dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coli 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II–dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur. PMID:23728172

  5. Screening of three Usher syndrome type II candidate genes

    SciTech Connect

    Bloemker, B.K.; Swaroop, A.; Kimberling, W.J.

    1994-09-01

    Usher syndrome type II (US2) is an autosomal recessive disorder that results in blindness due to retinitis pigmentosa and congenital hearing loss. The disease affects approximately 1 in 20,000 individuals in the general population and is responsible for over 50% of all cases of deafness with blindness. The underlying US2 defect is unknown. The US2 gene has been localized to the 1q41 region of chromosome 1 by linkage studies. Three genes previously localized to 1q were analyzed to assess their candidacy as the US2 gene. These were evaluated by PCR assays using DNA from a YAC contig spanning the US2 region on chromosome 1. The first gene evaluated was the human choroideremia-like gene (hCHML), which had been mapped to chromosome 1q. The sequence on 1q is a homologue of the human choroideremia gene on chromosome X. Choroideremia is a degenerative disorder causing ocular pathology similar to that observed in US2 patients. Therefore, hCHML is a candidate for the US2 gene. Two cDNAs (A and B) from an enriched human retinal pigment epithelium library have been mapped to 1q41 by in situ hybridization. Both cDNAs are considered good candidates. The hCHML and cDNA A were ruled out as candidates for the US2 gene based on negative results from PCR assays performed on YACs spanning the US2 region. cDNA B could not be ruled out as a candidate for the US2 gene by these assays. Answers to many clinical questions regarding US2 will only be resolved after the gene is identified and characterized. Eventually, understanding the function and expression of the US2 gene will provide a basis for the development of therapy.

  6. Overview of BioCreative II gene mention recognition

    PubMed Central

    Smith, Larry; Tanabe, Lorraine K; Ando, Rie Johnson nee; Kuo, Cheng-Ju; Chung, I-Fang; Hsu, Chun-Nan; Lin, Yu-Shi; Klinger, Roman; Friedrich, Christoph M; Ganchev, Kuzman; Torii, Manabu; Liu, Hongfang; Haddow, Barry; Struble, Craig A; Povinelli, Richard J; Vlachos, Andreas; Baumgartner, William A; Hunter, Lawrence; Carpenter, Bob; Tsai, Richard Tzong-Han; Dai, Hong-Jie; Liu, Feng; Chen, Yifei; Sun, Chengjie; Katrenko, Sophia; Adriaans, Pieter; Blaschke, Christian; Torres, Rafael; Neves, Mariana; Nakov, Preslav; Divoli, Anna; Maña-López, Manuel; Mata, Jacinto; Wilbur, W John

    2008-01-01

    Nineteen teams presented results for the Gene Mention Task at the BioCreative II Workshop. In this task participants designed systems to identify substrings in sentences corresponding to gene name mentions. A variety of different methods were used and the results varied with a highest achieved F1 score of 0.8721. Here we present brief descriptions of all the methods used and a statistical analysis of the results. We also demonstrate that, by combining the results from all submissions, an F score of 0.9066 is feasible, and furthermore that the best result makes use of the lowest scoring submissions. PMID:18834493

  7. State-of-the-art human gene therapy: part II. Gene therapy strategies and clinical applications.

    PubMed

    Wang, Dan; Gao, Guangping

    2014-09-01

    In Part I of this Review (Wang and Gao, 2014), we introduced recent advances in gene delivery technologies and explained how they have powered some of the current human gene therapy applications. In Part II, we expand the discussion on gene therapy applications, focusing on some of the most exciting clinical uses. To help readers to grasp the essence and to better organize the diverse applications, we categorize them under four gene therapy strategies: (1) gene replacement therapy for monogenic diseases, (2) gene addition for complex disorders and infectious diseases, (3) gene expression alteration targeting RNA, and (4) gene editing to introduce targeted changes in host genome. Human gene therapy started with the simple idea that replacing a faulty gene with a functional copy can cure a disease. It has been a long and bumpy road to finally translate this seemingly straightforward concept into reality. As many disease mechanisms unraveled, gene therapists have employed a gene addition strategy backed by a deep knowledge of what goes wrong in diseases and how to harness host cellular machinery to battle against diseases. Breakthroughs in other biotechnologies, such as RNA interference and genome editing by chimeric nucleases, have the potential to be integrated into gene therapy. Although clinical trials utilizing these new technologies are currently sparse, these innovations are expected to greatly broaden the scope of gene therapy in the near future.

  8. Molecular characterization of chicken class II transactivator gene.

    PubMed

    Nikbakht Brujeni, Gholamreza; Khosravi, Mohammad

    2015-01-01

    Class II transactivator (CIITA) is an effective transcriptional factor regulating various genes in the immune system. Since the detection of CIITA in 1993, there has been considerable progress toward understanding its role as an activator of MHC II genes in human and mouse; however, there is little knowledge of this gene in other animals such as chicken. Molecular characterization of the chicken CIITA gene transcript was performed to determine its sequence and expression in different tissues. The CIITA cDNA was first generated through reverse transcriptase-polymerase chain reaction (RT-PCR) from Cobb chicken spleen cell RNA, using oligonucleotide primers based on the predicted cDNA sequence. The effect of the immune system stimulation on the CIITA gene expression in kidney, liver, thymus, and spleen were assessed by semi-quantitative RT-PCR analysis. A partial cDNA sequence (1,688 bp) encoding part of the NACHT domain followed by seven of the transactivator and one of the NLS domains were obtained. Comparison of the deduced amino acid sequence with other CIITAs reveals high level of similarities in amino acid composition, secondary structure and phosphorylation sites. Furthermore, in comparison to the Red Jungle Fowl (RJF) sequence, we found 17 single nucleotide polymorphisms in Cobb broiler chicken, ten of which were reported for the first time. Gene expression analysis indicated that CIITA RNA amounts increased in all the examined tissues following stimulation with Brucella antigen. This investigation may indicate that CIITA molecule has an important role in the chicken immune responses as well as human and other animals.

  9. Chromosomal localization of human RNA polymerase II subunit genes

    SciTech Connect

    Acker, J.; Wintzerith, M.; Vigneron, M.; Kedinger, C. ); Mattei, M.G.; Roeckel, N.; Depetris, D. )

    1994-04-01

    The eukaryotic DNA-dependent RNA polymerase II (or B) is composed of 10 to 14 polypeptides ranging from 220 to 10 kDa. To gain further insight into the molecular structure and function of these subunits, the authors have undertaken the molecular cloning of nucleotide sequences corresponding to the human enzyme. The cDNAs of five subunits (hRPB220, hRPB140, hRPB33, hRPB25, and hRPB14.5) have been isolated. Using in situ hybridization, they show that the genes of these subunits have distinct chromosomal locations (17p13, 4q12, 16q13-q21, 19p13.3, and 19q12, respectively). Thus, if assembly of active polymerase molecules requires coordinated expression from these independent genes, mechanisms that ensure tight coregulation of the corresponding promoters must exist. 20 refs., 2 figs., 1 tab.

  10. Polycomb recruitment at the Class II transactivator gene.

    PubMed

    Boyd, Nathaniel H; Morgan, Julie E; Greer, Susanna F

    2015-10-01

    The Class II Transactivator (CIITA) is the master regulator of Major Histocompatibility Class II (MHC II) genes. Transcription of CIITA through the IFN-γ inducible CIITA promoter IV (CIITA pIV) during activation is characterized by a decrease in trimethylation of histone H3 lysine 27 (H3K27me3), catalyzed by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). While EZH2 is the known catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is present at the inactive CIITA pIV, the mechanism of PRC2 recruitment to mammalian promoters remains unknown. Here we identify two DNA-binding proteins, which interact with and regulate PRC2 recruitment to CIITA pIV. We demonstrate Yin Yang 1 (YY1) and Jumonji domain containing protein 2 (JARID2) are binding partners along with EZH2 in mammalian cells. Upon IFN-γ stimulation, YY1 dissociates from CIITA pIV while JARID2 binding to CIITA pIV increases, suggesting novel roles for these proteins in regulating expression of CIITA pIV. Knockdown of YY1 and JARID2 yields decreased binding of EZH2 and H3K27me3 at CIITA pIV, suggesting important roles for YY1 and JARID2 at CIITA pIV. JARID2 knockdown also results in significantly elevated levels of CIITA mRNA upon IFN-γ stimulation. This study is the first to identify novel roles of YY1 and JARID2 in the epigenetic regulation of the CIITA pIV by recruitment of PRC2. Our observations indicate the importance of JARID2 in CIITA pIV silencing, and also provide a novel YY1-JARID2-PRC2 regulatory complex as a possible explanation of differential PRC2 recruitment at inducible versus permanently silenced genes.

  11. Co-segregation of a homozygous SMN1 deletion and a heterozygous PMP22 duplication in a patient.

    PubMed

    Fernández, Raquel M; Peciña, Ana; Muñoz-Cabello, Beatriz; Antiñolo, Guillermo; Borrego, Salud

    2016-09-01

    Despite co-segregation of two different genetic neurological disorders within a family is rare, clinicians should take into consideration this possibility in patients presenting with unusual complex phenotypes or with unexpected electrophysiological findings. Here, we report a Spanish 11-month-old patient with spinal muscular atrophy type 2 and Charcot-Marie-Tooth 1A. PMID:27648268

  12. Statistical analysis of atom probe data: detecting the early stages of solute clustering and/or co-segregation.

    PubMed

    Hyde, J M; Cerezo, A; Williams, T J

    2009-04-01

    Statistical analysis of atom probe data has improved dramatically in the last decade and it is now possible to determine the size, the number density and the composition of individual clusters or precipitates such as those formed in reactor pressure vessel (RPV) steels during irradiation. However, the characterisation of the onset of clustering or co-segregation is more difficult and has traditionally focused on the use of composition frequency distributions (for detecting clustering) and contingency tables (for detecting co-segregation). In this work, the authors investigate the possibility of directly examining the neighbourhood of each individual solute atom as a means of identifying the onset of solute clustering and/or co-segregation. The methodology involves comparing the mean observed composition around a particular type of solute with that expected from the overall composition of the material. The methodology has been applied to atom probe data obtained from several irradiated RPV steels. The results show that the new approach is more sensitive to fine scale clustering and co-segregation than that achievable using composition frequency distribution and contingency table analyses.

  13. cumA Multicopper Oxidase Genes from Diverse Mn(II)-Oxidizing and Non-Mn(II)-Oxidizing Pseudomonas Strains

    PubMed Central

    Francis, Chris A.; Tebo, Bradley M.

    2001-01-01

    A multicopper oxidase gene, cumA, required for Mn(II) oxidation was recently identified in Pseudomonas putida strain GB-1. In the present study, degenerate primers based on the putative copper-binding regions of the cumA gene product were used to PCR amplify cumA gene sequences from a variety of Pseudomonas strains, including both Mn(II)-oxidizing and non-Mn(II)-oxidizing strains. The presence of highly conserved cumA gene sequences in several apparently non-Mn(II)-oxidizing Pseudomonas strains suggests that this gene may not be expressed, may not be sufficient alone to confer the ability to oxidize Mn(II), or may have an alternative function in these organisms. Phylogenetic analysis of both CumA and 16S rRNA sequences revealed similar topologies between the respective trees, including the presence of several distinct phylogenetic clusters. Overall, our results indicate that both the cumA gene and the capacity to oxidize Mn(II) occur in phylogenetically diverse Pseudomonas strains. PMID:11526033

  14. Characterization and expression of MHC class II alpha and II beta genes in mangrove red snapper (Lutjanus argentimaculatus).

    PubMed

    Wang, Tianyan; Tan, Shangjin; Cai, Zhonghua

    2015-12-01

    The major histocompatibility complex (MHC) class II plays a key role in adaptive immunity by presenting foreign peptides to CD4(+) T cells and by triggering the adaptive immune response. While the structure and function of MHC class II have been well characterized in mammalian, limited research has been done on fishes. In this study, we characterized the gene structure and expression of MHC class II α (Lunar-DAA) and II β (Lunar-DAB) of mangrove red snapper (Lutjanus argentimaculatus). Both genes shared, respectively, a high similarity and typical features with other vertebrate MHC class II α and II β. The phylogenetic analysis of the deduced peptides revealed that both Lunar-DAA and Lunar-DAB were located in the teleost subclass. Western blotting analyses indicated that both MHC class II α and II β were expressed ubiquitously in immune-related cells, tissues and organs, and that MHC class II α and II β chains existed mainly as heterodimers. While it was highly expressed in gills, thymus, head kidney (HK), spleen, head kidney macrophage and spleen leucocytes, MHC class II β chain was expressed with a low abundance in skin, intestine, stomach and heart. The highest expression of MHC class II β in thymus confirmed the conclusion that thymus is one of the primary lymphoid organs in fishes. The detection of MHC class II αβ dimers in HK macrophages and spleen leucocytes indicated that HK macrophages and spleen leucocytes play a critical role in the adaptive immunity in fishes. All these results provide valuable information for understanding the structure of MHC class II α and II β and their function in immune responses.

  15. Characterization and expression of MHC class II alpha and II beta genes in mangrove red snapper (Lutjanus argentimaculatus).

    PubMed

    Wang, Tianyan; Tan, Shangjin; Cai, Zhonghua

    2015-12-01

    The major histocompatibility complex (MHC) class II plays a key role in adaptive immunity by presenting foreign peptides to CD4(+) T cells and by triggering the adaptive immune response. While the structure and function of MHC class II have been well characterized in mammalian, limited research has been done on fishes. In this study, we characterized the gene structure and expression of MHC class II α (Lunar-DAA) and II β (Lunar-DAB) of mangrove red snapper (Lutjanus argentimaculatus). Both genes shared, respectively, a high similarity and typical features with other vertebrate MHC class II α and II β. The phylogenetic analysis of the deduced peptides revealed that both Lunar-DAA and Lunar-DAB were located in the teleost subclass. Western blotting analyses indicated that both MHC class II α and II β were expressed ubiquitously in immune-related cells, tissues and organs, and that MHC class II α and II β chains existed mainly as heterodimers. While it was highly expressed in gills, thymus, head kidney (HK), spleen, head kidney macrophage and spleen leucocytes, MHC class II β chain was expressed with a low abundance in skin, intestine, stomach and heart. The highest expression of MHC class II β in thymus confirmed the conclusion that thymus is one of the primary lymphoid organs in fishes. The detection of MHC class II αβ dimers in HK macrophages and spleen leucocytes indicated that HK macrophages and spleen leucocytes play a critical role in the adaptive immunity in fishes. All these results provide valuable information for understanding the structure of MHC class II α and II β and their function in immune responses. PMID:26454477

  16. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  17. MHC evolution in three salmonid species: a comparison between class II alpha and beta genes.

    PubMed

    Gómez, Daniela; Conejeros, Pablo; Marshall, Sergio H; Consuegra, Sofia

    2010-08-01

    The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites. PMID:20521040

  18. MHC evolution in three salmonid species: a comparison between class II alpha and beta genes.

    PubMed

    Gómez, Daniela; Conejeros, Pablo; Marshall, Sergio H; Consuegra, Sofia

    2010-08-01

    The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.

  19. Major histocompatibility complex class II genes and systemic sclerosis.

    PubMed

    Briggs, D; Welsh, K I

    1991-11-01

    susceptibility to the disease is conferred by neutral residues (Val, Ser, Ala), at position 57 of the DQ beta chain, while Asp at this position correlates with resistance. A similar phenomenon has been described in rheumatoid arthritis. Although DR4 in general is associated with rheumatoid arthritis, it is heterogeneous, but a subtype of DR4 which is characterised by positively charged residues at positions 70 and 71 of the beta chains is not found in patients with rheumatoid arthritis (Wordsworth B P et al, unpublished data). A similar approach applied to the study of systemic sclerosis is likely to be similarly rewarding. The precise subtyping of the class II genes and the characterisation of their associated haplotypes is therefore required for a complete understanding of the contribution of the MHC to the disease. Additional genes linked to the MHC must not be overlooked, and are relevant to associations of haplotypes with the disease. Of particular interest are the recent reports of a new class of proteins, which are determined by genes in the MHC and which are considered to play a part in the assembly of the antigen peptide/MHC molecule complex. PMID:1750798

  20. Evolution of the primate cytochrome c oxidase subunit II gene.

    PubMed

    Adkins, R M; Honeycutt, R L

    1994-03-01

    We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. PMID:8006990

  1. A Caenorhabditis Elegans RNA Polymerase II Gene, Ama-1 Iv, and Nearby Essential Genes

    PubMed Central

    Rogalski, T. M.; Riddle, D. L.

    1988-01-01

    The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20° but are arrested as larvae at 25°, and two others are fertile at 20° and sterile at 25°. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25° is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four γ-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development. PMID:8608933

  2. Major histocompatibility complex class II A gene polymorphism in the striped bass

    SciTech Connect

    Hardee, J.J.; Godwin, U.; Benedetto, R.; McConnell, T.J.

    1995-02-01

    Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex (Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating on the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the {alpha}1 encoding domain. The encoded {alpha}2, transmembrane, and cytoplasmic regions of all seven striped bass genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass. 55 refs., 5 figs., 1 tab.

  3. Major histocompatibility complex class II A gene polymorphism in the striped bass.

    PubMed

    Hardee, J J; Godwin, U; Benedetto, R; McConnell, T J

    1995-01-01

    Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex (Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating in the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the alpha 1 encoding domain. The encoded alpha 2, transmembrane, and cytoplasmic regions of all seven striped bass genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass.

  4. Molecular cloning, expression, and evolution analysis of type II CHI gene from peanut (Arachis hypogaea L.).

    PubMed

    Liu, Yu; Zhao, Shuzhen; Wang, Jiangshan; Zhao, Chuanzhi; Guan, Hongshan; Hou, Lei; Li, Changsheng; Xia, Han; Wang, Xingjun

    2015-01-01

    Chalcone isomerase (CHI) plays critical roles in plant secondary metabolism, which is important for the interaction between plants and the environment. CHI genes are widely studied in various higher plants. However, little information about CHI genes is available in peanut. Based on conservation of CHI gene family, we cloned the peanut type II CHI gene (AhCHI II) cDNA and genome sequence. The amino acid sequence of peanut CHI II was highly homologous to type II CHI from other plant species. qRT-PCR results showed that peanut CHI II is mainly expressed in roots; however, peanut CHI I is mainly expressed in tissues with high content of anthocyanin. Gene duplication and gene cluster analysis indicated that CHI II was derived from CHI I 65 million years ago approximately. Our gene structure analysis results are not in agreement with the previous hypothesis that CHI II was derived from CHI I by the insertion of an intron into the first exon. Moreover, no positive selection pressure was found in CHIs, while, 32.1 % of sites were under neutral selection, which may lead to mutation accumulation and fixation during great changes of environment.

  5. Isolation of the human insulin-like growth factor genes: insulin-like growth factor II and insulin genes are contiguous.

    PubMed Central

    Bell, G I; Gerhard, D S; Fong, N M; Sanchez-Pescador, R; Rall, L B

    1985-01-01

    Overlapping recombinant clones that encompass the insulin-like growth factor (IGF) I and II genes have been isolated from a human genomic DNA library. Each gene is present once per haploid genome; the IGF-I gene spans greater than 35 kilobase pairs (kbp) and the IGF-II gene is at least 15 kbp. The exon-intron organization of these genes is similar, each having four exons, which is one more than the related insulin gene. Comparison of the restriction endonuclease cleavage maps of the IGF-II and insulin genes, including their flanking regions and hybridization with an IGF-II cDNA probe, revealed that they are adjacent to one another. The IGF-II and insulin genes have the same polarity and are separated by 12.6 kbp of intergenic DNA that includes a dispersed middle repetitive Alu sequence. The order of the genes is 5'-insulin-IGF-II-3'. Images PMID:3901002

  6. Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells.

    PubMed

    Chen, X L; Tummala, P E; Olbrych, M T; Alexander, R W; Medford, R M

    1998-11-01

    Monocyte infiltration into the vessel wall, a key initial step in the process of atherosclerosis, is mediated in part by monocyte chemoattractant protein-1 (MCP-1). Hypertension, particularly in the presence of an activated renin-angiotensin system, is a major risk factor for the development of atherosclerosis. To investigate a potential molecular basis for a link between hypertension and atherosclerosis, we studied the effects of angiotensin II (Ang II) on MCP-1 gene expression in rat aortic smooth muscle cells. Rat smooth muscle cells treated with Ang II exhibited a dose-dependent increase in MCP-1 mRNA accumulation that was prevented by the AT1 receptor antagonist losartan. Ang II also activated MCP-1 gene transcription. Inhibition of NADH/NADPH oxidase, which generates superoxide and H2O2, with diphenylene iodonium or apocynin decreased Ang II-induced MCP-1 mRNA accumulation. Induction of MCP-1 gene expression by Ang II was inhibited by catalase, suggesting a second messenger role for H2O2. The tyrosine kinase inhibitor genistein and the mitogen-activated protein kinase kinase inhibitor PD098059 inhibited Ang II-induced MCP-1 gene expression, consistent with a mitogen-activated protein kinase-dependent signaling mechanism. Ang II may thus promote atherogenesis by direct activation of MCP-1 gene expression in vascular smooth muscle cells.

  7. Transcriptional control of MHC class II gene expression during differentiation from B cells to plasma cells.

    PubMed

    Dellabona, P; Latron, F; Maffei, A; Scarpellino, L; Accolla, R S

    1989-04-15

    In this study we investigated the molecular mechanisms responsible for the extinction of the constitutive MHC class II gene expression of human B cells on somatic cell hybridization with murine plasmocytoma cells. We found that this event is due to trans-acting suppressor functions of mouse origin pre-existing in the plasmocytoma cells and acting at transcriptional level. Transcription of the entire family of human class II genes is suppressed, including genes as DO beta for which a distinct regulation of expression in B cells had been previously demonstrated. Suppression appears specific for class II genes because in the hybrids expression of MHC class I genes of mouse is unaffected and of human only partially reduced. Interestingly, also murine invariant chain gene is expressed in both parental plasmocytoma and hybrid cells although at reduced amounts as compared to a murine class II positive B cell line. The class II negative phenotype of hybrid cells and parental plasmocytoma cells is highly stable and unaffected by treatment with protein synthesis inhibitors, suggesting that the transcriptional suppressor function is not mediated by rapid, labile turning-over proteins. Possible mechanisms responsible for transcriptional regulation of MHC class II gene expression during terminal differentiation of B cells to plasma cells are discussed. PMID:2495328

  8. Chromosomal localization and structure of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.; Huberman, E. |; Collart, F.; Varkony, T.; Drabkin, H.

    1994-05-01

    We determined the chromosomal localization and structure of the gene encoding human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205), an enzyme associated with cellular proliferation, malignant transformation, and differentiation. Using polymerase chain reaction (PCR) primers specific for type II IMPDH, we screened a panel of human-Chinese hamster cell somatic hybrids and a separate deletion panel of chromosome 3 hybrids and localized the gene to 3p21.1{yields}p24.2. Two overlapping yeast artificial chromosome clones containing the full gene for type II IMPDH were isolated and a physical map of 117 kb of human genomic DNA in this region of chromosome 3 was constructed. The gene for type II IMPDH was localized and oriented on this map and found to span no more than 12.5 kb.

  9. Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.

    PubMed Central

    Chung, H M; Lee, M G; Dietrich, P; Huang, J; Van der Ploeg, L H

    1993-01-01

    Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I. Images PMID:8497277

  10. Topoisomerase I deficiency causes RNA polymerase II accumulation and increases AID abundance in immunoglobulin variable genes.

    PubMed

    Maul, Robert W; Saribasak, Huseyin; Cao, Zheng; Gearhart, Patricia J

    2015-06-01

    Activation-induced deaminase (AID) is a DNA cytosine deaminase that diversifies immunoglobulin genes in B cells. Recent work has shown that RNA polymerase II (Pol II) accumulation correlates with AID recruitment. However, a direct link between Pol II and AID abundance has not been tested. We used the DT40 B-cell line to manipulate levels of Pol II by decreasing topoisomerase I (Top1), which relaxes DNA supercoiling in front of the transcription complex. Top1 was decreased by stable transfection of a short hairpin RNA against Top1, which produced an accumulation of Pol II in transcribed genes, compared to cells transfected with sh-control RNA. The increased Pol II density enhanced AID recruitment to variable genes in the λ light chain locus, and resulted in higher levels of somatic hypermutation and gene conversion. It has been proposed by another lab that AID itself might directly suppress Top1 to increase somatic hypermutation. However, we found that in both AID(+/+) and AID(-/-) B cells from DT40 and mice, Top1 protein levels were identical, indicating that the presence or absence of AID did not decrease Top1 expression. Rather, our results suggest that the mechanism for increased diversity when Top1 is reduced is that Pol II accumulates and recruits AID to variable genes.

  11. In vivo cII, gpt, and Spi⁻ gene mutation assays in transgenic mice and rats.

    PubMed

    Manjanatha, Mugimane G; Cao, Xuefei; Shelton, Sharon D; Mittelstaedt, Roberta A; Heflich, Robert H

    2013-01-01

    Transgenic mutation assays are used to identify and characterize genotoxic hazards and for determining the mode of action for carcinogens. The three most popular transgenic mutational models are Big Blue® (rats or mice), Muta™ mouse (mice), and gpt-delta (rats or mice). The Big Blue® and Muta™ mouse models use the cII gene as a reporter of mutation whereas gpt-delta rodents use the gpt gene and the red/gam genes (Spi⁻ selection) as mutation reporter genes. Here we describe methodology for conducting mutation assays with these transgenes. Transgenes recovered from tissue DNA are packaged into infectious lambda phage, bacteria are infected with the phage, and cII-mutant and Spi⁻ plaques and gpt-mutant colonies are isolated using selective conditions and quantified. Selected mutants can be further analyzed for identification of small sequence alterations in the cII and gpt genes and large deletions at the Spi⁻ locus.

  12. A cII-dependent promoter is located within the Q gene of bacteriophage lambda.

    PubMed

    Hoopes, B C; McClure, W R

    1985-05-01

    We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis. The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ. We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition. We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage. The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C. The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.

  13. Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma.

    PubMed

    Mackay, Donna S; Bennett, Thomas M; Shiels, Alan

    2015-01-01

    Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic. PMID:26162006

  14. Six DNA polymorphisms in the low density lipoprotein receptor gene: their genetic relationship and an example of their use for identifying affected relatives of patients with familial hypercholesterolaemia.

    PubMed Central

    Humphries, S; King-Underwood, L; Gudnason, V; Seed, M; Delattre, S; Clavey, V; Fruchart, J C

    1993-01-01

    We have determined the relative allele frequency and estimated linkage disequilibrium between six DNA polymorphisms of the low density lipoprotein (LDL) receptor gene. Polymorphisms were detected using the enzymes SfaNI, TaqI, StuI, HincII, AvaII, and NcoI after DNA amplification by the polymerase chain reaction. Strong linkage disequilibrium was detected between many of the pair wise comparisons in a sample of 60 patients heterozygous for familial hypercholesterolaemia (FH). Using the enzymes HincII, NcoI, and SfaNI, 85% of patients were heterozygous for at least one polymorphism and thus potentially informative for cosegregation studies. The polymorphisms were used to follow the inheritance of the defective allele of the LDL receptor gene in the relatives of a patient with FH. Assays of LDL receptor activity on lymphoblastoid cell lines from two members of the family was used to confirm that the proband, but not the hypercholesterolaemic brother, had a defect in the LDL receptor. In the family, none of the children had inherited the allele of the LDL receptor gene inferred to be defective. The problems associated with this cosegregation approach to identify relatives of patients with a clinical diagnosis of FH are discussed. PMID:8098067

  15. Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

    PubMed Central

    Ambite, Ines; Lutay, Nataliya; Stork, Christoph; Dobrindt, Ulrich; Wullt, Björn; Svanborg, Catharina

    2016-01-01

    Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease–associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that

  16. Contrasting evolutionary histories of MHC class I and class II loci in grouse—Effects of selection and gene conversion

    USGS Publications Warehouse

    Minias, Piotr; Bateson, Zachary W; Whittingham, Linda A; Johnson, Jeff A.; Oyler-McCance, Sara J.; Dunn, Peter O

    2016-01-01

    Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

  17. Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes.

    PubMed

    Pan, Hongmiao; Xu, Joshua; Kweon, Oh-Gew; Zou, Wen; Feng, Jinhui; He, Gui-Xin; Cerniglia, Carl E; Chen, Huizhong

    2015-05-01

    We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes.

  18. Gene targeting in embryonic stem cells, II: conditional technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  19. DNA sequence of the Peromyscus leucopus MHC class II gene Aa (MhcPeleAa)

    SciTech Connect

    Crew, M.D.; Bates, L.M.

    1996-09-01

    The genus Peromyscus has been extensively studied by populations biologists and ecologists for over eighty years, with P. leucopus (the white-footed mouse) being one of the most intensively investigated species. Polymorphic major histocompatibility complex (MHC) genes have proven useful in population genetic studies and might be helpful in understanding the population dynamics of Peromyscus species which are ubiquitously distributed over North and Central America. Polymorphism of P. leucopus MHC (MhcPele) class II genes was evident by restriction fragment length polymorphism (RFLP) analyses using human and mouse probes and Pele class II loci exhibited degrees of polymorphism similar to H2 class II genes (A-like>E-like). 8 refs., 2 figs.

  20. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  1. Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination.

    PubMed

    Reynolds, David L; Hofmeister, Brigitte T; Cliffe, Laura; Siegel, T Nicolai; Anderson, Britta A; Beverley, Stephen M; Schmitz, Robert J; Sabatini, Robert

    2016-08-01

    The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription.

  2. Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination

    PubMed Central

    Reynolds, David L.; Hofmeister, Brigitte T.; Cliffe, Laura; Siegel, T. Nicolai; Anderson, Britta A.; Beverley, Stephen M.; Schmitz, Robert J.; Sabatini, Robert

    2016-01-01

    Summary The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription. PMID:27125778

  3. Acetylation of RNA Polymerase II Regulates Growth-Factor-Induced Gene Transcription in Mammalian Cells

    PubMed Central

    Schröder, Sebastian; Herker, Eva; Itzen, Friederike; He, Daniel; Thomas, Sean; Gilchrist, Daniel A.; Kaehlcke, Katrin; Cho, Sungyoo; Pollard, Katherine S.; Capra, John A.; Schnölzer, Martina; Cole, Philip A.; Geyer, Matthias; Bruneau, Benoit G.; Adelman, Karen; Ott, Melanie

    2014-01-01

    SUMMARY Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes. PMID:24207025

  4. KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

    PubMed Central

    Rahman, Mohammed Tanjimur; Nakayama, Kentaro; Rahman, Munmun; Katagiri, Hiroshi; Katagiri, Atsuko; Ishibashi, Tomoka; Ishikawa, Masako; Sato, Emi; Iida, Kouji; Nakayama, Naomi; Ishikawa, Noriyuki; Miyazaki, Kohji

    2013-01-01

    In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications. PMID:23820584

  5. Genetic polymorphism of estrogen receptor alpha gene in Egyptian women with type II diabetes mellitus

    PubMed Central

    Motawi, Tarek M.K.; El-Rehany, Mahmoud A.; Rizk, Sherine M.; Ramzy, Maggie M.; el-Roby, Doaa M.

    2015-01-01

    Estrogen might play an important role in type 2 diabetes mellitus pathogenesis. A number of polymorphisms have been reported in the estrogen receptor alpha gene including the XbaI and PvuII restriction enzyme polymorphisms. The aim of this study was to determine if ESRα gene polymorphisms are associated with type 2 diabetes mellitus and correlated with lipid profile. Ninety diabetic Egyptian patients were compared with forty healthy controls. ESRα genotyping of PvuII and XbaI was performed using restriction fragment length polymorphism analysis. Our study showed that there is more significant difference in the frequency of C and G polymorphic allele between patients and control groups in PvuII and XbaI respectively. Also carriers of minor C and G alleles of PvuII and XbaI gene polymorphisms were associated with increased fasting blood glucose and disturbance in lipid profile as there is an increase in total cholesterol, triglycerides and Low density lipoprotein. So findings of present study suggest the possibility that PvuII and XbaI polymorphisms in ERα are related to T2DM and with increased serum lipids among Egyptian population. PMID:26401488

  6. Availability of type II diabetic families for detection of diabetes susceptibility genes.

    PubMed

    Cook, J T; Page, R C; O'Rahilly, S; Levy, J; Holman, R; Barrow, B; Hattersley, A T; Shaw, A G; Wainscoat, J S; Turner, R C

    1993-10-01

    Type II diabetes is a familial disorder, as evidenced by the increased prevalence in monozygotic cotwins and first-degree relatives of affected subjects; however, its genetic etiology is largely unknown. Well-characterized pedigrees are an essential resource for the study of susceptibility genes for type II diabetes. This study describes a 5-yr search for type II diabetic families in Oxfordshire, U.K. We interviewed 950 type II diabetic subjects concerning the availability of first-degree relatives; 127 Caucasian families ascertained through a proband with type II diabetes were studied, and 589 first-degree relatives were characterized. Three large pedigrees with maturity-onset diabetes of the young, and 8 multiplex multigenerational type II diabetic pedigrees were identified. We identified 12 sib-pairs in which both siblings had type II diabetes; however, only 7 sib-pairs had both parents alive, and 2 of these had both parents affected. If one also considers one sib having diabetes and one sib having glucose intolerance as being an affected sib-pair, we identified 30 sib-pairs of which 7 had both parents affected and probably had bilineal inheritance. We identified 76 complete nuclear families with both parents and offspring available for study, but only 6 were of optimal structure for linkage analysis. In conclusion, multiplex pedigrees and type II diabetic sib-pairs with living parents are uncommon, and their ascertainment requires a substantial investment of resources. Large-scale collaborative multicenter initiatives would be needed to collect a large resource of family material for the study of susceptibility genes for type II diabetes.

  7. Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes.

    PubMed

    Santamaria, P; Boyce-Jacino, M T; Lindstrom, A L; Barbosa, J J; Faras, A J; Rich, S S

    1991-01-01

    Nucleic acid sequences of the second exons of HLA-DRB1, -DRB3/4/5, -DQB1, and -DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.

  8. Expression of class II cytokine genes in children's skin.

    PubMed

    Reemann, Paula; Reimann, Ene; Suutre, Siim; Paavo, Maarjaliis; Loite, Ulvi; Porosaar, Orm; Abram, Kristi; Silm, Helgi; Vasar, Eero; Kõks, Sulev; Kingo, Külli

    2014-07-01

    Immune regulation of the skin plays an important role in susceptibility and development of illnesses. The aim of our study was to localise the interleukin (IL)-10 family of cytokines, in children's skin and to determine possible age-related differences in the expression level. The mRNA expression level of IL10, IL19, IL20, IL22, IL24, IL26, IL28B, IL29 and their receptors IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL22RA2, IL28RA was compared in skin biopsies of children and adults and in childrens' skin cells by quantitative real-time PCR (qRT-PCR). Immunohistochemistry was performed to confirm the qRT-PCR findings. We found age-related differences in the expression of IL10RB, IL20, IL20RA, IL22RA1, IL22RA2, IL26 and IL28RA genes. Cell type-dependent expression of IL10 family cytokines was apparent in the skin. In addition to previously known differences in systemic immunological response of adults and children, the present results reveal differences in immune profile of adult and juvenile skin.

  9. Inflammatory bowel disease associations with HLA Class II genes

    SciTech Connect

    Castro, R.; Yang, H.; Targan, S.

    1994-09-01

    A PCR-SSOP assay has been used to analyze HLA-Class II DRB1 and DQB1 alleles in 378 Caucasians from a population in Southern California. The data has been analyzed separately for the Ashkenasi Jews and non-Jewish patients (n=286) and controls (n=92). Two common clinical forms of inflammatory bowel disease (IBD) have been studied: ulcerative colitis (UC) and Crohn`s disease (CD). In CD, we observed a susceptible effect with the rare DR1 allele - DRB*0103 [O.R.=4.56; 95% CI (0.96, 42.97); p=0.03]; a trend for an increase in DRB1*0103 was also observed in UC patients. A susceptible effect with DRB1*1502 [O.R.=5.20; 95% CI (1.10, 48.99); p=0.02] was observed in non-Jewish UC patients. This susceptible effect was restricted to UC ANCA-positive (antineutrophil cytoplasmic antibodies) patients. In addition, a significant association with DRB1*1101-DQB1*0301 [O.R.=9.46; 95% CI (1.30, 413.87); p=0.01] was seen with UC among non-Jewish patients: this haplotype was increased with CD among non-Jewish patients. Two protective haplotypes were detected among CD non-Jewish patients: DRB1*1301-DQB1*0603 [O.R.=0.34; 95% CI (0.09, 1.09); p=0.04], and DRB*0404-DQB1*0302 [O.R.=<0.08; 95% CI (0.0, 0.84); p=0.01]. When the same data were analyzed at the serology level, we observed a positive association in UC with DR2 [O.R.6.77; 95% CI (2.47, 22.95); p=2 x 10{sup -4}], and a positive association in CD with DR1 [O.R.=2.63; 95% CI (1.14, 6.62); p=0.01] consistent with previous reports. Thus, some IBD disease associations appear to be common to both UC and CD, while some are unique to one disease.

  10. Expression Regulation of Major Histocompatibility Complex Class I and Class II Encoding Genes

    PubMed Central

    van den Elsen, Peter J.

    2011-01-01

    Major histocompatibility complex (MHC)-I and MHC-II molecules play an essential role in the immune response to pathogens by virtue of their ability to present peptides to CD8+ and CD4+ T cells, respectively. Given this critical role, MHC-I and MHC-II genes are regulated in a tight fashion at the transcriptional level by a variety of transcription factors that interact with conserved cis-acting regulatory promoter elements. In addition to the activities of these regulatory factors, modification of chromatin also plays an essential role in the efficient transcription of these genes to meet with local requirement for an effective immune response. The focus of this review is on the transcription factors that interact with conserved cis-acting promoter elements and the epigenetic mechanisms that modulate induced and constitutive expression of these MHC genes. PMID:22566838

  11. Familial cosegregation of manic-depressive illness and a form of hereditary cerebellar ataxia

    SciTech Connect

    Piqueras, J.F.; Santos, J.; Puertollano, R.

    1995-06-19

    We report on a Spanish family with co-occurrence of manic-depression and a form of hereditary cerebellar ataxia. All affected individuals in the second generation showed cerebellar ataxia and manic-depression simultaneously. Since anticipation has been described in both disorders and the pattern of segregation may be autosomal as well as X-linked, we have searched for a possible involvement of two candidate genes which are located either on an autosome (SCA1) or on the X-chromosome (GABRA3). We concluded that expansion of trinucleotide repeats at SCA1 gene cannot be considered as a disease-causing mutation, and this gene should be initially discarded. 19 refs., 3 figs.

  12. In vitro secondary activation (memory effect) of avian vitellogenin II gene in isolated liver nuclei.

    PubMed Central

    Jost, J P; Moncharmont, B; Jiricny, J; Saluz, H; Hertner, T

    1986-01-01

    The vitellogenin II gene is specifically reactivated in vitro (secondary stimulation, memory effect) in purified liver nuclei that had ceased to express the gene in vivo a month after the roosters had received a single injection of estradiol (primary stimulation). The in vitro reactivation depends on the addition to the nuclei of nuclear and cytoplasmic extracts from estradiol-stimulated livers, polyamines (0.1-1.0 mM), and calmodulin (0.1 mM). Under identical incubation conditions the vitellogenin gene could not be reactivated in oviduct, embryonic, and immature chicken liver nuclei. Two other genes, those for ovalbumin and lysozyme, which are regulated by estradiol in the oviduct, could not be activated in the liver nuclei. The correct initiation of vitellogenin gene transcription in the liver nuclei was tested by primer extension studies. Addition of the antiestrogen tamoxifen (0.1 microM) to the system decreased vitellogenin mRNA synthesis by about 45% without affecting total RNA synthesis. Addition of quercetin (0.1 mM) and trans-flupenthixol (0.2 mM), inhibitors of nuclear protein kinase II and calmodulin-dependent kinase, respectively, inhibited the synthesis of vitellogenin mRNA by about 55% without affecting total RNA synthesis. The inhibitory effects of the antiestrogen and the kinase inhibitors were not additive, suggesting that both classes of inhibitor act on the same target or related targets. Depleting the estradiol receptors from the cell and nuclear extracts by means of estradiol-receptor antibodies covalently bound to Matrex beads reduced the stimulation of the vitellogenin gene by 40%. We conclude that in addition to the estradiol receptor and phosphorylation of nuclear protein(s) there are additional factors responsible for the in vitro secondary activation of the avian vitellogenin II gene. Images PMID:3455757

  13. Differential expression of CaMK-II genes during early zebrafish embryogenesis.

    PubMed

    Rothschild, Sarah C; Lister, James A; Tombes, Robert M

    2007-01-01

    CaMK-II is a highly conserved Ca(2+)/calmodulin-dependent protein kinase expressed throughout the lifespan of all vertebrates. During early development, CaMK-II regulates cell cycle progression and "non-canonical" Wnt-dependent convergent extension. In the zebrafish, Danio rerio, CaMK-II activity rises within 2 hr after fertilization. At the time of somite formation, zygotic expression from six genes (camk2a1, camk2b1, camk2g1, camk2g2, camk2d1, camk2d2) results in a second phase of increased activity. Zebrafish CaMK-II genes are 92-95% identical to their human counterparts in the non-variable regions. During the first three days of development, alternative splicing yields at least 20 splice variants, many of which are unique. Whole-mount in situ hybridization reveals that camk2g1 comprises the majority of maternal expression. All six genes are expressed strongly in ventral regions at the 18-somite stage. Later, camk2a1 is expressed in anterior somites, heart, and then forebrain. Camk2b1 is expressed in somites, mid- and forebrain, gut, retina, and pectoral fins. Camk2g1 appears strongly along the midline and then in brain, gut, and pectoral fins. Camk2g2 is expressed early in the midbrain and trunk and exhibits the earliest retinal expression. Camk2d1 is elevated early at somite boundaries, then epidermal tissue, while camk2d2 is expressed in discrete anterior locations, steadily increasing along either side of the dorsal midline and then throughout the brain, including the retina. These findings reveal a complex pattern of CaMK-II gene expression consistent with pleiotropic roles during development. PMID:17103413

  14. Molecular organization of the 5S rDNA gene type II in elasmobranchs

    PubMed Central

    Castro, Sergio I.; Hleap, Jose S.; Cárdenas, Heiber; Blouin, Christian

    2016-01-01

    ABSTRACT The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS. PMID:26488198

  15. Molecular organization of the 5S rDNA gene type II in elasmobranchs.

    PubMed

    Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

    2016-01-01

    The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

  16. Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

    PubMed

    Choe, Jin; Sasaki, Yoko; Zhou, Liming; Takai, Hideki; Nakayama, Yohei; Ogata, Yorimasa

    2016-09-01

    Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

  17. Evolution of major histocompatibility complex class I and class II genes in the brown bear

    PubMed Central

    2012-01-01

    Background Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia. PMID:23031405

  18. Mutational hot spot in the DSPP gene causing dentinogenesis imperfecta type II.

    PubMed

    Kim, Jung-Wook; Hu, Jan C-C; Lee, Jae-Il; Moon, Sung-Kwon; Kim, Young-Jae; Jang, Ki-Taeg; Lee, Sang-Hoon; Kim, Chong-Chul; Hahn, Se-Hyun; Simmer, James P

    2005-02-01

    The current system for the classification of hereditary defects of tooth dentin is based upon clinical and radiographic findings and consists of two types of dentin dysplasia (DD) and three types of dentinogenesis imperfecta (DGI). However, whether DGI type III should be considered a distinct phenotype or a variation of DGI type II is debatable. In the 30 years since the classification system was first proposed, significant advances have been made regarding the genetic etiologies of inherited dentin defects. DGI type II is recognized as an autosomal dominant disorder with almost complete penetrance and a low frequency of de novo mutations. We have identified a mutation (c.52G-->T, p.V18F) at the first nucleotide of exon 3 of the DSPP (dentin sialophosphoprotein) gene in a Korean family (de novo) and a Caucasian family. This mutation has previously been reported as causing DGI type II in a Chinese family. These findings suggest that this mutation site represents a mutational "hot spot" in the DSPP gene. The clinical and radiographic features of these two families include the classic phenotypes associated with both DGI type II and type III. Finding that a single mutation causes both phenotypic patterns strongly supports the conclusion that DGI type II and DGI type III are not separate diseases but rather the phenotypic variation of a single disease. We propose a modification of the current classification system such that the designation "hereditary opalescent dentin" or "DGI type II" should be used to describe both the DGI type II and type III phenotypes.

  19. Cloning, characterization, and regulation of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.A.; Huberman, E. |

    1997-01-01

    Human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. Regulated IMPDH activity is associated with cellular proliferation, transformation, and differentiation. The authors cloned and sequenced the entire gene for type II IMPDH and here provide details regarding the organization of the gene and the characterization of its promoter. The gene spans approximately 5 kb and is disrupted by 12 introns. The transcriptional start sites were determined by S1 nuclease mapping to be somewhat heterogeneous but predominated at 102 and 85 nucleotides from the translational initiation codon. Through the use of heterologous gene constructs and transient transfection assays, a minimal promoter from {minus}206 to {minus}85 was defined. This promoter is TATA-less and contains several transcription factor motifs including four potential Sp 1 binding sites. The minimal promoter is GC-rich (69%) and resembles a CpG island. Through the use of gel mobility shift assays, nuclear proteins were shown to specifically interact with this minimal promoter. Stable transfectants were used to demonstrate that the down-regulation of IMPDH gene expression in response to reduced cellular proliferation occurs by a transcriptional mechanism.

  20. Widely distributed mutations in the COL2A1 gene produce achondrogenesis type II/hypochondrogenesis.

    PubMed

    Körkkö, J; Cohn, D H; Ala-Kokko, L; Krakow, D; Prockop, D J

    2000-05-15

    The COL2A1 gene was assayed for mutations in genomic DNA from 12 patients with achondrogenesis type II/hypochondrogenesis. The exons and flanking sequences of the 54 exons in the COL2A1 gene were amplified by a series of specific primers using PCR. The PCR products were scanned for mutations by conformation sensitive gel electrophoresis, and PCR products that generated heteroduplex bands were then sequenced. Mutations in the COL2A1 gene were found in all 12 patients. Ten of the mutations were single base substitutions that converted a codon for an obligate glycine to a codon for an amino acid with a bulkier side chain. One of the mutations was a change in a consensus RNA splice site. Another was an 18-base pair deletion of coding sequences. The results confirmed previous indications that conformation sensitive gel electrophoresis is highly sensitive for detection of mutations in large and complex genes. They also demonstrate that most, if not all, patients with achondrogenesis type II/hypochondrogenesis have mutations in the COL2A1 gene. PMID:10797431

  1. Coordinate amplification of metallothionein I and II gene sequences in cadmium-resistant CHO variants

    SciTech Connect

    Hildebrand, C.E.; Crawford, B.D.; Enger, M.D.

    1983-01-01

    Cadmium-resistanc (Cd/sup r/) variants of the Chinese hamster cell line, CHO, have been derived by stepwise selection for growth in medium containing CdCl/sub 2/. These variants show coordinately increased production of both metallothionein (MT) I and II and were stably resistant to Cd/sup 2 +/ in the absence of continued selection. Genomic DNAs from these Cd/sup r/ sublines were analyzed for both MT gene copy number and MT gene organization, using cDNA sequence probes specific for each of the two Chinese hamster isometallothioneins. These analyses revealed coordinate amplification of MT I and II genes in all Cd/sup r/ variants which had increased copies of MT-encoding sequences. In situ hybridization of an MT-encoding probe to mitotic chromosomes of a Cd/sup r/ variant, which has amplified MT genes at least 14-fold, revealed a single chromosomal site of hybridization. These results suggest that the isoMTs constitute a functionally related gene cluster which amplifies coordinately in response to toxic metal stress.

  2. Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase.

    PubMed

    Colombo, S; Grisa, M; Tortora, P; Vanoni, M

    1994-01-01

    Fumarase catalyzes the interconversion of L-malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C-terminal deletion.

  3. Regulation of Nrf2-Mediated Phase II Detoxification and Anti-oxidant Genes

    PubMed Central

    Keum, Young-Sam

    2012-01-01

    The molecular mechanisms by which a variety of naturally-occurring dietary compounds exert chemopreventive effects have been a subject of intense scientific investigations. Induction of phase II detoxification and anti-oxidant enzymes through activation of Nrf2/ARE-dependent gene is recognized as one of the major cellular defense mechanisms against oxidative or xenobiotic stresses and currently represents a critical chemopreventive mechanism of action. In the present review, the functional significance of Keap1/Nrf2 protein module in regulating ARE-dependent phase II detoxification and anti-oxidant gene expression is discussed. The biochemical mechanisms underlying the phosphorylation and expression of Keap1/Nrf2 proteins that are controlled by the intracellular signaling kinases and ubiquitin-mediated E3 ligase system as well as control of nucleocytoplasmic translocation of Nrf2 by its innate nuclear export signal (NES) are described. PMID:24116287

  4. [Identification of a novel mutation of IDS gene from a Chinese pedigree with MPS II].

    PubMed

    GUO, Yi-Bin; PAN, Hong-Da; GUO, Chun-Miao; LI, Yong-Mei; CHEN, Lu-Ming

    2009-11-01

    The purpose of this study was to understand the molecular genetic mechanism of mucopolysaccharidosis type II (MPS II) and to provide a prerequisite for future prenatal gene diagnosis. A preliminary diagnosis was made by qualitative detection of Urinary Glycosaminoglycans of the suspected MPS II proband. Then, mutation detection was performed on the proband and his family members with PCR and direct sequencing of PCR products. After the novel mutation of c.876 del 2 in IDS gene was detected, sequence analysis was performed on exon 6 of IDS gene of the 135 cases, which consisted of 120 randomly selected normal controls, and other 15 patients with MPS I, IV, and VI other than MPS II. Besides, the patho-genicity of the novel mutation was analyzed with the following 2 methods: conservative analysis of the sequence of muta-tion spots of different species and the direct test of the IDS enzyme activity of the patient and his relative family members. The result of uroscopy of the proband was strong positive (GAGs +++). There was a novel deletion mutation of c.876-877 del TC in the coding region of exon 6 of IDS gene, which was a hemizygous mutation. However, the mutation of his mother and sister was a heterozygous mutation. Detection of the exon 6 of IDS gene showed that the mutation was not found among normal controls and other patients with MPS I, IV, and VI other than MPS II. Homology comparison of amino acid sequences from different species showed that the phenylalanine (F) glutamine (Q) of the mutation site of c.876-877 del TC located in p.292-293 was highly conserved. The activity of IDS enzyme of the proband was only 2.3 nmol/4 h/mL, which was much lower than normal; but the activity of IDS enzyme of his father, mother and sister was 641.9 nmol/4 h/mL, 95.8 nmol/4h/mL and 103.2 nmol/4h/mL, respectively. These results illustrated that the deletion and frame-shift mutation of c.876-877 del TC detected was a novel pathologic mutation, which was the underlying cause of

  5. Mutations in the human CYP11B2 (aldosterone synthase) gene causing corticosterone methyloxidase II deficiency.

    PubMed Central

    Pascoe, L; Curnow, K M; Slutsker, L; Rösler, A; White, P C

    1992-01-01

    Corticosterone methyloxidase II (CMO-II) deficiency is an autosomal recessive disorder of aldosterone biosynthesis, characterized by an elevated ratio of 18-hydroxycorticosterone to aldosterone in serum. It is genetically linked to the CYP11B1 and CYP11B2 genes that, respectively, encode two cytochrome P450 isozymes, P450XIB1 and P450XIB2. Whereas P450XIB1 only catalyzes hydroxylation at position 11 beta of 11-deoxycorticosterone and 11-deoxycortisol, P450XIB2 catalyzes the synthesis of aldosterone from deoxycorticosterone, a process that successively requires hydroxylation at positions 11 beta and 18 and oxidation at position 18. To determine the molecular genetic basis of CMO-II deficiency, seven kindreds of Iranian-Jewish origin were studied in which members suffered from CMO-II deficiency. No mutations were found in the CYP11B1 genes, but two candidate mutations, R181W and V386A, were found in the CYP11B2 genes. When these mutations were individually introduced into CYP11B2 cDNA and expressed in cultured cells, R181W reduced 18-hydroxylase and abolished 18-oxidase activities but left 11 beta-hydroxylase activity intact, whereas V386A caused a small but consistent reduction in the production of 18-hydroxycorticosterone. All individuals affected with CMO-II deficiency were homozygous for both mutations, whereas eight asymptomatic subjects were homozygous for R181W alone and three were homozygous for V386A alone. These findings confirm that P450XIB2 is the major enzyme mediating oxidation at position 18 in the adrenal and suggest that a small amount of residual activity undetectable in in vitro assays is sufficient to synthesize normal amounts of aldosterone. Images PMID:1594605

  6. The Riia Gene of Bacteriophage T4. II. Regulation of Its Messenger RNA Synthesis

    PubMed Central

    Daegelen, P.; Brody, E.

    1990-01-01

    When the rII genes are first introduced into cells which had been previously infected by T4 phage deleted for these genes, the kinetics of synthesis of rIIA and rIIB RNA are rapid and identical. We show that this rapid synthesis depends on a functional motA gene for rIIB, but not for rIIA, RNA synthesis. By primer-extension mapping of T4 messenger RNA, we find three promoters close to the rIIA gene. One of them is an early promoter just before the rIIA.1 gene; it is used under all conditions tested. Another is in the coding portion of the rIIA.1 gene; it is weak, primarily because of a 19-bp spacing between the -10 and -35 elements, and its use is stimulated by T4 functions. The third is a motA-dependent (middle) promoter which has an unusual CCCGCTT box at -33. We present results which suggest that none of these promoters is likely to be the site at which the motB and motC gene products exercise their major influence on rIIA RNA synthesis. PMID:2379818

  7. A molecular map of the chicken major histocompatibility complex: the class II beta genes are closely linked to the class I genes and the nucleolar organizer.

    PubMed Central

    Guillemot, F; Billault, A; Pourquié, O; Béhar, G; Chaussé, A M; Zoorob, R; Kreibich, G; Auffray, C

    1988-01-01

    We have cloned in a cosmid vector four DNA clusters covering 320 kb of the chicken MHC (B complex), including five class II (B-L) beta genes defining two related isotypic families. Additional B complex genes have been revealed using tissue-specific cDNA probes. A cosmid fragment has been used to isolate a cDNA for a class I (B-F) transcript. This transcript, that is by far the most divergent known member of the class I gene family, hybridized to six B-F genes present in the cosmids. One of the clusters was shown to contain two rRNA transcriptional units from the nucleolar organizer region (NOR), marking the telomeric boundary of the B complex. None of the other B complex genes hybridizes to, or has the transcriptional characteristics of mammalian MHC class II alpha or class III genes. The map we have obtained shows that the B complex does not contain well defined class I and class II regions since B-F and B-L beta genes are closely associated with unrelated genes. Moreover, class II beta genes are very closely linked to class I genes in two clusters, and to the NOR in a third one. Images PMID:3141149

  8. Association and cosegregation of stroke with impaired endothelium-dependent vasorelaxation in stroke prone, spontaneously hypertensive rats.

    PubMed Central

    Volpe, M; Iaccarino, G; Vecchione, C; Rizzoni, D; Russo, R; Rubattu, S; Condorelli, G; Ganten, U; Ganten, D; Trimarco, B; Lindpaintner, K

    1996-01-01

    While hypertension is a major risk factor for stroke, it is not its sole determinant. Despite similar blood pressures, spontaneously hypertensive rats (SHR) do not share the predisposition to cerebrovascular disease typical of stroke-prone spontaneously hypertensive rats (SHRSP). We investigated vascular function in male SHR and SHRSP as well as in SHRSP/SHR-F2 hybrid animals. Animals were maintained on the appropriate dietary regimen necessary for the manifestation of stroke. Among the hybrid animals, a group of stroke-prone and a group of stroke-resistant rats were selected. Blood pressure was similar in all groups. Endothelium-independent vascular reactivity tested on isolated rings of thoracic aorta and basilar artery after death showed similar contractile and dilatory responses to serotonin and nitroglycerin, respectively, in all groups. In contrast, endothelium-dependent relaxation, in response to acetylcholine or substance P, was markedly reduced in SHRSP compared with SHR. Similarly, reduced vasodilatory responses were present in aortae of F2 rats that had suffered a stroke when compared with SHR or F2 rats resistant to stroke. The observed association and cosegregation of stroke with significant and specific impairment of endothelium-dependent vasorelaxation among SHRSP and stroke-prone F2 hybrids, respectively, suggest a potential causal role of altered endothelium-dependent vascular relaxation in the pathogenesis of stroke. PMID:8755632

  9. Identification of 42 Genes Linked to Stage II Colorectal Cancer Metastatic Relapse

    PubMed Central

    Al-Temaimi, Rabeah A.; Tan, Tuan Zea; Marafie, Makia J.; Thiery, Jean Paul; Quirke, Philip; Al-Mulla, Fahd

    2016-01-01

    Colorectal cancer (CRC) is one of the leading causes of cancer mortality. Metastasis remains the primary cause of CRC death. Predicting the possibility of metastatic relapse in early-stage CRC is of paramount importance to target therapy for patients who really need it and spare those with low-potential of metastasis. Ninety-six stage II CRC cases were stratified using high-resolution array comparative genomic hybridization (aCGH) data based on a predictive survival algorithm and supervised clustering. All genes included within the resultant copy number aberrations were each interrogated independently at mRNA level using CRC expression datasets available from public repositories, which included 1820 colon cancers, and 167 normal colon tissues. Reduced mRNA expression driven by copy number losses and increased expression driven by copy number gains revealed 42 altered transcripts (29 reduced and 13 increased transcripts) associated with metastatic relapse, short disease-free or overall survival, and/or epithelial to mesenchymal transition (EMT). Resultant genes were classified based on gene ontology (GO), which identified four functional enrichment groups involved in growth regulation, genomic integrity, metabolism, and signal transduction pathways. The identified 42 genes may be useful for predicting metastatic relapse in stage II CRC. Further studies are necessary to validate these findings. PMID:27136531

  10. Genetic variation in V gene of class II Newcastle disease virus.

    PubMed

    Hao, Huafang; Chen, Shengli; Liu, Peng; Ren, Shanhui; Gao, Xiaolong; Wang, Yanping; Wang, Xinglong; Zhang, Shuxia; Yang, Zengqi

    2016-01-01

    The genetic variation and molecular evolution of the V gene of the class II Newcastle disease virus (NDV) isolates with genotypes I-XVIII were determined using bioinformatics. Results indicated that low homology existed in different genotype viruses, whereas high homology often for the same genotypes, exception may be existed within genotypes I, V, VI, and XII. Sequence analysis showed that the genetic variation of V protein was consistent with virus genotype, and specific signatures on the V protein for nine genotypes were identified. Phylogenetic analysis demonstrated that the phylogenetic trees were highly consistent between the V and F genes, with slight discrepancies in the sub-genotypes. Evolutionary rate analyses based on V and F genes revealed the evolution rates varied in genotypes. These data indicate that the genetic variation of V protein is genotype-related and will help in elucidating the molecular evolution of NDV.

  11. Suppressors of Mutations in the rII Gene of Bacteriophage T4 Affect Promoter Utilization

    PubMed Central

    Hall, Dwight H.; Snyder, Ronald D.

    1981-01-01

    Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in λ lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH2 reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L2 decreases the folate analog resistance and the inability of sip2 to grow on CTr5X. L2 itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L2 affects another regulatory gene related to the mot gene. PMID:7262547

  12. A class II KNOX gene, KNOX4, controls seed physical dormancy

    PubMed Central

    Chai, Maofeng; Zhou, Chuanen; Molina, Isabel; Fu, Chunxiang; Nakashima, Jin; Li, Guifen; Zhang, Wenzheng; Park, Jongjin; Tang, Yuhong; Jiang, Qingzhen

    2016-01-01

    Physical dormancy of seed is an adaptive trait that widely exists in higher plants. This kind of dormancy is caused by a water-impermeable layer that blocks water and oxygen from the surrounding environment and keeps embryos in a viable status for a long time. Most of the work on hardseededness has focused on morphological structure and phenolic content of seed coat. The molecular mechanism underlying physical dormancy remains largely elusive. By screening a large number of Tnt1 retrotransposon-tagged Medicago truncatula lines, we identified nondormant seed mutants from this model legume species. Unlike wild-type hard seeds exhibiting physical dormancy, the mature mutant seeds imbibed water quickly and germinated easily, without the need for scarification. Microscopic observations of cross sections showed that the mutant phenotype was caused by a dysfunctional palisade cuticle layer in the seed coat. Chemical analysis found differences in lipid monomer composition between the wild-type and mutant seed coats. Genetic and molecular analyses revealed that a class II KNOTTED-like homeobox (KNOXII) gene, KNOX4, was responsible for the loss of physical dormancy in the seeds of the mutants. Microarray and chromatin immunoprecipitation analyses identified CYP86A, a gene associated with cutin biosynthesis, as one of the downstream target genes of KNOX4. This study elucidated a novel molecular mechanism of physical dormancy and revealed a new role of class II KNOX genes. Furthermore, KNOX4-like genes exist widely in seed plants but are lacking in nonseed species, indicating that KNOX4 may have diverged from the other KNOXII genes during the evolution of seed plants. PMID:27274062

  13. Linkage of the gene that encodes the alpha 1 chain of type V collagen (COL5A1) to type II Ehlers-Danlos syndrome (EDS II).

    PubMed

    Loughlin, J; Irven, C; Hardwick, L J; Butcher, S; Walsh, S; Wordsworth, P; Sykes, B

    1995-09-01

    Ehlers-Danlos syndrome (EDS) is a group of heritable disorders of connective tissue with skin, ligaments and blood vessels being the main sites affected. The commonest variant (EDS II) exhibits an autosomal dominant mode of inheritance and is characterized by joint hypermobility, cigarette paper scars, lax skin and excessive bruising. As yet no gene has been linked to EDS II, nor has linkage been established to a specific region of the genome. However, several candidate genes encoding proteins of the extracellular matrix have been excluded. Using an intragenic simple sequence repeat polymorphism, we report linkage of the COL5A1 gene, which encodes the alpha 1(V) chain of type V collagen, to EDS II. A maximum LOD score (Zmax) for linkage of 8.3 at theta = 0.00 was generated for a single large pedigree.

  14. Cloning of the major histocompatibility complex class II promoter binding protein affected in a hereditary defect in class II gene regulation.

    PubMed Central

    Reith, W; Barras, E; Satola, S; Kobr, M; Reinhart, D; Sanchez, C H; Mach, B

    1989-01-01

    The regulation of major histocompatibility complex class II gene expression is directly involved in the control of normal and abnormal immune responses. In humans, HLA-DR, -DQ, and -DP class II heterodimers are encoded by a family of alpha- and beta-chain genes clustered in the major histocompatibility complex. Their expression is developmentally controlled and normally restricted to certain cell types. This control is mediated by cis-acting sequences in class II promoters and by trans-acting regulatory factors. Several nuclear proteins bind to class II promoter sequences. In a form of hereditary immunodeficiency characterized by a defect in a trans-acting regulatory factor controlling class II gene transcription, we have observed that one of these nuclear factors (RF-X) does not bind to its target sequence (the class II X box). A cDNA encoding RF-X was isolated by screening a phage expression library with an X-box binding-site probe. The recombinant protein has the binding specificity of RF-X, including a characteristic gradient of affinity for the X boxes of HLA-DR, -DP, and -DQ promoters. RF-X mRNA is present in the regulatory mutants, indicating a defect in the synthesis of a functional form of the RF-X protein. Images PMID:2498880

  15. Persistent Ehrlichia chaffeensis infection occurs in the absence of functional major histocompatibility complex class II genes

    NASA Technical Reports Server (NTRS)

    Ganta, Roman Reddy; Wilkerson, Melinda J.; Cheng, Chuanmin; Rokey, Aaron M.; Chapes, Stephen K.

    2002-01-01

    Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis. We investigated the impact of two genes that control macrophage and T-cell function on murine resistance to E. chaffeensis. Congenic pairs of wild-type and toll-like receptor 4 (tlr4)- or major histocompatibility complex class II (MHC-II)-deficient mice were used for these studies. Wild-type mice cleared the infection within 2 weeks, and the response included macrophage activation and the synthesis of E. chaffeensis-specific Th1-type immunoglobulin G response. The absence of a functional tlr4 gene depressed nitric oxide and interleukin 6 secretion by macrophages and resulted in short-term persistent infections for > or =30 days. In the absence of MHC-II alleles, E. chaffeensis infections persisted throughout the entire 3-month evaluation period. Together, these data suggest that macrophage activation and cell-mediated immunity, orchestrated by CD4(+) T cells, are critical for conferring resistance to E. chaffeensis.

  16. Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene.

    PubMed

    Longet, David; Pawlowski, Jan

    2007-08-01

    Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.

  17. Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

    PubMed Central

    Shin, Sanghyun; Mackintosh, Caroline A.; Lewis, Janet; Heinen, Shane J.; Radmer, Lorien; Dill-Macky, Ruth; Baldridge, Gerald D.; Zeyen, Richard J.; Muehlbauer, Gary J.

    2008-01-01

    Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions. PMID:18467324

  18. Guanylyl cyclase/natriuretic peptide receptor-A gene disruption causes increased adrenal angiotensin II and aldosterone levels.

    PubMed

    Zhao, Di; Vellaichamy, Elangovan; Somanna, Naveen K; Pandey, Kailash N

    2007-07-01

    Disruption of the guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) gene leads to elevated arterial blood pressure and congestive heart failure in mice lacking NPRA. This study was aimed at determining whether Npr1 (coding for GC-A/NPRA) gene copy number affects adrenal ANG II and aldosterone (Aldo) levels in a gene-dose-dependent manner in Npr1 gene-targeted mice. Adrenal ANG II and Aldo levels increased in 1-copy mice compared with 2-copy mice, but decreased in 3-copy and 4-copy mice. In contrast, renal ANG II levels decreased in 1-copy (25%), 3-copy (38%), and 4-copy (39%) mice compared with 2-copy mice. The low-salt diet stimulated adrenal ANG II and Aldo levels in 1-copy (20 and 2,441%), 2-copy (15 and 2,339%), 3-copy (20 and 424%), and 4-copy (31 and 486%) mice, respectively. The high-salt diet suppressed adrenal ANG II and Aldo levels in 1-copy (46 and 29%) and 2-copy (38 and 17%) mice. On the other hand, the low-salt diet stimulated renal ANG II levels in 1-copy (45%), 2-copy (45%), 3-copy (59%), and 4-copy (48%) mice. However, the high-salt diet suppressed renal ANG II levels in 1-copy (28%) and 2-copy (27%) mice. In conclusion, NPRA signaling antagonizes adrenal ANG II and Aldo levels in a gene-dose dependent manner. Increased adrenal ANG II and Aldo levels may play an important role in elevated arterial blood pressure and progressive hypertension, leading to renal and vascular injury in Npr1 gene-disrupted mice.

  19. Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus).

    PubMed

    Lau, Quintin; Jobbins, Sarah E; Belov, Katherine; Higgins, Damien P

    2013-01-01

    Major histocompatibility complex (MHC) class II molecules have an integral role in the adaptive immune response, as they bind and present antigenic peptides to T helper lymphocytes. In this study of koalas, species-specific primers were designed to amplify exon 2 of the MHC class II DA and DB genes, which contain much of the peptide-binding regions of the α and β chains. A total of two DA α1 domain variants and eight DA β1 (DAB), three DB α1 and five DB β1 variants were amplified from 20 koalas from two free-living populations from South East Queensland and the Port Macquarie region in northern New South Wales. We detected greater variation in the β1 than in the α1 domains as well as evidence of positive selection in DAB. The present study provides a springboard to future investigation of the role of MHC in disease susceptibility in koalas.

  20. Isolation and characterization of major histocompatibility complex class II B genes in cranes.

    PubMed

    Kohyama, Tetsuo I; Akiyama, Takuya; Nishida, Chizuko; Takami, Kazutoshi; Onuma, Manabu; Momose, Kunikazu; Masuda, Ryuichi

    2015-11-01

    In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution. PMID:26452363

  1. DMA and DMB are the only genes in the class II region of the human MHC needed for class II-associated antigen processing

    SciTech Connect

    Ceman, S.; Rudersdorf, R.A.; Petersen, J.M.

    1995-03-15

    Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0{degrees}, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the {approximately} 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy. 67 refs., 5 figs., 1 tab.

  2. Adventitial gene transfer of catalase attenuates angiotensin II-induced vascular remodeling.

    PubMed

    Liu, Cun-Fei; Zhang, Jia; Shen, Kai; Gao, Ping-Jin; Wang, Hai-Ya; Jin, Xin; Meng, Chao; Fang, Ning-Yuan

    2015-04-01

    Vascular adventitia and adventitia‑derived reactive oxygen species (ROS) contribute to vascular remodeling following vascular injury. A previous ex vivo study in adventitial fibroblasts showed that catalase, one of most important anti‑oxide enzymes, was downregulated by angiotensin II (AngII). The aim of the present study was to investigate whether adventitial gene transfer of catalase affects AngII‑induced vascular remodeling in vivo. Adenoviruses co‑expressing catalase and enhanced green fluorescent protein (eGFP) or expressing eGFP only were applied to the adventitial surface of common carotid arteries of Sprague‑Dawley rats. Alzet minipumps administering AngII (0.75 mg/kg/day) were then implanted subcutaneously for 14 days. Systolic blood pressure and biological parameters of vascular remodeling were measured in each group. Adventitial fibroblasts were cultured and p38 mitogen‑activated protein kinase (MAPK) phosphorylation was measured using western blot analysis. The results showed that adventitial gene transfer of catalase had no effect on AngII‑induced systolic blood pressure elevation. However, catalase adenovirus transfection significantly inhibited AngII‑induced media hypertrophy compared with that of the control virus (P<0.05). In addition, catalase transfection significantly attenuated AngII‑induced ROS generation, macrophage infiltration, collagen deposition and adventitial α‑smooth muscle actin expression. Furthermore, catalase transfection significantly inhibited the AngII‑induced increase in p38MAPK phosphorylation. In conclusion, the results of the present study demonstrated that adventitial gene transfer of catalase significantly attenuated AngII‑induced vascular remodeling in rats via inhibition of adventitial p38MAPK phosphorylation.

  3. Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Ma, Ruiqin; He, Feng; Wen, Haishen; Li, Jifang; Shi, Bao; Shi, Dan; Liu, Miao; Mu, Weijie; Zhang, Yuanqing; Hu, Jian; Han, Weiguo; Zhang, Jianan; Wang, Qingqing; Yuan, Yuren; Liu, Qun

    2012-03-01

    As a specific gene of fish, cytochrome P450c17-II ( CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder ( Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI ( P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

  4. Phylogenetic positions of insectivora in eutheria inferred from mitochondrial cytochrome c oxidase subunit II gene.

    PubMed

    Onuma, M; Kusakabe, T; Kusakabe, S

    1998-02-01

    For the elucidation of the phylogenetic position of insectivora in eutheria, we have sequenced the cytochrome c oxidase subunit II (COII) gene of mitochondria for three insectivoran species [musk screw (Suncus murinus), shrew mole (Urotrichus talpoides), Japanese mole (Mogera wogura)] and analyzed these amino acid sequences with neighbor-joining (NJ) method and maximum likelihood (ML) method. NJ analysis shows polyphyly of Insectivora and Chiroptera. Assuming that each of Primates, Ferungulata, Chiroptera, Insectivora and Rodentia is a monophyletic group, ML analysis suggests that Chiroptera is a sister group of Insectivora and that Ferungulata is the closest outgroup to the (Insectivora and Chiroptera) clade.

  5. A new point mutation in the ND1 mitochondrial gene identified in a type II diabetic patient

    SciTech Connect

    Kalinin, V.N.; Schmidt, W.; Olek, K.

    1995-08-01

    A novel mutation in a mitochondrial gene was identified in a patient with type II diabetes mellitus. G-to-A transition was localized at the nt3316 position of gene ND1 and resulted in alanine threonine replacement at position 4 of mitochondrial NAD-H-dehydrogenase. 6 refs., 2 figs.

  6. Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota).

    PubMed

    Kuduk, K; Johanet, A; Allainé, D; Cohas, A; Radwan, J

    2012-08-01

    The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.

  7. Molecular evolution of the capsid gene in human norovirus genogroup II

    PubMed Central

    Kobayashi, Miho; Matsushima, Yuki; Motoya, Takumi; Sakon, Naomi; Shigemoto, Naoki; Okamoto-Nakagawa, Reiko; Nishimura, Koichi; Yamashita, Yasutaka; Kuroda, Makoto; Saruki, Nobuhiro; Ryo, Akihide; Saraya, Takeshi; Morita, Yukio; Shirabe, Komei; Ishikawa, Mariko; Takahashi, Tomoko; Shinomiya, Hiroto; Okabe, Nobuhiko; Nagasawa, Koo; Suzuki, Yoshiyuki; Katayama, Kazuhiko; Kimura, Hirokazu

    2016-01-01

    Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10−3 substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>102) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans. PMID:27384324

  8. Tyrosinemia type II (Richner-Hanhart syndrome): a new mutation in the TAT gene.

    PubMed

    Culic, Vida; Betz, Regina C; Refke, Melanie; Fumic, Ksenija; Pavelic, Jasminka

    2011-01-01

    In the present study we report the clinical features and the molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in a young girl from Croatia with Richner-Hanhart syndrome, mainly suffering from photophobia, hyperkeratosis of the palmes and soles and slight neurological abnormalities. Sequencing analysis of the TAT gene revealed a novel homozygous missense mutation c.1250G>A (p.R417Q) in exon 12, and herewith confirmed the clinical diagnosis. Showing the first symptoms in babyhood, at the age of 8 years it was for the first time clinically diagnosed that the patient suffers from tyrosinemia type II and a therapy with tyrosine and phenylalanine reduced diet has been started successfully. All symptoms disappeared within 2-4 weeks. Since that time, we have been following the girl until today for more than ten years. She is in a good condition, and attends the normal high school program.

  9. Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations

    USGS Publications Warehouse

    Bowen, L.; Aldridge, B.M.; Miles, A.K.; Stott, J.L.

    2006-01-01

    The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters. ?? 2006 Blackwell Munksgaard.

  10. Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes

    PubMed Central

    Kumar, Rahul

    2016-01-01

    Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species.

  11. Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes

    PubMed Central

    Kumar, Rahul

    2016-01-01

    Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species. PMID:27602045

  12. Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes.

    PubMed

    Kumar, Rahul

    2016-01-01

    Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species. PMID:27602045

  13. Molecular polymorphism and expression analysis of MHC class II B gene from red sea bream (Chrysophrys major).

    PubMed

    Chen, Song-Lin; Zhang, Yu-Xi; Xu, Mei-Yu; Ji, Xiang-Shan; Yu, Guo-Cai; Dong, Cheng-Fang

    2006-01-01

    MHC class II (major histocompatibility complex class II) plays an important role in the immune response of vertebrates. Its function is to present antigenic peptides to the T-cell receptor. In order to study the function and molecular polymorphism of class II B gene in fish, we have isolated cDNAs encoding class II B from spleen cDNA library of red sea bream (Chrysophrys major) by using EST sequencing, and examined genomic organization, molecular polymorphism and expression of red sea bream class II B gene. As in other vertebrates, five exons and four introns were identified in red sea bream class II B gene. Seven class II B alleles were identified from seven individuals of red sea bream. The deduced amino acid sequence of red sea bream MHC class II B 1(Chma-DAB*0101) had 87.1, 85.1, 87.1, 90.4, 87.1, 90.8% identity with those of red sea bream class II B 2, 3, 4, 5, 6, 7(Chma-DAB*0201-Chma-DAB*0701), respectively, and had 75.2, 74.5, 55.9, 55.1, 34.3 and 30.4% identity with those of striped sea bass, cichlid, rainbow trout, Atlantic salmon, mouse and human, respectively. Four different class II B alleles were observed in a single individual and two different 3' untranslated region (3' UTR) sequences from this individual may infer the existence of two loci at least. Semi-quantitative RT-PCR demonstrated that high expression was detected in liver, head kidney, kidney, intestine, gill, stomach, hear and spleen, low expression in muscle and blood. Challenge of red sea bream with the pathogenic bacteria, Vibrio anguillarum, resulted in a significant decrease in the expression of MHC class II B mRNA from 5 to 72 h after infection in liver, spleen, head kidney and intestine, followed by a recovery to normal level after 96 h.

  14. A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II.

    PubMed

    Kim, J W; Nam, S H; Jang, K T; Lee, S H; Kim, C C; Hahn, S H; Hu, J C C; Simmer, J P

    2004-08-01

    The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188C-->G, IVS2-3C-->G) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.

  15. A 3' splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II.

    PubMed

    Jin, Ping; Hao, Jing-Wen; Chen, Ke; Dong, Chang-Sheng; Yang, You-Bo; Mo, Zhao-Hui

    2013-10-10

    The purpose of this study was to identify the underlying genetic cause in a four generation Chinese family diagnosed with mucopolysaccharidosis type II. Peripheral blood samples were collected from family members and the iduronate-2-sulfatase (IDS) gene was analyzed by DNA sequencing. The impact of IDS mutations on mRNA transcription was determined by quantitative real-time RT-PCR (qRT-PCR) in both patients as well as in healthy control samples. In addition, RT-PCR was performed to confirm the characteristics of a found mutation located in non-canonical splicing site. A 3' splice site mutation c.880-8A>G (IVS 6-8A>G) was identified in two members of the analyzed MPS II family and sequencing of RT-PCR products showed that this mutation activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in an exon 7 frameshift introducing a premature stop codon and shorter peptide chain. In addition, qRT-PCR products from the two patients showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression in healthy control samples, possibly as a result of nonsense-mediated mRNA decay. In conclusion, in this study, we have identified an IDS gene splice mutation which is associated with clinically attenuated MPS II phenotype. In addition, our study accentuates the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only gDNA, the link between genotype and phenotype may have been misinterpreted.

  16. Murine neuroblastoma vaccines produced by retroviral transfer of MHC class II genes.

    PubMed

    Hock, R A; Reynolds, B D; Tucker-McClung, C L; Heuer, J G

    1996-01-01

    Malignant tumors express tumor-related antigens, but effective antitumor immunity does not occur in the primary host. One hypothesis is that there is insufficient stimulation of T-cell responses due to ineffective antigen presentation. An approach to overcome these deficiencies is to modify tumor cells to express major histocompatibility complex (MHC) class II genes and thus facilitate the presentation of antigens directly by tumor cells. Our experiments with a murine neuroblastoma cell line (neuro-2a) transduced with DR (xenogeneic), 1-Ab (allogeneic), or 1-Ak (syngeneic) MHC class II genes support this notion. The relative potencies of the modified neuro-2a to induce immunity to unmodified neuro-2a were neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak. Modified neuro-2a also could stimulate naive splenocyte proliferation in vitro. The relative magnitude of the proliferative responses seen after stimulation with modified tumor cells was neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak > unmodified neuro-2a. Hence, the tumor cell-induced splenocyte proliferative responses observed in vitro correlate with the effectiveness of the tumor cell vaccines to induce antitumor immunity in vivo. These data show that the expression of exogenous MHC class II on tumor cells is a potent stimulus for specific antitumor immunity. Because of the correlation of the in vivo and in vitro immune responses to modified tumor cells, the tumor-induced lymphocyte proliferation assay may be useful in evaluating tumor cell vaccines produced by additional genetic modifications of tumor cells.

  17. Early diagnosis of and surgical strategy for adrenal medullary disease in MEN II gene carriers

    SciTech Connect

    Jansson, S.; Tisell, L.E.; Fjaelling, M.L.; Lindberg, S.; Jacobsson, L.; Zachrisson, B.F.

    1988-01-01

    Sixteen multiple endocrine neoplasia type II (MEN II) gene carriers--12 who had undergone thyroidectomy because of medullary carcinoma of the thyroid and 4 whose thyroid glands had been removed because of C cell hyperplasia--were examined for the presence of pheochromocytomas. No patient had sought medical advice for pheochromocytoma symptoms. Fourteen patients had MEN IIa syndromes, one patient had a MEN IIb and another patient had a mixed syndrome of von Recklinghausen's neurofibromatosis and MEN II. Eight patients had undergone unilateral adrenalectomy for pheochromocytoma 11 +/- 4 years before. The patients underwent clinical examination, determination of the urinary excretion of catecholamines and metabolites, and /sup 131/I-metaiodobenzylguanidine (/sup 131/I-MIBG) and CAT scans. /sup 131/I-MIBG scanning was performed with images 1, 4, and 7 days after the radionuclide injection. In seven of eight patients who had undergone unilateral adrenalectomies, the /sup 131/I-MIBG scans showed accumulation of the radionuclide in the remaining adrenal gland. Bilateral adrenal accumulation of the radionuclide was demonstrated in seven of eight MEN IIa gene carriers who had not undergone adrenalectomy. Five patients, two of whom had undergone adrenalectomy, were found to have unilateral pheochromocytomas less than 2 cm in diameter. Only one of these five patients had an elevated excretion of urinary catecholamines. Between day 4 and day 7 after /sup 131/I-MIBG injection, adrenal glands with pheochromocytomas increased their relative accumulation of the radionuclide significantly more (p less than 0.02) than did adrenal glands without any demonstrable pheochromocytomas. All the pheochromocytomas were viewed by means of CAT scans.

  18. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

    SciTech Connect

    Gilmour, D.S.; Lis, J.T.

    1986-11-01

    By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

  19. Chicken ovalbumin upstream promoter transcription factor II regulates renin gene expression.

    PubMed

    Mayer, Sandra; Roeser, Marc; Lachmann, Peter; Ishii, Sumiyashi; Suh, Jae Mi; Harlander, Sabine; Desch, Michael; Brunssen, Coy; Morawietz, Henning; Tsai, Sophia Y; Tsai, Ming-Jer; Hohenstein, Bernd; Hugo, Christian; Todorov, Vladimir T

    2012-07-13

    This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.

  20. Chicken Ovalbumin Upstream Promoter Transcription Factor II Regulates Renin Gene Expression*

    PubMed Central

    Mayer, Sandra; Roeser, Marc; Lachmann, Peter; Ishii, Sumiyashi; Suh, Jae Mi; Harlander, Sabine; Desch, Michael; Brunssen, Coy; Morawietz, Henning; Tsai, Sophia Y.; Tsai, Ming-Jer; Hohenstein, Bernd; Hugo, Christian; Todorov, Vladimir T.

    2012-01-01

    This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression. PMID:22645148

  1. Distribution and origin of bovine major histocompatibility complex class II DQA1 genes in Japan.

    PubMed

    Takeshima, S; Chen, S; Miki, M; Kado, M; Aida, Y

    2008-09-01

    We sequenced the major histocompatibility complex (MHC) class II DQA1 gene in 352 Japanese cattle (95 Japanese Black, 91 Holstein, 102 Japanese Shorthorn and 64 Jersey cattle) using a new sequence-based typing method. In total, 19 bovine MHC (BoLA)-DQA1 alleles, of which two were novel alleles, were detected. The Holstein, Jersey, Japanese Shorthorn and Japanese Black breeds had 13, 12, 10 and 15 alleles, respectively. The dendrogram that was constructed by the neighbor-joining method on the basis of the DQA1 gene allele frequencies of the four Japanese cattle breeds showed that the Holstein and Japanese Black breeds were closest to each other, with Jersey being farther from these two breeds than Japanese Shorthorn. In addition, Wu-Kabat analysis showed that the DQA1 alleles of the Holstein and Japanese Black were the most and least polymorphic, respectively. Phylogenetic analyses indicated that the DQA1 gene of Bovidae such as cattle, sheep, bison and goat were more similar to pig SLA-DQA genes than to human HLA-DQA1 and dog DLA-DQA genes. The cattle, goat, bison, sheep, human and pig DQA1 molecules had similar rates of amino acid sequence polymorphism, but the distribution of their polymorphic residues differed from that in the dog DQA1 protein. However, the Bovidae DQA1 molecule had more polymorphic residues than the human, pig and dog DQA molecules at two regions, namely positions 52-53 and 65-66. This indicates that the Bovidae DQA1 locus is more polymorphic than the DQA loci of other species.

  2. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  3. Cooperativity between the J and S elements of class II major histocompatibility complex genes as enhancers in normal and class II- negative patient and mutant B cell lines

    PubMed Central

    1995-01-01

    The class II major histocompatibility complex genes all contain in their proximal promoters three cis-elements called S, X, and Y that are conserved in both sequence and position, and a fourth element, J, conserved in sequence but not in position. J, X, and Y and, to some extent, S, have been shown to be functionally important in regulation of expression of these genes. In the present study, a protein factor that binds cooperatively to the S plus J elements of the promoter of the class II major histocompatibility complex gene DPA has been detected. Moreover, functional cooperativity between S and J in activation of the enhancerless -40 interferon-beta (-40 IFN-beta) promoter has been demonstrated. Finally, the latter assay appears to subdivide complementation group A of class II negative human B cell lines that includes both mutants generated in vitro and cells from patients with the bare lymphocyte syndrome (type II). In three of these cell lines, the enhancerless -40 IFN-beta promoter containing the S plus J elements was functionally active, while in the others it was inactive. PMID:7790817

  4. Identification and mapping of two divergent, unlinked major histocompatibility complex class II B genes in Xiphophorus fishes.

    PubMed Central

    McConnell, T J; Godwin, U B; Norton, S F; Nairn, R S; Kazianis, S; Morizot, D C

    1998-01-01

    We have isolated two major histocompatibility complex (MHC) class II B genes from the inbred fish strain Xiphophorus maculatus Jp 163 A. We mapped one of these genes, designated here as DXB, to linkage group III, linked to a malic enzyme locus, also syntenic with human and mouse MHC. Comparison of genomic and cDNA clones shows the gene consists of six exons and five introns. The encoded beta1 domain has three amino acids deleted and a cytoplasmic tail nine amino acids longer than in other teleost class II beta chains, more similar to HLA-DRB, clawed frog Xela-F3, and nurse shark Gici-B. Key residues for disulfide bonds, glycosylation, and interaction with alpha chains are conserved. These same features are also present in a swordtail (Xiphophorus helleri) genomic DXB PCR clone. A second type of class II B clone was amplified by PCR from X. maculatus and found to be orthologous to class II genes identified in other fishes. This DAB-like gene is 63% identical to the X. maculatus DXB sequence in the conserved beta2-encoding exon and was mapped to new unassigned linkage group LG U24. The DXB gene, then, represents an unlinked duplicated locus not previously identified in teleosts. PMID:9691047

  5. Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy

    PubMed Central

    Stubbs, Sarah H.; Conrad, Nicholas K.

    2015-01-01

    Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency. PMID:25477387

  6. Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy.

    PubMed

    Stubbs, Sarah H; Conrad, Nicholas K

    2015-01-01

    Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency.

  7. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes

  8. RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

    PubMed Central

    Glover-Cutter, Kira; Kim, Soojin; Espinosa, Joaquin; Bentley, David L.

    2010-01-01

    We investigated co-transcriptional recruitment of pre-mRNA processing factors to human genes. Capping factors associate with paused RNA pol II at the 5′ ends of quiescent genes. They also track throughout actively transcribed genes, and accumulate with paused polymerase in the 3′ flanking region. 3′ processing factors CstF and CPSF are maximally recruited 0.5-1.5 kb downstream of poly(A) sites where they coincide with capping factors, Spt5, and Ser2 hyperphosphorylated, paused pol II. 3′ end processing factors also localize at transcription start sites, and this early recruitment is enhanced after polymerase arrest with DRB. These results suggest that promoters may help specify recruitment of 3′ end processing factors. We propose a dual pausing model where elongation arrests near the transcription start site and in the 3′ flank to allow co-transcriptional processing by factors recruited to the pol II ternary complex. PMID:18157150

  9. Diversification of porcine MHC class II genes: evidence for selective advantage.

    PubMed

    Luetkemeier, Erin S; Malhi, Ripan S; Beever, Jonathan E; Schook, Lawrence B

    2009-02-01

    The major histocompatibility complex (MHC) is an immunological gene-dense region of high diversity in mammalian species. Sus scrofa was domesticated by at least six independent events over Eurasia during the Holocene period. It has been hypothesized that the level and distribution of MHC variation in pig populations reflect genetic selection and environmental influences. In an effort to define the complexity of MHC polymorphisms and the role of selection in the generation of class II gene diversity (DQB, DRB1, and pseudogene PsiDRB3), DNA from globally distributed unrelated domestic pigs of European and Asian origins and a Suidae out-group was analyzed. The number of pseudogene alleles identified (PsiDRB3 33) was greater than those found in the expressed genes (DQB 20 and DRB1 23) but the level of observed heterozygosity (PsiDRB3 0.452, DQB 0.732, and DRB1 0.767) and sequence diversity (PsiDRB3 0.029, DQB 0.062, and DRB1 0.074) were significantly lower in the pseudogene, respectively. The substitution ratios reflected an excess of d (N) (DQB 1.476, DRB1 1.724, and PsiDRB3 0.508) and the persistence of expressed gene alleles suggesting the influence of balancing selection, while the pseudogene was undergoing purifying selection. The lack of a clear MHC phylogeographic tree, coupled with close genetic distances observed between the European and Asian populations (DQB 0.047 and DRB1 0.063) suggested that unlike observations using mtDNA, the MHC diversity lacks phylogeographic structure and appears to be globally uniform. Taken together, these results suggest that, despite regional differences in selective breeding and environments, no skewing of MHC diversity has occurred.

  10. Diversification of porcine MHC class II genes: evidence for selective advantage.

    PubMed

    Luetkemeier, Erin S; Malhi, Ripan S; Beever, Jonathan E; Schook, Lawrence B

    2009-02-01

    The major histocompatibility complex (MHC) is an immunological gene-dense region of high diversity in mammalian species. Sus scrofa was domesticated by at least six independent events over Eurasia during the Holocene period. It has been hypothesized that the level and distribution of MHC variation in pig populations reflect genetic selection and environmental influences. In an effort to define the complexity of MHC polymorphisms and the role of selection in the generation of class II gene diversity (DQB, DRB1, and pseudogene PsiDRB3), DNA from globally distributed unrelated domestic pigs of European and Asian origins and a Suidae out-group was analyzed. The number of pseudogene alleles identified (PsiDRB3 33) was greater than those found in the expressed genes (DQB 20 and DRB1 23) but the level of observed heterozygosity (PsiDRB3 0.452, DQB 0.732, and DRB1 0.767) and sequence diversity (PsiDRB3 0.029, DQB 0.062, and DRB1 0.074) were significantly lower in the pseudogene, respectively. The substitution ratios reflected an excess of d (N) (DQB 1.476, DRB1 1.724, and PsiDRB3 0.508) and the persistence of expressed gene alleles suggesting the influence of balancing selection, while the pseudogene was undergoing purifying selection. The lack of a clear MHC phylogeographic tree, coupled with close genetic distances observed between the European and Asian populations (DQB 0.047 and DRB1 0.063) suggested that unlike observations using mtDNA, the MHC diversity lacks phylogeographic structure and appears to be globally uniform. Taken together, these results suggest that, despite regional differences in selective breeding and environments, no skewing of MHC diversity has occurred. PMID:19142631

  11. Population genetics and natural selection in the gene encoding the Duffy binding protein II in Iranian Plasmodium vivax wild isolates.

    PubMed

    Valizadeh, Vahideh; Zakeri, Sedigheh; Mehrizi, Akram Abouie; Djadid, Navid Dinparast

    2014-01-01

    Region II of Duffy binding protein (PvDBP-II) is one of the most promising blood-stage vaccine candidate antigens against Plasmodium vivax and having knowledge of the nature and genetic polymorphism of PvDBP-II among global P. vivax isolates is important for developing a DBP-based vaccine. By using PCR and sequencing, the present molecular population genetic approach was carried out to investigate sequence diversity and natural selection of dbp-II gene in 63 P. vivax isolates collected from unstable and low transmission malaria-endemic areas of Iran during 2008-2012. Also, phylogenetic analysis, the diversifying natural selection, and recombination across the pvdbp-II gene, including regions containing B-cell epitopes were analyzed using the DnaSP and MEGA4 programs. Twenty two single nucleotide polymorphisms (SNPs, including 20 non-synonymous and 2 synonymous) were identified in PvDBP-II, resulting in 16 different PvDBP-II haplotypes among the Iranian P. vivax isolates. High binding inhibitory B-cell epitope (H3) overlapping with intrinsically unstructured/disordered region (aa: 384-392) appeared to be highly polymorphic (D384G/E385K/ K386N/Q/R390H), and positive selective pressure acted on this region. Most of the polymorphic amino acids, which are located on the surface of the protein, are under selective pressure that implies increased recombination events and exposure to the human immune system. In summary, PvDBP-II gene displays genetic polymorphism among Iranian P. vivax isolates and it is under selective pressure. Mutations, recombination, and positive selection seem to play a role in the resulting genetic diversity, and phylogenetic analysis of DNA sequences demonstrates that Iranian isolates represent a sample of the global population. These results are useful for understanding the nature of the P. vivax population in Iran and also for development of PvDBP-II-based malaria vaccine.

  12. Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines.

    PubMed Central

    Yang, Z; Accolla, R S; Pious, D; Zegers, B J; Strominger, J L

    1988-01-01

    Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF. Images PMID:2458252

  13. Coordinated changes of histone modifications and HDAC mobilization regulate the induction of MHC class II genes by Trichostatin A

    PubMed Central

    2006-01-01

    The deacetylase inhibitor Trichostatin A (TSA) induces the transcription of the Major Histocompatibility Class II (MHC II) DRA gene in a way independent of the master coactivator CIITA. To analyze the molecular mechanisms by which this epigenetic regulator stimulates MHC II expression, we used chromatin immunoprecipitation (ChIP) assays to monitor the alterations in histone modifications that correlate with DRA transcription after TSA treatment. We found that a dramatic increase in promoter linked histone acetylation is followed by an increase in Histone H3 lysine 4 methylation and a decrease of lysine 9 methylation. Fluorescence recovery after photobleaching (FRAP) experiments showed that TSA increases the mobility of HDAC while decreasing the mobility of the class II enhanceosome factor RFX5. These data, in combination with ChIP experiments, indicate that the TSA-mediated induction of DRA transcription involves HDAC relocation and enhanceosome stabilization. In order to gain a genome-wide view of the genes responding to inhibition of deacetylases, we compared the transcriptome of B cells before and after TSA treatment using Affymetrix microarrays. This analysis showed that in addition to the DRA gene, the entire MHC II family and the adjacent histone cluster that are located in chromosome 6p21-22 locus are strongly induced by TSA. A complex pattern of gene reprogramming by TSA involves immune recognition, antiviral, apoptotic and inflammatory pathways and extends the rationale for using Histone Deacetylase Inhibitors (HDACi) to modulate the immune response. PMID:16452299

  14. Expression of Intratumoral IGF-II Is Regulated by the Gene Imprinting Status in Triple Negative Breast Cancer from Vietnamese Patients

    PubMed Central

    Radhakrishnan, Vinodh Kumar; Hernandez, Lorraine Christine; Anderson, Kendra; Tan, Qianwei; De León, Marino; De León, Daisy D.

    2015-01-01

    African American women suffer higher incidence and mortality of triple negative breast cancer (TNBC) than Caucasian women. TNBC is very aggressive, causing the worst clinical outcome. We previously demonstrated that tumors from these patients express high IGF-II and exhibit high activation of the IGF signaling pathways. IGF-II gene expression is imprinted (monoallelic), promotes tumor progression, and metastasis and regulates Survivin, a TNBC prognostic marker. Since BC mortality has increased among young Vietnamese women, we analyzed 48 (paired) TNBC samples from Vietnamese patients to assess IGF-II expression. We analyzed all samples by qrtPCR for identification of IGF-II heterozygosity and to determine allelic expression of the IGF-II gene. We also analyzed the tissues for proIGF-II and Survivin by RT-PCR and Western blotting. A total of 28 samples displayed IGF-II heterozygosity of which 78% were biallelic. Tumors with biallelic IGF-II gene expression exhibited the highest levels of proIGF-II and Survivin. Although 100% of these tissues corresponding normal samples were biallelic, they expressed significantly lower levels of or no proIGF-II and Survivin. Thus, IGF-II biallelic gene expression is differentially regulated in normal versus tumor tissues. We propose that intratumoral proIGF-II is dependent on the IGF-II gene imprinting status and it will promote a more aggressive TNBC. PMID:26448747

  15. Annotation of gene promoters by integrative data-mining of ChIP-seq Pol-II enrichment data

    PubMed Central

    2010-01-01

    Background Use of alternative gene promoters that drive widespread cell-type, tissue-type or developmental gene regulation in mammalian genomes is a common phenomenon. Chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) or massive parallel sequencing (ChIP-seq) are enabling genome-wide identification of active promoters in different cellular conditions using antibodies against Pol-II. However, these methods produce enrichment not only near the gene promoters but also inside the genes and other genomic regions due to the non-specificity of the antibodies used in ChIP. Further, the use of these methods is limited by their high cost and strong dependence on cellular type and context. Methods We trained and tested different state-of-art ensemble and meta classification methods for identification of Pol-II enriched promoter and Pol-II enriched non-promoter sequences, each of length 500 bp. The classification models were trained and tested on a bench-mark dataset, using a set of 39 different feature variables that are based on chromatin modification signatures and various DNA sequence features. The best performing model was applied on seven published ChIP-seq Pol-II datasets to provide genome wide annotation of mouse gene promoters. Results We present a novel algorithm based on supervised learning methods to discriminate promoter associated Pol-II enrichment from enrichment elsewhere in the genome in ChIP-chip/seq profiles. We accumulated a dataset of 11,773 promoter and 46,167 non-promoter sequences, each of length 500 bp, generated from RNA Pol-II ChIP-seq data of five tissues (Brain, Kidney, Liver, Lung and Spleen). We evaluated the classification models in building the best predictor and found that Bagging and Random Forest based approaches give the best accuracy. We implemented the algorithm on seven different published ChIP-seq datasets to provide a comprehensive set of promoter annotations for both protein-coding and non-coding genes in

  16. Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia type II.

    PubMed

    Hühn, R; Stoermer, H; Klingele, B; Bausch, E; Fois, A; Farnetani, M; Di Rocco, M; Boué, J; Kirk, J M; Coleman, R; Scherer, G

    1998-03-01

    Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG-->TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

  17. Estrogen receptor α gene PvuII polymorphism and coronary artery disease: a meta-analysis of 21 studies*

    PubMed Central

    Ding, Jie; Xu, Hui; Yin, Xiang; Zhang, Fu-rong; Pan, Xiao-ping; Gu, Yi-an; Chen, Jun-zhu; Guo, Xiao-gang

    2014-01-01

    The association between the estrogen receptor α gene (ESR1) PvuII polymorphism (c.454-397T>C) and coronary artery disease (CAD) is controversial. Thus, we conducted a meta-analysis to evaluate the relationship. Data were collected from 21 studies encompassing 9926 CAD patients and 16 710 controls. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the relationship between PvuII polymorphism and CAD. The polymorphism in control populations in all studies followed Hardy-Weinberg equilibrium. We found a significant association between ESR1 PvuII polymorphism and CAD risk in all subjects. When the data were stratified by region, a significant association between ESR1 PvuII polymorphism and CAD risk was observed in Asian populations but not in Western populations. The current study suggests that ESR1 PvuII polymorphism has an important role in CAD susceptibility. PMID:24599688

  18. RNA polymerase II mutants defective in transcription of a subset of genes.

    PubMed Central

    Scafe, C; Nonet, M; Young, R A

    1990-01-01

    Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species. Images PMID:2406558

  19. Mutation Update for COL2A1 Gene Variants Associated with Type II Collagenopathies.

    PubMed

    Barat-Houari, Mouna; Sarrabay, Guillaume; Gatinois, Vincent; Fabre, Aurélie; Dumont, Bruno; Genevieve, David; Touitou, Isabelle

    2016-01-01

    Mutations in the COL2A1 gene cause a spectrum of rare autosomal-dominant conditions characterized by skeletal dysplasia, short stature, and sensorial defects. An early diagnosis is critical to providing relevant patient care and follow-up, and genetic counseling to affected families. There are no recent exhaustive descriptions of the causal mutations in the literature. Here, we provide a review of COL2A1 mutations extracted from the Leiden Open Variation Database (LOVD) that we updated with data from PubMed and our own patients. Over 700 patients were recorded, harboring 415 different mutations. One-third of the mutations are dominant-negative mutations that affect the glycine residue in the G-X-Y repeats of the alpha 1 chain. These mutations disrupt the collagen triple helix and are common in achondrogenesis type II and hypochondrogenesis. The mutations resulting in a premature stop codon are found in less severe phenotypes such as Stickler syndrome. The p.(Arg275Cys) substitution is found in all patients with COL2A1-associated Czech dysplasia. LOVD-COL2A1 provides support and potential collaborative material for scientific and clinical projects aimed at elucidating phenotype-genotype correlation and differential diagnosis in patients with type II collagenopathies. PMID:26443184

  20. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis

    PubMed Central

    Piriya, P. Sobana; Vasan, P. Thirumalai; Padma, V. S.; Vidhyadevi, U.; Archana, K.; Vennison, S. John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production. PMID:22919503

  1. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis.

    PubMed

    Piriya, P Sobana; Vasan, P Thirumalai; Padma, V S; Vidhyadevi, U; Archana, K; Vennison, S John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production.

  2. Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes

    PubMed Central

    Sarter, Kerstin; Leimgruber, Elisa; Gobet, Florian; Agrawal, Vishal; Dunand-Sauthier, Isabelle; Barras, Emmanuèle; Mastelic-Gavillet, Béatris; Kamath, Arun; Fontannaz, Paola; Guéry, Leslie; Duraes, Fernanda do Valle; Lippens, Carla; Ravn, Ulla; Santiago-Raber, Marie-Laure; Magistrelli, Giovanni; Fischer, Nicolas; Siegrist, Claire-Anne; Hugues, Stéphanie

    2016-01-01

    Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class II expression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2−/− mice exhibited enhanced effector CD4+ and CD8+ T cell responses, impaired CD4+ regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell–mediated immunity. PMID:26809444

  3. Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding. alpha. -mannosidase II

    SciTech Connect

    Fukuda, M.N.; Masri, K.A. ); Dell, A. ); Luzzatto, L. ); Moremen, K.W. )

    1990-10-01

    Congenital dyserythropoietic anemia type II, or hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis test (HEMPAS), is a genetic anemia in humans inherited by an autosomally recessive mode. The enzyme defect in most HEMPAS patients has previously been proposed as a lowered activity of N-acetylglucosaminyltransferase II, resulting in a lack of polylactosamine on proteins and leading to the accumulation of polylactosaminyl lipids. A recent HEMPAS case, G.C., has now been analyzed by cell-surface labeling, fast-atom-bombardment mass spectrometry of glycopeptides, and activity assay of glycosylation enzymes. Significantly decreased glycosylation of polylactosaminoglycan proteins and incompletely processed asparagine-linked oligosaccharides were detected in the erythrocyte membranes of G.C. These results suggest that G.C. cells contain a mutation in {alpha}-ManII-encoding gene that results in inefficient expression of {alpha}-ManII mRNA, either through reduced transcription or message instability. This report demonstrates that HEMPAS is caused by a defective gene encoding an enzyme necessary for the synthesis of asparagine-linked oligosaccharides.

  4. Differentially expressed genes in autosomal dominant osteopetrosis type II osteoclasts reveal known and novel pathways for osteoclast biology.

    PubMed

    Coudert, Amélie E; Del Fattore, Andrea; Baulard, Céline; Olaso, Robert; Schiltz, Corinne; Collet, Corinne; Teti, Anna; de Vernejoul, Marie-Christine

    2014-03-01

    Autosomal dominant osteopetrosis type II (ADO II) is a rare, heritable bone disorder characterized by a high bone mass and insufficient osteoclast activity. Mutations in the CLCN7 gene have been reported to cause ADO II. To gain novel insights into the pathways dysregulated in ADOII osteoclasts, we identified changes in gene expression in osteoclasts from patients with a heterozygous mutation of CLCN7. To do this, we carried out a transcriptomic study comparing gene expression in the osteoclasts of patients with ADO II and healthy donors. Our data show that, according to our selection criteria, 182 genes were differentially expressed in osteoclasts from patients and controls. From the 18 displaying the highest change in microarray, we confirmed differential expression for seven by qPCR. Although two of them have previously been found to be expressed in osteoclasts (ITGB5 and SERPINE2), the other five (CES1 (carboxyl esterase 1), UCHL1 (ubiquitin carboxy-terminal esterase L1, also known as ubiquitin thiolesterase), WARS (tryptophanyl-tRNA synthetase), GBP4 (guanylate-binding protein 4), and PRF1) are not yet known to have a role in this cell type. At the protein level, we confirmed elevated expression of ITGB5 and reduced expression of WARS, PRF1, and SERPINE2. Transfection of ClC-7 harboring the G215R mutation into osteoclasts resulted in an increased ITGB5 and reduced PRF1 expression of borderline significance. Finally, we observed that the ADO II patients presented a normal or increased serum level of bone formation markers, demonstrating a coupling between dysfunctional osteoclasts and osteoblasts. Sphingosine kinase 1 mRNA was expressed at the same level in ADO II and control osteoclasts. In conclusion, these data suggest that in addition to an acidification dysfunction caused by the CLCN7 mutation, a change in ITGB5, PRF1, WARS, and SERPINE2 expression could be part of the osteoclastic phenotype of ADO II.

  5. Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation

    PubMed Central

    Tummala, Padmaja; Mali, Raghuveer S.; Guzman, Eduardo; Zhang, Xiao

    2010-01-01

    Purpose During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites. Methods High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10–12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT–PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d). Results ChIP-on-chip Pol-II peak

  6. Major histocompatibility complex haplotypes and class II genes in non-Jewish patients with pemphigus vulgaris

    SciTech Connect

    Ahmed, A.R. Center for Blood Research, Boston, MA American Red Cross Blood Services-Northeast Region, Dedham, MA ); Wagner, R.; Khatri, K.; Notani, G.; Awdeh, Z.; Alper, C.A. ); Yunis, E.J. American Red Cross Blood Services-Northeast Region, Dedham, MA )

    1991-06-01

    Previous studies demonstrated that HLA-DR4 was markedly increased among Ashkenazi Jewish patients with pemphigus vulgaris (PV), almost entirely as the common Jewish extended haplotype (HLA-B38, SC21, DR4, DQw8) or as the haplotype HLA-B35, SC31, DR4, DQw8, and that HLA-DR4, DQw8 was distributed among patients in a manner consistent with dominant expression of a class II (D-region or D-region-linked) susceptibility gene. In the present study of major histocompatibility complex (MHC) halotypes in 25 non-Jewish PV patients, DR4, DQw8 was found in 12 of the patients and DRw6, DQw5 was found in 15. Only 3 patients had neither. The non-Jewish patients were of more Southern European extraction than our controls. This suggests that there are two major MHC susceptibility alleles in American patients with PV. The more ancient apparently arose on a haplotype in the Jews, HLA-B38(35), SC21(SC31), DR4, DQw8, and spread to other populations largely as D-region segments. The other arose in or near Italy on the haplotype HLA-Bw55, SB45, DRw14, DQw5 amd has also partially fragmented so that many patients carry only DRw14, DQw5. The available data do not permit the specific localization of either the DR4, DQw8-or the DRw14, DQw5-linked susceptibility genes.

  7. Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II.

    PubMed

    Natt, E; Kida, K; Odievre, M; Di Rocco, M; Scherer, G

    1992-10-01

    Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame.

  8. Tissue-specific, inducible and functional expression of the E alpha d MHC class II gene in transgenic mice.

    PubMed Central

    Pinkert, C A; Widera, G; Cowing, C; Heber-Katz, E; Palmiter, R D; Flavell, R A; Brinster, R L

    1985-01-01

    We have introduced the class II E alpha d gene into (C57BL/6 X SJL) F2 mice which do not express their endogenous E alpha gene. The mRNA expression of the E alpha d gene shows the same tissue distribution as the endogenous class II genes except in the case of one mouse, which carried 19 copies of the E alpha d gene. In this mouse expression of E alpha d mRNA was seen in all tissues tested. Expression of the transgene was induced by gamma-interferon in isolated macrophages from the transgenic mice. In addition, fluorescence activated cell sorter (FACS) analysis, mixed lymphocyte response and antigen-presentation experiments showed that the product of the transferred gene is expressed on the cell surface and functions as a major histocompatibility complex restriction element. Transmission of the gene occurred only with female transgenic mice, all males were infertile or did not transmit the gene, suggesting an effect of the transferred DNA sequence on male reproductive function. Images Fig. 2. PMID:3935430

  9. Trans-species polymorphism and selection in the MHC class II DRA genes of domestic sheep.

    PubMed

    Ballingall, Keith T; Rocchi, Mara S; McKeever, Declan J; Wright, Frank

    2010-01-01

    Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the

  10. Trans-species polymorphism and selection in the MHC class II DRA genes of domestic sheep.

    PubMed

    Ballingall, Keith T; Rocchi, Mara S; McKeever, Declan J; Wright, Frank

    2010-06-30

    Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the

  11. A new set of differentially expressed signaling genes is early expressed in coffee leaf rust race II incompatible interaction.

    PubMed

    Diola, Valdir; Brito, Giovani G; Caixeta, Eveline T; Pereira, Luiz F P; Loureiro, Marcelo E

    2013-08-01

    New races of coffee rust are overcoming resistance genes available in germplasm and cultivated cultivars and bringing recently some coffee-producing countries in severe economic challenge. The objective of this study was to identify the genes that are linked to host resistance to the major coffee rust race II. In our study, we have identified and studied a segregating population that has a single monogenic resistant gene to coffee rust. Coffee leaves of parents, resistant, and susceptible genotypes of the F2 generation plants were inoculated with pathogen spores. A differential analysis was performed by combined cDNA-AFLP and bulk segregant analysis (BSA) in pooled samples collected 48 and 72 h postinoculation, increasing the selectiveness for differential gene expression. Of 108 differential expressed genes, between 33,000 gene fragments analyzed, 108 differential expressed genes were identified in resistant plants. About 20 and 22 % of these resistant-correlated genes are related to signaling and defense genes, respectively. Between signaling genes, the major subclass corresponds to receptor and resistant homolog genes, like nucleotide-binding site leucine-rich repeat (NBS-LRR), Pto-like, RLKs, Bger, and RGH1A, all not previously described in coffee rust responses. The second major subclass included kinases, where two mitogen-activated kinases (MAPK) are identified. Further gene expression analysis was performed for 21 selected genes by real-time PCR gene expression analysis at 0, 12, 24, 48, and 72 h postinoculation. The expression of genes involved in signaling and defense was higher at 24 and 72 h after inoculation, respectively. The NBS-LRR was the more differentially expressed gene between the signaling genes (four times more expressed in the resistant genotype), and thraumatin (PR5) was the more expressed between all genes (six times more expressed). Multivariate analysis reinforces the significance of the temporal separation of identified signaling and

  12. HIGH FREQUENCY GENETIC TRANSFORMATION OF CICHORIUM INTYBUS L. USING nptII GENE AS A SELECTIVE MARKER.

    PubMed

    Matvieieva, N; Shakhovsky, A; Kvasko, O; Kuchuk, N

    2015-01-01

    Cichorium intybus L. is an important vegetable crop used as salad (leaf form) and for the production of coffee substitutes (root form). At the same time these plants can also be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency Agrobacterium rhizogenes- or A. tumefaciens-mediated transformation of C. intybus L. for construction of transgenic "hairy" roots and plants. The used plasmids contained target human interferonifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpB(ΔTMD) fused gene and human telomerase reverse transcriptase h Tert gene. Using of nptII gene as a selective one was preferable to the bar gene for chicory. In this case the frequency of transgenic plants or "hairy" roots formation was significantly higher. Cultivation of explants on the medium with Basta in concentration 1-2 mg/l have led to plants death or to significant reduction of number of shoots formed. Frequency of "hairy" roots formation varied from 5.9 to 42.3% after A. rhizogenes-mediated transformation. Frequency of regeneration of transgenic plants varied from 10 to 86% after A. tumefaciens-mediated transformation. Both A. rhizogenes- and A. tumefaciens-mediated transformation frequency depended on the type of explants, roots or cotyledons, and vector used. Usage of A. tumefaciens carrying pCB064 plasmid (target esxA:fbpB(ΔTMD) fused gene and nptII selective gene) resulted in the most effective regeneration of transgenic plants with regeneration frequency up to 86%. In the case of chicory A. rhizogenes-mediated transformation the highest regeneration frequency up to 42.3% was demonstrated using p CB161 vector with ifn-α2b target gene and nptII selective gene. PMID:26419064

  13. Growth habit and photo-synthetic activity of shoot cultures of Medicago sativa L. transformed with the oryzacystatin II gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro maintained shoot cultures of alfalfa (Medicago sativa L. cv. Zajeÿarska 83) that were transformed with the oryzacystatin II (OCII) gene and propagated on growth regulator-free medium were subjected to analysis of morphological characteristics and photosynthetic activity. The most striking f...

  14. Specific suppression of major histocompatibility complex class I and class II genes in astrocytes by brain-enriched gangliosides

    PubMed Central

    1993-01-01

    The effect of brain-enriched gangliosides on constitutive and cytokine- inducible expression of major histocompatibility complex (MHC) class I and II genes in cultured astrocytes was studied. Before treatment with gangliosides, astrocytes expressed constitutive MHC class I but not class II molecules, however, the expression of both MHC class I and II cell surface molecules on astrocytes was induced to high levels by interferon gamma (IFN-gamma). Constitutive and IFN-gamma-inducible expression of MHC class I and II molecules was suppressed by treatment of astrocytes with exogenous bovine brain gangliosides in a dose- dependent manner. Constitutive and induced MHC class I and II mRNA levels were also suppressed by gangliosides, indicating control through transcriptional mechanisms. This was consistent with the ability of gangliosides to suppress the binding activity of transcription factors, especially NF-kappa B-like binding activity, important for the expression of both MHC class I and II genes. These studies may be important for understanding mechanisms of central nervous system (CNS)- specific regulation of major histocompatibility molecules in neuroectodermal cells and the role of gangliosides in regulating MHC- restricted antiviral and autoimmune responses within the CNS. PMID:8376939

  15. Phylogenetic relationships and protein modelling revealed two distinct subfamilies of group II HKT genes between crop and model grasses.

    PubMed

    Ariyarathna, H A Chandima K; Francki, Michael G

    2016-07-01

    Molecular evolution of large protein families in closely related species can provide useful insights on structural functional relationships. Phylogenetic analysis of the grass-specific group II HKT genes identified two distinct subfamilies, I and II. Subfamily II was represented in all species, whereas subfamily I was identified only in the small grain cereals and possibly originated from an ancestral gene duplication post divergence from the coarse grain cereal lineage. The core protein structures were highly analogous despite there being no more than 58% amino acid identity between members of the two subfamilies. Distinctly variable regions in known functional domains, however, indicated functional divergence of the two subfamilies. The subsets of codons residing external to known functional domains predicted signatures of positive Darwinian selection potentially identifying new domains of functional divergence and providing new insights on the structural function and relationships between protein members of the two subfamilies. PMID:27203707

  16. Selection and Trans-Species Polymorphism of Major Histocompatibility Complex Class II Genes in the Order Crocodylia

    PubMed Central

    Jaratlerdsiri, Weerachai; Isberg, Sally R.; Higgins, Damien P.; Miles, Lee G.; Gongora, Jaime

    2014-01-01

    Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II α and β evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II β diversity, whilst diversity within MHC class II α is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II α sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II α sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II β sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85–90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. PMID:24503938

  17. Alternative gene expression in type I and type II cells may enable further nuclear changes during conjugation of Blepharisma japonicum.

    PubMed

    Sugiura, Mayumi; Tanaka, Yuri; Suzaki, Toshinobu; Harumoto, Terue

    2012-03-01

    In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis.

  18. 'Energy expenditure genes' or 'energy absorption genes': a new target for the treatment of obesity and Type II diabetes.

    PubMed

    Braud, Sandrine; Ciufolini, Marco; Harosh, Itzik

    2010-12-01

    Several hundred genes associated or linked to obesity have been described in the scientific literature. Whereas many of these genes are potential targets for the treatment of obesity and associated conditions, none of them have permitted the developement of an efficient drug therapy. As proposed by the 'thrifty genotype' theory, obesity genes may have conferred an evolutionary advantage in times of food shortage through efficient energy exploitation, while 'lean' or 'energy expenditure' genes may have become very rare during the same periods. It is therefore a challenge to identify 'energy expenditure genes' or 'energy absorption genes,' whose mutations or single nucleotide polymorphisms do result in reduced energy intake. We submit that such 'energy absorption' or 'energy expenditure' genes (crucial genes) are potential new targets for the treatment of obesity. These genes can be identified in rare genetic diseases that produce a lean, failure-to-thrive, energy malabsorption or starvation phenotype.

  19. Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

    PubMed Central

    Acker, J; Wintzerith, M; Vigneron, M; Kedinger, C

    1993-01-01

    The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity. Images PMID:8265347

  20. Cloning and sequence determination of the Schizosaccharomyces pombe rpb1 gene encoding the largest subunit of RNA polymerase II.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ueshima, R; Ishihama, A

    1991-01-01

    The gene, rpb1, encoding the largest subunit of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB1, of Saccharomyces cerevisiae as a cross-hybridization probe. We have determined the complete sequence of this gene, and parts of PCR-amplified rpb1 cDNA. The predicted coding sequence, interrupted by six introns, encodes a polypeptide of 1,752 amino acid residues in length with a molecular weight of 194 kilodaltons. This polypeptide contains eight conserved structural domains characteristic of the largest subunit of RNA polymerases from other eukaryotes and, in addition, 29 repetitions of the C-terminal heptapeptide found in all the eukaryotic RNA polymerase II largest subunits so far examined. Images PMID:2011520

  1. RNA polymerase II pauses at the 5 prime end of the transcriptionally induced Drosophila hsp70 gene

    SciTech Connect

    O'Brien, T.; Lis, J.T. )

    1991-10-01

    An RNA polymerase II molecule is associated with the 5{prime} end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides. Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here the authors report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5{prime} end of hsp70 was transcribed approximately five times during the 25-min heat shock that they used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

  2. Feature Selection and Classification of MAQC-II Breast Cancer and Multiple Myeloma Microarray Gene Expression Data

    PubMed Central

    Liu, Qingzhong; Sung, Andrew H.; Chen, Zhongxue; Liu, Jianzhong; Huang, Xudong; Deng, Youping

    2009-01-01

    Microarray data has a high dimension of variables but available datasets usually have only a small number of samples, thereby making the study of such datasets interesting and challenging. In the task of analyzing microarray data for the purpose of, e.g., predicting gene-disease association, feature selection is very important because it provides a way to handle the high dimensionality by exploiting information redundancy induced by associations among genetic markers. Judicious feature selection in microarray data analysis can result in significant reduction of cost while maintaining or improving the classification or prediction accuracy of learning machines that are employed to sort out the datasets. In this paper, we propose a gene selection method called Recursive Feature Addition (RFA), which combines supervised learning and statistical similarity measures. We compare our method with the following gene selection methods: Support Vector Machine Recursive Feature Elimination (SVMRFE)Leave-One-Out Calculation Sequential Forward Selection (LOOCSFS)Gradient based Leave-one-out Gene Selection (GLGS) To evaluate the performance of these gene selection methods, we employ several popular learning classifiers on the MicroArray Quality Control phase II on predictive modeling (MAQC-II) breast cancer dataset and the MAQC-II multiple myeloma dataset. Experimental results show that gene selection is strictly paired with learning classifier. Overall, our approach outperforms other compared methods. The biological functional analysis based on the MAQC-II breast cancer dataset convinced us to apply our method for phenotype prediction. Additionally, learning classifiers also play important roles in the classification of microarray data and our experimental results indicate that the Nearest Mean Scale Classifier (NMSC) is a good choice due to its prediction reliability and its stability across the three performance measurements: Testing accuracy, MCC values, and AUC errors. PMID

  3. The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisin production.

    PubMed

    Brown, Daren W; Butchko, Robert A E; Busman, Mark; Proctor, Robert H

    2007-07-01

    Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Deltafum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

  4. Site-directed mutagenesis of the psbC gene of photosystem II: isolation and functional characterization of CP43-less photosystem II core complexes.

    PubMed

    Rögner, M; Chisholm, D A; Diner, B A

    1991-06-01

    Two mutants of Synechocystis PCC 6803 lacking the psbC gene product CP43 were constructed by site-directed mutagenesis. Analysis of cells and thylakoid membranes of these mutants indicates that PS II reaction centers accumulate to a concentration of about 10% of that of WT cells. PS II core complexes isolated from mutants lacking the CP43 subunit show light-driven electron transfer from the secondary electron donor Z to the primary quinone electron acceptor QA with a quantum yield similar to that of wild type, indicating that CP43 is not required for binding or function of QA. The use of mutants for the removal of CP43 thus avoids the loss of QA function associated with biochemical extraction of CP43 from intact core complexes. Both absorbance and fluorescence emission maxima of the mutant complexes show a blue shift in comparison to the WT PS II core complex, indicating that the absorbance spectrum of CP43 is red-shifted relative to that of the remainder of the core complex. The antenna size of these CP43-less complexes is about 70% of that of WT, indicating that approximately 15 chlorophyll molecules are bound by CP43. The molecular mass of the PS II complex, including the detergent shell, shifts from 310 +/- 15 kDa in WT to 285 +/- 15 kDa in the CP43-less mutants.

  5. Synonymous rates at the RpII215 gene of Drosophila: variation among species and across the coding region.

    PubMed Central

    Llopart, A; Aguadé, M

    1999-01-01

    The region encompassing the RpII215 gene that encodes the largest component of the RNA polymerase II complex (1889 amino acids) has been sequenced in Drosophila subobscura, D. madeirensis, D. guanche, and D. pseudoobscura. Nonsynonymous divergence estimates (Ka) indicate that this gene has a very low rate of amino acid replacements. Given its low Ka and constitutive expression, synonymous substitution rates are, however, unexpectedly high. Sequence comparisons have allowed the molecular clock hypothesis to be tested. D. guanche is an insular species and it is therefore expected to have a reduced effective size relative to D. subobscura. The significantly higher rate of synonymous substitutions detected in the D. guanche lineage could be explained if synonymous mutations behave as nearly neutral. Significant departure from the molecular clock hypothesis for synonymous and nonsynonymous substitutions was detected when comparing the D. subobscura, D. pseudoobscura, and D. melanogaster lineages. Codon bias and synonymous divergence between D. subobscura and D. melanogaster were negatively correlated across the RpII215 coding region, which indicates that selection coefficients for synonymous mutations vary across the gene. The C-terminal domain (CTD) of the RpII215 protein is structurally and functionally differentiated from the rest of the protein. Synonymous substitution rates were significantly different in both regions, which strongly indicates that synonymous mutations in the CTD and in the non-CTD regions are under detectably different selection coefficients. PMID:10224259

  6. A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

    PubMed Central

    Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

    2008-01-01

    A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

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

    PubMed

    Ando, A; Kawata, H; Murakami, T; Shigenari, A; Shiina, T; Sada, M; Tsuji, T; Toriu, A; Nakanishi, Y; Mitsuhashi, T; Sekikawa, K; Inoko, H

    2001-04-01

    cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM alpha 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 alpha 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.

  8. Plant genetic transformation efficiency of selected Malaysian rice based on selectable marker gene (hptII).

    PubMed

    Htwe, Nwe Nwe; Ling, Ho Chai; Zaman, Faridah Qamaruz; Maziah, Mahmood

    2014-04-01

    Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.

  9. Differential alleleic expression of the type II collagen gene (COL2A2) in osteoarthritic cartilage

    SciTech Connect

    Loughlin, J.; Irven, C.; Sykes, B.; Athanasou, N.; Carr, A.

    1995-05-01

    Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced <12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA. 31 refs., 4 figs., 3 tabs.

  10. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation.

    PubMed

    George, Amee J; Purdue, Brooke W; Gould, Cathryn M; Thomas, Daniel W; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A; Morgan, Kylie A; Simpson, Kaylene J; Thomas, Walter G; Hannan, Ross D

    2013-12-01

    The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  11. Occurrence of aminoglycoside phosphotransferase subclass I and II structural genes among Enterobacteriaceae spp. isolated from meat samples.

    PubMed

    Jayaratne, A H; Collins-Thompson, D L; Trevors, J T

    1990-08-01

    3'-Aminoglycoside phosphotransferase [APH(3')] enzymes are a group responsible for resistance to the antibiotics kanamycin (Km) and neomycin (Nm) in bacteria. Escherichia coli ECT24, originally isolated from a meat sample, harboured an 83-kb conjugative R-plasmid (pRPJ24) that carries transferable resistance to Km and Nm. Plasmid pRPJ24 was transferred by conjugation to Enterobacter cloacae 94R, which was used as the source of plasmid DNA in development of a probe for the Km-resistance determinant. Random cloning of BamHI and HindIII double-digest restriction fragments of pRPJ24 in the pUC18 vector plasmid produced clones resistant to both Nm and Km carrying a 1.9-kb DNA insert. Southern hybridization of pRPJ24 cloned chimeric plasmid DNA (pKPJ94) showed homology with the APH(3')II gene from transposon Tn5. A PstI digest of pKPJ94 produced a 920-bp fragment which hybridized with the APH(3')II structural gene, and was used as a DNA probe for the APH(3')II subclass gene. A 980-bp BamHI fragment from plasmid pGH54 carrying the APH(3')I gene from transposon Tn903 was used as a subclass I probe. Total DNA from 206 randomly screened Km-resistant Enterobacteriaceae isolates from raw ground beef and chicken meat samples were examined for the occurrence of APH(3') subclass I and II using non-radioactively-labelled DNA probes. Thirty-six percent and 60% of the isolates examined carried subclass I and II resistances, respectively, in the isolates from chicken meat samples. The corresponding values for bacterial strains from raw ground beef samples were 51% and 72%, respectively. Four percent of the resistant bacterial isolates from chicken samples did not display homology to either probe.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. ColoLipidGene: signature of lipid metabolism-related genes to predict prognosis in stage-II colon cancer patients

    PubMed Central

    Vargas, Teodoro; Moreno-Rubio, Juan; Herranz, Jesús; Cejas, Paloma; Molina, Susana; González-Vallinas, Margarita; Mendiola, Marta; Burgos, Emilio; Aguayo, Cristina; Custodio, Ana B.; Machado, Isidro; Ramos, David; Gironella, Meritxell; Espinosa-Salinas, Isabel; Ramos, Ricardo; Martín-Hernández, Roberto; Risueño, Alberto; De Las Rivas, Javier; Reglero, Guillermo; Yaya, Ricardo; Fernández-Martos, Carlos; Aparicio, Jorge; Maurel, Joan; Feliu, Jaime; de Molina, Ana Ramírez

    2015-01-01

    Lipid metabolism plays an essential role in carcinogenesis due to the requirements of tumoral cells to sustain increased structural, energetic and biosynthetic precursor demands for cell proliferation. We investigated the association between expression of lipid metabolism-related genes and clinical outcome in intermediate-stage colon cancer patients with the aim of identifying a metabolic profile associated with greater malignancy and increased risk of relapse. Expression profile of 70 lipid metabolism-related genes was determined in 77 patients with stage II colon cancer. Cox regression analyses using c-index methodology was applied to identify a metabolic-related signature associated to prognosis. The metabolic signature was further confirmed in two independent validation sets of 120 patients and additionally, in a group of 264 patients from a public database. The combined analysis of these 4 genes, ABCA1, ACSL1, AGPAT1 and SCD, constitutes a metabolic-signature (ColoLipidGene) able to accurately stratify stage II colon cancer patients with 5-fold higher risk of relapse with strong statistical power in the four independent groups of patients. The identification of a group of 4 genes that predict survival in intermediate-stage colon cancer patients allows delineation of a high-risk group that may benefit from adjuvant therapy, and avoids the toxic and unnecessary chemotherapy in patients classified as low-risk group. PMID:25749516

  13. Three Classes of Plasmid (47–63 kb) Carry the Type B Neurotoxin Gene Cluster of Group II Clostridium botulinum

    PubMed Central

    Carter, Andrew T.; Austin, John W.; Weedmark, Kelly A.; Corbett, Cindi; Peck, Michael W.

    2014-01-01

    Pulsed-field gel electrophoresis and DNA sequence analysis of 26 strains of Group II (nonproteolytic) Clostridium botulinum type B4 showed that 23 strains carried their neurotoxin gene cluster on a 47–63 kb plasmid (three strains lacked any hybridization signal for the neurotoxin gene, presumably having lost their plasmid). Unexpectedly, no neurotoxin genes were found on the chromosome. This apparent constraint on neurotoxin gene transfer to the chromosome stands in marked contrast to Group I C. botulinum, in which neurotoxin gene clusters are routinely found in both locations. The three main classes of type B4 plasmid identified in this study shared different regions of homology, but were unrelated to any Group I or Group III plasmid. An important evolutionary aspect firmly links plasmid class to geographical origin, with one class apparently dominant in marine environments, whereas a second class is dominant in European terrestrial environments. A third class of plasmid is a hybrid between the other two other classes, providing evidence for contact between these seemingly geographically separated populations. Mobility via conjugation has been previously demonstrated for the type B4 plasmid of strain Eklund 17B, and similar genes associated with conjugation are present in all type B4 plasmids now described. A plasmid toxin–antitoxin system pemI gene located close to the neurotoxin gene cluster and conserved in each type B4 plasmid class may be important in understanding the mechanism which regulates this unique and unexpected bias toward plasmid-borne neurotoxin genes in Group II C. botulinum type B4. PMID:25079343

  14. Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

    PubMed Central

    Bartolomei, M S; Corden, J L

    1987-01-01

    RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene. Images PMID:3821724

  15. Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response

    PubMed Central

    Liu, Ming-Kun; Lin, Jhe-Jhih; Chen, Chung-Yung; Kuo, Szu-Cheng; Wang, Yu-Ming; Chan, Hong-Lin; Wu, Tzong Yuan

    2016-01-01

    BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy. PMID:27314325

  16. Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations.

    PubMed

    Alves, S; Mangas, M; Prata, M J; Ribeiro, G; Lopes, L; Ribeiro, H; Pinto-Basto, J; Lima, M Reis; Lacerda, L

    2006-12-01

    Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104-2AG, c.241-2A>G, c.241-1G>A, c.418+1G>A, c.880-8AG and c.1181-1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations.

  17. Familial 5q11.2 → q13.3 Segmental Duplication Cosegregating With Multiple Anomalies, Including Schizophrenia

    PubMed Central

    McGillivray, Barbara C.; Bassett, Anne S.; Langlois, Sylvie; Pantzar, Tapio; Wood, Stephen

    2011-01-01

    We report on 2 relatives with a segmental duplication of 5q11.2 → 13.3. The phenotype is surprisingly limited for the degree of chromosome imbalance, the propositus presenting with schizophrenia. Using RFLP markers, we have shown that the gene for HEXB lies within the duplicated region. We suggest this region as a candidate region for the location of a single major gene which predisposes to schizophrenia and which may be assessed by linkage analysis. PMID:1967903

  18. The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

    PubMed Central

    Padilla-Mejía, Norma E.; Florencio-Martínez, Luis E.; Moreno-Campos, Rodrigo; Vizuet-de-Rueda, Juan C.; Cevallos, Ana M.; Hernández-Rivas, Rosaura; Manning-Cela, Rebeca

    2014-01-01

    Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes. PMID:25548151

  19. Functional analysis of the class II hydrophobin gene HFB2-6 from the biocontrol agent Trichoderma asperellum ACCC30536.

    PubMed

    Huang, Ying; Mijiti, Gulijimila; Wang, Zhiying; Yu, Wenjing; Fan, Haijuan; Zhang, Rongshu; Liu, Zhihua

    2015-02-01

    A class II hydrophobin gene, HFB2-6, was cloned from Trichoderma asperellum ACCC30536 and its biocontrol function was studied. According to our previous transcriptome data, six of the eight class II hydrophobin genes were obviously differential expression in four inducing conditions, especially the gene HFB2-6. Moreover, HFB2-6 proven to be differentially transcribed under eight different treatments. HFB2-6 transcripts were up-regulated under 1% Alternaria alternata cell wall and 5% A. alternata fermentation liquid treatments, and by nutritional stress conditions, suggesting that HFB2-6 plays roles in interactions with both biotic and abiotic environmental conditions. HFB2-6 expression was down-regulated under 1% poplar leaf powder culture conditions, but its expression was up-regulated under 1% poplar root powder, indicating that HFB2-6 has a function in root colonization. Furthermore, the recombinant hydrophobin rHFB2-6 was successfully expressed in Escherichia coli BL21-HFB2-6 and purified from the recombinant strain. Genes related to both the jasmonic acid and salicylic acid signal transduction pathways were up-regulated by interaction with renatured rHFB2-6. The ORCA3 (octadecanoid-derivative responsive Catharanthus AP2-domain) gene of the poplar jasmonic acid signal transduction pathway showed a peak expression of 4.48 times at 2 h, and the peak expression of PR1 (pathogenesis-related protein gene) in the salicylic acid signal transduction pathway was 4.58 times at 72 h. Two genes, MP (monopteros) and GH3.17 (auxin original response gene), in the auxin signal transduction pathway were also up-regulated after induction with rHFB2-6, indicating that rHFB2-6 can promote poplar growth and confer broad-spectrum resistance to pathogens.

  20. Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1.

    PubMed

    Hosted, T J; Rochefort, D A; Benson, D R

    1993-06-01

    Frankia alni CpI1 has two glutamine synthetases (GSs), GSI and GSII. The GSI gene (glnA) was isolated from a cosmid library of F. alni CpI1 DNA by heterologous probing with glnA from Streptomyces coelicolor. The glnA gene was shown to be located upstream of the GSII gene (glnII) by DNA-DNA hybridization. The nucleotide sequences of the 1,422-bp CpI1 glnA gene and of the 449-bp intervening region between glnA and glnII were determined, and the glnA amino acid sequence was deduced. In common with GSIs from other organisms, CpI1 GSI contains five conserved regions near the active site and a conserved tyrosine at the adenylylation site. F. alni CpI1 glnA complemented the glutamine growth requirement of the Escherichia coli glnA deletion strain YMC11 but only when expressed from an E. coli lac promoter. While the functional significance of maintaining two GSs adjacent to one another remains unclear, this arrangement in F. alni provides support for the recently proposed origin of GSI and GSII as resulting from a gene duplication early in the evolution of life. PMID:8099074

  1. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.

    PubMed

    Fox, Barbara A; Falla, Alejandra; Rommereim, Leah M; Tomita, Tadakimi; Gigley, Jason P; Mercier, Corinne; Cesbron-Delauw, Marie-France; Weiss, Louis M; Bzik, David J

    2011-09-01

    Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

  2. The great diversity of major histocompatibility complex class II genes in Philippine native cattle.

    PubMed

    Takeshima, S N; Miyasaka, T; Polat, M; Kikuya, M; Matsumoto, Y; Mingala, C N; Villanueva, M A; Salces, A J; Onuma, M; Aida, Y

    2014-12-01

    Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle. PMID:25606401

  3. The great diversity of major histocompatibility complex class II genes in Philippine native cattle.

    PubMed

    Takeshima, S N; Miyasaka, T; Polat, M; Kikuya, M; Matsumoto, Y; Mingala, C N; Villanueva, M A; Salces, A J; Onuma, M; Aida, Y

    2014-12-01

    Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle.

  4. The great diversity of major histocompatibility complex class II genes in Philippine native cattle

    PubMed Central

    Takeshima, S.N.; Miyasaka, T.; Polat, M.; Kikuya, M.; Matsumoto, Y.; Mingala, C.N.; Villanueva, M.A.; Salces, A.J.; Onuma, M.; Aida, Y.

    2014-01-01

    Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle. PMID:25606401

  5. Major histocompatibility complex haplotypes and class II genes in non-Jewish patients with pemphigus vulgaris.

    PubMed Central

    Ahmed, A R; Wagner, R; Khatri, K; Notani, G; Awdeh, Z; Alper, C A; Yunis, E J

    1991-01-01

    Previous studies demonstrated that HLA-DR4 was markedly increased among Ashkenazi Jewish patients with pemphigus vulgaris (PV), almost entirely as the common Jewish extended haplotype [HLA-B38, SC21, DR4, DQw8] or as the haplotype HLA-B35, SC31, DR4, DQw8, and that HLA-DR4, DQw8 was distributed among patients in a manner consistent with dominant expression of a class II (D-region or D-region-linked) susceptibility gene. In the present study of major histocompatibility complex (MHC) haplotypes in 25 non-Jewish PV patients, DR4, DQw8 was found in 12 of the patients and DRw6, DQw5 was found in 15. Only 3 patients had neither. Only 1 of the DR4, DQw8 haplotypes was [HLA-B38, SC21, DR4, DQw8] and 2 were HLA-B35, SC31, DR4, DQw8; most were the presumed fragments (SC31, DR4, DQw8) or (SC21, DR4, DQw8) or DR4, DQw8 with some other complotype. Of the patients with DRw6, DQw5, all were DRw14, DQw5, and 6 had a rare Caucasian haplotype, HLA-Bw55, SB45, DRw14, DQw5. Four of 6 of these were found in patients of Italian extraction, as was the 1 normal example. The non-Jewish patients were of more Southern European extraction than our controls. This suggests that there are two major MHC susceptibility alleles in American patients with PV. The more ancient apparently arose on a haplotype in the Jews, HLA-B38(35), SC21(SC31), DR4, DQw8, and spread to other populations largely as D-region segments. The other arose in or near Italy on the haplotype HLA-Bw55, SB45, DRw14, DQw5 and has also partially fragmented so that many patients carry only DRw14, DQw5. The available data do not permit the specific localization of either the DR4, DQw8- or the DRw14, DQw5-linked susceptibility genes. Images PMID:1675792

  6. Characterization of a type II collagen gene (COL2A1) mutation identified in cultured chondrocytes from human hypochondrogenesis.

    PubMed Central

    Horton, W A; Machado, M A; Ellard, J; Campbell, D; Bartley, J; Ramirez, F; Vitale, E; Lee, B

    1992-01-01

    A subtle mutation in the type II collagen gene COL2A1 was detected in a case of human hypochondrogenesis by using a chondrocyte culture system and PCR-cDNA scanning analysis. Chondrocytes obtained from cartilage biopsies were dedifferentiated and expanded in monolayer culture and then redifferentiated by culture over agarose. Single-strand conformation polymorphism and direct sequencing analysis identified a G----A transition, resulting in a glycine substitution at amino acid 574 of the pro alpha 1(II) collagen triple-helical domain. Morphologic assessment of cartilage-like structures produced in culture and electrophoretic analysis of collagens synthesized by the cultured chondrocytes suggested that the glycine substitution interferes with conversion of type II procollagen to collagen, impairs intracellular transport and secretion of the molecule, and disrupts collagen fibril assembly. This experimental approach has broad implications for the investigation of human chondrodysplasias as well as human chondrocyte biology. Images PMID:1374906

  7. A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle.

    PubMed

    Saini, S S; Hein, W R; Kaushik, A

    1997-06-01

    In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice

  8. A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.

    PubMed

    Wang, HaoYang; Hou, YanNing; Cui, YingXia; Huang, YuFeng; Shi, YiChao; Xia, XinYi; Lu, HongYong; Wang, YunHua; Li, XiaoJun

    2009-03-01

    Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

  9. HLA-class II genes in Mexican Amerindian Mayas: relatedness with Guatemalan Mayans and other populations.

    PubMed

    Vargas-Alarcón, Gilberto; Granados, Julio; Pérez-Hernández, Nonanzit; Rodríguez-Pérez, José Manuel; Canto-Cetina, Thelma; Coral-Vázquez, Ramón Mauricio; Areces, Cristina; Gómez-Prieto, Pablo; Arnaiz-Villena, Antonio

    2011-01-01

    We analyzed the HLA class II allele frequencies in 50 healthy unrelated Mayan individuals. The relationship with other worldwide populations was studied by using HLA data from 71 different populations. The most frequent alleles were HLA-DRB1*04, HLA-DRB1*01, HLA-DQB1*0302 and HLA-DQB1*0501. When comparisons with other Mexican Amerindian groups were made, some differences were observed. Mayans showed an increased frequency of HLA-DRB1*01 when compared to Nahuas, Mayos, Teenek and Mazatecans (p < 0.05), whereas the HLA-DRB1*04 was increased in Mayans when compared to Nahuas (p < 0.05). The analysis of HLA-DQB1 alleles showed an increased frequency of DQB1*0302 in Mayans when compared to Nahuas and Mazatecans (p < 0.05), whereas the frequency of HLA-DQB1*0301 was decreased in Mayans when compared to Nahuas, Mayos, Teenek and Mazatecans (p < 0.05). Decreased frequency of HLA-DQB1*0501 in Mayans when compared to Nahuas was found. Neighbour Joining dendrogram shows that Mexican Mayans are genetically close to some of the most ancient groups living in Mexico and some South American Amerindians. However, Guatemalan Mayans do not cluster together with Mexican Mayas showing that languages do not correlate with genes, particularly in Amerindians. The data corroborate the restricted polymorphism of HLA-DRB1 and DQB1 alleles and the high frequency of HLA-DRB1*04 and HLA-DQB1*0302 in Mayans from Mexico.

  10. Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences.

    PubMed

    Azevedo, M I; Botton, S A; Pereira, D I B; Robe, L J; Jesus, F P K; Mahl, C D; Costa, M M; Alves, S H; Santurio, J M

    2012-09-14

    Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum. PMID:22483240

  11. Dominant mutations in the type II collagen gene, COL2A1, produce spondyloepimetaphyseal dysplasia, Strudwick type.

    PubMed

    Tiller, G E; Polumbo, P A; Weis, M A; Bogaert, R; Lachman, R S; Cohn, D H; Rimoin, D L; Eyre, D R

    1995-09-01

    The chondrodysplasias are a heterogeneous group of disorders characterized by abnormal growth or development of cartilage. Current classification is based on mode of inheritance as well as clinical, histologic, and/or radiographic features. A clinical spectrum of chondrodysplasia phenotypes, ranging from mild to perinatal lethal, is due to defects in the gene for type II collagen, COL2A1. This spectrum includes Stickler syndrome, Kniest dysplasia, spondyloepiphyseal dysplasia congenita (SEDC), achondrogenesis type II, and hypochondrogenesis. Individuals affected with these disorders exhibit abnormalities of the growth plate, nucleus pulposus, and vitreous humor, which are tissues that contain type II collagen. The Strudwick type of spondyloepimetaphyseal dysplasia (SEMD) is characterized by disproportionate short stature, pectus carinatum, and scoliosis, as well as dappled metaphyses (which are not seen in SEDC). The phenotype was first described by Murdoch and Walker in 1969, and a series of 14 patients was later reported by Anderson et al. The observation of two affected sibs born to unaffected parents led to the classification of SEMD Strudwick as an autosomal recessive disorder. We now describe the biochemical characterization of defects in alpha 1(II) collagen in three unrelated individuals with SEMD Strudwick, each of which is due to heterozygosity for a unique mutation in COL2A1. Our data support the hypothesis that some cases, if not all cases, of this distinctive chondrodysplasia result from dominant mutations in COL2A1, thus expanding the clinical spectrum of phenotypes associated with this gene. PMID:7550321

  12. Dominant mutations in the type II collagen gene, COL2A1, produce spondyloepimetaphyseal dysplasia, Strudwick type.

    PubMed

    Tiller, G E; Polumbo, P A; Weis, M A; Bogaert, R; Lachman, R S; Cohn, D H; Rimoin, D L; Eyre, D R

    1995-09-01

    The chondrodysplasias are a heterogeneous group of disorders characterized by abnormal growth or development of cartilage. Current classification is based on mode of inheritance as well as clinical, histologic, and/or radiographic features. A clinical spectrum of chondrodysplasia phenotypes, ranging from mild to perinatal lethal, is due to defects in the gene for type II collagen, COL2A1. This spectrum includes Stickler syndrome, Kniest dysplasia, spondyloepiphyseal dysplasia congenita (SEDC), achondrogenesis type II, and hypochondrogenesis. Individuals affected with these disorders exhibit abnormalities of the growth plate, nucleus pulposus, and vitreous humor, which are tissues that contain type II collagen. The Strudwick type of spondyloepimetaphyseal dysplasia (SEMD) is characterized by disproportionate short stature, pectus carinatum, and scoliosis, as well as dappled metaphyses (which are not seen in SEDC). The phenotype was first described by Murdoch and Walker in 1969, and a series of 14 patients was later reported by Anderson et al. The observation of two affected sibs born to unaffected parents led to the classification of SEMD Strudwick as an autosomal recessive disorder. We now describe the biochemical characterization of defects in alpha 1(II) collagen in three unrelated individuals with SEMD Strudwick, each of which is due to heterozygosity for a unique mutation in COL2A1. Our data support the hypothesis that some cases, if not all cases, of this distinctive chondrodysplasia result from dominant mutations in COL2A1, thus expanding the clinical spectrum of phenotypes associated with this gene.

  13. Nucleotide polymorphism at the RpII215 gene in Drosophila subobscura. Weak selection on synonymous mutations.

    PubMed Central

    Llopart, A; Aguadé, M

    2000-01-01

    Nucleotide variation in an 8.1-kb fragment encompassing the RpII215 gene, which encodes the largest subunit of the RNA polymerase II complex, is analyzed in a sample of 11 chromosomes from a natural population of Drosophila subobscura. No amino acid polymorphism was detected among the 157 segregating sites. The observed numbers of preferred and unpreferred derived synonymous mutations can be explained by neutral mutational processes. In contrast, preferred mutations segregate at significantly higher frequency than unpreferred mutations, suggesting the action of natural selection. The polymorphism to divergence ratio is different for preferred and unpreferred changes, in agreement with their beneficial and deleterious effects on fitness, respectively. Preferred and unpreferred codons are nonrandomly distributed in the RpII215 gene, leading to a heterogeneous distribution of polymorphic to fixed synonymous differences across this coding region. This intragenic variation of the polymorphism/divergence ratio cannot be explained by different patterns of gene expression, mutation, or recombination rates, and therefore it indicates that selection coefficients for synonymous mutations can vary extensively across a coding region. The application of nucleotide composition stationarity tests in coding and flanking noncoding regions, assumed to behave neutrally, allows the detection of the action of natural selection when stationarity holds in the noncoding region. PMID:10880485

  14. MHC class II genes in the European badger (Meles meles): characterization, patterns of variation, and transcription analysis.

    PubMed

    Sin, Yung Wa; Dugdale, Hannah L; Newman, Chris; Macdonald, David W; Burke, Terry

    2012-04-01

    The major histocompatibility complex (MHC) comprises many genes, some of which are polymorphic with numerous alleles. Sequence variation among alleles is most pronounced in exon 2 of the class II genes, which encodes the α1 and β1 domains that form the antigen-binding site (ABS) for the presentation of peptides. The MHC thus plays an important role in pathogen defense. European badgers (Meles meles) are a good species in which to study the MHC, as they harbor a variety of pathogens. We present the first characterization of MHC class II genes, isolated from genomic DNA (gDNA) and complementary DNA (cDNA), in the European badger. Examination of seven individuals revealed four DRB, two DQB, two DQA, and two DRA putatively functional gDNA sequences. All of these sequences, except DRA, exhibited high variability in exon 2; DRB had the highest variability. The ABS codons demonstrated high variability, due potentially to balancing selection, while non-ABS codons had lower variability. Positively selected sites were detected in DRB and DQA. Phylogenetic analysis demonstrated trans-species polymorphism of class II genes. Comparison with cDNA from whole blood revealed that only DRB had a transcription pattern reflecting the alleles that were present in the gDNA, while the other three genes had disparities between gDNA and cDNA. Only one sequence was transcribed, even though two gDNA sequences were present, from each of both DQB and DRA. Our characterization of badger MHC sequences forms a basis for further studies of MHC variability, mate choice, and pathogen resistance in this, and other, species. PMID:22038175

  15. Quantitative resistance to late blight from Solanum berthaultii cosegregates with RPi-ber: insights in stability through isolates and environment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic resistance is a valuable tool in the fight against late blight of potatoes, but little is known about the stability and specificity of quantitative resistance including the effect of defeated major resistance genes. In the present study, we investigated the effect of different isolates of Ph...

  16. Analysis of apoB and apoC-II gene polymorphism in random sample and CHD patients from Moscow

    SciTech Connect

    Pogoda, T.V.; Nikonova, A.; Perova, N.V.

    1994-09-01

    We have analyzed the allele frequency distributions of the 3{prime} apoB gene minisatellite and apoC-II gene microsatellite in random sample of coronary heart disease (CHD) patients. For this purpose we used the PCR technique followed by high-resolution PAGE. It was revealed that the apoB allele, harboring 30 repeats (apoB 30), as well as the apoC-II allele harboring 30 repeats (apoC-II 30), were less frequent in patients at the same time as the frequency of the apoB 32 and apoC-II 17 alleles was greater in patients. The greater frequency of apoB alleles which were larger in size than apoB 46 (defined as `long` - L) was observed in patients with high apoB levels (>160mg dl). The analysis of apoB genotype distribution showed that in a random sample the most common genotype was apoB 34,36 (a combination of the most frequent alleles in the random sample). In patients with high apoB levels, it was twice less frequent, and the most common genotype was apoB 36,L (43% versus 12% in the random sample). Analysis of data on a lipid spectrum of subjects from the random sample with different apoB and apoC-II 17 alleles were associated with atherogenic shifts in the lipid profile, at the same time as apoB 30 and apoC-II 30 alleles - with an apparently favorable lipid profile. The increment of the disease-related risk was observed for subjects with a combination of apoB 32 allele or apoB 36,L genotype with the apoC-11 17 allele. Alternatively, combination of these apoB variants with the apoC-II 30 allele resulted in decreased related risk. In conclusion, simultaneous analysis of two candidate gene variants demonstrated interaction in their influence on the lipid spectrum.

  17. Cosegregation of the mitochondrial DNA A1555G and G4309A mutations results in deafness and mitochondrial myopathy.

    PubMed

    Campos, Y; García, A; López, A; Jiménez, S; Rubio, J C; Del Hoyo, P; Bustos, F; Martín, M A; Cabello, A; Ricoy, J R; Arenas, J

    2002-02-01

    We report a patient with progressive external ophthalmoplegia (PEO), exercise intolerance, and deafness after aminoglycoside exposure, harboring two pathogenic mutations in her mtDNA: an A1555G in the 12S rRNA gene and a G4309A in the tRNA(Ile) gene. Muscle histochemistry showed abundant ragged-red fibers, and biochemistry revealed normal respiratory chain function. The A1555G mutation was homoplasmic in blood from the proband and from all maternal relatives. The G4309A mutation was abundant in the proband's muscle, less abundant in her blood, still less abundant in the mother's blood, and absent in blood from other maternal relatives. Family members were asymptomatic. Our data suggest that the former mutation resulted in aminoglycoside-induced deafness and the latter caused PEO plus exercise intolerance.

  18. Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II Mage proteins.

    PubMed

    López-Sánchez, Noelia; González-Fernández, Zaira; Niinobe, Michio; Yoshikawa, Kazuaki; Frade, José María

    2007-07-18

    In mammals, the type II melanoma antigen (Mage) protein family is constituted by at least 10 closely related members that are expressed in different tissues, including the nervous system. These proteins are believed to regulate cell cycle withdrawal, neuronal differentiation, and apoptosis. However, the analysis of their specific function has been complicated by functional redundancy. In accordance with previous studies in teleosts and Drosophila, we present evidence that only one mage gene exists in genomes from protists, fungi, plants, nematodes, insects, and nonmammalian vertebrates. We have identified the chicken mage gene and cloned the cDNA encoding the chick Mage protein (CMage). CMage shares close homology with the type II Mage protein family, and, as previously shown for the type II Mage proteins Necdin and Mage-G1, it can interact with the transcription factor E2F-1. CMage is expressed in specific regions of the developing nervous system including the retinal ganglion cell layer, the ventral horn of the spinal cord, and the dorsal root ganglia, coinciding with the expression of the neurotrophin receptor p75 (p75(NTR)) in these regions. We show that the intracellular domain of p75(NTR) can interact with both CMage and Necdin, thus preventing the binding of the latter proteins to the transcription factor E2F-1, and facilitating the proapoptotic activity of E2F-1 in N1E-115 differentiating neurons. The presence of a single mage gene in the chicken genome, together with the close functional resemblance between CMage and Necdin, makes this species ideal to further analyze signal transduction through type II Mage proteins.

  19. Intratracheal administration of a nanoparticle-based therapy with the angiotensin II type 2 receptor gene attenuates lung cancer growth.

    PubMed

    Kawabata, Atsushi; Baoum, Abdulgader; Ohta, Naomi; Jacquez, Stephanie; Seo, Gwi-Moon; Berkland, Cory; Tamura, Masaaki

    2012-04-15

    Targeted gene delivery, transfection efficiency, and toxicity concerns remain a challenge for effective gene therapy. In this study, we dimerized the HIV-1 TAT peptide and formulated a nanoparticle vector (dTAT NP) to leverage the efficiency of this cell-penetrating strategy for tumor-targeted gene delivery in the setting of intratracheal administration. Expression efficiency for dTAT NP-encapsulated luciferase or angiotensin II type 2 receptor (AT2R) plasmid DNA (pDNA) was evaluated in Lewis lung carcinoma (LLC) cells cultured in vitro or in vivo in orthotopic tumor grafts in syngeneic mice. In cell culture, dTAT NP was an effective pDNA transfection vector with negligible cytotoxicity. Transfection efficiency was further increased by addition of calcium and glucose to dTAT/pDNA NP. In orthotopic tumor grafts, immunohistochemical analysis confirmed that dTAT NP successfully delivered pDNA to the tumor, where it was expressed primarily in tumor cells along with the bronchial epithelium. Notably, gene expression in tumor tissues persisted at least 14 days after intratracheal administration. Moreover, bolus administration of dTAT NP-encapsulated AT2R or TNF-related apoptosis-inducing ligand (TRAIL) pDNA markedly attenuated tumor growth. Taken together, our findings offer a preclinical proof-of-concept for a novel gene delivery system that offers an effective intratracheal strategy for administering lung cancer gene therapy.

  20. Transcriptome-Wide Survey and Expression Profile Analysis of Putative Chrysanthemum HD-Zip I and II Genes.

    PubMed

    Song, Aiping; Li, Peiling; Xin, Jingjing; Chen, Sumei; Zhao, Kunkun; Wu, Dan; Fan, Qingqing; Gao, Tianwei; Chen, Fadi; Guan, Zhiyong

    2016-01-01

    The homeodomain-leucine zipper (HD-Zip) transcription factor family is a key transcription factor family and unique to the plant kingdom. It consists of a homeodomain and a leucine zipper that serve in combination as a dimerization motif. The family can be classified into four subfamilies, and these subfamilies participate in the development of hormones and mediation of hormone action and are involved in plant responses to environmental conditions. However, limited information on this gene family is available for the important chrysanthemum ornamental species (Chrysanthemum morifolium). Here, we characterized 17 chrysanthemum HD-Zip genes based on transcriptome sequences. Phylogenetic analyses revealed that 17 CmHB genes were distributed in the HD-Zip subfamilies I and II and identified two pairs of putative orthologous proteins in Arabidopsis and chrysanthemum and four pairs of paralogous proteins in chrysanthemum. The software MEME was used to identify 7 putative motifs with E values less than 1e-3 in the chrysanthemum HD-Zip factors, and they can be clearly classified into two groups based on the composition of the motifs. A bioinformatics analysis predicted that 8 CmHB genes could be targeted by 10 miRNA families, and the expression of these 17 genes in response to phytohormone treatments and abiotic stresses was characterized. The results presented here will promote research on the various functions of the HD-Zip gene family members in plant hormones and stress responses. PMID:27196930

  1. Abscisic Acid Mediates Wound Induction but Not Developmental-Specific Expression of the Proteinase Inhibitor II Gene Family.

    PubMed Central

    Pena-Cortes, H; Willmitzer, L; Sanchez-Serrano, JJ

    1991-01-01

    The expression of the potato and tomato proteinase inhibitor II (pin2) gene family is subject to both developmental and environmental control, being constitutively expressed in potato tubers while only being present in the foliage of the potato or tomato plants after mechanical damage. There is evidence that the phytohormone abscisic acid (ABA) is involved in this wound induction of pin2 gene expression. This paper describes experiments that demonstrate that ABA is able to induce the expression of the pin2 gene family, both locally and systemically, at physiological concentrations. The significance of the ABA involvement in the pin2 induction upon wounding has been further strengthened by analyzing the expression of a pin2 promoter-[beta]-glucuronidase gene fusion in transgenic ABA-deficient mutant potato plants. We have analyzed the developmental regulation of pin2 gene expression in wild-type and ABA-deficient potato and tomato plants. The pin2 mRNA level is identical in mutant and wild-type parental Solanum phureja tubers. In addition, evidence is presented for pin2 also being constitutively expressed at certain stages in the development of both tomato and potato flowers. Again, the ABA deficiency appears to have little influence in this tissue-specific expression in the mutants. These results suggest the action of separate pathways for the developmental and environmental regulation of pin2 gene expression. PMID:12324624

  2. Transcriptome-Wide Survey and Expression Profile Analysis of Putative Chrysanthemum HD-Zip I and II Genes

    PubMed Central

    Song, Aiping; Li, Peiling; Xin, Jingjing; Chen, Sumei; Zhao, Kunkun; Wu, Dan; Fan, Qingqing; Gao, Tianwei; Chen, Fadi; Guan, Zhiyong

    2016-01-01

    The homeodomain-leucine zipper (HD-Zip) transcription factor family is a key transcription factor family and unique to the plant kingdom. It consists of a homeodomain and a leucine zipper that serve in combination as a dimerization motif. The family can be classified into four subfamilies, and these subfamilies participate in the development of hormones and mediation of hormone action and are involved in plant responses to environmental conditions. However, limited information on this gene family is available for the important chrysanthemum ornamental species (Chrysanthemum morifolium). Here, we characterized 17 chrysanthemum HD-Zip genes based on transcriptome sequences. Phylogenetic analyses revealed that 17 CmHB genes were distributed in the HD-Zip subfamilies I and II and identified two pairs of putative orthologous proteins in Arabidopsis and chrysanthemum and four pairs of paralogous proteins in chrysanthemum. The software MEME was used to identify 7 putative motifs with E values less than 1e-3 in the chrysanthemum HD-Zip factors, and they can be clearly classified into two groups based on the composition of the motifs. A bioinformatics analysis predicted that 8 CmHB genes could be targeted by 10 miRNA families, and the expression of these 17 genes in response to phytohormone treatments and abiotic stresses was characterized. The results presented here will promote research on the various functions of the HD-Zip gene family members in plant hormones and stress responses. PMID:27196930

  3. Congenital Dyserythropoietic Anemia Type II: molecular analysis and expression of the SEC23B Gene

    PubMed Central

    2011-01-01

    Background Congenital dyserythropoietic anemia type II (CDAII), the most common form of CDA, is an autosomal recessive condition. CDAII diagnosis is based on invasive, expensive, and time consuming tests that are available only in specialized laboratories. The recent identification of SEC23B mutations as the cause of CDAII opens new possibilities for the molecular diagnosis of the disease. The aim of this study was to characterize molecular genomic SEC23B defects in 16 unrelated patients affected by CDAII and correlate the identified genetic alterations with SEC23B transcript and protein levels in erythroid precursors. Methods SEC23B was sequenced in 16 patients, their relatives and 100 control participants. SEC23B transcript level were studied by quantitative PCR (qPCR) in peripheral erythroid precursors and lymphocytes from the patients and healthy control participants. Sec23B protein content was analyzed by immunoblotting in samples of erythroblast cells from CDAII patients and healthy controls. Results All of the investigated cases carried SEC23B mutations on both alleles, with the exception of two patients in which a single heterozygous mutation was found. We identified 15 different SEC23B mutations, of which four represent novel mutations: p.Gln214Stop, p.Thr485Ala, p.Val637Gly, and p.Ser727Phe. The CDAII patients exhibited a 40-60% decrease of SEC23B mRNA levels in erythroid precursors when compared with the corresponding cell type from healthy participants. The largest decrease was observed in compound heterozygote patients with missense/nonsense mutations. In three patients, Sec23B protein levels were evaluated in erythroid precursors and found to be strictly correlated with the reduction observed at the transcript level. We also demonstrate that Sec23B mRNA expression levels in lymphocytes and erythroblasts are similar. Conclusions In this study, we identified four novel SEC23B mutations associated with CDAII disease. We also demonstrate that the genetic

  4. Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II

    SciTech Connect

    De Paepe, A.; Nuytinck, L.; Naeyaert, J.M.

    1997-03-01

    The Ehlers-Danlos syndrome (EDS) is a heterogeneous connective-tissue disorder of which at least nine subtypes are recognized. Considerable clinical overlap exists between the EDS I and II subtypes, suggesting that both are allelic disorders. Recent evidence based on linkage and transgenic mice studies suggest that collagen V is causally involved in human EDS. Collagen V forms heterotypic fibrils with collagen I in many tissues and plays an important role in collagen I fibrillogenesis. We have identified a mutation in COL5A1, the gene encoding the pro{alpha}1(V) collagen chain, segregating with EDS I in a four-generation family. The mutation causes the substitution of the most 5{prime} cysteine residue by a serine within a highly conserved sequence of the pro{alpha}1(V) C-propeptide domain and causes reduction of collagen V by preventing incorporation of the mutant pro{alpha}1 (V) chains in the collagen V trimers. In addition, we have detected splicing defects in the COL5A1 gene in a patient with EDS I and in a family with EDS II. These findings confirm the causal role of collagen V in at least a subgroup of EDS I, prove that EDS I and II are allelic conditions, and represent a, so far, unique example of a human collagen disorder caused by substitution of a highly conserved cysteine residue in the C-propeptide domain of a fibrillar collagen. 30 refs., 6 figs., 2 tabs.

  5. Association of high CD4-positive T cell infiltration with mutations in HLA class II-regulatory genes in microsatellite-unstable colorectal cancer.

    PubMed

    Surmann, Eva-Maria; Voigt, Anita Y; Michel, Sara; Bauer, Kathrin; Reuschenbach, Miriam; Ferrone, Soldano; von Knebel Doeberitz, Magnus; Kloor, Matthias

    2015-03-01

    Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9%), 3 (6.7%), and 1 (2.2%) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.

  6. The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

    PubMed Central

    Robbins, A K; Dorney, D J; Wathen, M W; Whealy, M E; Gold, C; Watson, R J; Holland, L E; Weed, S D; Levine, M; Glorioso, J C

    1987-01-01

    We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions

  7. Chromatin features, RNA polymerase II and the comparative expression of lens genes encoding crystallins, transcription factors, and autophagy mediators

    PubMed Central

    Sun, Jian; Rockowitz, Shira; Chauss, Daniel; Wang, Ping; Kantorow, Marc; Zheng, Deyou

    2015-01-01

    Purpose Gene expression correlates with local chromatin structure. Our studies have mapped histone post-translational modifications, RNA polymerase II (pol II), and transcription factor Pax6 in lens chromatin. These data represent the first genome-wide insights into the relationship between lens chromatin structure and lens transcriptomes and serve as an excellent source for additional data analysis and refinement. The principal lens proteins, the crystallins, are encoded by predominantly expressed mRNAs; however, the regulatory mechanisms underlying their high expression in the lens remain poorly understood. Methods The formaldehyde-assisted identification of regulatory regions (FAIRE-Seq) was employed to analyze newborn lens chromatin. ChIP-seq and RNA-seq data published earlier (GSE66961) have been used to assist in FAIRE-seq data interpretation. RNA transcriptomes from murine lens epithelium, lens fibers, erythrocytes, forebrain, liver, neurons, and pancreas were compared to establish the gene expression levels of the most abundant mRNAs versus median gene expression across other differentiated cells. Results Normalized RNA expression data from multiple tissues show that crystallins rank among the most highly expressed genes in mammalian cells. These findings correlate with the extremely high abundance of pol II all across the crystallin loci, including crystallin genes clustered on chromosomes 1 and 5, as well as within regions of “open” chromatin, as identified by FAIRE-seq. The expression levels of mRNAs encoding DNA-binding transcription factors (e.g., Foxe3, Hsf4, Maf, Pax6, Prox1, Sox1, and Tfap2a) revealed that their transcripts form “clusters” of abundant mRNAs in either lens fibers or lens epithelium. The expression of three autophagy regulatory mRNAs, encoding Tfeb, FoxO1, and Hif1α, was found within a group of lens preferentially expressed transcription factors compared to the E12.5 forebrain. Conclusions This study reveals novel features of

  8. RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast.

    PubMed

    Nakazawa, Norihiko; Sajiki, Kenichi; Xu, Xingya; Villar-Briones, Alejandro; Arakawa, Orie; Yanagida, Mitsuhiro

    2015-06-01

    Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP sequencing (ChIP-seq). We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage and heat-shock-inducible genes, we show that pol II-mediated transcripts cause condensin accumulation. First, condensin's enrichment on mitotically activated genes was abolished by deleting the sep1(+) gene that encodes an M-phase-specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat-shock protein genes in interphase and even during mid-mitosis. In interphase, condensin accumulates preferentially during the postreplicative phase. Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation. PMID:25847133

  9. Evolutionary dynamics of the mS952 intron: a novel mitochondrial group II intron encoding a LAGLIDADG homing endonuclease gene.

    PubMed

    Mullineux, Sahra-Taylor; Willows, Karla; Hausner, Georg

    2011-06-01

    Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element. PMID:21479820

  10. Isolation and functional characterization of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II.

    PubMed

    Robledo-Ortiz, Claudia I; Flores-Carreón, Arturo; Hernández-Cervantes, Arturo; Álvarez-Vargas, Aurelio; Lee, Keunsook K; Díaz-Jiménez, Diana F; Munro, Carol A; Cano-Canchola, Carmen; Mora-Montes, Héctor M

    2012-08-01

    The N-linked glycosylation is a ubiquitous protein modification in eukaryotic cells. During the N-linked glycan synthesis, the precursor Glc(3)Man(9)GlcNAc(2) is processed by endoplasmic reticulum (ER) glucosidases I, II and α1,2-mannosidase, before transporting to the Golgi complex for further structure modifications. In fungi of medical relevance, as Candida albicans and Aspergillus, it is well known that ER glycosidases are important for cell fitness, cell wall organization, virulence, and interaction with the immune system. Despite this, little is known about these enzymes in Sporothrix schenckii, the causative agent of human sporotrichosis. This limited knowledge is due in part to the lack of a genome sequence of this organism. In this work we used degenerate primers and inverse PCR approaches to isolate the open reading frame of S. schenckii ROT2, the encoding gene for α subunit of ER glucosidase II. This S. schenckii gene complemented a Saccharomyces cerevisiae rot2Δ mutant; however, when expressed in a C. albicans rot2Δ mutant, S. schenckii Rot2 partially increased the levels of α-glucosidase activity, but failed to restore the N-linked glycosylation defect associated to the mutation. To our knowledge, this is the first report where a gene involved in protein N-linked glycosylation is isolated from S. schenckii.

  11. Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum.

    PubMed

    Giacani, Lorenzo; Lukehart, Sheila; Centurion-Lara, Arturo

    2007-11-01

    In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with the green fluorescent protein reporter gene (GFP), and promoter activity was measured both as fluorescence emission from Escherichia coli cultures transformed with the different plasmid constructs and using quantitative RT-PCR. For tprJ, G and E-derived clones, fluorescence was significantly higher with constructs containing eight Gs or fewer, while plasmids containing the same promoters with none or more Gs gave modest or no signal above the background. In contrast, tprF/I-derived clones induced similar levels of fluorescence regardless of the number of Gs within the promoter. GFP mRNA quantification showed that all of the promoters induced measurable transcription of the GFP gene; however, only for Subfamily II promoters was message synthesis inversely correlated to the number of Gs in the construct.

  12. Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum

    PubMed Central

    Giacani, Lorenzo; Lukehart, Sheila; Centurion-Lara, Arturo

    2010-01-01

    In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with the green fluorescent protein reporter gene (GFP), and promoter activity was measured both as fluorescence emission from Escherichia coli cultures transformed with the different plasmid constructs and using quantitative RT-PCR. For tprJ, G and E-derived clones, fluorescence was significantly higher with constructs containing eight Gs or fewer, while plasmids containing the same promoters with none or more Gs gave modest or no signal above the background. In contrast, tprF/I-derived clones induced similar levels of fluorescence regardless of the number of Gs within the promoter. GFP mRNA quantification showed that all of the promoters induced measurable transcription of the GFP gene; however, only for Subfamily II promoters was message synthesis inversely correlated to the number of Gs in the construct. PMID:17683506

  13. Splitting hares and tortoises: a classification of neuronal immediate early gene transcription based on poised RNA polymerase II.

    PubMed

    Saha, R N; Dudek, S M

    2013-09-01

    Immediate early transcription is an integral part of the neuronal response to environmental stimulation and serves many brain processes including development, learning, triggers of programmed cell death, and reaction to injury and drugs. Following a stimulus, neurons express a select few genes within a short period of time without undergoing de novo protein translation. Referred to as the 'gateway to genetic response', these immediate early genes (IEGs) are either expressed within a few minutes of stimulation or later within the hour. In neuronal IEGs that are expressed rapidly, productive elongation in response to neuronal activity is jump-started by constitutive transcription initiation together with RNA polymerase II stalling in the vicinity of the promoter. IEGs expressed later in the hour do not depend on this mechanism. On the basis of this Polymerase II poising, we propose that the immediate early genes can be grouped in two distinct classes: the rapid and the delayed IEGs. The possible biological relevance of these classes in neurons is discussed.

  14. Restriction fragment length polymorphism of the human insulin gene region among type II diabetic Mexican-Americans and Tunisians.

    PubMed

    Frazier, M L; Ferrell, R E; Arem, R; Chamakhi, S; Field, J B

    1986-06-01

    The human insulin gene is flanked by a polymorphic locus that is located approximately 500 base pairs (bp) from the 5' end of the point where transcription begins (Bell et al. 1981; Bell et al, 1982). Its occurrence is due to an insertion-deletion region which gives rise to two major classes of alleles: those containing small insertions of 0-600 bp and those containing larger insertions of 1,600-2,200 bp (Owerbach and Nerup, 1982). Insertions of 600-1,600 bp are rare (Rotwein et al., 1983). The larger insertions have previously been reported to be associated with type 2 diabetes (Owerbach and Nerup, 1982). We have conducted studies on a Mexican-American population in Starr County, Texas (98% Mexican-American) and a Tunisian population in Tunis, Tunisia, to determine if the frequency distribution of these classes of insulin gene alleles are similar to the previously reported frequency distributions and if any of the classes of alleles are associated with type 2 diabetes in these populations. We conclude that none of the classes of insulin gene alleles are associated with type II diabetes among Mexican-Americans or Tunisians, and that the frequency distributions of the insulin gene alleles do not vary significantly between the Tunisians, Mexican-Americans, or the aggregate data resulting from combining the insulin gene frequencies of several of the populations described thus far (Bell et al., 1984). PMID:3016846

  15. Genetic basis of neural tube defects. II. Genes correlated with folate and methionine metabolism.

    PubMed

    Gos, Monika; Szpecht-Potocka, Agnieszka

    2002-01-01

    Effective supplementation with folate, which prevents neural tube defect (NTD) occurrence, and high homocysteine levels in the blood of NTD children's mothers suggest that genes involved in folate and homocysteine metabolism can be involved in NTD aetiology. Genes encoding methylenetetrahydrofolate reductase (MTHFR) or methylenetetrahydrofolate dehydrogenase (MTHFD) belong to the first group. Genes encoding methionine synthase (MTR), its regulator - methionine synthase reductase (MTRR) and also cystathionine synthase (CBS) can be included in the second group. We present a current list of the folate and homocysteine metabolism genes that are known to be involved in NTD and pay special attention to primary and secondary NTD prevention.

  16. Molecular characterization of swine leukocyte antigen (SLA) class II genes in outbred pig populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune, disease and vaccine responses. Thus, understanding how SLA gene polymorphism affects immunity, especially in outbred pig populations with a diverse genetic background, requires accu...

  17. NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene.

    PubMed

    Torres, Leuridan Cavalcante; Soares, Diogo Cordeiro de Queiroz; Kulikowski, Leslie Domenici; Franco, Jose Francisco; Kim, Chong Ae

    2014-10-01

    The mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal storage disorders that are clinically characterized by abnormalities in multiple organ systems and reduced life expectancy. Whereas the lysosome is essential to the functioning of the immune system, some authors suggest that the MPS patients have abnormalities in the immune system similar to the patients with primary immunodeficiency. In this study, we evaluated 8 male MPS type II patients of the same family with novel mutation in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency.

  18. Frequent intragenic deletion of the P gene in Tanzanian patients with Type II oculocutaneous albinism (OCA2)

    SciTech Connect

    Spritz, R.; Fukai, K.; Holmes, S.A.

    1995-06-01

    Type II oculocutaneous albinism (OCA2) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in the skin, hair, and eyes. OCA2, which results from mutations of the P gene, is the most frequent type of albinism in African and African-American patients. OCA2 is especially frequent in Tanzania, where it occurs with an incidence of {approximately}1/1,400. We have identified abnormalities of the P gene in each of 13 unrelated patients with OCA2 from Tanzania. One of these, a deletion of exon 7, is strongly predominant, accounting for {approximately}77% of mutant alleles in this group of patients. 20 refs., 2 figs.

  19. Characterization of MHC class I and II genes in a subantarctic seabird, the blue petrel, Halobaena caerulea (Procellariiformes).

    PubMed

    Strandh, Maria; Lannefors, Mimi; Bonadonna, Francesco; Westerdahl, Helena

    2011-10-01

    The great polymorphism observed in the major histocompatibility complex (MHC) genes is thought to be maintained by pathogen-mediated selection possibly combined with MHC-disassortative mating, guided by MHC-determined olfactory cues. Here, we partly characterize the MHC class I and II B of the blue petrel, Halobaena caerulea (Procellariiformes), a bird with significant olfactory abilities that lives under presumably low pathogen burdens in Subantarctica. Blue petrels are long-lived, monogamous birds which suggest the necessity of an accurate mate choice process. The species is ancestral to songbirds (Passeriformes; many MHC loci), although not to gamefowls (Galliformes; few MHC loci). Considering the phylogenetic relationships and the low subantarctic pathogen burden, we expected few rather than many MHC loci in the blue petrel. However, when we analysed partial MHC class I and class II B cDNA and gDNA sequences we found evidence for as many as at least eight MHC class I loci and at least two class II B loci. These class I and II B sequences showed classical MHC characteristics, e.g. high nucleotide diversity, especially in putative peptide-binding regions where signatures of positive selection was detected. Trans-species polymorphism was found between MHC class II B sequences of the blue petrel and those of thin-billed prion, Pachyptila belcheri, two species that diverged ∼25 MYA. The observed MHC allele richness in the blue petrel may well serve as a basis for mate choice, especially since olfactory discrimination of MHC types may be possible in this species. PMID:21607694

  20. A specific collagen type II gene (COL2A1) mutation presenting as spondyloperipheral dysplasia

    SciTech Connect

    Zabel, B.; Hilbert, K.; Spranger, J.; Winterpacht, A.; Stoeb, H.; Superti-Furga, A.

    1996-05-03

    We report on a patient with a skeletal dysplasia characterized by short stature, spondylo-epiphyseal involvement, and brachydactyly E-like changes. This condition has been described as spondyloperipheral dysplasia and the few published cases suggest autosomal dominant inheritance with considerable clinical variability. We found our sporadic case to be due to a collagen type II defect resulting from a specific COL2A1 mutation. This mutation is the first to be located at the C-terminal outside the helical domain of COL2A1. A frameshift as consequence of a 5 bp duplication in exon 51 leads to a stop codon. The resulting truncated C-propeptide region seems to affect helix formation and produces changes of chondrocyte morphology, collagen type II fibril structure and cartilage matrix composition. Our case with its distinct phenotype adds another chondrodysplasia to the clinical spectrum of type II collagenopathies. 16 refs., 4 figs.

  1. Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

    PubMed

    Kikkawa, Eri F; Tsuda, Tomi T; Sumiyama, Daisuke; Naruse, Taeko K; Fukuda, Michio; Kurita, Masanori; Wilson, Rory P; LeMaho, Yvon; Miller, Gary D; Tsuda, Michio; Murata, Koichi; Kulski, Jerzy K; Inoko, Hidetoshi

    2009-05-01

    The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

  2. Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

    PubMed

    Yang, Liming; Quan, Shuo; Nasjletti, Alberto; Laniado-Schwartzman, Michal; Abraham, Nader G

    2004-06-01

    The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (P<0.05) in LXSN rats (23+/-1, 37+/-2, and 52+/-2 at 0.5, 2, and 10 ng). Importantly, treating LSN-HHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli.

  3. Genes Involved in Interleukin-1 Receptor Type II Activities Are Associated With Asthmatic Phenotypes

    PubMed Central

    Madore, Anne-Marie; Vaillancourt, Vanessa T.; Bouzigon, Emmanuelle; Sarnowski, Chloé; Monier, Florent; Dizier, Marie-Hélène; Demenais, Florence

    2016-01-01

    Purpose Interleukin-1 (IL-1) plays a key role in inflammation and immunity and its decoy receptor, IL-1R2, has been implicated in transcriptomic and genetic studies of asthma. Methods Two large asthma family collections, the French-Canadian Saguenay—Lac-St-Jean (SLSJ) study and the French Epidemiological Study on the Genetics and Environment of Asthma (EGEA), were used to investigate the association of SNPs in 10 genes that modulate IL-1R2 activities with asthma, allergic asthma, and atopy. Gene-gene interactions were also tested. Results One SNP in BACE2 was associated with allergic asthma in the SLSJ study and replicated in the EGEA study before statistical correction for multiple testing. Additionally, two SNPs in the MMP2 gene were replicated in both studies prior to statistical correction and reached significance in the combined analysis. Moreover, three gene-gene interactions also survived statistical correction in the combined analyses (BACE1-IL1RAP in asthma and allergic asthma and IL1R1-IL1RAP in atopy). Conclusions Our results highlight the relevance of genes involved in the IL-1R2 activity in the context of asthma and asthma-related traits. PMID:27334786

  4. Characterization of a class II pilin expression locus from Neisseria meningitidis: evidence for increased diversity among pilin genes in pathogenic Neisseria species.

    PubMed Central

    Aho, E L; Botten, J W; Hall, R J; Larson, M K; Ness, J K

    1997-01-01

    Strains of Neisseria meningitidis elaborate one of two classes of pili. Meningococcal class I pili have many features in common with pili produced by N. gonorrhoeae, including the ability to bind monoclonal antibody SM1 and a common gene and protein structure consisting of conserved, semivariable, and hypervariable regions. Class II pili are SM1 nonreactive and display smaller subunit molecular weights than do gonococcal or meningococcal class I pili. In this study, we have determined the N-terminal amino acid sequence for class II pilin and isolated the expression locus encoding class II pilin from N. meningitidis FAM18. Meningococcal class II pilin displays features typical of type IV pili and shares extensive amino acid identity with the N-terminal conserved regions of other neisserial pilin proteins. However, the deduced class II pilin sequence displays several unique features compared with previously reported meningococcal class I and gonococcal pilin sequences. Class II pilin lacks several conserved peptide regions found within the semivariable and hypervariable regions of other neisserial pilins and displays a large deletion in a hypervariable region of the protein believed to be exposed on the pilus face in gonococcal pili. DNA sequence comparisons within all three regions of the coding sequence also suggest that the meningococcal class II pilin gene is the most dissimilar of the three types of neisserial pilE loci. Additionally, the class II locus fails to display flanking-sequence homology to class I and gonococcal genes and lacks a downstream Sma/Cla repeat sequence, a feature present in all other neisserial pilin genes examined to date. These data indicate meningococcal class II pili represent a structurally distinct class of pili and suggest that relationships among pilin genes in pathogenic Neisseria do not necessarily follow species boundaries. PMID:9199428

  5. DNA methylation status of cyp17-II gene correlated with its expression pattern and reproductive endocrinology during ovarian development stages of Japanese flounder (Paralichthys olivaceus).

    PubMed

    Ding, YuXia; He, Feng; Wen, HaiShen; Li, JiFang; Ni, Meng; Chi, MeiLi; Qian, Kun; Bu, Yan; Zhang, DongQian; Si, YuFeng; Zhao, JunLi

    2013-09-15

    Cytochrome P450c17-II (cyp17-II, 17α-hydroxylase) is responsible for the production of steroid hormones during oocyte maturation in vertebrates. The comparative expression pattern of cyp17-II gene during the gonadal development stages will provide important insights into its function of gonadal development. In addition, epigenetic modification especially DNA methylation plays a vital role in regulation of gene expression. The adult female Japanese flounder at different ovarian development stage (from stages II to V) was obtained in this experiment. The expression of cyp17-II gene in the ovary of Japanese flounder during the gonadal development stages was measured by quantitative PCR. Reproductive traits included gonadosomatic index (GSI), plasma estradiol-17β (E2) and testosterone (T) were also measured. Moreover, whole CpG dinucleotides methylation status of the two CpG rich regions in cyp17-II coding region was detected by bisulfate sequencing. In the ovary, the cyp17-II gene had the lowest mRNA expression at the early ovarian development stage, but then increased afterward. The variation trends of T and E2 level were consistent with the cyp17-II expression pattern in ovary. In contrast, the whole methylation levels of each CpG rich region (exon 4 and 6) in cyp17-II coding region were declined from stages II to IV, then increased at stage V. The methylation levels of whole CpG sites in each CpG rich region were inversely correlated with the values of ovarian cyp17-II gene expression, T and E2 level, and GSI. Based on the present study, we proposed that cyp17-II may regulate the level of steroid hormone, and then stimulate the oocyte growth and maturation. The cyp17-II gene transcriptional activity was possibly affected by the methylation level of CpG rich regions in coding region. These findings will help in the study of the molecular mechanism of fish reproduction and endocrine physiology.

  6. Heligmosomoides polygyrus infection is associated with lower MHC class II gene expression in Apodemus flavicollis: indication for immune suppression?

    PubMed

    Axtner, Jan; Sommer, Simone

    2011-12-01

    Due to their key role in recognizing foreign antigens and triggering the subsequent immune response the genes of the major histocompatibility complex (MHC) provide a potential target for parasites to attack in order to evade detection and expulsion from the host. A diminished MHC gene expression results in less activated T cells and might serve as a gateway for pathogens and parasites. Some parasites are suspected to be immune suppressors and promote co-infections of other parasites even in other parts of the body. In our study we found indications that the gut dwelling nematode Heligmosomoides polygyrus might exert a systemic immunosuppressive effect in yellow-necked mice (Apodemus flavicollis). The amount of hepatic MHC class II DRB gene RNA transcripts in infected mice was negatively associated with infection intensity with H. polygyrus. The hepatic expression of immunosuppressive cytokines, such as transforming growth factor β and interleukin 10 was not associated with H. polygyrus infection. We did not find direct positive associations of H. polygyrus with other helminth species. But the prevalence and infection intensity of the nematodes Syphacia stroma and Trichuris muris were higher in multiple infected individuals. Furthermore, our data indicated antagonistic effects in the helminth community of A. flavicollis as cestode infection correlated negatively with H. polygyrus and helminth species richness. Our study shows that expression analyses of immune relevant genes can also be performed in wildlife, opening new aspects and possibilities for future ecological and evolutionary research. PMID:21983561

  7. Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis.

    PubMed

    Gowda, Chandrika S; Song, Chunhua; Ding, Yali; Kapadia, Malika; Dovat, Sinisa

    2016-03-01

    Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros' DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL. PMID:26912004

  8. Genetic variation of the major histocompatibility complex (MHC class II B gene) in the threatened Hume's pheasant, Syrmaticus humiae.

    PubMed

    Chen, Weicai; Bei, Yongjian; Li, Hanhua

    2015-01-01

    Major histocompatibility complex (MHC) genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB) exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae), which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ⁄ dS ratio at putative antigen-binding sites (ABS) was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation.

  9. Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Colasuonno, Pasqualina; Uauy, Cristobal; Beckles, Diane M.; Dubcovsky, Jorge

    2016-01-01

    Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat. PMID:26924849

  10. Characterization of class II β chain major histocompatibility complex genes in a family of Hawaiian honeycreepers: 'amakihi (Hemignathus virens).

    PubMed

    Jarvi, Susan I; Bianchi, Kiara R; Farias, Margaret Em; Txakeeyang, Ann; McFarland, Thomas; Belcaid, Mahdi; Asano, Ashley

    2016-07-01

    Hawaiian honeycreepers (Drepanidinae) have evolved in the absence of mosquitoes for over five million years. Through human activity, mosquitoes were introduced to the Hawaiian archipelago less than 200 years ago. Mosquito-vectored diseases such as avian malaria caused by Plasmodium relictum and Avipoxviruses have greatly impacted these vulnerable species. Susceptibility to these diseases is variable among and within species. Due to their function in adaptive immunity, the role of major histocompatibility complex genes (Mhc) in disease susceptibility is under investigation. In this study, we evaluate gene organization and levels of diversity of Mhc class II β chain genes (exon 2) in a captive-reared family of Hawaii 'amakihi (Hemignathus virens). A total of 233 sequences (173 bp) were obtained by PCR+1 amplification and cloning, and 5720 sequences were generated by Roche 454 pyrosequencing. We report a total of 17 alleles originating from a minimum of 14 distinct loci. We detected three linkage groups that appear to represent three distinct haplotypes. Phylogenetic analysis revealed one variable cluster resembling classical Mhc sequences (DAB) and one highly conserved, low variability cluster resembling non-classical Mhc sequences (DBB). High net evolutionary divergence values between DAB and DBB resemble that seen between chicken BLB system and YLB system genes. High amino acid identity among non-classical alleles from 12 species of passerines (DBB) and four species of Galliformes (YLB) was found, suggesting that these non-classical passerine sequences may be related to the Galliforme YLB sequences. PMID:26971289

  11. Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

    PubMed Central

    McCarthy, James K.; Tebo, Bradley M.

    2013-01-01

    Bacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in Pseudomonas putida GB-1 has long remained unknown. To identify the P. putida GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG, mofA, moxA, and mopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11 Pseudomonas species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes. PMID:23124227

  12. TALE-PvuII fusion proteins--novel tools for gene targeting.

    PubMed

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  13. Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution II: dynamics

    PubMed Central

    Keränen, Soile VE; Fowlkes, Charless C; Luengo Hendriks, Cris L; Sudar, Damir; Knowles, David W; Malik, Jitendra; Biggin, Mark D

    2006-01-01

    Background To accurately describe gene expression and computationally model animal transcriptional networks, it is essential to determine the changing locations of cells in developing embryos. Results Using automated image analysis methods, we provide the first quantitative description of temporal changes in morphology and gene expression at cellular resolution in whole embryos, using the Drosophila blastoderm as a model. Analyses based on both fixed and live embryos reveal complex, previously undetected three-dimensional changes in nuclear density patterns caused by nuclear movements prior to gastrulation. Gene expression patterns move, in part, with these changes in morphology, but additional spatial shifts in expression patterns are also seen, supporting a previously proposed model of pattern dynamics based on the induction and inhibition of gene expression. We show that mutations that disrupt either the anterior/posterior (a/p) or the dorsal/ventral (d/v) transcriptional cascades alter morphology and gene expression along both the a/p and d/v axes in a way suggesting that these two patterning systems interact via both transcriptional and morphological mechanisms. Conclusion Our work establishes a new strategy for measuring temporal changes in the locations of cells and gene expression patterns that uses fixed cell material and computational modeling. It also provides a coordinate framework for the blastoderm embryo that will allow increasingly accurate spatio-temporal modeling of both the transcriptional control network and morphogenesis. PMID:17184547

  14. Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

    PubMed

    Monerawela, Chandre; James, Tharappel C; Wolfe, Kenneth H; Bond, Ursula

    2015-03-01

    Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts.

  15. Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; Mullen, Susan; Tan, Tzer Han; Jung, Yoon J.; Cissé, Ibrahim I.

    2016-01-01

    Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging. PMID:27782203

  16. MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris).

    PubMed

    Pokorny, Ina; Sharma, Reeta; Goyal, Surendra Prakash; Mishra, Sudanshu; Tiedemann, Ralph

    2010-10-01

    Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.

  17. MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris).

    PubMed

    Pokorny, Ina; Sharma, Reeta; Goyal, Surendra Prakash; Mishra, Sudanshu; Tiedemann, Ralph

    2010-10-01

    Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal. PMID:20821315

  18. Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-gamma-inducible MHC-II gene expression.

    PubMed

    Zika, Eleni; Fauquier, Lucas; Vandel, Laurence; Ting, Jenny P-Y

    2005-11-01

    Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-gamma. IFN-gamma activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-gamma-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, CARM1, and the acetyltransferase cyclic-AMP response element binding (CREB)-binding protein (CBP) during this process. CARM1 synergizes with CIITA in activating MHC-II transcription and synergy is abrogated when an arginine methyltransferase-defective CARM1 mutant is used. Protein-arginine methyltransferase 1 has much less effect on MHC-II transcription. Specific RNA interference reduced CARM1 expression as well as MHC-II expression. The recruitment of CARM1 to the promoter requires endogenous CIITA and results in methylation of histone H3-R17; hence, CIITA is an upstream regulator of histone methylation. Previous work has shown that CARM1 can methylate CBP at three arginine residues. Using wild-type CBP and a mutant of CBP lacking the CARM1-targeted arginine residues (R3A), we show that arginine methylation of CBP is required for IFN-gamma induction of MHC-II. A kinetic analysis shows that CIITA, CARM1, and H3-R17 methylation all precede CBP loading on the MHC-II promoter during IFN-gamma treatment. These results suggest functional and temporal relationships among CIITA, CARM1, and CBP for IFN-gamma induction of MHC-II.

  19. Genetic analysis of ryanodine receptor 1 gene and carnitine palmitoyltransferase II gene: an autopsy case of neuroleptic malignant syndrome related to vegetamin.

    PubMed

    Matsusue, Aya; Hara, Kenji; Kageura, Mitsuyoshi; Kashiwagi, Masayuki; Lu, Wang; Ishigami, Akiko; Gotohda, Takako; Tokunaga, Itsuo; Nisimura, Akiyoshi; Sugimura, Tomoko; Kubo, Shin-ichi

    2009-04-01

    We report an autopsy case of a man in his forties who died 2 days after taking an overdose of vegetamin. The autopsy findings were as follows: externally, the upper epidermis of some parts of the body had become loosened. The epidermis was easily detached from the dermis using the fingers. Viscous fluid adhered around the nose and mouth. The brain was edematous and weighed 1520 g. Skeletal muscle was discolored. The urine was a slightly red-tinged yellow. The organs showed congestion. Urine tests: urea nitrogen: 1.95 g/day; creatinine: 0.66 g/day; urine myoglobin: 1100 ng/mL. Blood level of drugs: phenobarbital: 38.2 microg/ml; promethazine: 2.22 microg/ml; chlorpromazine: 0.96 microg/ml. Immunohistochemistry identified myoglobin in the kidney. From these findings, his cause of death was considered to be vegetamin-induced neuroleptic malignant syndrome and rhabdomyolysis. Mutation of the ryanodine receptor 1 gene is associated with malignant hyperthermia. However, there was no mutation which causes amino acid substitution in the three hot-spot regions of the ryanodine receptor 1 gene. Partial deficiency of carnitine palmitoyltransferase II is the commonest cause of recurrent rhabdomyolysis in adults. The subject was found to be heterozygous for an amino acid exchange in exon 4, (1203)G-->A causing a (368)Val-->Ile amino acid substitution. It is necessary to examine other candidate gene mutations. PMID:19269221

  20. Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast*

    PubMed Central

    Ansari, Suraiya A.; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z.; Rode, Kara A.; Barber, Wesley T.; Ellis, Laura C.; LaPorta, Erika; Orzechowski, Amanda M.; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H.

    2014-01-01

    Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. PMID:24727477

  1. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  2. ASTRAL-II: coalescent-based species tree estimation with many hundreds of taxa and thousands of genes

    PubMed Central

    Mirarab, Siavash; Warnow, Tandy

    2015-01-01

    Motivation: The estimation of species phylogenies requires multiple loci, since different loci can have different trees due to incomplete lineage sorting, modeled by the multi-species coalescent model. We recently developed a coalescent-based method, ASTRAL, which is statistically consistent under the multi-species coalescent model and which is more accurate than other coalescent-based methods on the datasets we examined. ASTRAL runs in polynomial time, by constraining the search space using a set of allowed ‘bipartitions’. Despite the limitation to allowed bipartitions, ASTRAL is statistically consistent. Results: We present a new version of ASTRAL, which we call ASTRAL-II. We show that ASTRAL-II has substantial advantages over ASTRAL: it is faster, can analyze much larger datasets (up to 1000 species and 1000 genes) and has substantially better accuracy under some conditions. ASTRAL’s running time is O(n2k|X|2), and ASTRAL-II’s running time is O(nk|X|2), where n is the number of species, k is the number of loci and X is the set of allowed bipartitions for the search space. Availability and implementation: ASTRAL-II is available in open source at https://github.com/smirarab/ASTRAL and datasets used are available at http://www.cs.utexas.edu/~phylo/datasets/astral2/. Contact: smirarab@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26072508

  3. Clinical usefulness of gene-expression profile to rule out acute rejection after heart transplantation: CARGO II

    PubMed Central

    Crespo-Leiro, Maria G.; Stypmann, Jörg; Schulz, Uwe; Zuckermann, Andreas; Mohacsi, Paul; Bara, Christoph; Ross, Heather; Parameshwar, Jayan; Zakliczyński, Michal; Fiocchi, Roberto; Hoefer, Daniel; Colvin, Monica; Deng, Mario C.; Leprince, Pascal; Elashoff, Barbara; Yee, James P.; Vanhaecke, Johan

    2016-01-01

    Aims A non-invasive gene-expression profiling (GEP) test for rejection surveillance of heart transplant recipients originated in the USA. A European-based study, Cardiac Allograft Rejection Gene Expression Observational II Study (CARGO II), was conducted to further clinically validate the GEP test performance. Methods and results Blood samples for GEP testing (AlloMap®, CareDx, Brisbane, CA, USA) were collected during post-transplant surveillance. The reference standard for rejection status was based on histopathology grading of tissue from endomyocardial biopsy. The area under the receiver operating characteristic curve (AUC-ROC), negative (NPVs), and positive predictive values (PPVs) for the GEP scores (range 0–39) were computed. Considering the GEP score of 34 as a cut-off (>6 months post-transplantation), 95.5% (381/399) of GEP tests were true negatives, 4.5% (18/399) were false negatives, 10.2% (6/59) were true positives, and 89.8% (53/59) were false positives. Based on 938 paired biopsies, the GEP test score AUC-ROC for distinguishing ≥3A rejection was 0.70 and 0.69 for ≥2–6 and >6 months post-transplantation, respectively. Depending on the chosen threshold score, the NPV and PPV range from 98.1 to 100% and 2.0 to 4.7%, respectively. Conclusion For ≥2–6 and >6 months post-transplantation, CARGO II GEP score performance (AUC-ROC = 0.70 and 0.69) is similar to the CARGO study results (AUC-ROC = 0.71 and 0.67). The low prevalence of ACR contributes to the high NPV and limited PPV of GEP testing. The choice of threshold score for practical use of GEP testing should consider overall clinical assessment of the patient's baseline risk for rejection. PMID:26746629

  4. Schistosoma mansoni TGF-β Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene

    PubMed Central

    Osman, Ahmed; Niles, Edward G; Verjovski-Almeida, Sergio; LoVerde, Philip T

    2006-01-01

    Members of transforming growth factor-beta (TGF-β) superfamily play pivotal roles in development in multicellular organisms. We report the functional characterization of the Schistosoma mansoni type II receptor (SmTβRII). Mining of the S. mansoni expressed sequence tag (EST) database identified an EST clone that shows homology to the kinase domain of type II receptors from different species. The amplified EST sequence was used as a probe to isolate a cDNA clone spanning the entire coding region of a type II serine/threonine kinase receptor. The interaction of SmTβRII with SmTβRI was elucidated and shown to be dependent on TGF-β ligand binding. Furthermore, in the presence of human TGF-β1, SmTβRII was able to activate SmTβRI, which in turn activated SmSmad2 and promoted its interaction with SmSmad4, proving the transfer of the signal from the receptor complex to the Smad proteins. Gynaecophoral canal protein (GCP), whose expression in male worms is limited to the gynaecophoric canal, was identified as a potential TGF-β target gene in schistosomes. Knocking down the expression of SmTβRII using short interfering RNA molecules (siRNA) resulted in a concomitant reduction in the expression of GCP. These data provide evidence for the direct involvement of SmTβRII in mediating TGF-β–induced activation of the TGF-β target gene, SmGCP, within schistosome parasites. The results also provide additional evidence for a role for the TGF-β signaling pathway in male-induced female reproductive development. PMID:16789838

  5. Human class II major histocompatibility complex gene transfer into murine neuroblastoma leads to loss of tumorigenicity, immunity against subsequent tumor challenge, and elimination of microscopic preestablished tumors.

    PubMed

    Hock, R A; Reynolds, B D; Tucker-McClung, C L; Kwok, W W

    1995-01-01

    Immunological recognition of transformed cells is critically important to limit tumor development and proliferation. Because established tumors have escaped immune recognition and elimination, novel strategies to enhance antitumor immunity have been developed. A unique approach has used the introduction of genes encoding major histocompatibility complex (MHC) antigens into tumor cells. Experiments in mice have shown that the expression of syngeneic class II MHC antigens in tumor cells completely abrogates tumorigenicity and induces tumor-specific immunity. In this study we sought to determine whether a more effective antitumor immune response would be generated by introducing xenogeneic class II MHC genes into tumor cells. To address this question we used recombinant retroviruses to express human class II MHC genes in a highly malignant murine neuroblastoma cell line, Neuro-2a. We found that normal mice inoculated with Neuro-2a expressing the human class II MHC antigen did not develop tumors and were immune to subsequent challenge with unmodified Neuro-2a cells. In addition, mice bearing small established Neuro-2a tumors were cured by vaccination with Neuro-2a expressing human class II MHC. We hypothesize that a similar approach using retroviral-mediated transduction of class II MHC genes into human tumor cells may be an effective alternative to current cancer treatment.

  6. IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor

    PubMed Central

    Versteyhe, Soetkin; Klaproth, Birgit; Borup, Rehannah; Palsgaard, Jane; Jensen, Maja; Gray, Steven G.; De Meyts, Pierre

    2013-01-01

    Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor. PMID:23950756

  7. Coxiella burnetii Nine Mile II proteins modulate gene expression of monocytic host cells during infection

    PubMed Central

    2010-01-01

    Background Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV) of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection. Results We have identified 36 host cell genes that are specifically regulated when de novo C. burnetii protein synthesis occurs during infection using comparative microarray analysis. Two parallel sets of infected and uninfected THP-1 cells were grown for 48 h followed by the addition of chloramphenicol (CAM) to 10 μg/ml in one set. Total RNA was harvested at 72 hpi from all conditions, and microarrays performed using Phalanx Human OneArray™ slides. A total of 784 (mock treated) and 901 (CAM treated) THP-1 genes were up or down regulated ≥2 fold in the C. burnetii infected vs. uninfected cell sets, respectively. Comparisons between the complementary data sets (using >0 fold), eliminated the common gene expression changes. A stringent comparison (≥2 fold) between the separate microarrays revealed 36 host cell genes modulated by C. burnetii protein synthesis. Ontological analysis of these genes identified the innate immune response, cell death and proliferation, vesicle trafficking and development, lipid homeostasis, and cytoskeletal organization as predominant cellular functions modulated by C. burnetii protein synthesis. Conclusions Collectively, these data indicate that C. burnetii proteins actively regulate the expression of specific host cell genes and pathways. This is in addition to host cell genes that respond to the presence of the pathogen whether or not

  8. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    SciTech Connect

    Benson, D.L.; Isackson, P.J.; Hendry, S.H.; Jones, E.G. )

    1991-06-01

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate.

  9. Loss of DNase II function in the gonad is associated with a higher expression of antimicrobial genes in Caenorhabditis elegans.

    PubMed

    Yu, Hsiang; Lai, Huey-Jen; Lin, Tai-Wei; Chen, Chang-Shi; Lo, Szecheng J

    2015-08-15

    Three waves of apoptosis shape the development of Caenorhabditis elegans. Although the exact roles of the three DNase II genes (nuc-1, crn-6 and crn-7), which are known to mediate degradation of apoptotic DNA, in the embryonic and larval phases of apoptosis have been characterized, the DNase II acting in the third wave of germ cell apoptosis remains undetermined. In the present study, we performed in vitro and in vivo assays on various mutant nematodes to demonstrate that NUC-1 and CRN-7, but not CRN-6, function in germ cell apoptosis. In addition, in situ DNA-break detection and anti-phosphorylated ERK (extracellular-signal-regulated kinase) staining illustrated the sequential and spatially regulated actions of NUC-1 and CRN-7, at the pachytene zone of the gonad and at the loop respectively. In line with the notion that UV-induced DNA fragment accumulation in the gonad activates innate immunity responses, we also found that loss of NUC-1 and CRN-7 lead to up-regulation of antimicrobial genes (abf-2, spp-1, nlp-29, cnc-2, and lys-7). Our observations suggest that an incomplete digestion of DNA fragments resulting from the absence of NUC-1 or CRN-7 in the gonad could induce the ERK signalling, consequently activating antimicrobial gene expression. Taken together, the results of the present study demonstrate for the first time that nuc-1 and crn-7 play a role in degrading apoptotic DNA in distinct sites of the gonad, and act as negative regulators of innate immunity in C. elegans.

  10. A Gene of the β3-Glycosyltransferase Family Encodes N-Acetylglucosaminyltransferase II Function in Trypanosoma brucei*

    PubMed Central

    Damerow, Manuela; Graalfs, Frauke; Güther, M. Lucia S.; Mehlert, Angela; Izquierdo, Luis; Ferguson, Michael A. J.

    2016-01-01

    The bloodstream form of the human pathogen Trypanosoma brucei expresses oligomannose, paucimannose, and complex N-linked glycans, including some exceptionally large poly-N-acetyllactosamine-containing structures. Despite the presence of complex N-glycans in this organism, no homologues of the canonical N-acetylglucosaminyltransferase I or II genes can be found in the T. brucei genome. These genes encode the activities that initiate the elaboration of the Manα1–3 and Manα1–6 arms, respectively, of the conserved trimannosyl-N-acetylchitobiosyl core of N-linked glycans. Previously, we identified a highly divergent T. brucei N-acetylglucosaminyltransferase I (TbGnTI) among a set of putative T. brucei glycosyltransferase genes belonging to the β3-glycosyltransferase superfamily (Damerow, M., Rodrigues, J. A., Wu, D., Güther, M. L., Mehlert, A., and Ferguson, M. A. (2014) J. Biol. Chem. 289, 9328–9339). Here, we demonstrate that TbGT15, another member of the same β3-glycosyltransferase family, encodes an equally divergent N-acetylglucosaminyltransferase II (TbGnTII) activity. In contrast to multicellular organisms, where GnTII activity is essential, TbGnTII null mutants of T. brucei grow in culture and are still infectious to animals. Characterization of the large poly-N-acetyllactosamine containing N-glycans of the TbGnTII null mutants by methylation linkage analysis suggests that, in wild-type parasites, the Manα1–6 arm of the conserved trimannosyl core may carry predominantly linear poly-N-acetyllactosamine chains, whereas the Manα1–3 arm may carry predominantly branched poly-N-acetyllactosamine chains. These results provide further detail on the structure and biosynthesis of complex N-glycans in an important human pathogen and provide a second example of the adaptation by trypanosomes of β3-glycosyltransferase family members to catalyze β1–2 glycosidic linkages. PMID:27189951

  11. Targeted gene knockdown in zebrafish reveals distinct intraembryonic functions for insulin-like growth factor II signaling.

    PubMed

    White, Yvonne A R; Kyle, Joshua T; Wood, Antony W

    2009-09-01

    IGF-II is the predominant IGF ligand regulating prenatal growth in all vertebrates, including humans, but its central role in placental development has confounded efforts to fully elucidate its functions within the embryo. Here we use a nonplacental model vertebrate (zebrafish) to interrogate the intraembryonic functions of IGF-II signaling. The zebrafish genome contains two coorthologs of mammalian IGF2 (igf2a, igf2b), which exhibit distinct patterns of expression during embryogenesis. Expression of igf2a mRNA is restricted to the notochord, primarily during segmentation/neurulation. By contrast, igf2b mRNA is expressed in midline tissues adjacent to the notochord, with additional sites of expression in the ventral forebrain, and the pronephros. To identify their intraembryonic functions, we suppressed the expression of each gene with morpholino oligonucleotides. Knockdown of igf2a led to defects in dorsal midline development, characterized by delayed segmentation, notochord undulations, and ventral curvature. Similarly, suppression of igf2b led to defects in dorsal midline development but also induced ectopic fusion of the nephron primordia, and defects in ventral forebrain development. Subsequent onset of severe body edema in igf2b, but not igf2a morphants, further suggested a distinct role for igf2b in development of the embryonic kidney. Simultaneous knockdown of both genes increased the severity of dorsal midline defects, confirming a conserved role for both genes in dorsal midline development. Collectively, these data provide evidence that the zebrafish orthologs of IGF2 function in dorsal midline development during segmentation/neurulation, whereas one paralog, igf2b, has evolved additional, distinct functions during subsequent organogenesis.

  12. Genomic organization and expression analysis of mouse kynurenine aminotransferase II, a possible factor in the pathophysiology of Huntington's disease.

    PubMed

    Yu, P; Mosbrook, D M; Tagle, D A

    1999-09-01

    Decreased levels of the endogenous neuroprotectant kynurenic acid (KYNA) have been observed in the brain of Huntington's Disease (HD) patients and may be related to neuronal loss in this disorder. This reduction may be caused by a dysfunction of kynurenine aminotransferase II (KAT II), the major enzyme responsible for the synthesis of KYNA in the brain. Towards understanding the role of KAT II in HD, we isolated and characterized the cDNA sequence and determined the genomic organization of mouse KAT II (mKat-2). The full length mKat-2 cDNA is 1812 bp, encoding 425 amino acids, and shares 89.9% amino acid similarity with the rat Kat-2 sequence. The gene for mKat-2 is composed of 13 exons divided by 12 intronic sequences. Northern blot analysis demonstrated that mKat-2 mRNA is mainly expressed in kidney and liver. RT-PCR showed mKat-2 expression in the brain starting from at least d11 of embryonic development. An alternative isoform mKat-2beta, derived from the usage of novel exons, shows a different expression pattern from mKat-2. Western blot analysis of various mouse tissues shows a 40-kDa protein in brain, heart, kidney, and liver. In the kidney and liver an additional 45-kDa isoform was detected. Use of the BSS chromosomal mapping panel from The Jackson Laboratory indicates that the mKat-2 gene co-segregates with polymorphic markers D8Mit129 and D8Mit128 on mouse Chr 8. Knowledge of the genomic organization, the isoform tissue-specific expression patterns, the chromosomal localization of mKat-2, and the reagents generated here, will provide the tools for further studies and allow generation and characterization of mice that are nullizygous for mKat-2.

  13. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

    SciTech Connect

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu; Oh, Jae-Wook; Song, Hyuk; Kim, Jae-Hwan; Kim, Jin-Hoi

    2011-07-01

    Highlights: {yields} The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. {yields} The core promoter was located in the 5F-1. {yields} Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. {yields} These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

  14. Polymorphisms in Factor II and Factor V thrombophilia genes among Circassians in Jordan.

    PubMed

    Dajani, R; Arafat, A; Hakooz, N; Al-Abbadi, Z; Yousef, Al-Motassem; El Khateeb, M; Quadan, F

    2013-01-01

    Thrombosis is a major cause of morbidity and mortality worldwide. Genetic factors are one component of thrombosis. We studied the prevalence of two mutations that are known risk factors in the pathogenesis of arterial and venous thrombosis in the genetically isolated Circassian population in Jordan. Factor II G20210A and Factor V Leiden single nucleotide polymorphisms were analysed by polymerase chain reaction and restriction fragment length polymorphism method in 104 random unrelated subjects from the Circassian population in Jordan. The prevalence rates among the Circassian population in Jordan for Factor II G20210A was 12.2% and for Factor V Leiden was 7.7%. We have shown that the population is in Hardy-Weinberg equilibrium and that the prevalences of both mutations are within the range of other ethnic groups. This is the first study to describe Circassian health related genetic characteristics in Jordan. Such population-based studies will contribute to understanding the interaction between genetic and environmental risk factors. It will remain to be seen whether carriers of Factor II G20210A and Factor V Leiden are more likely to develop thrombosis. This issue should be studied in the future to determine the need for screening of these mutations particularly in thrombophilia patients.

  15. Expanding our understanding of sequence-function relationships of type II polyketide biosynthetic gene clusters: bioinformatics-guided identification of Frankiamicin A from Frankia sp. EAN1pec.

    PubMed

    Ogasawara, Yasushi; Yackley, Benjamin J; Greenberg, Jacob A; Rogelj, Snezna; Melançon, Charles E

    2015-01-01

    A large and rapidly increasing number of unstudied "orphan" natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase α/β sequence characteristics in the genomes of Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of Frankia gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes. PMID:25837682

  16. Expanding our Understanding of Sequence-Function Relationships of Type II Polyketide Biosynthetic Gene Clusters: Bioinformatics-Guided Identification of Frankiamicin A from Frankia sp. EAN1pec

    PubMed Central

    Ogasawara, Yasushi; Yackley, Benjamin J.; Greenberg, Jacob A.; Rogelj, Snezna; Melançon, Charles E.

    2015-01-01

    A large and rapidly increasing number of unstudied “orphan” natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase α/β sequence characteristics in the genomes of Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of Frankia gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes. PMID:25837682

  17. [Influence of mutant genes on crystallin synthesis in the forming mouse lens. II. Fidget and ocular retardation genes].

    PubMed

    Iakovlev, M I; Platonov, E S; Koniukhov, B V

    1977-01-01

    The beginning of synthesis and the localization of alpha- and gamma-crystallins in the developing lenses of the 10-13 and 15 days old mouse embryos of the genotypes fi/fi +/+, +/+ or/or, fi/fi or/or and +/+ +/+ were studied by means of indirect immunofluorescence. The synthesis of crystallins in the mutant embryos with the exception of the embryo +/+ or/or was shown to begin somewhat later than in the normal ones but to proceed in all defective lenses, irrespective of the degree of defect. Hence, the activation of the genes controlling the synthesis of alpha-crystallins begins at the early stages of lens development and the synthesis of these proteins proceeds even during the abnormal with the slowing down of the formation of primary lens fibers. In the cases of strong defects of morphogenesis in the fi/fi +/+ and, especially, fi/fi or/or, embryos gamma-crystallins were not detected. The synthesis of gamma-crystallins appears to begin at the final stages of lens fiber differentiation.

  18. The subsidiary GntII system for gluconate metabolism in Escherichia coli: alternative induction of the gntV gene.

    PubMed

    Gómez, Keyla M; Rodríguez, Andrea; Rodriguez, Yesseima; Ramírez, Alvaro H; Istúriz, Tomás

    2011-01-01

    Two systems are involved in the transport and phosphorylation of gluconate in Escherichia coli. GntI, the main system, consists of high and low-affinity gluconate transporters and a thermoresistant gluconokinase for its phosphorylation. The corresponding genes, gntT, gntU and gntK at 76.5 min, are induced by gluconate. GntII, the subsidiary system, includes IdnT and GntV, which duplicate activities of transport and phosphorylation of gluconate, respectively. Gene gntV at 96.8 min is divergently transcribed from the idnDOTR operon involved in L-idonate metabolism. These genetic elements are induced by the substrate or 5-keto-D-gluconate. Because gntV is also induced in cells grown in gluconate, it was of interest to investigate its expression in this condition. E. coli gntK, idnOokan mutants were constructed to study this question. These idnO kan-cassete inserted mutants, unable to convert gluconate to 5-keto-D-gluconate, permitted examining gntV expression in the absence of this inducer and demonstrating that it is not required when the cells grow in gluconate. The results suggest that E. coli gntV gene is alternatively induced by 5-keto-D-gluconate or gluconate in cells cultivated either in idonate or gluconate. In this way, the control of gntV expression would seem to be involved in the efficient utilization of these substrates.

  19. QTL mapping of genetic determinants of lipoprotein metabolism in mice: Mutations of the apolipoprotein A-II gene affecting lipoprotein turnover

    SciTech Connect

    Weinreb, A.; Purcell-Huynh, D.A.; Castellani, L.W.

    1994-09-01

    Cholesterol and lipoproteins represent important risk factors for atherosclerosis. In order to better understand the genes involved in determining lipoprotein levels, quantitative trait locus (QTL) mapping was performed using a cross between NZB and SM/J mice. Significant LOD scores for loci determining total cholesterol, HDL cholesterol, LDL and VLDL cholesterol, triglycerides, free fatty acids, and apolipoprotein A-II (apoA-II) were obtained. NZB mice have a 7-10 fold higher apoA-II level SM/J. LOD scores of 19.6 (chow) and 10.3 (high fat) were obtained at the apoA-II gene locus. Comparison of apoA-II levels by apoA-II genotype reveals that {approximately}30% of the variance in apoA-II levels can be accounted for by differences within the apoA-II gene. Northern analysis of mRNA from NZB and SM/J mice fed a high fat diet failed to show any significant differences in mRNA levels. The rates of apoA-II protein synthesis relative to total protein synthesis between the two strains were similar, with a rate of 0.16% for NZB and 0.18% for SM/J. Sequencing of NZB and SM/J apoA-II cDNAs revealed a pro5 to gln5 substitution in SM/J. Therefore, differences in the apoA-II levels between NZB and SM/J may be partly due to a structural difference in apoA-II resulting in an increased rate of apoA-II clearance in SM/J. A coincident QTL for HDL at the same chromosome 1 locus suggests that a structural difference in apoA-II may be affecting the rate of HDL clearance. It is of interest to note that the pro5 to gln5 substitution leads to apoA-II amyloid deposition in the SAM mouse.

  20. Interpreting In vitro Data for Priortizing Environmental Chemical Concerns: II. Trout and Human ER Gene Expression

    EPA Science Inventory

    A series of p-alkylphenols (p-APs), and select alkoxy, phenyl- and ortho-substituted phenols, were tested in rainbow trout- and human- estrogen receptor (rtER, hER)-mediated gene activation. Chemicals previously tested in ER binding assays optimized for detection of low affinity ...

  1. Mutation in collagen II alpha 1 isoforms delineates Stickler and Wagner syndrome phenotypes

    PubMed Central

    Tran-Viet, Khanh-Nhat; Soler, Vincent; Quiette, Valencia; Powell, Caldwell; Yanovitch, Tammy; Metlapally, Ravikanth; Luo, Xiaoyan; Katsanis, Nicholas; Nading, Erica

    2013-01-01

    Purpose Stickler syndrome is an arthro-ophthalmopathy with phenotypic overlap with Wagner syndrome. The common Stickler syndrome type I is inherited as an autosomal dominant trait, with causal mutations in collagen type II alpha 1 (COL2A1). Wagner syndrome is associated with mutations in versican (VCAN), which encodes for a chondroitin sulfate proteoglycan. A three-generation Caucasian family variably diagnosed with either syndrome was screened for sequence variants in the COL2A1 and VCAN genes. Methods Genomic DNA samples derived from saliva were collected from all family members (six affected and four unaffected individuals). Complete sequencing of COL2A1 and VCAN was performed on two affected individuals. Direct sequencing of remaining family members was conducted if the discovered variants followed segregation. Results A base-pair substitution (c.258C>A) in exon 2 of COL2A1 cosegregated with familial disease status. This known mutation occurs in a highly conserved site that causes a premature stop codon (p.C86X). The mutation was not seen in 1,142 ethnically matched control DNA samples. Conclusions Premature stop codons in COL2A1 exon 2 lead to a Stickler syndrome type I ocular-only phenotype with few or no systemic manifestations. Mutation screening of COL2A1 exon 2 in families with autosomal dominant vitreoretinopathy is important for accurate clinical diagnosis. PMID:23592912

  2. Transcriptional up-regulation of antioxidant genes by PPAR{delta} inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells

    SciTech Connect

    Kim, Hyo Jung; Ham, Sun Ah; Paek, Kyung Shin; Hwang, Jung Seok; Jung, Si Young; Kim, Min Young; Jin, Hanna; Kang, Eun Sil; Woo, Im Sun; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Han, Chang Woo; Seo, Han Geuk

    2011-03-25

    Research highlights: {yields} Activation of PPAR{delta} by GW501516 significantly inhibited Ang II-induced premature senescence in hVSMCs. {yields} Agonist-activated PPAR{delta} suppressed generation of Ang II-triggered ROS with a concomitant reduction in DNA damage. {yields} GW501516 up-regulated expression of antioxidant genes, such as GPx1, Trx1, Mn-SOD and HO-1. {yields} Knock-down of these antioxidant genes abolished the effects of GW501516 on ROS production and premature senescence. -- Abstract: This study evaluated peroxisome proliferator-activated receptor (PPAR) {delta} as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPAR{delta} by GW501516, a specific agonist of PPAR{delta}, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPAR{delta} suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPAR{delta}-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

  3. Characterization of type I and II procollagen α1 chain in Amur sturgeon (Acipenser schrenckii) and comparison of their gene expression.

    PubMed

    Zhang, Xi; Azuma, Noriko; Hagihara, Seishi; Adachi, Shinji; Ura, Kazuhiro; Takagi, Yasuaki

    2016-03-15

    To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the proα chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the proα chains revealed that type I and II collagen share the basic structure of the proα chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons.

  4. Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus).

    PubMed

    Gasper, J S; Shiina, T; Inoko, H; Edwards, S V

    2001-07-01

    Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.

  5. A novel mutation in the DSPP gene associated with dentinogenesis imperfecta type II.

    PubMed

    Lee, S-K; Lee, K-E; Jeon, D; Lee, G; Lee, H; Shin, C-U; Jung, Y-J; Lee, S-H; Hahn, S-H; Kim, J-W

    2009-01-01

    Hereditary dentin defects are divided into dentinogenesis imperfecta and dentin dysplasia. We identified a family segregating severe dentinogenesis imperfecta. The kindred spanned four generations and showed an autosomal-dominant pattern of inheritance. The proband was a child presenting with a severely affected primary dentition, with wide-open pulp chambers and multiple pulp exposures, resembling a DGI type III (DGI-III) pattern. We hypothesized that a mutation in the DSPP gene is responsible for this severe phenotype. Mutational analyses revealed a novel mutation (c.53T>A, p.V18D) near the intron-exon boundary in the third exon of the DSPP gene. We analyzed the effect of the mutation by means of an in vitro splicing assay, which revealed that the mutation did not affect pre-mRNA splicing. Further studies are needed for a better understanding of the nature of the disease and the development of an appropriate treatment strategy.

  6. Coptotermes gestroi (Isoptera: Rhinotermitidae) in Brazil: possible origins inferred by mitochondrial cytochrome oxidase II gene sequences.

    PubMed

    Martins, C; Fontes, L R; Bueno, O C; Martins, V G

    2010-09-01

    The Asian subterranean termite, Coptotermes gestroi, originally from northeast India through Burma, Thailand, Malaysia, and the Indonesian archipelago, is a major termite pest introduced in several countries around the world, including Brazil. We sequenced the mitochondrial COII gene from individuals representing 23 populations. Phylogenetic analysis of COII gene sequences from this and other studies resulted in two main groups: (1) populations of Cleveland (USA) and four populations of Malaysia and (2) populations of Brazil, four populations of Malaysia, and one population from each of Thailand, Puerto Rico, and Key West (USA). Three new localities are reported here, considerably enlarging the distribution of C. gestroi in Brazil: Campo Grande (state of Mato Grosso do Sul), Itajaí (state of Santa Catarina), and Porto Alegre (state of Rio Grande do Sul).

  7. HLA class II genes modulate vaccine-induced antibody responses to affect HIV-1 acquisition

    PubMed Central

    Prentice, Heather A.; Tomaras, Georgia D.; Geraghty, Daniel E.; Apps, Richard; Fong, Youyi; Ehrenberg, Philip K.; Rolland, Morgane; Kijak, Gustavo H.; Krebs, Shelly J.; Nelson, Wyatt; DeCamp, Allan; Shen, Xiaoying; Yates, Nicole L.; Zolla-Pazner, Susan; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Ferrari, Guido; Juliana McElrath, M.; Montefiori, David C.; Bailer, Robert T.; Koup, Richard A.; O’Connell, Robert J.; Robb, Merlin L.; Michael, Nelson L.; Gilbert, Peter B.; Kim, Jerome H.; Thomas, Rasmi

    2016-01-01

    In the RV144 vaccine trial, two antibody responses were found to correlate with HIV-1 acquisition. Because human leukocyte antigen (HLA) class II–restricted CD4+ T cells are involved in antibody production, we tested whether HLA class II genotypes affected HIV-1–specific antibody levels and HIV-1 acquisition in 760 individuals. Indeed, antibody responses correlated with acquisition only in the presence of single host HLA alleles. Envelope (Env)–specific immunoglobulin A (IgA) antibodies were associated with increased risk of acquisition specifically in individuals with DQB1*06. IgG antibody responses to Env amino acid positions 120 to 204 were higher and were associated with decreased risk of acquisition and increased vaccine efficacy only in the presence of DPB1*13. Screening IgG responses to overlapping peptides spanning Env 120–204 and viral sequence analysis of infected individuals defined differences in vaccine response that were associated with the presence of DPB1*13 and could be responsible for the protection observed. Overall, the underlying genetic findings indicate that HLA class II modulated the quantity, quality, and efficacy of antibody responses in the RV144 trial. PMID:26180102

  8. Non-neutral evolution and reciprocal monophyly of two expressed Mhc class II B genes in Leach's storm-petrel.

    PubMed

    Dearborn, Donald C; Gager, Andrea B; Gilmour, Morgan E; McArthur, Andrew G; Hinerfeld, Douglas A; Mauck, Robert A

    2015-02-01

    The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach's storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7%). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species. PMID:25416539

  9. Association of Variants in Genes Related to the Immune Response and Obesity with Benign Prostatic Hyperplasia in CLUE II

    PubMed Central

    Lopez, David S.; Peskoe, Sarah B.; Tsilidis, Konstantinos K.; Hoffman-Bolton, Judy; Helzlsouer, Kathy J.; Isaacs, William B.; Smith, Michael W.; Platz, Elizabeth A.

    2014-01-01

    BACKGROUND Chronic inflammation and obesity may contribute to the genesis or progression of benign prostatic hyperplasia (BPH) and BPH-associated lower urinary tract symptoms (LUTS). The influence of variants in genes related to these states on BPH has not been studied extensively. Thus, we evaluated the association of 17 single nucleotide polymorphisms (SNPs) in immune response genes (IL1B, IL6, IL8, IL10, TNF, CRP, TLR4, RNASEL) and genes involved in obesity, including insulin regulation (LEP, ADIPOQ, PPARG, TCF7L2), with BPH. METHODS BPH cases (N=568) and age-frequency matched controls (N=568) were selected from among adult male CLUE II cohort participants who responded in 2000 to a mailed questionnaire. BPH was defined as BPH surgery, use of BPH medications, or symptomatic BPH (American Urological Association Symptom Index Score ≥15). Controls were men who had not had BPH surgery, did not use BPH medications, and whose symptom score was ≤7. Age-adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. RESULTS None of the candidate SNPs was statistically significantly associated with BPH. However, we could not rule out possible weak associations for CRP rs1205 (1082C>T), ADIPOQ rs1501299 (276C>A), PPARG rs1801282 (-49C>G), and TCF7L2 rs7903146 (47833T>C). After summing risk alleles, men with ≥4 had an increased BPH risk compared with those with ≤1 (OR, 1.78; 95% CI, 1.10-2.89; Ptrend=0.006). CONCLUSION SNPs in genes related to immune response and obesity, especially in combination, may be associated with BPH. PMID:25224558

  10. Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

    PubMed

    Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Uzgun, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

    2009-12-31

    phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

  11. Analyses of RNA Polymerase II genes from free-living protists: phylogeny, long branch attraction, and the eukaryotic big bang.

    PubMed

    Dacks, Joel B; Marinets, Alexandra; Ford Doolittle, W; Cavalier-Smith, Thomas; Logsdon, John M

    2002-06-01

    The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant obstacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Polymerase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Ochromonas danica (a heterokont); we have also analyzed the RPB1 gene from the nucleomorph (nm) genome of Guillardia theta (a cryptomonad). Using a variety of phylogenetic methods our analysis shows that RPB1s from Giardia intestinalis and Trichomonas vaginalis are probably subject to intense LBA effects. Thus, the deep branching of these taxa on RPB1 trees is questionable and should not be interpreted as evidence favoring their early divergence. Similar effects are discernable, to a lesser extent, with the Mastigamoeba invertens RPB1 sequence. Upon removal of the outgroup and these problematic sequences, analyses of the remaining RPB1s indicate some resolution among major eukaryotic groups. The most robustly supported higher-level clades are the opisthokonts (animals plus fungi) and the red algae plus the cryptomonad nm-the latter result gives added support to the red algal origin of cryptomonad chloroplasts. Clades comprising Dictyostelium discoideum plus Acanthamoeba castellanii (Amoebozoa) and Ochromonas plus Plasmodium falciparum (chromalveolates) are consistently observed and moderately supported. The clades supported by our RPB1 analyses are congruent with other data, suggesting that bona fide phylogenetic relationships are being resolved. Thus, the RPB1 gene has apparently retained some phylogenetically meaningful signal, making it worthwhile to obtain sequences from more diverse protist taxa. Additional RPB1 data, especially in combination with other genes, should provide further resolution of branching orders among protist

  12. Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism.

    PubMed

    Eimes, John A; Townsend, Andrea K; Sepil, Irem; Nishiumi, Isao; Satta, Yoko

    2015-01-01

    A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of

  13. Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism

    PubMed Central

    Townsend, Andrea K.; Sepil, Irem; Nishiumi, Isao; Satta, Yoko

    2015-01-01

    A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of

  14. Molecular evidence of RNA polymerase II gene reveals the origin of worldwide cultivated barley

    PubMed Central

    Wang, Yonggang; Ren, Xifeng; Sun, Dongfa; Sun, Genlou

    2016-01-01

    The origin and domestication of cultivated barley have long been under debate. A population-based resequencing and phylogenetic analysis of the single copy of RPB2 gene was used to address barley domestication, to explore genetic differentiation of barley populations on the worldwide scale, and to understand gene-pool exchanges during the spread and subsequent development of barley cultivation. Our results revealed significant genetic differentiation among three geographically distinct wild barley populations. Differences in haplotype composition among populations from different geographical regions revealed that modern cultivated barley originated from two major wild barley populations: one from the Near East Fertile Crescent and the other from the Tibetan Plateau, supporting polyphyletic origin of cultivated barley. The results of haplotype frequencies supported multiple domestications coupled with widespread introgression events that generated genetic admixture between divergent barley gene pools. Our results not only provide important insight into the domestication and evolution of cultivated barley, but also enhance our understanding of introgression and distinct selection pressures in different environments on shaping the genetic diversity of worldwide barley populations, thus further facilitating the effective use of the wild barley germplasm. PMID:27786300

  15. The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

    PubMed

    Erra-Pujada, M; Debeire, P; Duchiron, F; O'Donohue, M J

    1999-05-01

    The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.

  16. Maturation in Larch : II. Effects of Age on Photosynthesis and Gene Expression in Developing Foliage.

    PubMed

    Hutchison, K W; Sherman, C D; Weber, J; Smith, S S; Singer, P B; Greenwood, M S

    1990-11-01

    The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO(2) per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased

  17. TinII intron, an enhancer to affect the function of the cytoplasmic male sterility related gene T in Brassica juncea.

    PubMed

    Jin, ZhuPing; Wu, LingLing; Cao, JiaShu; Chen, ZhuJun; Pei, YanXi

    2013-12-01

    The T gene, which was cloned from the mitochondria of tumorous stem mustard (Brassica juncea var. tumida), is a cytoplasmic male sterility (CMS)-related gene that can produce two transcripts, T1170 and T1243. The latter is transcribed with the uncleaved intron TinII. In our previous study, transgenic Arabidopsis thaliana plants over-expressing the T1243 transcript (OE-T1243) showed a severe male-sterile phenotype, whereas OE-T1170 plants did not. However, the functional mechanism of the T gene in B. Juncea remained unknown. In this study, microscopic analyses of paraffin sections of anthers confirmed that OE-T1243 plants did not produce normal pollen, whereas OE-T1170 plants did. We analyzed the transcription of 15 anther development-related genes and found that transcript levels of nozzle/sporocyteless and barely any meristem 1 and 2 were markedly lower in OE-T1243 plants than those in wild type, while the transcript levels of these genes in OE-T1170 plants were unchanged. To investigate the potential roles of TinII, we inserted the TinII sequence upstream of a minimal region (-60) of the cauliflower mosaic virus 35S promoter fused to the 5' end of the β-glucuronidase (GUS) reporter gene. Analysis of the transgenic plants suggested that TinII acted as an enhancer to significantly increase GUS expression. The potential action mechanism is that the TinII intron acts as an enhancer to affect the function of the CMS-related gene T. PMID:24302291

  18. Group II Intron-Mediated Trans-Splicing in the Gene-Rich Mitochondrial Genome of an Enigmatic Eukaryote, Diphylleia rotans.

    PubMed

    Kamikawa, Ryoma; Shiratori, Takashi; Ishida, Ken-Ichiro; Miyashita, Hideaki; Roger, Andrew J

    2016-02-01

    Although mitochondria have evolved from a single endosymbiotic event, present day mitochondria of diverse eukaryotes display a great range of genome structures, content and features. Group I and group II introns are two features that are distributed broadly but patchily in mitochondrial genomes across branches of the tree of eukaryotes. While group I intron-mediated trans-splicing has been reported from some lineages distantly related to each other, findings of group II intron-mediated trans-splicing has been restricted to members of the Chloroplastida. In this study, we found the mitochondrial genome of the unicellular eukaryote Diphylleia rotans possesses currently the second largest gene repertoire. On the basis of a probable phylogenetic position of Diphylleia, which is located within Amorphea, current mosaic gene distribution in Amorphea must invoke parallel gene losses from mitochondrial genomes during evolution. Most notably, although the cytochrome c oxidase subunit (cox) 1 gene was split into four pieces which located at a distance to each other, we confirmed that a single mature mRNA that covered the entire coding region could be generated by group II intron-mediated trans-splicing. This is the first example of group II intron-mediated trans-splicing outside Chloroplastida. Similar trans-splicing mechanisms likely work for bipartitely split cox2 and nad3 genes to generate single mature mRNAs. We finally discuss origin and evolution of this type of trans-splicing in D. rotans as well as in eukaryotes. PMID:26833505

  19. Group II Intron-Mediated Trans-Splicing in the Gene-Rich Mitochondrial Genome of an Enigmatic Eukaryote, Diphylleia rotans

    PubMed Central

    Kamikawa, Ryoma; Shiratori, Takashi; Ishida, Ken-Ichiro; Miyashita, Hideaki; Roger, Andrew J.

    2016-01-01

    Although mitochondria have evolved from a single endosymbiotic event, present day mitochondria of diverse eukaryotes display a great range of genome structures, content and features. Group I and group II introns are two features that are distributed broadly but patchily in mitochondrial genomes across branches of the tree of eukaryotes. While group I intron-mediated trans-splicing has been reported from some lineages distantly related to each other, findings of group II intron-mediated trans-splicing has been restricted to members of the Chloroplastida. In this study, we found the mitochondrial genome of the unicellular eukaryote Diphylleia rotans possesses currently the second largest gene repertoire. On the basis of a probable phylogenetic position of Diphylleia, which is located within Amorphea, current mosaic gene distribution in Amorphea must invoke parallel gene losses from mitochondrial genomes during evolution. Most notably, although the cytochrome c oxidase subunit (cox) 1 gene was split into four pieces which located at a distance to each other, we confirmed that a single mature mRNA that covered the entire coding region could be generated by group II intron-mediated trans-splicing. This is the first example of group II intron-mediated trans-splicing outside Chloroplastida. Similar trans-splicing mechanisms likely work for bipartitely split cox2 and nad3 genes to generate single mature mRNAs. We finally discuss origin and evolution of this type of trans-splicing in D. rotans as well as in eukaryotes. PMID:26833505

  20. De novo mutation in the DSPP gene associated with dentinogenesis imperfecta type II in a Japanese family.

    PubMed

    Kida, Miyuki; Tsutsumi, Tomonori; Shindoh, Masanobu; Ikeda, Hisami; Ariga, Tadashi

    2009-12-01

    Dentinogenesis imperfecta (DGI) type II is one of the most common dominantly inherited dentin defects, in which both the primary and permanent teeth are affected. Here, we report a Japanese family with autosomal-dominant DGI type II, including both molecular genetic defects and pathogenesis with histological analysis. Mutation analysis revealed a mutation (c.53T>A, p.V18D, g.1192T>A) involving the second nucleotide of the first codon within exon 3 of the dentin sialophosphoprotein (DSPP) gene. This mutation has previously been reported in a Korean family. Thus far, 24 allelic DSPP mutations have been reported, and this is the seventh mutation involving the DSPP V18 residue. Among those, only one other was shown to be caused by a de novo mutation, and that mutation also affected the V18 amino acid residue. The DSPP V18 residue is highly conserved among other mammalian species. These findings thus suggest that the V18 amino acid might be a sensitive mutational hot spot, playing a critical role in the pathogenesis of DGI.

  1. DNA methylation of angiotensin II receptor gene in nonalcoholic steatohepatitis-related liver fibrosis

    PubMed Central

    Asada, Kiyoshi; Aihara, Yosuke; Takaya, Hiroaki; Noguchi, Ryuichi; Namisaki, Tadashi; Moriya, Kei; Uejima, Masakazu; Kitade, Mitsuteru; Mashitani, Tsuyoshi; Takeda, Kosuke; Kawaratani, Hideto; Okura, Yasushi; Kaji, Kosuke; Douhara, Akitoshi; Sawada, Yasuhiko; Nishimura, Norihisa; Seki, Kenichiro; Mitoro, Akira; Yamao, Junichi; Yoshiji, Hitoshi

    2016-01-01

    AIM To clarify whether Agtr1a methylation is involved in the development of nonalcoholic steatohepatitis (NASH)-related liver fibrosis in adult rats. METHODS A choline-deficient amino acid (CDAA) diet model was employed for methylation analysis of NASH-related liver fibrosis. Agtr1a methylation levels were measured in the livers of CDAA- and control choline-sufficient amino acid (CSAA)-fed rats for 8 and 12 wk using quantitative methylation-specific PCR. Hepatic stellate cells (HSCs) were isolated by collagenase digestion of the liver, followed by centrifugation of the crude cell suspension through a density gradient. Agtr1a methylation and its gene expression were also analyzed during the activation of HSCs. RESULTS The mean levels of Agtr1a methylation in the livers of CDAA-fed rats (11.5% and 18.6% at 8 and 12 wk, respectively) tended to be higher (P = 0.06 and 0.09, respectively) than those in the livers of CSAA-fed rats (2.1% and 5.3% at 8 and 12 wk, respectively). Agtr1a was not methylated at all in quiescent HSCs, but was clearly methylated in activated HSCs (13.8%, P < 0.01). Interestingly, although Agtr1a was hypermethylated, the Agtr1a mRNA level increased up to 2.2-fold (P < 0.05) in activated HSCs compared with that in quiescent HSCs, suggesting that Agtr1a methylation did not silence its expression but instead had the potential to upregulate its expression. These findings indicate that Agtr1a methylation and its upregulation of gene expression are associated with the development of NASH-related liver fibrosis. CONCLUSION This is the first study to show that DNA methylation is potentially involved in the regulation of a renin-angiotensin system-related gene expression during liver fibrosis. PMID:27729955

  2. Gene mapping in marsupials and monotremes. II. Assignments to the X chromosome of dasyurid marsupials.

    PubMed

    Dobrovic, A; Marshall Graves, J A

    1986-01-01

    Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.

  3. Genome sequence of Fulvimarina pelagi HTCC2506T, a Mn(II)-oxidizing alphaproteobacterium possessing an aerobic anoxygenic photosynthetic gene cluster and Xanthorhodopsin.

    PubMed

    Kang, Ilnam; Oh, Hyun-Myung; Lim, Seung-Il; Ferriera, Steve; Giovannoni, Stephen J; Cho, Jang-Cheon

    2010-09-01

    Fulvimarina pelagi is a Mn(II)-oxidizing marine heterotrophic bacterium in the order Rhizobiales. Here we announce the draft genome sequence of F. pelagi HTCC2506(T), which was isolated from the Sargasso Sea by using dilution-to-extinction culturing. The genome sequence contained a xanthorhodopsin gene as well as a photosynthetic gene cluster, which suggests the coexistence of two different phototrophic mechanisms in a single microorganism. PMID:20639329

  4. Carbon nanotubes part II: a remarkable carrier for drug and gene delivery

    PubMed Central

    Karimi, Mahdi; Solati, Navid; Ghasemi, Amir; Estiar, Mehrdad Asghari; Hashemkhani, Mahshid; Kiani, Parnian; Mohamed, Elmira; Saeidi, Ahad; Taheri, Mahdiar; Avci, Pinar; Aref, Amir R; Amiri, Mohammad; Baniasadi, Fazel; Hamblin, Michael R

    2015-01-01

    Introduction Carbon nanotubes (CNT) have recently been studied as novel and versatile drug and gene delivery vehicles. When CNT are suitably functionalized, they can interact with various cell types and are taken up by endocytosis. Areas covered Anti-cancer drugs cisplatin and doxorubicin have been delivered by CNT, as well as methotrexate, taxol and gemcitabine. The delivery of the antifungal compound amphotericin B and the oral administration of erythropoietin have both been assisted using CNT. Frequently, targeting moieties such as folic acid, epidermal growth factor or various antibodies are attached to the CNT-drug nanovehicle. Different kinds of functionalization (e.g., polycations) have been used to allow CNT to act as gene delivery vectors. Plasmid DNA, small interfering RNA and micro-RNA have all been delivered by CNT vehicles. Significant concerns are raised about the nanotoxicology of the CNT and their potentially damaging effects on the environment. Expert opinion CNT-mediated drug delivery has been studied for over a decade, and both in vitro and in vivo studies have been reported. The future success of CNTs as vectors in vivo and in clinical application will depend on achievement of efficacious therapy with minimal adverse effects and avoidance of possible toxic and environmentally damaging effects. PMID:25613837

  5. [Somatic hypermutagenesis in immunoglobulin genes. II. Properties of somatic mutations and clonal selection].

    PubMed

    Rogozin, I B; Solov'ev, V V

    1989-01-01

    Analysis of the collection of 203 somatic mutations in immunoglobulin genes was carried out. It was shown, that the high frequency of these mutations in CDRs of V-genes may be connected with the high concentration of repeats in these regions. In addition, the observed clusterization of mutations may emerge from simultaneous correction of several pertubations of complementarity in the heteroduplex, formed by the repeat regions. It was revealed, that somatic mutations in FRs are characterized by reliably smaller changes of some important amino acid physical-chemical properties than in CDRs. These data obviously indicate the occurrence of B-lymphocytes clonal selection. Analysis of synonymous substitutions has shown, that stabilizing selection seems to provide the conservatism of FRs (it leads to the conservation of the protein three-dimensional structure) and movement selection may provide the proliferation of B-lymphocytes with considerable changes in CDRs, if these mutations improve antigens binding. Preferential fixation of transitions in comparison with transversions, particularly expressed in FRs, may also be connected with the fact, that transitions lead to smaller changes of amino acid physical-chemical properties and they are rejected by selection to a smaller extent.

  6. Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis.

    PubMed

    Tomiyama, Shiori; Nakamachi, Tomoya; Uchiyama, Minoru; Matsuda, Kouhei; Konno, Norifumi

    2015-05-15

    Urotensin II (UII) exhibits diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response via the UII receptor (UTR) in mammals. However, in amphibians the function of the UII-UTR system remains unknown. In the present study, we investigated the potential immune function of UII using leukocytes isolated from the African clawed frog, Xenopus laevis. Stimulation of male frogs with lipopolysaccharide increased mRNA expression of UII and UTR in leukocytes, suggesting that inflammatory stimuli induce activation of the UII-UTR system. Migration assays showed that both UII and UII-related peptide enhanced migration of leukocytes in a dose-dependent manner, and that UII effect was inhibited by the UTR antagonist urantide. Inhibition of Rho kinase with Y-27632 abolished UII-induced migration, suggesting that it depends on the activation of RhoA/Rho kinase. Treatment of isolated leukocytes with UII increased the expression of several cytokine genes including tumor necrosis factor-α, interleukin-1β, and macrophage migration inhibitory factor, and the effects were abolished by urantide. These results suggest that in amphibian leukocytes the UII-UTR system is involved in the activation of leukocyte migration and cytokine gene expression in response to inflammatory stimuli.

  7. Gene expression in the DpnI and DpnII restriction enzyme systems of Streptococcus pneumoniae

    SciTech Connect

    Lacks, S.A.; Sabelnikov, A.G.; Chen, Jau-Der; Greenberg, B.

    1992-12-31

    Although a number of bacterial species are naturally transformable, that is, their cells are able to take up external DNA in substantial amounts and integrate it into the chromosome without artificial manipulation of the cell surface, Streptococcus pneumoniae, the first species in which this phenomenon was detected, remains a prototype of such transformation. Cells of S. pneumonias also contain potent restriction endonucleases able to severely restrict DNA introduced during viral infection. Our current understanding of the genetic basis of the complementary DpnI and DpnII restriction systems and of the biochemistry of their component enzymes are briefly reviewed. The manner in which these enzymes impinge on the transfer of chromosomal genes and of plasmeds will be examined in detail. It will be seen that far from acting against foreign DNA in general, the restriction systems seem to be designed to exclude only infecting viral DNA The presence of complementary restriction systems in different cells of S. pneumonias enhances their effectiveness in blocking viral infection and promoting species survival. This enhanced effectiveness requires the expression of alternative restriction systems. Therefore, the ability of the cells to transfer the restriction enzyme genes and to regulate their expression are important for survival of the species.

  8. A Novel Frameshift Mutation of the USH2A Gene in a Korean Patient with Usher Syndrome Type II.

    PubMed

    Boo, Sung Hyun; Song, Min-Jung; Kim, Hee-Jin; Cho, Yang-Sun; Chu, Hosuk; Ko, Moon-Hee; Chung, Won-Ho; Kim, Jong-Won; Hong, Sung Hwa

    2013-03-01

    Usher syndrome type II (USH2) is the most common form of Usher syndrome, characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa. It has been shown that mutations in the USH2A gene are responsible for USH2. The authors herein describe a 34-year-old Korean woman with the typical clinical manifestation of USH2; she had bilateral hearing disturbance and progressive visual deterioration, without vestibular dysfunction. Molecular genetic study of the USH2A gene revealed a novel frameshift mutation (c.2310delA; Glu771LysfsX17). She was heterozygous for this mutation, and no other mutation was found in USH2A, suggesting the possibility of an intronic or large genomic rearrangement mutation. To the best of our knowledge, this is the first report of a genetically confirmed case of USH2 in Korea. More investigations are needed to delineate genotype-phenotype correlations and ethnicity-specific genetic background of Usher syndrome. PMID:23526569

  9. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    SciTech Connect

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  10. Dissection of immune gene networks in primary melanoma tumors critical for antitumor surveillance of patients with stage II-III resectable disease.

    PubMed

    Sivendran, Shanthi; Chang, Rui; Pham, Lisa; Phelps, Robert G; Harcharik, Sara T; Hall, Lawrence D; Bernardo, Sebastian G; Moskalenko, Marina M; Sivendran, Meera; Fu, Yichun; de Moll, Ellen H; Pan, Michael; Moon, Jee Young; Arora, Sonali; Cohain, Ariella; DiFeo, Analisa; Ferringer, Tammie C; Tismenetsky, Mikhail; Tsui, Cindy L; Friedlander, Philip A; Parides, Michael K; Banchereau, Jacques; Chaussabel, Damien; Lebwohl, Mark G; Wolchok, Jedd D; Bhardwaj, Nina; Burakoff, Steven J; Oh, William K; Palucka, Karolina; Merad, Miriam; Schadt, Eric E; Saenger, Yvonne M

    2014-08-01

    Patients with resected stage II-III cutaneous melanomas remain at high risk for metastasis and death. Biomarker development has been limited by the challenge of isolating high-quality RNA for transcriptome-wide profiling from formalin-fixed and paraffin-embedded (FFPE) primary tumor specimens. Using NanoString technology, RNA from 40 stage II-III FFPE primary melanomas was analyzed and a 53-immune-gene panel predictive of non-progression (area under the curve (AUC)=0.920) was defined. The signature predicted disease-specific survival (DSS P<0.001) and recurrence-free survival (RFS P<0.001). CD2, the most differentially expressed gene in the training set, also predicted non-progression (P<0.001). Using publicly available microarray data from 46 primary human melanomas (GSE15605), a coexpression module enriched for the 53-gene panel was then identified using unbiased methods. A Bayesian network of signaling pathways based on this data identified driver genes. Finally, the proposed 53-gene panel was confirmed in an independent test population of 48 patients (AUC=0.787). The gene signature was an independent predictor of non-progression (P<0.001), RFS (P<0.001), and DSS (P=0.024) in the test population. The identified driver genes are potential therapeutic targets, and the 53-gene panel should be tested for clinical application using a larger data set annotated on the basis of prospectively gathered data.

  11. Identification of Type II Interferon Receptors in Geese: Gene Structure, Phylogenetic Analysis, and Expression Patterns

    PubMed Central

    Zhou, Hao; Chen, Shun; Qi, Yulin; Zhou, Qin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-01-01

    Interferon γ receptor 1 (IFNGR1) and IFNGR2 are two cell membrane molecules belonging to class II cytokines, which play important roles in the IFN-mediated antiviral signaling pathway. Here, goose IFNGR1 and IFNGR2 were cloned and identified for the first time. Tissue distribution analysis revealed that relatively high levels of goose IFNγ mRNA transcripts were detected in immune tissues, including the harderian gland, cecal tonsil, cecum, and thymus. Relatively high expression levels of both IFNGR1 and IFNGR2 were detected in the cecal tonsil, which implicated an important role of IFNγ in the secondary immune system of geese. No specific correlation between IFNγ, IFNGR1, and IFNGR2 expression levels was observed in the same tissues of healthy geese. IFNγ and its cognate receptors showed different expression profiles, although they appeared to maintain a relatively balanced state. Furthermore, the agonist R848 led to the upregulation of goose IFNγ but did not affect the expression of goose IFNGR1 or IFNGR2. In summary, trends in expression of goose IFNγ and its cognate receptors showed tissue specificity, as well as an age-related dependency. These findings may help us to better understand the age-related susceptibility to pathogens in birds. PMID:26345454

  12. Identification of Type II Interferon Receptors in Geese: Gene Structure, Phylogenetic Analysis, and Expression Patterns.

    PubMed

    Zhou, Hao; Chen, Shun; Qi, Yulin; Zhou, Qin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-01-01

    Interferon γ receptor 1 (IFNGR1) and IFNGR2 are two cell membrane molecules belonging to class II cytokines, which play important roles in the IFN-mediated antiviral signaling pathway. Here, goose IFNGR1 and IFNGR2 were cloned and identified for the first time. Tissue distribution analysis revealed that relatively high levels of goose IFNγ mRNA transcripts were detected in immune tissues, including the harderian gland, cecal tonsil, cecum, and thymus. Relatively high expression levels of both IFNGR1 and IFNGR2 were detected in the cecal tonsil, which implicated an important role of IFNγ in the secondary immune system of geese. No specific correlation between IFNγ, IFNGR1, and IFNGR2 expression levels was observed in the same tissues of healthy geese. IFNγ and its cognate receptors showed different expression profiles, although they appeared to maintain a relatively balanced state. Furthermore, the agonist R848 led to the upregulation of goose IFNγ but did not affect the expression of goose IFNGR1 or IFNGR2. In summary, trends in expression of goose IFNγ and its cognate receptors showed tissue specificity, as well as an age-related dependency. These findings may help us to better understand the age-related susceptibility to pathogens in birds.

  13. Midkine, a newly discovered regulator of the renin-angiotensin pathway in mouse aorta: significance of the pleiotrophin/midkine developmental gene family in angiotensin II signaling.

    PubMed

    Ezquerra, Laura; Herradon, Gonzalo; Nguyen, Trang; Silos-Santiago, Inmaculada; Deuel, Thomas F

    2005-07-29

    We previously demonstrated that pleiotrophin (PTN the protein, Ptn the gene) highly regulates the levels of expression of the genes encoding the proteins of the renin-angiotensin pathway in mouse aorta. We now demonstrate that the levels of expression of these same genes are significantly regulated in mouse aorta by the PTN family member midkine (MK the protein, Mk the gene); a 3-fold increase in expression of renin, an 82-fold increase in angiotensinogen, a 6-fold decrease in the angiotensin converting enzyme, and a 6.5-fold increase in the angiotensin II type 1 and a 9-fold increase in the angiotensin II type 2 receptor mRNAs were found in Mk-/- mouse aorta in comparison with the wild type (WT, +/+). The results in Mk-/- mice are remarkably similar to those previously reported in Ptn-/- mouse aorta, with the single exception of that the levels of the angiotensinogen gene expression in Ptn-/- mice are equal to those in WT+/+ mouse aorta, and thus, in contrast to Mk gene expression unaffected by levels of Ptn gene expression. The data indicate that MK and PTN share striking but not complete functional redundancy. These data support potentially high levels importance of MK and the MK/PTN developmental gene family in downstream signals initiated by angiotensin II either in development or in the many pathological conditions in which MK expression levels are increased, such as atherosclerosis and many human neoplasms that acquire constitutive endogenous Mk gene expression by mutation during tumor progression and potentially provide a target through the renin-angiotensin pathway to treat advanced malignancies.

  14. Development of a simultaneous high resolution typing method for three SLA class II genes, SLA-DQA, SLA-DQB1, and SLA-DRB1 and the analysis of SLA class II haplotypes.

    PubMed

    Le, MinhThong; Choi, Hojun; Choi, Min-Kyeung; Cho, Hyesun; Kim, Jin-Hoi; Seo, Han Geuk; Cha, Se-Yeon; Seo, Kunho; Dadi, Hailu; Park, Chankyu

    2015-06-15

    The characterization of the genetic variations of major histocompatibility complex (MHC) is essential to understand the relationship between the genetic diversity of MHC molecules and disease resistance and susceptibility in adaptive immunity. We previously reported the development of high-resolution individual locus typing methods for three of the most polymorphic swine leukocyte antigens (SLA) class II loci, namely, SLA-DQA, SLA-DQB1, and SLA-DRB1. In this study, we extensively modified our previous protocols and developed a method for the simultaneous amplification of the three SLA class II genes and subsequent analysis of individual loci using direct sequencing. The unbiased and simultaneous amplification of alleles from the all three hyper-polymorphic and pseudogene containing genes such as MHC genes is extremely challenging. However, using this method, we demonstrated the successful typing of SLA-DQA, SLA-DQB1, and SLA-DRB1 for 31 selected individuals comprising 26 different SLA class II haplotypes which were identified from 700 animals using the single locus typing methods. The results were identical to the known genotypes from the individual locus typing. The new method has significant benefits over the individual locus typing, including lower typing cost, use of less biomaterial, less effort and fewer errors in handling large samples for multiple loci. We also extensively characterized the haplotypes of SLA class II genes and reported three new haplotypes. Our results should serve as a basis to investigate the possible association between polymorphisms of MHC class II and differences in immune responses to exogenous antigens.

  15. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

    SciTech Connect

    Vandenberg, P.; Khillan, J.S.; Prockop, D.J.; Helminen, H.; Kontusaari, S.; Ala-Kokko, L. )

    1991-09-01

    A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

  16. Gene dysregulation is restored in the Parkinson’s disease MPTP neurotoxic mice model upon treatment of the therapeutic drug CuII(atsm)

    PubMed Central

    Cheng, Lesley; Quek, Camelia Y. J.; Hung, Lin W.; Sharples, Robyn A.; Sherratt, Nicki A.; Barnham, Kevin J.; Hill, Andrew F.

    2016-01-01

    The administration of MPTP selectively targets the dopaminergic system resulting in Parkinsonism-like symptoms and is commonly used as a mice model of Parkinson’s disease. We previously demonstrated that the neuroprotective compound CuII(atsm) rescues nigral cell loss and improves dopamine metabolism in the MPTP model. The mechanism of action of CuII(atsm) needs to be further defined to understand how the compound promotes neuronal survival. Whole genome transcriptomic profiling has become a popular method to examine the relationship between gene expression and function. Substantia nigra samples from MPTP-lesioned mice were evaluated using whole transcriptome sequencing to investigate the genes altered upon CuII(atsm) treatment. We identified 143 genes affected by MPTP lesioning that are associated with biological processes related to brain and cognitive development, dopamine synthesis and perturbed synaptic neurotransmission. Upon CuII(atsm) treatment, the expression of 40 genes involved in promoting dopamine synthesis, calcium signaling and synaptic plasticity were restored which were validated by qRT-PCR. The study provides the first detailed whole transcriptomic analysis of pathways involved in MPTP-induced Parkinsonism. In addition, we identify key therapeutic pathways targeted by a potentially new class of neuroprotective agents which may provide therapeutic benefits for other neurodegenerative disorders. PMID:26928495

  17. Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents

    PubMed Central

    Selvin, Joseph; Sathiyanarayanan, Ganesan; Lipton, Anuj N.; Al-Dhabi, Naif Abdullah; Valan Arasu, Mariadhas; Kiran, George S.

    2016-01-01

    The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products. PMID:26903957

  18. Extending the rapeseed gene pool with resynthesized Brassica napus II: Heterosis.

    PubMed

    Girke, Andreas; Schierholt, Antje; Becker, Heiko C

    2012-04-01

    Hybrid breeding relies on the combination of parents from two differing heterotic groups. However, the genetic diversity in adapted oilseed rape breeding material is rather limited. Therefore, the use of resynthesized Brassica napus as a distant gene pool was investigated. Hybrids were derived from crosses between 44 resynthesized lines with a diverse genetic background and two male sterile winter oilseed rape tester lines. The hybrids were evaluated together with their parents and check cultivars in 2 years and five locations in Germany. Yield, plant height, seed oil, and protein content were monitored, and genetic distances were estimated with molecular markers (127 polymorphic RFLP fragments). Resynthesized lines varied in yield between 40.9 dt/ha and 21.5 dt/ha, or between 85.1 and 44.6% of check cultivar yields. Relative to check cultivars, hybrids varied from 91.6 to 116.6% in yield and from 94.5 to 103.3% in seed oil content. Mid-parent heterosis varied from -3.5 to 47.2% for yield. The genetic distance of parental lines was not significantly correlated with heterosis or hybrid yield. Although resynthesized lines do not meet the elite rapeseed standards, they are a valuable source for hybrid breeding due to their large distance from present breeding material and their high heterosis when combined with European winter oilseed rape.

  19. Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

    SciTech Connect

    Gonzalez-Gay, M.A.; Zanelli, E.; Krco, C.J.

    1995-05-01

    Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E{beta}{sup d} molecule prevents CIA development in susceptible H2{sup q} mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F{sub 1} mice, only H2Eb{sup d} and H2Eb{sup s} molecules showed protection. Using recombinant B10.RDD (Eb{sup d/b}) mice, we found that CIA protection was mediated by the first domain of the E{beta}{sup d} molecule. Using peptides covering the third hypervariable region of the E{beta} chain, we found a perfect correlation between presentation of E{beta} peptides by the H2A{sup q} molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E{beta} peptides for the H2A{sup q} molecule. 35 refs., 2 figs., 3 tabs.

  20. HLA class II genes in Latvian patients with juvenile rheumatoid arthritis.

    PubMed

    Rumba, I; Denisova, A; Sochnev, A; Nilsson, B; Sanjeevi, C B

    1997-01-01

    PCR-based HLA genotyping was used to analyze the association of HLA-DR and -DQ genes in 127 juvenile rheumatoid arthritis patients and 111 population-based controls from Latvia. The results show DQA1*03 to be positively associated in overall patients and DRB1*01-DQA1*0101-DQB1*0501 to be negatively associated with JRA in overall patients and in polyarthritis patients compared to controls. These data indicate the immunogenetic heterogeneity in the JRA patients, in the disease subgroups and in different ethnic groups. Rheumatoid factor (RF) was assayed in patients (n = 119) and controls (n = 98). RF was present in patients (7/119, 6%) compared to controls (5/98, 5%). None of the DQA1, DQB1 alleles, DQ and DR-DQ haplotypes was associated in seropositive patients compared to seropositive controls. DR1-DQ5 (DQA1*0101-B*0501) was decreased in seronegative patients (11/111, 10%) compared to seronegative controls (24/105, 23%), but the difference was not significant after correction of the p value.

  1. Influence of Intron II microsatellite polymorphism in human toll-like receptor 2 gene in leprosy.

    PubMed

    Suryadevara, Naveen Chandra; Neela, Venkata Sanjeev Kumar; Devalraju, Kamakshi Prudhula; Jain, Suman; SivaSai, Krovvidi S R; Valluri, Vijaya Lakshmi; Jonnalagada, Subbanna; Anandaraj, M P J S

    2013-08-01

    Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy. PMID:23619473

  2. Genomics and polymorphism of Agph-DAB1, an Mhc class II B gene in red-winged blackbirds (Agelaius phoeniceus).

    PubMed

    Edwards, S V; Gasper, J; March, M

    1998-03-01

    To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II B genes, except that the introns are surprisingly large, ranging from 98 to over 600 bp, making this the longest avian class II B gene to date. Using primers targeted toward the introns flanking the peptide-binding region (PBR), we amplified the entirety of the second exon and determined nucleotide sequences of 41 PCR products from eight individual blackbirds. The 10 sequence types found, among which were two probable pseudogene sequences, exhibit the classic hallmarks for evolution of PBRs, namely, an excess of nonsynonymous over synonymous substitutions and evidence of gene conversion events in polymorphic subdomains. Despite these patterns and our use of intron primers, the distribution of sequences among individuals suggests that more than one locus was amplified in most individuals, and the bushlike tree of sequences provides little information as to locus-specific clusters. These results imply a complex history of gene conversion, recent duplication, or possibly, concerted evolution among multiple loci, although Agph-DAB1, the first genomic Mhc sequence from a bird other than chicken, provides important clues in the quest for locus-specific Mhc primers in birds.

  3. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    PubMed

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  4. Prevalence of a plasmid-mediated type II dihydrofolate reductase gene among trimethoprim-resistant urinary pathogens in Greek hospitals.

    PubMed

    Tsakris, A; Vatopoulos, A C; Johnson, A P; Pitt, T L; Legakis, N J; Tzouvelekis, L S

    1992-04-01

    The genetic basis of trimethoprim resistance was examined in 24 Klebsiella pneumoniae, 27 Enterobacter cloacae, five Enterobacter aerogenes and nine Serratia marcescens urinary isolates from five hospitals in Greece. Analysis of the 65 isolates by serotyping and phage-typing identified 53 distinct strains. Thirty-eight isolates (15 K. pneumoniae, 19 E. cloacae, two E. aerogenes and two S. marcescens) hybridized with a probe specific for a gene encoding type II dihydrofolate reductase (DHFR). Three of the K. pneumoniae and four of the E. cloacae isolates which reacted with this probe also hybridized with probes specific for type I DHFR and transposon Tn7. Two E. cloacae isolates hybridized only with the probe for type I DHFR, while a further three isolates hybridized only with the type I DHFR and Tn7 probes. None of the isolates hybridized with a probe for type V DHFR. The plasmids in transconjugants derived from 40 isolates were analysed by digestion with restriction enzymes and Southern blotting. Eighteen (45%) of the donors (12 K. pneumoniae and 6 E. cloacae) produced transconjugants containing plasmids of about 95 kb in size, while transconjugants from the other donors had plasmids in the range 100-185 kb. Of the 18 transconjugants containing a 95 kb plasmid, 15 had similar restriction endonuclease digest patterns, although they varied in terms of the range of antimicrobial resistances which they encoded. When EcoRI digests of these 15 plasmids were hybridized with the type II DHFR probe, a 23 kb common band reacted with the probe.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Schisandra Chinensis Baillon regulates the gene expression of phase II antioxidant/detoxifying enzymes in hepatic damage induced rats

    PubMed Central

    Jang, Han I; Do, Gyeong-Min; Lee, Hye Min; Ok, Hyang Mok; Shin, Jae-Ho

    2014-01-01

    BACKGROUND/OBJECTIVES This study investigated the antioxidant activities and hepatoprotective effects of Schisandra chinensis Baillon extract (SCE) against tert-butyl hydroperoxide (t-BHP)-induced oxidative hepatic damage in rats. MATERIALS/METHODS Sprague-Dawley (SD) rats were pretreated with SCE (300, 600, and 1,200 mg/kg BW) or saline once daily for 14 consecutive days. On day 14, each animal, except those belonging to the normal control group, were injected with t-BHP (0.8 mmol/kg BW/i.p.), and all of the rats were sacrificed 16 h after t-BHP injection. RESULTS Although no significant differences in AST and ALT levels were observed among the TC and SCE groups, the high-dose SCE group showed a decreasing tendency compared to the TC group. However, erythrocyte SOD activity showed a significant increase in the low-dose SCE group compared with the TC group. On the other hand, no significant differences in hepatic total glutathione (GSH) level, glutathione reductase (GR), and glutathione peroxidase (GSH-Px) activities were observed among the TC and SCE groups. Hepatic histopathological evaluation revealed that pretreatment with SCE resulted in reduced t-BHP-induced incidence of lesions, such as neutrophil infiltration, swelling of liver cells, and necrosis. In particular, treatment with a high dose of SCE resulted in induction of phase II antioxidant/detoxifying enzyme expression, such as glutathione S-transferase (GST) and glutamate-cysteine ligase catalytic subunit (GCLC). CONCLUSIONS Based on these results, we conclude that SCE exerts protective effects against t-BHP induced oxidative hepatic damage through the reduction of neutrophil infiltration, swelling of liver cells, and necrosis. In addition, SCE regulates the gene expression of phase II antioxidant/detoxifying enzymes independent of hepatic antioxidant enzyme activity. PMID:24944771

  6. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    PubMed

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  7. A dendritic nano-sized hexanuclear ruthenium(II) complex as a one- and two-photon luminescent tracking non-viral gene vector

    PubMed Central

    Qiu, Kangqiang; Yu, Bole; Huang, Huaiyi; Zhang, Pingyu; Huang, Juanjuan; Zou, Shanshan; Chen, Yu; Ji, Liangnian; Chao, Hui

    2015-01-01

    Fluorescent tracking gene delivery could provide us with a better understanding of the critical steps in the transfection process. However, for in vivo tracking applications, a small diameter (<10 nm) is one of the rigorous requirements for tracking vectors. Herein, we have demonstrated a new paradigm for two-photon tracking gene delivery based on a dendritic nano-sized hexanuclear ruthenium(II) polypyridyl complex. Because this metallodendrimer has a multivalent periphery, the complex, which is 6.1 nm, showed high stability and excellent dispersibility and could stepwise condense DNA in vitro. With the outstanding photochemical properties of Ru(II) polypyridyl, this complex could track gene delivery in vivo using one- and two-photon imaging. PMID:26185052

  8. Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

    PubMed

    Tollenaere, C; Bryja, J; Galan, M; Cadet, P; Deter, J; Chaval, Y; Berthier, K; Ribas Salvador, A; Voutilainen, L; Laakkonen, J; Henttonen, H; Cosson, J-F; Charbonnel, N

    2008-09-01

    We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system. PMID:18624885

  9. Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias.

    PubMed

    Hästbacka, J; Superti-Furga, A; Wilcox, W R; Rimoin, D L; Cohn, D H; Lander, E S

    1996-02-01

    Atelosteogenesis type II (AO II) is a neonatally lethal chondrodysplasia whose clinical and histological characteristics resemble those of another chondrodysplasia, the much less severe diastrophic dysplasia (DTD). The similarity suggests a shared pathogenesis involving lesions in the same biochemical pathway and perhaps the same gene. DTD is caused by mutations in the recently identified diastrophic dysplasia sulfate-transporter gene (DTDST). Here, we report that AOII patients also have DTDST mutations, which lead to defective uptake of inorganic sulfate and insufficient sulfation of macromolecules by patient mesenchymal cells in vitro. Together with our recent observation that a third even more severe chondrodysplasia, achondrogenesis type IB, is also caused by mutations in DTDST, these results demonstrate a phenotypic series of three chondrodysplasias of increasing severity caused by lesions in a single sulfate-transporter gene. The severity of the phenotype appears to be correlated with the predicted effect of the mutations on the residual activity of the DTDST protein. PMID:8571951

  10. Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

    PubMed

    Tollenaere, C; Bryja, J; Galan, M; Cadet, P; Deter, J; Chaval, Y; Berthier, K; Ribas Salvador, A; Voutilainen, L; Laakkonen, J; Henttonen, H; Cosson, J-F; Charbonnel, N

    2008-09-01

    We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

  11. MHC II-β chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

    PubMed

    Picchietti, S; Abelli, L; Guerra, L; Randelli, E; Proietti Serafini, F; Belardinelli, M C; Buonocore, F; Bernini, C; Fausto, A M; Scapigliati, G

    2015-02-01

    MHC II-β chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-β expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-β expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-β, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes.

  12. Pharmacokinetics and pharmacodynamics of phase II drug metabolizing/antioxidant enzymes gene response by anticancer agent sulforaphane in rat lymphocytes.

    PubMed

    Wang, Hu; Khor, Tin Oo; Yang, Qian; Huang, Ying; Wu, Tien-Yuan; Saw, Constance Lay-Lay; Lin, Wen; Androulakis, Ioannis P; Kong, Ah-Ng Tony

    2012-10-01

    This study assesses the pharmacokinetics (PK) and pharmacodynamics (PD) of Nrf2-mediated increased expression of phase II drug metabolizing enzymes (DME) and antioxidant enzymes which represents an important component of cancer chemoprevention in rat lymphocytes following intravenous (iv) administration of an anticancer phytochemical sulforaphane (SFN). SFN was administered intravenously to four groups of male Sprague-Dawley JVC rats each group comprising four animals. Blood samples were drawn at selected time points. Plasma were obtained from half of each of the blood samples and analyzed using a validated LC-MS/MS method. Lymphocytes were collected from the remaining blood samples using Ficoll-Paque Plus centrifuge medium. Lymphocyte RNAs were extracted and converted to cDNA, quantitative real-time PCR analyses were performed, and fold changes were calculated against those at time zero for the relative expression of Nrf2-target genes of phase II DME/antioxidant enzymes. PK-PD modeling was conducted based on Jusko's indirect response model (IDR) using GastroPlus and bootstrap method. SFN plasma concentration declined biexponentially and the pharmacokinetic parameters were generated. Rat lymphocyte mRNA expression levels showed no change for GSTM1, SOD, NF-κB, UGT1A1, or UGT1A6. Moderate increases (2-5-fold) over the time zero were seen for HO-1, Nrf2, and NQO1, and significant increases (>5-fold) for GSTT1, GPx1, and Maf. PK-PD analyses using GastroPlus and the bootstrap method provided reasonable fitting for the PK and PD profiles and parameter estimates. Our present study shows that SFN could induce Nrf2-mediated phase II DME/antioxidant mRNA expression for NQO1, GSTT1, Nrf2, GPx, Maf, and HO-1 in rat lymphocytes after iv administration, suggesting that Nrf2-mediated mRNA expression in lymphocytes may serve as surrogate biomarkers. The PK-PD IDR model simultaneously linking the plasma concentrations of SFN and the PD response of lymphocyte mRNA expression is

  13. The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochrondrial maxi-circle DNA.

    PubMed Central

    Hensgens, L A; Brakenhoff, J; De Vries, B F; Sloof, P; Tromp, M C; Van Boom, J H; Benne, R

    1984-01-01

    A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes. Images PMID:6093040

  14. Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors

    SciTech Connect

    Gicquel, C.; Schneid, H.; Le Bouc, Y.; Bertagna, X.; Francillard-Leblond, M.; Luton, J.P.; Girard, F.

    1994-06-01

    Little is known about the pathophysiology of sporadic adrenocortical tumors in adults. Because loss of heterozygosity at the 11p15 locus has been described in childhood tumors, particularly in adrenocortical tumors associated with the Beckwith-Wiedemann syndrome, and because insulin-like growth factor-II (IGF-II) is a crucial regulator of fetal adrenal growth, the authors looked for structural analysis at the 11p15 locus and IGF-II gene expression in 23 sporadic adrenocortical adult tumors: 6 carcinomas (5 with Cushing`s syndrome and 1 nonsecreting) and 17 benign adenomas (13 with Cushing`s syndrome, 1 pure androgen secreting, and 3 nonsecreting). Twenty-one patients were informative at the 11p15 locus, and six (four carcinomas and two adenomas) of them (28.5%) exhibited 11p15 structural abnormalities in tumor DNA (five, a uniparental disomy and one, a mosaicism). In a single case that could be further studied, a paternal isodisomy was observed. Very high IGF-II mRNA contents were detected in seven tumors (30%; 5 of the 6 carcinomas and 2 of the 17 adenomas). They were particularly found in tumors with uniparental disomy at the 11p15 locus. Overall, a strong correlation existed between IGF-II mRNA contents and DNA demethylation at the IGF-II locus. These data show that genetic alterations involving the 11p15 locus were highly frequent in malignant tumors, but found only in rare adenomas. These results in combination with evidence for overexpression of IGF-II from the 11p15.5 locus suggest that abnormalities in structure and/or expression of the IGF-II gene play a role as a late event of a multistep process of tumorigenesis. 58 refs., 6 figs., 4 tabs.

  15. Angiotensin II modulates interleukin-1{beta}-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-{kappa}B crosstalk

    SciTech Connect

    Xu, Shanqin; Zhi, Hui; Hou, Xiuyun; Jiang, Bingbing

    2011-07-08

    Highlights: {yields} We examine how angiotensin II modulates ERK-NF-{kappa}B crosstalk and gene expression. {yields} Angiotensin II suppresses IL-1{beta}-induced prolonged ERK and NF-{kappa}B activation. {yields} ERK-RSK1 signaling is required for IL-1{beta}-induced prolonged NF-{kappa}B activation. {yields} Angiotensin II modulates NF-{kappa}B responsive genes via regulating ERK-NF-{kappa}B crosstalk. {yields} ERK-NF-{kappa}B crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1{beta}-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-{kappa}B, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1{beta}-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1{beta}, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE{sup -/-}) mice. VCAM-1 and iNOS expression were higher in ApoE{sup -/-} than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE{sup -/-} mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin

  16. A susceptibility gene for kidney disease in an obese mouse model of type II diabetes maps to chromosome 8

    PubMed Central

    Chua, Streamson; Li, Yifu; Liu, Shun Mei; Liu, Ruijie; Chan, Ka Tak; Martino, Jeremiah; Zheng, Zongyu; Susztak, Katalin; D'Agati, Vivette D; Gharavi, Ali G.

    2014-01-01

    Most mouse models of diabetes do not fully reproduce features of human diabetic nephropathy, limiting their utility in inferring mechanisms of human disease. Here we performed detailed phenotypic and genetic characterization of leptin-receptor (Lepr) deficient mice on the FVB/NJ background (FVBdb/db), an obese model of type II diabetes, to determine their suitability to model human diabetic nephropathy. These mice have sustained hyperglycemia, significant albuminuria and characteristic diabetic renal findings including mesangial sclerosis and nodular glomerulosclerosis after 6 months of age. In contrast, equally obese, hyperglycemic Lepr/Sur1 deficient C57BL/6J (Sur1 has defective insulin secretion) mice have minimal evidence of nephropathy. A genome-wide scan in 165 Lepr deficient backcross progeny derived from FVB/NJ and C57BL/6J identified a major locus influencing nephropathy and albuminuria on chromosome 8B1-C5 (Dbnph1 locus, peak lod score 5.0). This locus was distinct from those contrasting susceptibility to beta cell hypertrophy and HIV-nephropathy between the same parental strains, indicating specificity to diabetic kidney disease. Genome-wide expression profiling showed that high and low risk Dbnph1 genotypes were associated with significant enrichment for oxidative phosphorylation and lipid clearance, respectively; molecular pathways shared with human diabetic nephropathy. Hence, we found that the FVBdb/db mouse recapitulates many clinical, histopathological and molecular features of human diabetic nephropathy. Identifying underlying susceptibility gene(s) and downstream dysregulated pathways in these mice may provide insight into the disease pathogenesis in humans. PMID:20520596

  17. Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.

    PubMed

    Rimoldi, Valeria; Straniero, Letizia; Asselta, Rosanna; Mauri, Lucia; Manfredini, Emanuela; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Soldà, Giulia; Primignani, Paola

    2014-03-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants. PMID:24361966

  18. The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats

    SciTech Connect

    Katayama, Seiichi . E-mail: katayama@ankaken.co.jp; Ashizawa, Koji; Gohma, Hiroshi; Fukuhara, Tadahiro; Narumi, Kazunori; Tsuzuki, Yasuhiro; Tatemoto, Hideki; Nakada, Tadashi; Nagai, Kenji

    2006-12-15

    The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17{alpha}-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ER{alpha}-selective agonist), diarylpropionitrile (DPN, an ER{beta}-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE in the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ER{alpha}-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ER{beta}-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

  19. Swine leukocyte antigen class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) polymorphism and genotyping in Guizhou minipigs.

    PubMed

    Liu, Z Z; Xia, J H; Xin, L L; Wang, Z G; Qian, L; Wu, S G; Yang, S L; Li, K

    2015-11-30

    The swine leukocyte antigen (SLA) complex harbors highly polymorphic gene clusters encoding glycoproteins that are involved in responses to vaccines, infectious disease, and production performance. Pigs with well-defined SLA class II genes are useful for the study of disease, immunology, and vaccines. In this study, we analyzed four SLA class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) in 22 founder Guizhou minipigs using a sequence-based typing method. Twelve alleles were detected, compared with the SLA class II allele sequences in the GenBank, and one of twelve alleles was found to be novel in Guizhou minipigs. There are four SLA II haplotypes, and one of them has been previously reported in Meishan pigs. Furthermore, based on sequence information of these alleles, we developed a simple SLA typing method implemented to SLA-typing for unknown offspring of Guizhou minipigs, relying on designed twelve sequence specific primers that could discriminate between each other. According to the combination of sequence-based typing and PCR-SSP, we were able to rapidly check SLA typing of Guizhou breeding stock and identified four SLA haplotypes in the herd. Therefore, SLA-defined Guizhou minipigs will be useful as animal models for xenotransplantation and immunological research.

  20. Sequence Analysis of Inducible Prophage phIS3501 Integrated into the Haemolysin II Gene of Bacillus thuringiensis var israelensis ATCC35646

    PubMed Central

    Moumen, Bouziane; Nguen-The, Christophe; Sorokin, Alexei

    2012-01-01

    Diarrheic food poisoning by bacteria of the Bacillus cereus group is mostly due to several toxins encoded in the genomes. One of them, cytotoxin K, was recently identified as responsible for severe necrotic syndromes. Cytotoxin K is similar to a class of proteins encoded by genes usually annotated as haemolysin II (hlyII) in the majority of genomes of the B. cereus group. The partially sequenced genome of Bacillus thuringiensis var israelensis ATCC35646 contains several potentially induced prophages, one of them integrated into the hlyII gene. We determined the complete sequence and established the genomic organization of this prophage-designated phIS3501. During induction of excision of this prophage with mitomycin C, intact hlyII gene is formed, thus providing to cells a genetic ability to synthesize the active toxin. Therefore, this prophage, upon its excision, can be implicated in the regulation of synthesis of the active toxin and thus in the virulence of bacterial host. A generality of selection for such systems in bacterial pathogens is indicated by the similarity of this genetic arrangement to that of Staphylococcus aureus  β-haemolysin. PMID:22567391

  1. Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur

    SciTech Connect

    Klebig, M.L.; Woychik, R.P.; Wilkinson, J.E.; Geisler, J.G. |

    1995-05-23

    Mice that carry the lethal yellow (A{sup y}) or viable yellow (A{sup vy}) mutation, two dominant mutations of the agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant {open_quotes}obese yellow{close_quotes} a-locus mutations suggested that ectopic expression of the normal agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an agouti cDNA clone encoding the normal agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants. 42 refs., 5 figs.

  2. TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum ssp. pallidum.

    PubMed

    Giacani, Lorenzo; Godornes, Charmie; Puray-Chavez, Maritza; Guerra-Giraldez, Cristina; Tompa, Martin; Lukehart, Sheila A; Centurion-Lara, Arturo

    2009-06-01

    Transcriptional regulation in Treponema pallidum ssp. pallidum is poorly understood, primarily because this organism cannot be cultivated in vitro or genetically manipulated. We have recently shown a phase variation mechanism controlling transcription initiation of Subfamily II tpr (T. pallidumrepeat) genes (tprE, tprG and tprJ), a group of virulence factor candidates. Furthermore, the same study suggested that additional mechanisms might influence the level of transcription of these tprs. The T. pallidum genome sequence has revealed a few open reading frames with similarity to known bacterial transcription factors, including four catabolite activator protein homologues. In this work, sequences matching the Escherichia coli cAMP receptor protein (CRP) binding motif were identified in silico upstream of tprE, tprG and tprJ. Using elecrophoretic mobility shift assay and DNaseI footprinting assay, recombinant TP0262, a T. pallidum CRP homologue, was shown to bind specifically to amplicons obtained from the tpr promoters containing putative CRP binding motifs. Using a heterologous reporter system, binding of TP0262 to these promoters was shown to either increase (tprE and tprJ) or decrease (tprG) tpr promoter activity. This is the first characterization of a T. pallidum transcriptional modulator that influences tpr promoter activity.

  3. A novel type II collagen gene mutation in a family with spondyloepiphyseal dysplasia and extensive intrafamilial phenotypic diversity.

    PubMed

    Nakashima, Yasuharu; Sakamoto, Yuma; Nishimura, Gen; Ikegawa, Shiro; Iwamoto, Yukihide

    2016-01-01

    The purpose of this study was to describe a family with spondyloepiphyseal dysplasia caused by a novel type II collagen gene (COL2A1) mutation and the family's phenotypic diversity. Clinical and radiographic examinations of skeletal dysplasia were conducted on seven affected family members across two generations. The entire coding region of COL2A1, including the flanking intron regions, was analyzed with PCR and direct sequencing. The stature of the subjects ranged from extremely short to within normal height range. Hip deformity and advanced osteoarthritis were noted in all the subjects, ranging from severe coxa plana to mild acetabular dysplasia. Atlantoaxial subluxation combined with a hypoplastic odontoid process was found in three of the subjects. Various degrees of platyspondyly were confirmed in all subjects. Genetically, a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala) was identified in all the affected family members; however, it was not present in the one unaffected family member tested. We described a family with spondyloepiphyseal dysplasia and a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala). Phenotypes were diverse even among individuals with the same mutation and within the same family. PMID:27274858

  4. A novel type II collagen gene mutation in a family with spondyloepiphyseal dysplasia and extensive intrafamilial phenotypic diversity

    PubMed Central

    Nakashima, Yasuharu; Sakamoto, Yuma; Nishimura, Gen; Ikegawa, Shiro; Iwamoto, Yukihide

    2016-01-01

    The purpose of this study was to describe a family with spondyloepiphyseal dysplasia caused by a novel type II collagen gene (COL2A1) mutation and the family’s phenotypic diversity. Clinical and radiographic examinations of skeletal dysplasia were conducted on seven affected family members across two generations. The entire coding region of COL2A1, including the flanking intron regions, was analyzed with PCR and direct sequencing. The stature of the subjects ranged from extremely short to within normal height range. Hip deformity and advanced osteoarthritis were noted in all the subjects, ranging from severe coxa plana to mild acetabular dysplasia. Atlantoaxial subluxation combined with a hypoplastic odontoid process was found in three of the subjects. Various degrees of platyspondyly were confirmed in all subjects. Genetically, a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala) was identified in all the affected family members; however, it was not present in the one unaffected family member tested. We described a family with spondyloepiphyseal dysplasia and a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala). Phenotypes were diverse even among individuals with the same mutation and within the same family. PMID:27274858

  5. TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription

    PubMed Central

    Montes, Marta; Cloutier, Alexandre; Sánchez-Hernández, Noemí; Michelle, Laetitia; Lemieux, Bruno; Blanchette, Marco; Hernández-Munain, Cristina; Chabot, Benoit

    2012-01-01

    Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-xS 5′ splice site. PMID:22158966

  6. Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas reinhardtii.

    PubMed

    Gimpel, Javier A; Nour-Eldin, Hussam H; Scranton, Melissa A; Li, Daphne; Mayfield, Stephen P

    2016-07-15

    Oxygenic photosynthesis provides the energy to produce all food and most of the fuel on this planet. Photosystem II (PSII) is an essential and rate-limiting component of this process. Understanding and modifying PSII function could provide an opportunity for optimizing photosynthetic biomass production, particularly under specific environmental conditions. PSII is a complex multisubunit enzyme with strong interdependence among its components. In this work, we have deleted the six core genes of PSII in the eukaryotic alga Chlamydomonas reinhardtii and refactored them in a single DNA construct. Complementation of the knockout strain with the core PSII synthetic module from three different green algae resulted in reconstitution of photosynthetic activity to 85, 55, and 53% of that of the wild-type, demonstrating that the PSII core can be exchanged between algae species and retain function. The strains, synthetic cassettes, and refactoring strategy developed for this study demonstrate the potential of synthetic biology approaches for tailoring oxygenic photosynthesis and provide a powerful tool for unraveling PSII structure-function relationships.

  7. CNS-directed gene therapy for the treatment of neurologic and somatic mucopolysaccharidosis type II (Hunter syndrome)

    PubMed Central

    Motas, Sandra; Haurigot, Virginia; Garcia, Miguel; Marcó, Sara; Ribera, Albert; Roca, Carles; Sánchez, Víctor; Molas, Maria; Bertolin, Joan; Maggioni, Luca; León, Xavier; Ruberte, Jesús; Bosch, Fatima

    2016-01-01

    Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9-Ids) to the cerebrospinal fluid of MPSII mice with already established disease. Treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of brain transcriptomic signature, and disappearance of neuroinflammation. Moreover, our vector also transduced the liver, providing a peripheral source of therapeutic protein that corrected storage pathology in visceral organs, with evidence of cross-correction of nontransduced organs by circulating enzyme. Importantly, AAV9-Ids-treated MPSII mice showed normalization of behavioral deficits and considerably prolonged survival. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter syndrome patients with cognitive impairment. PMID:27699273

  8. CNS-directed gene therapy for the treatment of neurologic and somatic mucopolysaccharidosis type II (Hunter syndrome)

    PubMed Central

    Motas, Sandra; Haurigot, Virginia; Garcia, Miguel; Marcó, Sara; Ribera, Albert; Roca, Carles; Sánchez, Víctor; Molas, Maria; Bertolin, Joan; Maggioni, Luca; León, Xavier; Ruberte, Jesús; Bosch, Fatima

    2016-01-01

    Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9-Ids) to the cerebrospinal fluid of MPSII mice with already established disease. Treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of brain transcriptomic signature, and disappearance of neuroinflammation. Moreover, our vector also transduced the liver, providing a peripheral source of therapeutic protein that corrected storage pathology in visceral organs, with evidence of cross-correction of nontransduced organs by circulating enzyme. Importantly, AAV9-Ids-treated MPSII mice showed normalization of behavioral deficits and considerably prolonged survival. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter syndrome patients with cognitive impairment.

  9. Domain structures and molecular evolution of class I and class II major histocompatibility gene complex (MHC) products deduced from amino acid and nucleotide sequence homologies

    NASA Astrophysics Data System (ADS)

    Ohnishi, Koji

    1984-12-01

    Domain structures of class I and class II MHC products were analyzed from a viewpoint of amino acid and nucleotide sequence homologies. Alignment statistics revealed that class I (transplantation) antigen H chains consist of four mutually homologous domains, and that class II (HLA-DR) antigen β and α chains are both composed of three mutually homologous ones. The N-terminal three and two domains of class I and class II (both β and α) gene products, respectively, all of which being ˜90 residues long, were concluded to be homologous to β2-microglobulin (β2M). The membraneembedded C-terminal shorter domains of these MHC products were also found to be homologous to one another and to the third domain of class I H chains. Class I H chains were found to be more closely related to class II α chains than to class II β chains. Based on these findings, an exon duplication history from a common ancestral gene encoding a β2M-like primodial protein of one-domain-length up to the contemporary MHC products was proposed.

  10. Expression and purification of SfaX(II), a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli.

    PubMed

    Paracuellos, Patricia; Ohman, Anders; Sauer-Eriksson, A Elisabeth; Uhlin, Bernt Eric

    2012-12-01

    Pathogenic Escherichia coli strains commonly harbor genes involved in formation of fimbriae, such as the sfa(II) fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. The sfaX(II) gene, located at the distal end of the sfa(II) operon, was recently shown to play a role in controlling virulence-related gene expression in extraintestinal pathogenic E. coli (ExPEC). Until now, detailed characterization of the SfaX(II) protein has been hampered by difficulties in obtaining large quantities of soluble protein. By a rational modeling approach, we engineered a Cys70Ser mutation, which successfully improved solubility of the protein. Here, we present the expression, purification, and initial characterization of the recombinant SfaX(IIC70S) mutant. The protein was produced in E. coli BL21 (DE3) cells grown in autoinduction culture media. The plasmid vector harbored DNA encoding the SfaX(IIC70S) protein N-terminally fused with a six histidine (H6) sequence followed by a ZZ tag (a derivative of the Staphylococcus protein A) (H6-ZZ tag). The H6-ZZ tag was cleaved off with Tobacco Etch Virus (TEV) protease and the 166 amino acid full-length homo-dimeric protein was purified using affinity and size-exclusion chromatography. Electrophoretic mobility gel shift assays and atomic force microscopy demonstrated that the protein possesses DNA-binding properties, suggesting that the transcriptional regulatory activity of SfaX(II) can be mediated via direct binding to DNA. PMID:23022032

  11. Towards the simplification of MHC typing protocols: targeting classical MHC class II genes in a passerine, the pied flycatcher Ficedula hypoleuca

    PubMed Central

    2010-01-01

    Background Major Histocompatibility Complex (MHC) has drawn the attention of evolutionary biologists due to its importance in crucial biological processes, such as sexual selection and immune response in jawed vertebrates. However, the characterization of classical MHC genes subjected to the effects of natural selection still remains elusive in many vertebrate groups. Here, we have tested the suitability of flanking intron sequences to guide the selective exploration of classical MHC genes driving the co-evolutionary dynamics between pathogens and their passerine (Aves, Order Passeriformes) hosts. Findings Intronic sequences flanking the usually polymorphic exon 2 were isolated from different species using primers sitting on conserved coding regions of MHC class II genes (β chain). Taking the pied flycatcher Ficedula hypoleuca as an example, we demonstrate that careful primer design can evade non-classical MHC gene and pseudogene amplification. At least four polymorphic and expressed loci were co-replicated using a single pair of primers in five non-related individuals (N = 28 alleles). The cross-amplification and preliminary inspection of similar MHC fragments in eight unrelated songbird taxa suggests that similar approaches can also be applied to other species. Conclusions Intron sequences flanking the usually polymorphic exon 2 may assist the specific investigation of classical MHC class II B genes in species characterized by extensive gene duplication and pseudogenization. Importantly, the evasion of non-classical MHC genes with a more specific function and non-functional pseudogenes may accelerate data collection and diminish lab costs. Comprehensive knowledge of gene structure, polymorphism and expression profiles may be useful not only for the selective examination of evolutionarily relevant genes but also to restrict chimera formation by minimizing the number of co-amplifying loci. PMID:20815923

  12. Myocyte-specific enhancer factor 2C: a novel target gene of miR-214-3p in suppressing angiotensin II-induced cardiomyocyte hypertrophy

    PubMed Central

    Tang, Chun-Mei; Liu, Fang-zhou; Zhu, Jie-Ning; Fu, Yong-Heng; Lin, Qiu-Xiong; Deng, Chun-Yu; Hu, Zhi-Qin; Yang, Hui; Zheng, Xi-Long; Cheng, Jian-Ding; Wu, Shu-Lin; Shan, Zhi-Xin

    2016-01-01

    The role of microRNA-214-3p (miR-214-3p) in cardiac hypertrophy was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced mouse cardiac hypertrophy. In mice with either Ang-II infusion or transverse aortic constriction (TAC) model, miR-214-3p expression was markedly decreased in the hypertrophic myocardium. Down-regulation of miR-214-3p was observed in the myocardium of patients with cardiac hypertrophy. Expression of miR-214-3p was upregulated in Ang-II-induced hypertrophic neonatal mouse ventricular cardiomyocytes. Cardiac hypertrophy was attenuated in Ang-II-infused mice by tail vein injection of miR-214-3p. Moreover, miR-214-3p inhibited the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) in Ang-II-treated mouse cardiomyocytes in vitro. Myocyte-specific enhancer factor 2C (MEF2C), which was increased in Ang-II-induced hypertrophic mouse myocardium and cardiomyocytes, was identified as a target gene of miR-214-3p. Functionally, miR-214-3p mimic, consistent with MEF2C siRNA, inhibited cell size increase and protein expression of ANP and β-MHC in Ang-II-treated mouse cardiomyocytes. The NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in cardiomyocytes. Taken together, our results revealed that MEF2C is a novel target of miR-214-3p, and attenuation of miR-214-3p expression may contribute to MEF2Cexpressionin cardiac hypertrophy. PMID:27796324

  13. Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures.

    PubMed

    Charity, J A; Holland, L; Grace, L J; Walter, C

    2005-02-01

    An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.

  14. The Increasing Importance of Gene-Based Analyses

    PubMed Central

    Cirulli, Elizabeth T.

    2016-01-01

    In recent years, genome and exome sequencing studies have implicated a plethora of new disease genes with rare causal variants. Here, I review 150 exome sequencing studies that claim to have discovered that a disease can be caused by different rare variants in the same gene, and I determine whether their methods followed the current best-practice guidelines in the interpretation of their data. Specifically, I assess whether studies appropriately assess controls for rare variants throughout the entire gene or implicated region as opposed to only investigating the specific rare variants identified in the cases, and I assess whether studies present sufficient co-segregation data for statistically significant linkage. I find that the proportion of studies performing gene-based analyses has increased with time, but that even in 2015 fewer than 40% of the reviewed studies used this method, and only 10% presented statistically significant co-segregation data. Furthermore, I find that the genes reported in these papers are explaining a decreasing proportion of cases as the field moves past most of the low-hanging fruit, with 50% of the genes from studies in 2014 and 2015 having variants in fewer than 5% of cases. As more studies focus on genes explaining relatively few cases, the importance of performing appropriate gene-based analyses is increasing. It is becoming increasingly important for journal editors and reviewers to require stringent gene-based evidence to avoid an avalanche of misleading disease gene discovery papers. PMID:27055023

  15. Interactive roles of NPR1 gene-dosage and salt diets on cardiac angiotensin II, aldosterone and pro-inflammatory cytokines levels inmutantmice

    PubMed Central

    Zhao, Di; Das, Subhankar; Pandey, Kailash N.

    2015-01-01

    Objective The objective of the present study was to elucidate the interactive roles of guanylyl cyclase/natriuretic peptide receptor-A (NPRA) gene (Npr1) and salt diets on cardiac angiotensin II (ANG II), aldosterone and proinflammatory cytokines levels in Npr1 gene-targeted (1-copy, 2-copy, 3-copy, 4-copy) mice. Methods Npr1 genotypes included 1-copy gene-disrupted heterozygous (+/−), 2-copy wild-type (+/+), 3-copy gene-duplicated heterozygous (++/+) and 4-copy gene-duplicated homozygous (++/++) mice. Animals were fed low, normal and high-salt diets. Plasma and cardiac levels of ANG II, aldosterone and pro-inflammatory cytokines were determined. Results With a high-salt diet, cardiac ANG II levels were increased (+) in 1-copy mice (13.7 ± 2.8 fmol/mg protein, 111%) compared with 2-copy mice (6.5 ± 0.6), but decreased (−) in 4-copy (4.0 ± 0.5, 38%) mice. Cardiac aldosterone levels were increased (+) in 1-copy mice (80 ± 4 fmol/mg protein, 79%) compared with 2-copy mice (38 ± 3). Plasma tumour necrosis factor alpha was increased (+) in 1-copy mice (30.27 ± 2.32 pg/ml, 38%), compared with 2-copy mice (19.36 ± 2.49, 24%), but decreased (−) in 3-copy (11.59 ± 1.51, 12%) and 4-copy (7.13 ± 0.52, 22%) mice. Plasma interleukin (IL)-6 and IL-1α levels were also significantly increased (+) in 1-copy compared with 2-copy mice but decreased (−) in 3-copy and 4-copy mice. Conclusion These results demonstrate that a high-salt diet aggravates cardiac ANG II, aldosterone and proinflammatory cytokine levels in Npr1 gene-disrupted 1-copy mice, whereas, in Npr1 gene-duplicated (3-copy and 4-copy) mice, high salt did not render such elevation, suggesting the potential roles of Npr1 against salt loading. PMID:23188418

  16. Mutations in the MGAT2 gene controlling complex N-glycan synthesis cause carbohydrate-deficient glycoprotein syndrome type II, an autosomal recessive disease with defective brain development

    SciTech Connect

    Tan, J.; Schachter, H.; Dunn, J.

    1996-10-01

    Carbohydrate-deficient glycoprotein syndrome (CDGS) type II is a multisystemic congenital disease with severe involvement of the nervous system. Two unrelated CDGS type II patients are shown to have point mutations (one patient having Ser{r_arrow}Phe and the other having His{r_arrow}Arg) in the catalytic domain of the gene MGAT2, encoding UDP-GlcNAc:{alpha}-6-D-mannoside {Beta}-1,2-N-ace-tylglucosaminyltransferase II (GnT II), an enzyme essential for biosynthesis of complex Asn-linked glycans. Both mutations caused both decreased expression of enzyme protein in a baculovirus/insect cell system and inactivation of enzyme activity. Restriction-endonuclease analysis of DNA from 23 blood relatives of one of these patients showed that 13 donors were heterozygotes; the other relatives and 21 unrelated donors were normal homozygotes. All heterozygotes showed a significant reduction (33%-68%) in mononuclear-cell GnT II activity. The data indicate that CDGS type II is an autosomal recessive disease and that complex Asn-linked glycans are essential for normal neurological development. 38 refs., 4 figs., 1 tab.

  17. Structural analysis of the regulatory elements of the type-II procollagen gene. Conservation of promoter and first intron sequences between human and mouse.

    PubMed Central

    Vikkula, M; Metsäranta, M; Syvänen, A C; Ala-Kokko, L; Vuorio, E; Peltonen, L

    1992-01-01

    Transcription of the type-II procollagen gene (COL2A1) is very specifically restricted to a limited number of tissues, particularly cartilages. In order to identify transcription-control motifs we have sequenced the promoter region and the first intron of the human and mouse COL2A1 genes. With the assumption that these motifs should be well conserved during evolution, we have searched for potential elements important for the tissue-specific transcription of the COL2A1 gene by aligning the two sequences with each other and with the available rat type-II procollagen sequence for the promoter. With this approach we could identify specific evolutionarily well-conserved motifs in the promoter area. On the other hand, several suggested regulatory elements in the promoter region did not show evolutionary conservation. In the middle of the first intron we found a cluster of well-conserved transcription-control elements and we conclude that these conserved motifs most probably possess a significant function in the control of the tissue-specific transcription of the COL2A1 gene. We also describe locations of additional, highly conserved nucleotide stretches, which are good candidate regions in the search for binding sites of yet-uncharacterized cartilage-specific transcription regulators of the COL2A1 gene. PMID:1637314

  18. Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice.

    PubMed

    Jennings, Brett L; Moore, Joseph A; Pingili, Ajeeth K; Estes, Anne M; Fang, Xiao R; Kanu, Alie; Gonzalez, Frank J; Malik, Kafait U

    2015-05-01

    Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg(-1)·min(-1)) infused by miniosmotic pumps for 2 wk in female Cyp1b1(+/+) mice did not alter water consumption, urine output, Na(+) excretion, osmolality, or protein excretion. However, in Cyp1b1(-/-) mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na(+) excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-β in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17β-estradiol metabolite 2-methoxyestradiol in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17β-estradiol.

  19. Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice

    PubMed Central

    Jennings, Brett L.; Moore, Joseph A.; Pingili, Ajeeth K.; Estes, Anne M.; Fang, Xiao R.; Kanu, Alie; Gonzalez, Frank J.

    2015-01-01

    Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg−1·min−1) infused by miniosmotic pumps for 2 wk in female Cyp1b1+/+ mice did not alter water consumption, urine output, Na+ excretion, osmolality, or protein excretion. However, in Cyp1b1−/− mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na+ excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-β in Cyp1b1−/− but not Cyp1b1+/+ mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17β-estradiol metabolite 2-methoxyestradiol in Cyp1b1−/− but not Cyp1b1+/+ mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17β-estradiol. PMID:25694484

  20. A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells

    SciTech Connect

    Singer-Sam, J.; Dai, A.; Riggs, A.D. ); Goldstein, L.; Gartler, S.M. )

    1992-02-15

    Hpa II site H8 is in the CpG-rich 5{prime} untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG island' whose methylation pattern is perfectly correlated with transcriptional silence of this gene. The authors measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. They conclude that site H8 is the unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

  1. The human tyrosine aminotransferase gene: characterization of restriction fragment length polymorphisms and haplotype analysis in a family with tyrosinemia type II.

    PubMed

    Westphal, E M; Natt, E; Grimm, T; Odievre, M; Scherer, G

    1988-07-01

    Deficiency in hepatic tyrosine aminotransferase (TAT) causes tyrosinemia type II, an autosomal recessively inherited disorder. Using a TAT cosmid clone, we have identified an MspI restriction fragment length polymorphism (RFLP) 5' to the TAT gene, with allele frequencies of 0.63 and 0.37. Analysis of the cloned maternal and paternal TAT alleles from a patient with tyrosinemia type II led to the identification of a HaeIII RFLP at the 3' end of the TAT gene, with allele frequencies of 0.94 and 0.06. The two RFLPs are 27 kb apart and in no allelic association. From haplotype frequencies, a polymorphism information content (PIC) value of 0.44 was obtained. The two RFLPs have allowed the unambiguous identification of the mutant TAT alleles in the patient's pedigree by haplotype analysis.

  2. Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection ▿ †

    PubMed Central

    Fox, Barbara A.; Falla, Alejandra; Rommereim, Leah M.; Tomita, Tadakimi; Gigley, Jason P.; Mercier, Corinne; Cesbron-Delauw, Marie-France; Weiss, Louis M.; Bzik, David J.

    2011-01-01

    Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission. PMID:21531875

  3. Sequence polymorphism of two major histocompatibility (MH) class II B genes and their association with Vibrio anguillarum infection in half-smooth tongue sole ( Cynoglossus semilaevis)

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Zhang, Quanqi; Yu, Yan; Li, Shuo; Zhong, Qiwang; Sun, Yeying; Wang, Zhigang; Qi, Jie; Zhai, Jieming; Wang, Xubo

    2011-11-01

    Major histocompatibility complex (MHC) class II B molecules play an important role in the adaptive immune response in fish. Previous study has reported that two highly polymorphic class II B genes, Cyse-DAB and Cyse-DBB exist in half-smooth tongue sole ( Cynoglossus semilaevis). In this study, the polymorphism within exon 2 of the class II B genes following bacterial challenge was evaluated. Two hundred C. semilaevis individuals were injected intraperitoneally with Vibrio anguillarum. Muscle tissue from the first 20 dead and 20 of the survivors was collected for genotyping. Sixty alleles from the 40 individuals were isolated, of which 32 belonged to Cyse-DAB and 28 belonged to Cyse-DBB. The rate of d N (non-synonymous substitution) was higher than that of d S (synonymous substitution) in the PBRs (peptide binding residues) of both class II B genes. Conversely, the rate of d S was higher than d N in the non-PBRs and the complete exon 2 sequence. Thus, the results suggest that positive selection has occurred in the PBRs and purifying selection in the non-PBRs and exon 2. Thirteen class II B alleles were used to study the association between alleles and resistance to infection. Though not significant, alleles Cyse-DAB*0601, Cyse-DAB*0706, and Cyse-DBB*0101, Cyse-DBB*1301 were only found in surviving individuals and may represent alleles that have resistance against V. anguillarum infection. Alleles Cyse-DAB*0701 and Cyse-DAB*1301 were significantly more prevalent in dead individuals than in surviving ones and may represent alleles that are associated with increased susceptibility to V. anguillarum infection.

  4. Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole ( Cynoglossus semilaevis)

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Wang, Xubo; Zhang, Quanqi; Wang, Zhigang; Qi, Jie; Yi, Qilin; Liu, Zhipeng; Wang, Yanan; Yu, Haiyang

    2012-03-01

    Major histocompatibility complex class II antigens are important in vertebrate immune system. In the present study, the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole ( Cynoglossus semilaevis), and its open reading frame (ORF) polymorphism was studied. The whole cDNA sequence was 992 bp in length, including the ORF with 717 bp. Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/ amino acids in specific positions. Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA. Four Cyse-DAA alleles were observed in one individual, and three to five Cyse-DBA alleles were observed in each of the three detected individuals, which indicated that at least two loci existed in each gene. Moreover, in order to study the function of the alleles in resistance to infection, 200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis. Fifty-six alleles were identified among the 40 individuals. Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA. Eighteen alleles were selected for studying their function in resistance to infection. Alleles Cyse-DAA*0201, Cyse-DAA*1101, Cyse-DBA*0401, Cyse-DBA*1102, Cyse-DBA*1801 and Cyse-DBA*2201 were identified only in surviving individuals, while alleles Cyse- DAA*0901, Cyse-DBA*1101 and Cyse-DBA*1401 occurred more frequently in dead individuals. This study confirmed the existence and polymorphism of two class II A genes as well as the relationship between alleles of class II A genes and disease susceptibility/ resistance in half-smooth tongue sole.

  5. Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices

    PubMed Central

    Corradi, Nicolas; Sanders, Ian R

    2006-01-01

    Background The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. Results In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. Conclusion On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence that structural genomic

  6. Dual Activation of TRIF and MyD88 Adaptor Proteins by Angiotensin II Evokes Opposing Effects on Pressure, Cardiac Hypertrophy and Inflammatory Gene Expression

    PubMed Central

    Singh, Madhu V.; Cicha, Michael Z.; Meyerholz, David K.; Chapleau, Mark W.; Abboud, François M.

    2015-01-01

    Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors (TLRs) are key determinants of the immunological outcome through their pro-inflammatory response. TLR activated signaling pathways utilize several adaptor proteins of which adaptor proteins MyD88 and TRIF define two major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)-induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression (Tnfa, Nox4 and Agtr1a) was significantly greater in the heart and kidney of MyD88-deficient mice compared with wild type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. On the other hand, in mice with non-functional TRIF (Trifmut mice), Ang II induced hypertension and cardiac hypertrophy were abrogated, and pro-inflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a pro-inflammatory innate immune response, causing hypertension, and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88 dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses. PMID:26195481

  7. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    PubMed Central

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E.

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD. PMID:24701363

  8. A Case of Transforming Growth Factor-β-Induced Gene-Related Oculorenal Syndrome: Granular Corneal Dystrophy Type II with a Unique Nephropathy

    PubMed Central

    Iwafuchi, Yoichi; Morioka, Tetsuo; Oyama, Yuko; Nozu, Kandai; Iijima, Kazumoto; Narita, Ichiei

    2016-01-01

    Many types of inherited renal diseases have ocular features that occasionally support a diagnosis. The following study describes an unusual example of a 40-year-old woman with granular corneal dystrophy type II complicated by renal involvement. These two conditions may coincidentally coexist; however, there are some reports that demonstrate an association between renal involvement and granular corneal dystrophy type II. Granular corneal dystrophy type II is caused by a mutation in the transforming growth factor-β-induced (TGFBI) gene. The patient was referred to us because of the presence of mild proteinuria without hematuria that was subsequently suggested to be granular corneal dystrophy type II. A kidney biopsy revealed various glomerular and tubular basement membrane changes and widening of the subendothelial space of the glomerular basement membrane by electron microscopy. However, next-generation sequencing revealed that she had no mutation in a gene that is known to be associated with monogenic kidney diseases. Conversely, real-time polymerase chain reaction, using a simple buccal swab, revealed TGFBI heteromutation (R124H). The TGFBI protein plays an important role in cell-collagen signaling interactions, including extracellular matrix proteins which compose the renal basement membrane. This mutation can present not only as corneal dystrophy but also as renal disease. TGFBI-related oculorenal syndrome may have been unrecognized. It is difficult to diagnose this condition without renal electron microscopic studies. To the best of our knowledge, this is the first detailed report of nephropathy associated with a TGFBI mutation. PMID:27781206

  9. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease.

    PubMed

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E; Ortiz, Genaro Gabriel

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  10. Sequence, internal homology and high-level expression of the gene for a DNA-(cytosine N4)-methyltransferase, M.Pvu II.

    PubMed Central

    Tao, T; Walter, J; Brennan, K J; Cotterman, M M; Blumenthal, R M

    1989-01-01

    The base sequence of the pvuIIM gene has been determined. This gene codes for a DNA-(cytosine N4)-methyltransferase, M.Pvu II. The base sequence contains a single large open reading frame that predicts a 38.3kDa polypeptide, consistent with experimental data. The pvuIIM gene contains some sequences common to DNA methyltransferases in general, but includes none of the sequences specifically conserved among DNA-(cytosine 5)-methyltransferases. The pvuIIM sequence also reveals an internal homology at the amino acid level, each half of which spans over 100 amino acids and is itself homologous to the sequences of some DNA-(adenine N6)-methyltransferases. A derivative of the pvuIIM plasmid was constructed to allow high-level production of M.Pvu II. Specifically, the composite Ptac promoter was inserted 5' to pvuIIM, intervening DNA was deleted, and the resulting construct was used to transform an mcrB laclq strain of Escherichia coli. When this transformant was induced with isopropyl-B-D-galactopyranoside (IPTG), growth rapidly ceased and M.Pvu II accumulated to the point of comprising over 10% of the total soluble protein. Images PMID:2662138

  11. Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (Huso huso) and the disruptive effect of methylmercury on gene expression.

    PubMed

    Gharaei, Ahmad; Mahboudi, Fereidoun; Esmaili-Sari, Abbas; Edalat, Rozita; Adeli, Ahmad; Keyvanshokooh, Saeed

    2010-09-01

    Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg(-1) [control]; 0.76 mg kg(-1) [low]; 7.8 mg kg(-1) [medium]; 16.22 mg kg(-1) [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.

  12. Glu-47, which forms a salt bridge between neurophysin-II and arginine vasopressin, is deleted in patients with familial central diabetes insipidus

    SciTech Connect

    Yuasa, Hiromitsu; Ito, Masafumi; Nagasaki, Hiroshi; Oiso, Yutaka; Saito, Hidehiko ); Miyamoto, S.; Sasaki, N. )

    1993-09-01

    The arginine vasopressin (AVP) gene was sequenced in a pedigree with familial central diabetes insipidus (DI). When polymerase chain reaction-amplified DNAs from affected subjects were subjected to polyacrylamide gel electrophoresis, fragments including exon 2 displayed two additional, slower migrating bands. These extra bands represented DNA heteroduplexes, indicating that there was a deletion or insertion mutation in exon 2. As the region with such a mutation was identified by direct sequence analysis, polymerase chain reaction-amplified fragments including the region were subcloned and sequenced. A 3-basepair deletion (AGG) out of two consecutive AGG sequences (nucleotides 1824-1829) was identified in one of two alleles. The cosegregation of the mutation with the DI phenotype in the family was confirmed by restriction enzyme analyses. This mutation should yield an abnormal AVP precursor lacking Glu[sup 47] in its neurophysin-II (NP) moiety. Since Glu[sup 47] is essential for NP molecules to form a salt bridge with AVP, it is very likely that the function of NP as a carrier protein for AVP would be impaired. The authors suggest that AVP would undergo accelerated proteolytic degradation, and this mechanism would be involved in the pathogenesis of DI in this pedigree. 34 refs., 4 figs., 2 tabs.

  13. Multiple endocrine neoplasia (MEN) II

    MedlinePlus

    Sipple syndrome; MEN II; Pheochromocytoma - MEN II; Thyroid cancer - pheochromocytoma; Parathyroid cancer - pheochromocytoma ... The cause of MEN II is a defect in a gene called RET. This defect causes many tumors to appear in the same ...

  14. Use of meta-analysis to combine candidate gene association studies: application to study the relationship between the ESR PvuII polymorphism and sow litter size.

    PubMed

    Alfonso, Leopoldo

    2005-01-01

    This article investigates the application of meta-analysis on livestock candidate gene effects. The PvuII polymorphism of the ESR gene is used as an example. The association among ESR PvuII alleles with the number of piglets born alive and total born in the first (NBA1, TNB1) and later parities (NBA, TNB) is reviewed by conducting a meta-analysis of 15 published studies including 9329 sows. Under a fixed effects model, litter size values were significantly lower in the "AA" genotype groups when compared with "AB" and "BB" homozygotes. Under the random effects model, the results were similar although differences between "AA" and "AB" genotype groups were not clearly significant for NBA and TNB. Nevertheless, the most noticeable result was the high and significant heterogeneity estimated among studies. This heterogeneity could be assigned to error sampling, genotype by environment interaction, linkage or epistasis, as referred to in the literature, but also to the hypothesis of population admixture/stratification. It is concluded that meta-analysis can be considered as a helpful analytical tool to synthesise and discuss livestock candidate gene effects. The main difficulty found was the insufficient information on the standard errors of the estimated genotype effects in several publications. Consequently, the convenience of publishing the standard errors or the concrete P-values instead of the test significance level should be recommended to guarantee the quality of candidate gene effect meta-analyses.

  15. Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription.

    PubMed

    Ido, Ayaka; Iwata, Shinya; Iwata, Yuka; Igarashi, Hisako; Hamada, Takahiro; Sonobe, Seiji; Sugiura, Masahiro; Yukawa, Yasushi

    2016-02-01

    In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin). PMID:26662274

  16. Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker.

    PubMed

    Chakraborty, M; Sairam Reddy, P; Laxmi Narasu, M; Krishna, Gaurav; Rana, Debashis

    2016-01-01

    Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l(-1)) and Paramomycin (70 mg l(-1)) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe. PMID:27186018

  17. The relation of the XbaI and PvuII polymorphisms of the estrogen receptor gene and the CAG repeat polymorphism of the androgen receptor gene to peak bone mass and bone turnover rate among young healthy men.

    PubMed

    Välimäki, Ville-Valtteri; Piippo, Kirsi; Välimäki, Stiina; Löyttyniemi, Eliisa; Kontula, Kimmo; Välimäki, Matti J

    2005-12-01

    The genes coding for estrogen receptor-alpha (ER-alpha) and androgen receptors (AR) are potential candidates for the regulation of bone mass and turnover, which may contribute to both the achievement of peak bone mass and bone loss after completion of growth. The present study was aimed at elucidating the role of two restriction fragment lengths (XbaI and PvuII) polymorphisms of the ER gene and the CAG repeat polymorphism of the AR gene as determinants of peak bone mass in men; special attention was paid to the interaction between serum free estradiol (E2) levels and the XbaI and PvuII genotypes. A cross-sectional study, with data on lifestyle factors collected retrospectively, was performed in 234 young men, aged 18.3 to 20.6 years. Of the men, 184 were recruits of the Finnish Army and 50 were men of similar age who had postponed their military service for reasons not related to health. Bone mineral content (BMC), density (BMD) and scan area were measured in the lumbar spine and upper femur by dual-energy X-ray absorptiometry (DXA). The bone turnover rate was assessed by measuring serum type I procollagen aminoterminal propeptide (PINP) and tartrate-resistant acid phosphatase 5b (TRACP5b) as well as urinary excretion of type I collagen aminoterminal telopeptide (NTX). After adjusting for age, height, weight, exercise, smoking, calcium and alcohol intake, BMC, scan area and BMD at all measurement sites were similar for the different XbaI and PvuII genotypes of the ER and independent of the number of the CAG repeats of the AR gene. No association was found between free E2 levels and bone parameters among any genotype group of the XbaI and PvuII polymorphisms. Except for urinary NTX, which showed a tendency to higher values for the xx (P=0.08) and pp (P=0.10) genotypes of the ER, bone turnover markers were not related to the genotypes studied. Our study does not support the view that the XbaI and PvuII polymorphisms of the ER gene and the CAG polymorphism of the AR

  18. The human gene (CSNK2A1) coding for the casein kinase II subunit [alpha] is located on chromosome 20 and contains tandemly arranged Alu repeats

    SciTech Connect

    Wirkner, U.; Lichter, P.; Pyerin, W. ); Voss, H.; Ansorge, W. )

    1994-01-15

    The authors have isolated and characterized an 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit [alpha] gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII[alpha]. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII[alpha] gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats. 50 refs., 5 figs., 1 tab.

  19. Biologic Determinants of Tumor Recurrence in Stage II Colon Cancer: Validation Study of the 12-Gene Recurrence Score in Cancer and Leukemia Group B (CALGB) 9581

    PubMed Central

    Venook, Alan P.; Niedzwiecki, Donna; Lopatin, Margarita; Ye, Xing; Lee, Mark; Friedman, Paula N.; Frankel, Wendy; Clark-Langone, Kim; Millward, Carl; Shak, Steven; Goldberg, Richard M.; Mahmoud, Najjia N.; Warren, Robert S.; Schilsky, Richard L.; Bertagnolli, Monica M.

    2013-01-01

    Purpose A greater understanding of the biology of tumor recurrence should improve adjuvant treatment decision making. We conducted a validation study of the 12-gene recurrence score (RS), a quantitative assay integrating stromal response and cell cycle gene expression, in tumor specimens from patients enrolled onto Cancer and Leukemia Group B (CALGB) 9581. Patients and Methods CALGB 9581 randomly assigned 1,713 patients with stage II colon cancer to treatment with edrecolomab or observation and found no survival difference. The analysis reported here included all patients with available tissue and recurrence (n = 162) and a random (approximately 1:3) selection of nonrecurring patients. RS was assessed in 690 formalin-fixed paraffin-embedded tumor samples with quantitative reverse transcriptase polymerase chain reaction by using prespecified genes and a previously validated algorithm. Association of RS and recurrence was analyzed by weighted Cox proportional hazards regression. Results Continuous RS was significantly associated with risk of recurrence (P = .013) as was mismatch repair (MMR) gene deficiency (P = .044). In multivariate analyses, RS was the strongest predictor of recurrence (P = .004), independent of T stage, MMR, number of nodes examined, grade, and lymphovascular invasion. In T3 MMR-intact (MMR-I) patients, prespecified low and high RS groups had average 5-year recurrence risks of 13% (95% CI, 10% to 16%) and 21% (95% CI, 16% to 26%), respectively. Conclusion The 12-gene RS predicts recurrence in stage II colon cancer in CALGB 9581. This is consistent with the importance of stromal response and cell cycle gene expression in colon tumor recurrence. RS appears to be most discerning for patients with T3 MMR-I tumors, although markers such as grade and lymphovascular invasion did not add value in this subset of patients. PMID:23530100

  20. Expression of a higher plant psbA gene in Synechocystis 6803 yields a functional hybrid photosystem II reaction center complex.

    PubMed Central

    Nixon, P J; Rögner, M; Diner, B A

    1991-01-01

    The psbA gene codes for the D1 polypeptide of the photosystem II reaction center complex and is found in all photosynthetic organisms that carry out oxygenic photosynthesis. Here we describe the construction and characterization of a strain of the cyanobacterium Synechocystis sp PCC 6803 in which the three endogenous psbA genes are replaced by a single psbA gene from the chloroplast genome of the higher plant Poa annua. The resulting chimeric strain, KWPAS, grows photoautotrophically with a doubling time of 26 hours compared with 20 hours for wild-type Synechocystis 6803. The mutant oxidizes water to oxygen at light-saturated rates comparable with wild type, despite differences in 15% of the primary structure of D1 between these species. RNA gel blot analysis indicates the presence in KWPAS of a psbA transcript of approximately 1.25 kilobases, consistent with the chloroplast promoter also acting as a promoter in Synechocystis. By using antibodies specific for the carboxyl-terminal extension of the D1 polypeptide of higher plants, we showed that the D1 polypeptide synthesized by KWPAS is post-translationally modified at the carboxyl terminus, probably through processing. A detailed biophysical analysis of the chimeric photosystem II complex indicated that the rates of forward electron transfer are similar to wild type. The rates of charge recombination between the donor and acceptor sides of the reaction center are, however, accelerated by as much as a factor of nine (QA- to S2) and are the most likely explanation for the lower rate of photoautotrophic growth in the mutant. We conclude that the psbA gene from a higher plant can be expressed in cyanobacteria and its product processed and assembled into a functional chimeric photosystem II reaction center. PMID:1840918

  1. [Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana].

    PubMed

    Huang, Jian-Song; Zhan, Jin-Biao; Zou, Yuan; Feng, Wei-Hong

    2006-07-01

    The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.

  2. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells

    PubMed Central

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  3. Association between Estrogen Receptor-α Gene XbaI and PvuII Polymorphisms and Periodontitis Susceptibility: A Meta-Analysis

    PubMed Central

    Weng, Hong; Zhang, Chao; Hu, Yuan-Yuan; Yuan, Rui-Xia; Zuo, Hong-Xia; Yan, Jin-Zhu; Niu, Yu-Ming

    2015-01-01

    Background. Certain studies have previously explored the association between the estrogen receptor-α (ER-α) gene polymorphisms and periodontitis susceptibility, although the current results are controversial. The present study, using meta-analysis, aimed to investigate the nature of the genetic susceptibility of the ER-α for developing periodontitis. Methods. A comprehensive literature search of PubMed, Embase, CNKI, and Wanfang databases was conducted up to January 8, 2015. Statistical manipulation was performed using Stata version 13.0 software. Odds ratios (ORs) and corresponding 95% confident intervals (CIs) were calculated to estimate the association in five genetic models. Results. A total of 17 eligible case-control studies from seven identified publications consisting of nine studies for the XbaI polymorphism and eight studies for the PvuII polymorphism were included in the meta-analysis. We found elevated risk of periodontitis in XbaI XX genotype carriers. Moreover, subgroup analyses demonstrated increased risk for chronic periodontitis of XbaI XX genotype carriers, specifically in the Chinese Han female population. No significant association was observed between PvuII polymorphism and periodontitis. Conclusion. Current evidence indicated that the homozygote (XX) genotype of ER-α gene XbaI polymorphism, but not PvuII mutation, may increase the risk of chronic periodontitis, specifically in the Chinese Han female population. PMID:26688601

  4. Effects of alien and intraspecies cytoplasms on manifestation of nuclear genes for wheat resistance to brown rust: II. Specificity of cytoplasm influence on different Lr genes

    SciTech Connect

    Voluevich, E.A.; Buloichik, A.A.; Palilova, A.N.

    1995-04-01

    Specificity of expression of the major nuclear genes Lr to two brown rust clones in hybrids with the same maternal cytoplasm was analyzed. It was evaluated by a resistant: susceptible ratio in the F{sub 2}. Reciprocal hybrids were obtained from the cross between the progeny of homozygous susceptible plants of the cultivar Penjamo 62 and its alloplasmatic lines carrying cytoplasms of Triticum dicoccoides var. fulvovillosum, Aegilops squarrosa var. typical, Agropyron trichophorum, and isogenic lines of the cultivar Thatcher (Th) with the Lr1, Lr9, Lr15, and Lr19 genes. It was shown that the effect of the Lr1 gene in the cytoplasm of cultivar Thatcher and in eu-, and alloplasmatic forms of Penjamo 62 was less expressed than that of other Lr genes. Cytoplasm of the alloplasmatic line (dicoccoides)-Penjamo 62 was the only exception: in the F{sub 2}, hybrids with Th (Lr1) had a higher yield of resistant forms than those with Th (Lr15). In the hybrid combinations studied, expression and/or transmission of the Lr19 gene was more significant than that of other genes. This gene had no advantages over Lr15 and Lr19 only in cytoplasm of the alloplasmatic line (squarrosa)-Penjamo 62. In certain hybrid cytoplasms, the display of the Lr1, Lr15, and Lr19 genes, in contrast to Lr9, varied with the virulence of the pathogen clones. 15 refs., 5 tabs.

  5. Transcription analysis of class II human leukocyte antigen genes from normal and immunodeficient B lymphocytes, using polymerase chain reaction.

    PubMed Central

    Bull, M; van Hoef, A; Gorski, J

    1990-01-01

    The RNA transcript levels of all human leukocyte antigen class II loci were determined from class II congenital immunodeficient B cells by polymerase chain reaction amplification of cDNA. No mRNA was observed under conditions in which 0.01% normal levels could be visualized. Pre-mRNA could be amplified from normal B cells but not from immunodeficient B cells, indicating a transcription defect. Images PMID:2113177

  6. A nonsense nucleotide substitution in the oculocutaneous albinism II gene underlies the original pink-eyed dilution allele (Oca2(p)) in mice.

    PubMed

    Shoji, Haruka; Kiniwa, Yukiko; Okuyama, Ryuhei; Yang, Mu; Higuchi, Keiichi; Mori, Masayuki

    2015-01-01

    The original pink-eyed dilution (p) on chromosome 7 is a very old spontaneous mutation in mice. The oculocutaneous albinism II (Oca2) gene has previously been identified as the p gene. Oca2 transcripts have been shown to be absent in the skin of SJL/J mice with the original p mutant allele (Oca2(p)); however, the molecular genetic lesion underlying the original Oca2(p) allele has never been reported. The NCT mouse (commonly known as Nakano cataract mouse) has a pink-eyed dilution phenotype, which prompted us to undertake a molecular genetic analysis of the Oca2 gene of this strain. Our genetic linkage analysis suggests that the locus for the pink-eyed dilution phenotype of NCT is tightly linked to the Oca2 locus. PCR cloning and nucleotide sequence analysis indicates that the NCT mouse has a nonsense nucleotide substitution at exon 7 of the Oca2 gene. Examination of three mouse strains (NZW/NSlc, SJL/J, and 129X1/SvJJmsSlc) with the original Oca2(p) allele revealed the presence of a nonsense nucleotide substitution identical to that in the NCT strain. RT-PCR analysis revealed that the Oca2 transcripts were absent in the skin of NCT mice, suggesting intervention of the nonsense-mediated mRNA decay pathway. Collectively, the data in this study indicate that the nonsense nucleotide substitution in the Oca2 gene underlies the Oca2(p) allele. Our data also indicate that the NCT mouse can be used not only as a cataract model, but also as a model for human type II oculocutaneous albinism.

  7. Pharmacodynamics of dietary phytochemical indoles I3C and DIM: Induction of Nrf2-mediated phase II drug metabolizing and antioxidant genes and synergism with isothiocyanates.

    PubMed

    Saw, Constance Lay-Lay; Cintrón, Melvilí; Wu, Tien-Yuan; Guo, Yue; Huang, Ying; Jeong, Woo-Sik; Kong, Ah-Ng Tony

    2011-07-01

    The antioxidant response element (ARE) is a critical regulatory element for the expression of many phase II drug metabolizing enzymes (DME), phase III transporters and antioxidant enzymes, mediated by the transcription factor Nrf2. The aim of this study was to examine the potential activation and synergism of Nrf2-ARE-mediated transcriptional activity between four common phytochemicals present in cruciferous vegetables; the indoles: indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM); and the isothiocyanates (ITCs): phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). The cytotoxicity of the compounds was determined in a human liver hepatoma cell line (HepG2-C8). The combination index was calculated to assess the synergistic effects on the induction of ARE-mediated gene expressions. Quantitative real-time polymerase chain reaction (qPCR) was employed to measure the mRNA expressions of Nrf2 and Nrf2-mediated genes. I3C and DIM showed less cytotoxicity than SFN and PEITC. Compared with I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after combined treatments of 6.25 µm I3C + 1 µm SFN, 6.25 µm I3C + 1 µm PEITC and 6.25 µm DIM + 1 µm PEITC, while an additive effect was observed for 6.25 µm DIM + 1 µm SFN. Induction of endogenous Nrf2, phase II genes (GSTm2, UGT1A1 and NQO1) and antioxidant genes (HO-1 and SOD1) was also observed. In summary, the indole I3C or DIM alone could induce or syngergistically induce in combination with the ITCs SFN or PEITC, Nrf2-ARE-mediated gene expression, which could potentially enhance cancer chemopreventive activity. PMID:21656528

  8. A nonsense nucleotide substitution in the oculocutaneous albinism II gene underlies the original pink-eyed dilution allele (Oca2(p)) in mice.

    PubMed

    Shoji, Haruka; Kiniwa, Yukiko; Okuyama, Ryuhei; Yang, Mu; Higuchi, Keiichi; Mori, Masayuki

    2015-01-01

    The original pink-eyed dilution (p) on chromosome 7 is a very old spontaneous mutation in mice. The oculocutaneous albinism II (Oca2) gene has previously been identified as the p gene. Oca2 transcripts have been shown to be absent in the skin of SJL/J mice with the original p mutant allele (Oca2(p)); however, the molecular genetic lesion underlying the original Oca2(p) allele has never been reported. The NCT mouse (commonly known as Nakano cataract mouse) has a pink-eyed dilution phenotype, which prompted us to undertake a molecular genetic analysis of the Oca2 gene of this strain. Our genetic linkage analysis suggests that the locus for the pink-eyed dilution phenotype of NCT is tightly linked to the Oca2 locus. PCR cloning and nucleotide sequence analysis indicates that the NCT mouse has a nonsense nucleotide substitution at exon 7 of the Oca2 gene. Examination of three mouse strains (NZW/NSlc, SJL/J, and 129X1/SvJJmsSlc) with the original Oca2(p) allele revealed the presence of a nonsense nucleotide substitution identical to that in the NCT strain. RT-PCR analysis revealed that the Oca2 transcripts were absent in the skin of NCT mice, suggesting intervention of the nonsense-mediated mRNA decay pathway. Collectively, the data in this study indicate that the nonsense nucleotide substitution in the Oca2 gene underlies the Oca2(p) allele. Our data also indicate that the NCT mouse can be used not only as a cataract model, but also as a model for human type II oculocutaneous albinism. PMID:25736709

  9. Construction of a YAC contig and STS map spanning at least 10 cM in 1q41, the critical region of Usher II gene

    SciTech Connect

    Wang, J.Y.; Zhen, D.K.; Li, B.F.

    1994-09-01

    Usher syndrome is an autosomal recessive disorder causing congenital hearing loss, progressive retinitis pigmentosa and vestibular dysfunction. The Usher syndrome is both clinically and genetically heterogeneous. At least three genetic types of Usher syndrome are know to exist. The Usher II (USH2) syndrome has originally been linked to 1q41 between D1S70 and D1S81. more recently its location was refined and placed between D1S217 and D1S229. We have constructed a YAC contig containing 23 clones and a minimum of 10 Mbp of human DNA. A total of three NotI linking clones, fourteen polymorphic microsatelite markers, eight YAC end clones and twenty lambda and cosmid subclones have been used to order the YACs and assess their integrity. The YAC subclones were used to reassess the location of the USH2 gene. Seven CpG islands have already been identified in the region. Several potential exons have been identified by exon amplification in the cosmid subclones. This map of overlapping clones, the set of densely spaced physical markers and potential exons will promote our understanding of the 1q1 region, its associated genes and eventually the gene mutated in Usher syndrome type II.

  10. Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): Evidence for a phenotypic series involving three chondrodysplasias

    SciTech Connect

    Haestbacka, J.; Lander, E.S.; Superti-Furga, A.

    1996-02-01

    Atelosteogenesis type II (AO II) is a neonatally lethal chondrodysplasia whose clinical and histological characteristics resemble those of another chondrodysplasia, the much less severe diastrophic dysplasia (DTD). The similarity suggests a shared pathogenesis involving lesions in the same biochemical pathway and perhaps the same gene. DTD is caused by mutations in the recently identified diastrophic dysplasia sulfate-transporter gene (DTDST). Here, we report that AOII patients also have DTDST mutations, which lead to defective uptake of inorganic sulfate and insufficient sulfation of macromolecules by patient mesenchymal cells in vitro. Together with our recent observation that a third even more severe chondrodysplasia, achondrogenesis type IB, is also caused by mutations in DTDST, these results demonstrate a phenotypic series of three chondrodysplasias of increasing severity caused by lesions in a single sulfate-transporter gene. The severity of the phenotype appears to be correlated with the predicted effect of the mutations on the residual activity of the DTDST protein. 24 refs., 6 figs., 1 tab.

  11. Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias.

    PubMed Central

    Hästbacka, J.; Superti-Furga, A.; Wilcox, W. R.; Rimoin, D. L.; Cohn, D. H.; Lander, E. S.

    1996-01-01

    Atelosteogenesis type II (AO II) is a neonatally lethal chondrodysplasia whose clinical and histological characteristics resemble those of another chondrodysplasia, the much less severe diastrophic dysplasia (DTD). The similarity suggests a shared pathogenesis involving lesions in the same biochemical pathway and perhaps the same gene. DTD is caused by mutations in the recently identified diastrophic dysplasia sulfate-transporter gene (DTDST). Here, we report that AOII patients also have DTDST mutations, which lead to defective uptake of inorganic sulfate and insufficient sulfation of macromolecules by patient mesenchymal cells in vitro. Together with our recent observation that a third even more severe chondrodysplasia, achondrogenesis type IB, is also caused by mutations in DTDST, these results demonstrate a phenotypic series of three chondrodysplasias of increasing severity caused by lesions in a single sulfate-transporter gene. The severity of the phenotype appears to be correlated with the predicted effect of the mutations on the residual activity of the DTDST protein. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 3 Figure 6 PMID:8571951

  12. Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene.

    PubMed

    Kugler, W; Pekrun, A; Laspe, P; Erdlenbruch, B; Lakomek, M

    2001-04-01

    Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001. PMID:11295830

  13. Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes

    PubMed Central

    Sun, Xiaoru; Zheng, Minghuan; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Dragos; Chen, Chengshui; Chen, Luonan; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC-specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL) and CD8+ T cells from lungs (T-LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up-regulated and 70% down-regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over-expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types. PMID:24826900

  14. Monilophyte mitochondrial rps1 genes carry a unique group II intron that likely originated from an ancient paralog in rpl2.

    PubMed

    Knie, Nils; Grewe, Felix; Knoop, Volker

    2016-09-01

    Intron patterns in plant mitochondrial genomes differ significantly between the major land plant clades. We here report on a new, clade-specific group II intron in the rps1 gene of monilophytes (ferns). This intron, rps1i25g2, is strikingly similar to rpl2i846g2 previously identified in the mitochondrial rpl2 gene of seed plants, ferns, and the lycophyte Phlegmariurus squarrosus Although mitochondrial ribosomal protein genes are frequently subject to endosymbiotic gene transfer among plants, we could retrieve the mitochondrial rps1 gene in a taxonomically wide sampling of 44 monilophyte taxa including basal lineages such as the Ophioglossales, Psilotales, and Marattiales with the only exception being the Equisetales (horsetails). Introns rps1i25g2 and rpl2i846g2 were likewise consistently present with only two exceptions: Intron rps1i25g2 is lost in the genus Ophioglossum and intron rpl2i846g2 is lost in Equisetum bogotense Both intron sequences are moderately affected by RNA editing. The unprecedented primary and secondary structure similarity of rps1i25g2 and rpl2i846g2 suggests an ancient retrotransposition event copying rpl2i846g2 into rps1, for which we suggest a model. Our phylogenetic analysis adding the new rps1 locus to a previous data set is fully congruent with recent insights on monilophyte phylogeny and further supports a sister relationship of Gleicheniales and Hymenophyllales. PMID:27354706

  15. Gonadotropin-I and -II subunit gene expression of male striped bass (Morone saxatilis) after gonadotropin-releasing hormone analogue injection: quantitation using an optimized ribonuclease protection assay.

    PubMed

    Hassin, S; Gothilf, Y; Blaise, O; Zohar, Y

    1998-05-01

    In fish, both gonadotropin (GtH)-I and -II are involved in the spermatogenic process, but the differential regulation of these hormones by GnRH is still poorly understood. To gain further insight into the GnRH regulation of GtH-I and -II gene expression in the male striped bass, we have developed and optimized a ribonuclease protection assay for the simultaneous measurement of all GtH subunit mRNAs in a single pituitary gland. The RNA extraction protocol enables the determination of GtH protein content in the same sample, thus enhancing the power of the method. Maturing striped bass males were injected intramuscularly with [D-Ala6,Pro9Net]-LHRH (GnRHa) and sampled at 6 and 24 h postinjection. The mRNA levels of the alpha subunit and GtH-IIbeta increased after 6 h (4- and 6-fold, respectively), while the GtH-Ibeta mRNA levels increased only 2-fold after 24 h. Interestingly, GnRHa stimulation caused a significant increase in beta-actin mRNA levels. GnRHa treatment also resulted in a 2-fold decrease in pituitary GtH-II content, associated with a dramatic increase of plasma GtH-II levels from undetectable levels (< 0.2 ng/ml) to 13+/-2 ng/ml after 6 h. These results demonstrate that both GtH-Ibeta and -Ilbeta are expressed during striped bass spermatogenesis and that the two genes are subjected to differential regulation by GnRHa.

  16. Fetal thymus graft prevents age-related hearing loss and up regulation of the IL-1 receptor type II gene in CD4(+) T cells.

    PubMed

    Iwai, Hiroshi; Inaba, Muneo

    2012-09-15

    We found that rejuvenation of the recipient immunity by inoculation of young CD4(+) T cells or a fetal thymus graft led to down regulation of the interleukin 1 receptor type II (IL-1R2) gene in CD4(+) T cells and reduced age-related hearing loss and degeneration of the spiral ganglion in SAMP1 mice, a murine model of human senescence. Our studies on the relationship between age-related systemic immune dysfunctions and neurodegeneration mechanisms open up new avenues of treatment of neurosenescence, including presbycusis, for which there is no effective therapy.

  17. Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene

    PubMed Central

    Henderson, Heather H.; Timberlake, Kensey B.; Austin, Zoe A.; Badani, Hussain; Sanford, Bridget; Tremblay, Keriann; Baird, Nicholas L.; Jones, Kenneth; Rovnak, Joel; Frietze, Seth; Gilden, Don

    2015-01-01

    ABSTRACT Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5P) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2P)-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. IMPORTANCE Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of m

  18. Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    PubMed

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].

  19. The class II HD-ZIP JAIBA gene is involved in meristematic activity and important for gynoecium and fruit development in Arabidopsis

    PubMed Central

    Zúñiga-Mayo, Victor M.; Marsch-Martínez, Nayelli; de Folter, Stefan

    2012-01-01

    Development and patterning of the gynoecium – and later the fruit – must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In a recent work we reported the functional characterization of the class II HD-ZIP JAIBA (JAB) gene. Mutant jab plants showed sporophytic defects in male and female reproductive development, and combined with the mutant crabs claw (crc) caused defects in the floral meristem (FM) determination process and gynoecium medial tissue development. Furthermore, the JAB protein interacted with transcription factors known to regulate meristematic activity, fruit development and FM determinacy. Preliminary results presented here suggest a genetic interaction between JAB and the gene SHOOT MERISTEMLESS (STM). PMID:22951401

  20. The class II HD-ZIP JAIBA gene is involved in meristematic activity and important for gynoecium and fruit development in Arabidopsis.

    PubMed

    Zúñiga-Mayo, Victor M; Marsch-Martínez, Nayelli; de Folter, Stefan

    2012-11-01

    Development and patterning of the gynoecium - and later the fruit - must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In a recent work we reported the functional characterization of the class II HD-ZIP JAIBA (JAB) gene. Mutant jab plants showed sporophytic defects in male and female reproductive development, and combined with the mutant crabs claw (crc) caused defects in the floral meristem (FM) determination process and gynoecium medial tissue development. Furthermore, the JAB protein interacted with transcription factors known to regulate meristematic activity, fruit development and FM determinacy. Preliminary results presented here suggest a genetic interaction between JAB and the gene SHOOT MERISTEMLESS (STM). PMID:22951401

  1. HincII and KpnI RFLPs for laminin B1 (LAMB1) gene on chromosome 7

    SciTech Connect

    Ardinger, H.H.; Rose, K.M.; Murray, J.C. ); Yamada, Y. )

    1988-09-12

    p1236 was isolated from a human endothelial cell cDNA library in {lambda}gt11. It is a 1.2 kb insert subcloned into the EcoRI site of pUC19. HincII identifies a two allele polymorphism with bands of 7 or 5.8 and 1.2 kb. KpnI identifies a two allele polymorphism with bands of 40 or 25 and 15 kb. The probe was localized to 7q31 using in situ hybridization. Co-dominant segregation was shown in 3 (hincII) and 7(KpnI) families. A rare polymorphism with BamHI was observed in one person. Linkage disequilibrium appears to be present between HincII and KpnI alleles. A double digest of SacI with KpnI allows better visualization of the KpnI bands.

  2. The QTL within the H2 Complex Involved in the Control of Tuberculosis Infection in Mice Is the Classical Class II H2-Ab1 Gene

    PubMed Central

    Logunova, Nadezhda; Korotetskaya, Maria; Polshakov, Vladimir; Apt, Alexander

    2015-01-01

    The level of susceptibility to tuberculosis (TB) infection depends upon allelic variations in numerous interacting genes. In our mouse model system, the whole-genome quantitative trait loci (QTLs) scan revealed three QTLs involved in TB control on chromosomes 3, 9, and in the vicinity of the H2 complex on chromosome 17. For the present study, we have established a panel of new congenic, MHC-recombinant mouse strains bearing differential small segments of chromosome 17 transferred from the TB-susceptible I/St (H2 j) strain onto the genetic background of TB-resistant C57BL/6 (B6) mice (H2 b). This allowed narrowing the QTL interval to 17Ch: 33, 77–34, 34 Mb, containing 36 protein-encoding genes. Cloning and sequencing of the H2 j allelic variants of these genes demonstrated profound polymorphic variations compare to the H2 b haplotype. In two recombinant strains, B6.I-249.1.15.100 and B6.I-249.1.15.139, recombination breakpoints occurred in different sites of the H2-Aβ 1 gene (beta-chain of the Class II heterodimer H2-A), providing polymorphic variations in the domain β1 of the Aβ-chain. These variations were sufficient to produce different TB-relevant phenotypes: the more susceptible B6.I-249.1.15.100 strain demonstrated shorter survival time, more rapid body weight loss, higher mycobacterial loads in the lungs and more severe lung histopathology compared to the more resistant B6.I-249.1.15.139 strain. CD4+ T cells recognized mycobacterial antigens exclusively in the context of the H2-A Class II molecule, and the level of IFN-γ-producing CD4+ T cells in the lungs was significantly higher in the resistant strain. Thus, we directly demonstrated for the first time that the classical H2- Ab1 Class II gene is involved in TB control. Molecular modeling of the H2-Aj product predicts that amino acid (AA) substitutions in the Aβ-chain modify the motif of the peptide–MHC binding groove. Moreover, unique AA substitutions in both α- and β-chains of the H2-Aj molecule

  3. MHC class II transactivator represses human IL-4 gene transcription by interruption of promoter binding with CBP/p300, STAT6 and NFAT1 via histone hypoacetylation

    PubMed Central

    Zhou, Xiaorong; Jiang, Yang; Lu, Liming; Ding, Qing; Jiao, Zhijun; Zhou, Yun; Xin, Lijun; Chou, Kuang-Yen

    2007-01-01

    In addition to its property of enhancing major histocompatibility complex (MHC) class II expression, the class II transactivator (CIITA) was recently demonstrated to be involved in T helper type 1/type 2 (Th1/Th2) differentiation by regulating interleukin-4 (IL-4) gene transcription. There was however, controversy regarding whether CIITA promotes or suppresses IL-4 expression in the experiments with transgenic mice. To clarify the discrepancy by using simpler experimental systems, human Jurkat T cells that express IL-4 but not interferon-γ, even if stimulated with phorbol 12-myristate 13-acetate plus ionomycin, were used for CIITA transfection. Significant suppression of IL-4 gene expression was demonstrated. Simultaneously, histones H3 and H4 in the IL-4 promoter were hypoacetylated. The suppression could be totally reversed by the histone deacetylatase inhibitor trichostatin A. Furthermore, the IL-4 expression was determined in primarily established human Th1/Th2 cells to which CIITA small interference RNA (siRNA) had been introduced. A substantially increased level of IL-4 was recorded in the CIITA siRNA-transfected Th1 cells, which was in parallel with significantly enhanced acetylation in histone H3 of the IL-4 promoter. Chromatin immunoprecipitation analysis indicated that CIITA abrogated the binding of coactivator CBP/p300 and transcription factors STAT6/NFAT1 to IL-4 promoter in the CIITA-transfected cells. In conclusion, CIITA was active in the repression of transcription activation of human IL-4 gene in both the T-cell line and the primary human CD4 T cells by preventing transcription factors from binding to IL-4 promoter through histone hypoacetylation. Our data confirm a potential significant role of CIITA in controlling Th1/Th2 differentiation via modulation of IL-4 gene activation. PMID:17645498

  4. Immunohistochemical expression of p53 in breast carcinoma is associated with the intron 1 BglII polymorphism of the p53 gene.

    PubMed

    Trejo-Becerril, C; Sarmiento, R G; Abad, M M; Ichaso, N; Delgado, R; Cruz, J J; Dueñas-González, A

    2000-09-18

    Breast carcinoma is a public health problem worldwide. It is known that both genetic and environmental factors are important for breast carcinogenesis and that structural and/or functional alterations at p53 gene are commonly observed in breast tumors. In addition, polymorphisms of several genes in either their coding or non-coding sequences have been found related to cancer risk and/or clinicopathological characteristics of tumors. In this study we have evaluated the intron 1 BglII polymorphism of the p53 gene with a PCR-based approach in 117 cases of breast cancer and 102 healthy women and its association with the immunohistochemical expression of p53 in the tumors. The results showed that the presence of the polymorphism (allele 2) is highly associated with the tumor expression of p53 (p<0.0001) and that there is a trend for increased frequency of allele 2 in cases than in controls (p=0.2376). These data suggest that the germ-line variation in the intron 1 of the p53 gene could produce functional or structural changes of the protein that is reflected by its abnormal expression.

  5. Identification of a Novel Gene (HSN2) Causing Hereditary Sensory and Autonomic Neuropathy Type II through the Study of Canadian Genetic Isolates

    PubMed Central

    Lafrenière, Ronald G.; MacDonald, Marcia L. E.; Dubé, Marie-Pierre; MacFarlane, Julie; O’Driscoll, Mary; Brais, Bernard; Meilleur, Sébastien; Brinkman, Ryan R.; Dadivas, Owen; Pape, Terry; Platon, Christèle; Radomski, Chris; Risler, Jenni; Thompson, Jay; Guerra-Escobio, Ana-Maria; Davar, Gudarz; Breakefield, Xandra O.; Pimstone, Simon N.; Green, Roger; Pryse-Phillips, William; Goldberg, Y. Paul; Younghusband, H. Banfield; Hayden, Michael R.; Sherrington, Robin; Rouleau, Guy A.; Samuels, Mark E.

    2004-01-01

    Hereditary sensory and autonomic neuropathy (HSAN) type II is an autosomal recessive disorder characterized by impairment of pain, temperature, and touch sensation owing to reduction or absence of peripheral sensory neurons. We identified two large pedigrees segregating the disorder in an isolated population living in Newfoundland and performed a 5-cM genome scan. Linkage analysis identified a locus mapping to 12p13.33 with a maximum LOD score of 8.4. Haplotype sharing defined a candidate interval of 1.06 Mb containing all or part of seven annotated genes, sequencing of which failed to detect causative mutations. Comparative genomics revealed a conserved ORF corresponding to a novel gene in which we found three different truncating mutations among five families including patients from rural Quebec and Nova Scotia. This gene, termed “HSN2,” consists of a single exon located within intron 8 of the PRKWNK1 gene and is transcribed from the same strand. The HSN2 protein may play a role in the development and/or maintenance of peripheral sensory neurons or their supporting Schwann cells. PMID:15060842

  6. Genetic variation of the MHC class II DRB genes in the Japanese weasel, Mustela itatsi, endemic to Japan, compared with the Siberian weasel, Mustela sibirica.

    PubMed

    Nishita, Y; Abramov, A V; Kosintsev, P A; Lin, L-K; Watanabe, S; Yamazaki, K; Kaneko, Y; Masuda, R

    2015-12-01

    Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship.

  7. Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves.

    PubMed

    Arbanasić, H; Huber, Đ; Kusak, J; Gomerčić, T; Hrenović, J; Galov, A

    2013-01-01

    The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved. PMID:23134500

  8. Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.

    PubMed Central

    Ohta, K; Beall, D S; Mejia, J P; Shanmugam, K T; Ingram, L O

    1991-01-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA). PMID:2059047

  9. Identification of a novel gene (HSN2) causing hereditary sensory and autonomic neuropathy type II through the Study of Canadian Genetic Isolates.

    PubMed

    Lafreniere, Ronald G; MacDonald, Marcia L E; Dube, Marie-Pierre; MacFarlane, Julie; O'Driscoll, Mary; Brais, Bernard; Meilleur, Sebastien; Brinkman, Ryan R; Dadivas, Owen; Pape, Terry; Platon, Christele; Radomski, Chris; Risler, Jenni; Thompson, Jay; Guerra-Escobio, Ana-Maria; Davar, Gudarz; Breakefield, Xandra O; Pimstone, Simon N; Green, Roger; Pryse-Phillips, William; Goldberg, Y Paul; Younghusband, H Banfield; Hayden, Michael R; Sherrington, Robin; Rouleau, Guy A; Samuels, Mark E

    2004-05-01

    Hereditary sensory and autonomic neuropathy (HSAN) type II is an autosomal recessive disorder characterized by impairment of pain, temperature, and touch sensation owing to reduction or absence of peripheral sensory neurons. We identified two large pedigrees segregating the disorder in an isolated population living in Newfoundland and performed a 5-cM genome scan. Linkage analysis identified a locus mapping to 12p13.33 with a maximum LOD score of 8.4. Haplotype sharing defined a candidate interval of 1.06 Mb containing all or part of seven annotated genes, sequencing of which failed to detect causative mutations. Comparative genomics revealed a conserved ORF corresponding to a novel gene in which we found three different truncating mutations among five families including patients from rural Quebec and Nova Scotia. This gene, termed "HSN2," consists of a single exon located within intron 8 of the PRKWNK1 gene and is transcribed from the same strand. The HSN2 protein may play a role in the development and/or maintenance of peripheral sensory neurons or their supporting Schwann cells.

  10. Genetic variation of the MHC class II DRB genes in the Japanese weasel, Mustela itatsi, endemic to Japan, compared with the Siberian weasel, Mustela sibirica.

    PubMed

    Nishita, Y; Abramov, A V; Kosintsev, P A; Lin, L-K; Watanabe, S; Yamazaki, K; Kaneko, Y; Masuda, R

    2015-12-01

    Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship. PMID:26593752

  11. Dilatative uropathy as a manifestation of neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin-neurophysin-II gene.

    PubMed

    Lindenthal, V; Mainberger, A; Morris-Rosendahl, D J; Löning, L; Mayer, W; Müller, H L

    2013-12-01

    Polydypsia and polyuria are frequent symptoms in patients with sellar masses caused by neurohypophyseal diabetes insipidus. Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI), a disorder caused by mutations in the arginine vasopressin (AVP) -neurophysin II (NPII) gene, should be considered as a rare differential diagnosis. A delayed diagnosis bears the risk of life-threatening electrolyte imbalances and permanent urinary tract damage, leading to impaired quality of life.We present a Caucasian kindred of at least 4 generations with FNDI.Clinical histories, endocrine para-meters, and results of molecular analyses of the AVP gene are presented with a review of the literature on diabetes insipidus (DI) related urinary tract dilatation.Polyuria and polydipsia were only reported based on explicit and thorough interrogation after more than 4 years of clinical follow-up. A novel heterozygous mutation in the AVP gene was found in all examined symptomatic subjects (c.1-33_c.4del37nt). A literature review revealed that non-obstructive hydronephrosis (NOH) is a rare but known complication of DI.Since increased fluid intake is often a typical familial pattern in adFNDI, it is frequently missed as being pathologic in affected patients, therefore a detailed clinical history of drinking volumes is of critical importance. AVP gene testing is an important component in the confirmation of the diagnosis. Otherwise unexplainable NOH should lead to further investigations and evaluation of rare diseases like FNDI.

  12. Zinc site redesign in T4 gene 32 protein: structure and stability of cobalt(II) complexes formed by wild-type and metal ligand substitution mutants.

    PubMed

    Guo, J; Giedroc, D P

    1997-01-28

    Phage T4 gene 32 protein (gp32) is a zinc metalloprotein which binds cooperatively and preferentially to single-stranded nucleic acids and functions as a replication and recombination accessory protein. Zn(II) coordination by gp32 employs a His-Cys3 metal ligand donor set derived from the His64-X12-Cys77-X9-Cys87-X2-Cys90 sequence in the ssDNA-binding core domain of the molecule. Crystallographic studies reveal that His64 and Cys77 are derived from two independent beta-strands within a distorted three-stranded beta-sheet and are relatively more buried from solvent than are Cys87 and Cys90, which are positioned immediately before and within, respectively, an alpha-helix. In an effort to understand the origin of the stability of the metal complex, we have employed an anaerobic optical spectroscopic, competitive metal binding assay to determine the coordination geometry and association constants (Ka) for the binding of Co(II) to wild-type gp32 and a series of zinc ligand substitution mutants. At pH 7.5, 25 degrees C, wild-type gp32 binds Co(II) with a Ka approximately 1 x 10(9) M-1. Competition experiments reveal that Ka for Zn(II) is 3.0 (+/-1.0) x 10(11) M-1. We find that all non-native metal complexes retain tetrahedral or distorted tetrahedral coordination geometry but are greatly destabilized in a manner essentially of whether a new protein-derived coordination bond is formed (e.g., in H64C gp32) or not. Co(II) binding isotherms obtained for three His64 substitution mutants, H64C, H64D, and H64N gp32s, suggest that each mutant forms a dimeric Cys4 tetrathiolate intermediate complex at limiting [Co(II)]f, each then rearranges at high [Co(II)]f to form a monomolecular site of the expected geometry and Ka approximately 1 x 10(4) M-1. Like the His64 mutants, C77A gp32 appears to form at least two types of complexes over the course of a Co(II) titration: one with octahedral coordination geometry formed at low [Co(II)]f, with a second tetrahedral or five

  13. Impaired growth and development of Colorado potato beetle larvae on potato plants overexpressing the oryzacystatin II gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant proteinase inhibitors are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. Oryzacystatins I and II (OCI and OCII) show potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate ...

  14. The gene encoding gamma-glutamyl transpeptidase II in the fission yeast is regulated by oxidative and metabolic stress.

    PubMed

    Kang, Hyun-Jung; Kim, Byung-Chul; Park, Eun-Hee; Ahn, Kisup; Lim, Chang-Jin

    2005-09-30

    gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2.) catalyzes the transfer of the gamma-glutamyl moiety from gamma-glutamylcontaining compounds, notably glutathione (GSH), to acceptor amino acids and peptides. A second gene (GGTII) encoding GGT was previously isolated and characterized from the fission yeast Schizosaccharomyces pombe. In the present work, the GGTII-lacZ fusion gene was constructed and used to study the transcriptional regulation of the S. pombe GGTII gene. The synthesis of beta-galactosidase from the GGTII-lacZ fusion gene was significantly enhanced by NO-generating SNP and hydrogen peroxide in the wildtype yeast cells. The GGTII mRNA level was increased in the wild-type S. pombe cells treated with SNP. However, the induction by SNP was abolished in the Pap1-negative S. pombe cells, implying that the induction by SNP of GGTII is mediated by Pap1. Fermentable carbon sources, such as glucose (at low concentrations), lactose and sucrose, as a sole carbon source, enhanced the synthesis of beta-galactosidase from the GGTII-lacZ fusion gene in wildtype KP1 cells but not in Pap1-negative cells. Glycerol, a non-fermentable carbon source, was also able to induce the synthesis of beta-galactosidase from the fusion gene, but other non-fermentable carbon sources such as acetate and ethanol were not. Transcriptional induction of the GGTII gene by fermentable carbon sources was also confirmed by increased GGTII mRNA levels in the yeast cells grown with them. Nitrogen starvation was also able to induce the synthesis of beta-galactosidase from the GGTII-lacZ fusiongene in a Pap1-dependent manner. On the basis of the results, it is concluded that the S. pombe GGTII gene is regulated by oxidative and metabolic stress.

  15. Inherited and de novo deletion of the tyrosine aminotransferase gene locus at 16q22.1----q22.3 in a patient with tyrosinemia type II.

    PubMed

    Natt, E; Westphal, E M; Toth-Fejel, S E; Magenis, R E; Buist, N R; Rettenmeier, R; Scherer, G

    1987-12-01

    Tyrosinemia II is an autosomal-recessively inherited condition caused by deficiency in the liver-specific enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5). We have restudied a patient with typical symptoms of tyrosinemia II who in addition suffers from multiple congenital anomalies including severe mental retardation. Southern blot analysis using a human TAT cDNA probe revealed a complete deletion of both TAT alleles in the patient. Molecular and cytogenetic analysis of the patient and his family showed one deletion to be maternally inherited, extending over at least 27 kb and including the complete TAT structural gene, whereas loss of the second TAT allele results from a small de novo interstitial deletion, del 16 (pter----q22.1::q22.3----qter), in the paternally inherited chromosome 16. Three additional loci previously assigned to 16q22 were studied in our patient: haptoglobin (HP), lecithin: cholesterol acyltransferase (LCAT), and the metallothionein gene cluster MT1,MT2. Of these three markers, only the HP locus was found to be codeleted with the TAT locus on the del(16) chromosome.

  16. CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.

    PubMed

    Baliardini, Cecilia; Meyer, Claire-Lise; Salis, Pietrino; Saumitou-Laprade, Pierre; Verbruggen, Nathalie

    2015-09-01

    Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants. PMID:26162428

  17. Breed relationships facilitate fine-mapping studies: A 7.8-kb deletion cosegregates with Collie eye anomaly across multiple dog breeds

    PubMed Central

    Parker, Heidi G.; Kukekova, Anna V.; Akey, Dayna T.; Goldstein, Orly; Kirkness, Ewen F.; Baysac, Kathleen C.; Mosher, Dana S.; Aguirre, Gustavo D.; Acland, Gregory M.; Ostrander, Elaine A.

    2007-01-01

    The features of modern dog breeds that increase the ease of mapping common diseases, such as reduced heterogeneity and extensive linkage disequilibrium, may also increase the difficulty associated with fine mapping and identifying causative mutations. One way to address this problem is by combining data from multiple breeds segregating the same trait after initial linkage has been determined. The multibreed approach increases the number of potentially informative recombination events and reduces the size of the critical haplotype by taking advantage of shortened linkage disequilibrium distances found across breeds. In order to identify breeds that likely share a trait inherited from the same ancestral source, we have used cluster analysis to divide 132 breeds of dog into five primary breed groups. We then use the multibreed approach to fine-map Collie eye anomaly (cea), a complex disorder of ocular development that was initially mapped to a 3.9-cM region on canine chromosome 37. Combined genotypes from affected individuals from four breeds of a single breed group significantly narrowed the candidate gene region to a 103-kb interval spanning only four genes. Sequence analysis revealed that all affected dogs share a homozygous deletion of 7.8 kb in the NHEJ1 gene. This intronic deletion spans a highly conserved binding domain to which several developmentally important proteins bind. This work both establishes that the primary cea mutation arose as a single disease allele in a common ancestor of herding breeds as well as highlights the value of comparative population analysis for refining regions of linkage. PMID:17916641

  18. Breed relationships facilitate fine-mapping studies: a 7.8-kb deletion cosegregates with Collie eye anomaly across multiple dog breeds.

    PubMed

    Parker, Heidi G; Kukekova, Anna V; Akey, Dayna T; Goldstein, Orly; Kirkness, Ewen F; Baysac, Kathleen C; Mosher, Dana S; Aguirre, Gustavo D; Acland, Gregory M; Ostrander, Elaine A

    2007-11-01

    The features of modern dog breeds that increase the ease of mapping common diseases, such as reduced heterogeneity and extensive linkage disequilibrium, may also increase the difficulty associated with fine mapping and identifying causative mutations. One way to address this problem is by combining data from multiple breeds segregating the same trait after initial linkage has been determined. The multibreed approach increases the number of potentially informative recombination events and reduces the size of the critical haplotype by taking advantage of shortened linkage disequilibrium distances found across breeds. In order to identify breeds that likely share a trait inherited from the same ancestral source, we have used cluster analysis to divide 132 breeds of dog into five primary breed groups. We then use the multibreed approach to fine-map Collie eye anomaly (cea), a complex disorder of ocular development that was initially mapped to a 3.9-cM region on canine chromosome 37. Combined genotypes from affected individuals from four breeds of a single breed group significantly narrowed the candidate gene region to a 103-kb interval spanning only four genes. Sequence analysis revealed that all affected dogs share a homozygous deletion of 7.8 kb in the NHEJ1 gene. This intronic deletion spans a highly conserved binding domain to which several developmentally important proteins bind. This work both establishes that the primary cea mutation arose as a single disease allele in a common ancestor of herding breeds as well as highlights the value of comparative population analysis for refining regions of linkage.

  19. CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.1[OPEN

    PubMed Central

    Baliardini, Cecilia; Meyer, Claire-Lise; Salis, Pietrino; Saumitou-Laprade, Pierre; Verbruggen, Nathalie

    2015-01-01

    Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium2+/hydrogen+ antiporter, cation/hydrogen+ exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants. PMID:26162428

  20. An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy.

    PubMed Central

    Clark, K M; Taylor, R W; Johnson, M A; Chinnery, P F; Chrzanowska-Lightowlers, Z M; Andrews, R M; Nelson, I P; Wood, N W; Lamont, P J; Hanna, M G; Lightowlers, R N; Turnbull, D M

    1999-01-01

    A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation. PMID:10205264

  1. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis

    PubMed Central

    2014-01-01

    Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA

  2. Cosegregation of intragenic markers with a novel mutation that causes Crigler-Najjar syndrome type I: Implication in carrier detection and prenatal diagnosis

    SciTech Connect

    Moghrabi, N.; Clarke, D.J.; Burchell, B.; Boxer, M. )

    1993-09-01

    Crigler-Najjar syndrome type I (CN-1) is a familial disorder characterized by severe unconjugated hyperbilirubinemia and jaundice and leads to kernicterus, neurological damage, and eventual death unless treated with liver transplantation. Previous reports identified mutations in the UGT1 gene complex to be the cause of the disease. The total absence of all phenol/bilirubin UGT (UDP-glucuronosyl transferase) proteins and their activities in liver homogenate of a CN-1 patient was determined by enzymological and immunochemical analysis. A novel homozygous nonsense mutation (CGA [yields] TGA) was identified in the patient by the combined techniques of PCR and direct sequenching. This mutation was located in exon 3 of the constant region in the gene complex which is common to all phenol and bilirubin UGTs. The segregation of the mutation in the patient's family was analyzed and confirmed the recessive nature of the disease. Newly developed intragenic polymorphic probes (UGT1[sup *]4 and UGT-Const) were used on Southern blots of MspI-digested genomic DNA of the patient and his family. The segregation of haplotypes with the mutation for the patient and his family revealed the allele identified by the A1-B1-C2 haplotype to be carrying the mutation. The risk of recombination occurring is negligible, because of the intragenic nature of the probes. This study demonstrates the potential usefulness of these probes in carrier detection and prenatal/presymptomatic diagnosis. 36 refs., 5 figs., 1 tab.

  3. A unique restriction site in the flaA gene allows rapid differentiation of group I and group II Clostridium botulinum strains by PCR-restriction fragment length polymorphism analysis.

    PubMed

    Paul, Catherine J; Tran, Shulin; Tam, Kevin J; Austin, John W

    2007-09-01

    Clostridium botulinum produces the potent botulinum neurotoxin, the causative agent of botulism. Based on distinctive physiological traits, strains of C. botulinum can be divided into four groups: however, only groups I and II are associated with human illness. Alignment of the flaA gene sequences from 40 group I and 40 group II strains identified a single BsrG1 restriction cut site that was present at base pair 283 in all group II flaA sequences and was not found in any group I sequence. The flaA gene was amplified by rapid colony PCR from 22 group I strains and 18 group II strains and digested with BsrGI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with BsrGI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with BsrGI restriction digest of the flaA gene at 60 degrees C is a significant improvement over current methods, such as meat digestion or amplified fragment length polymorphism, as a strain can be identified as either group I or group II in under 5 h when starting with a visible plated C. botulinum colony.

  4. Analysis of P gene mutations in patients with type II (tyrosinase-positive) oculocutaneous albinism (OCA2)

    SciTech Connect

    Lee, S.T.; Nicholls, R.D.; Schnur, R. ||

    1994-09-01

    OCA2 is an autosomal recessive disorder in which the biosynthesis of melanin pigment is greatly reduced in the skin, hair, and eyes. Recently, we showed that OCA2 results from mutations of the P gene, in chromosome segment 15q11-q13. In addition to OCA2, mutations of P account for OCA associated with the Prader-Willi syndrome and some cases of {open_quotes}autosomal recessive ocular albinism{close_quotes} (AROA). We have now studied 38 unrelated patients with various forms of OCA2 or AROA from a variety of different ethnic groups. None of these patients had detectable abnormalities of the tyrosinase (TYR) gene. Among 8 African-American patients with OCA2 we observed apparent locus homogeneity. We detected abnormalities of the P gene in all 8 patients, including 12 different mutations and deletions, most of which are unique to this group and none of which is predominant. In contrast, OCA2 in other populations appears to be genetically heterogeneous. Among 21 Caucasian patients we detected abnormalities of the P gene in only 8, comprising 9 different point mutations and deletions, some of which also occurred among the African-American patients. Among 3 Middle-Eastern, 3 Indo-Pakistani, and 3 Asian patients we detected mutations of the P gene in only one from each group. In a large Indo-Pakistani kindred with OCA2 we have excluded both the TYR and P genes on the basis of genetic linkage. The prevalence of mutations of the P gene thus appears to be much higher among African-Americans with OCA2 than among patients from other ethnic groups. The incidence of OCA2 in some parts of equatorial Africa is extremely high, as frequent as 1 per 1100, and the disease has been linked to P in South African Bantu. The eventual characterization of P gene mutations in Africans will be informative with regard to the origins of P gene mutations in African-American patients.

  5. Identification of three new mutations in the NADH-cytochrome b5 reductase gene responsible for recessive congenital methemoglobinemia type II

    SciTech Connect

    Mota-Vieira, L.; Kaplan, J.C.; Kahn, A.; Leroux, A.

    1994-09-01

    Recessive congenital methemoglobinemia (RCM; McKusick N{degrees}25800) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases: in type I form, cyanosis is the only symptom and the enzyme is only defective in red blood cells; in type II form, cyanosis is associated with severe mental retardation and neurological impairment and the enzyme defect is systemic. We have identified three new molecular defects in two unrelated patients with type II RCM. A homozygous C{r_arrow}T transition in codon 218 (Arg) was detected in the cDNA of one patient, resulting in a premature stop codon (TGA) in exon 8. Restriction enzyme analysis of genomic DNA confirmed the homozygosity of the propositus and heterozygosity for an identical defect in both parents. The second patient was found to be a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation (T{r_arrow}C) with substitution of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele showed a 3 bp deletion of nucleotides 815-817 of the cDNA. The CTG ATG sequence at position 814-819 in exon 9 coding for Leu-271 and Met-272 was replaced by the CTG triplet, with conservation of the Leu-271 and loss of the Met-272. To our knowledge, these are the first examples of a homozygous nonsense mutation and of a compound heterozygous mutation detected in the cytb5r gene. This finding supports the diversity of genetic defects in the cytb5r gene leading to the severe form of the disease.

  6. A phase I/II clinical trial of beta-globin gene therapy for beta-thalassemia.

    PubMed

    Bank, Arthur; Dorazio, Ronald; Leboulch, Philippe

    2005-01-01

    Recent success in the long-term correction of mouse models of human beta-thalassemia and sickle cell anemia by lentiviral vectors and evidence of high gene transfer and expression in transduced human hematopoietic cells have led to a first clinical trial of gene therapy for the disease. A LentiGlobin vector containing a beta-globin gene (beta(A-T87Q)) that produces a hemoglobin (Hbbeta(A-T87Q)) that can be distinguished from normal hemoglobin will be used. The LentiGlobin vector is self-inactivating and contains large elements of the beta-globin locus control region as well as chromatin insulators and other features that should prevent untoward events. The study will be done in Paris with Eliane Gluckman as the principal investigator and Philippe Leboulch as scientific director. PMID:16339679

  7. Characterization and evolution of major histocompatibility complex class II genes in the aye-aye, Daubentonia madagascariensis.

    PubMed

    Go, Yasuhiro; Rakotoarisoa, Gilbert; Kawamoto, Yoshi; Shima, Taizo; Koyama, Naoki; Randrianjafy, Albert; Mora, Roger; Hirai, Hirohisa

    2005-04-01

    Major histocompatibility complex genes (Mhc-DQB and Mhc-DRB) were sequenced in seven aye-ayes (Daubentonia madagascariecsis), which is an endemic and endangered species in Madagascar. An aye-aye from a north-eastern population showed genetic relatedness to individuals of a north-western population and had a somewhat different repertoire from another north-eastern individual. These observations suggest that the extent of genetic variation in Mhc genes is not excessively small in the aye-aye in spite of recent rapid destruction of their habitat by human activities. In light of Mhc gene evolution, trans-species and allelic polymorphisms can be estimated to have been retained for more than 50 Ma (million years) based on the time scale of lemur evolution. PMID:15322927

  8. CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments.

    PubMed

    Zinzow-Kramer, W M; Long, A B; Youngblood, B A; Rosenthal, K M; Butler, R; Mohammed, A-U-R; Skountzou, I; Ahmed, R; Evavold, B D; Boss, J M

    2012-06-01

    Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

  9. Cosegregation of intragenic markers with a novel mutation that causes Crigler-Najjar syndrome type I: implication in carrier detection and prenatal diagnosis.

    PubMed Central

    Moghrabi, N; Clarke, D J; Burchell, B; Boxer, M

    1993-01-01

    Crigler-Najjar syndrome type 1 (CN-1) is a familial disorder characterized by severe unconjugated hyperbilirubinemia and jaundice and leads to kernicterus, neurological damage, and eventual death unless treated with liver transplantation. Previous reports identified mutations in the UGT1 gene complex to be the cause of the disease. The total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate of a CN-1 patient was determined by enzymological and immunochemical analysis. A novel homozygous nonsense mutation (CGA-->TGA) was identified in the patient by the combined techniques of PCR and direct sequencing. This mutation was located in exon 3 of the constant region in the gene complex which is common to all phenol and bilirubin UGTs. The segregation of the mutation in the patient's family was analyzed and confirmed the recessive nature of the disease. Newly developed intragenic polymorphic probes (UGT1* 4 and UGT-Const) were used on Southern blots of MspI-digested genomic DNA of the patient and his family. The segregation of individual alleles within the family was observed from haplotypes generated. Comparison of the segregation of haplotypes with the mutation for the patient and his family revealed the allele identified by the A1-B1-C2 haplotype to be carrying the mutation. The risk of recombination occurring is negligible, because of the intragenic nature of the probes. This study demonstrates the potential usefulness of these probes in carrier detection and prenatal/presymptomatic diagnosis. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8102509

  10. Biotechnology and genetic engineering in the new drug development. Part II. Monoclonal antibodies, modern vaccines and gene therapy.

    PubMed

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

  11. Demand theory of gene regulation. II. Quantitative application to the lactose and maltose operons of Escherichia coli.

    PubMed

    Savageau, M A

    1998-08-01

    Induction of gene expression can be accomplished either by removing a restraining element (negative mode of control) or by providing a stimulatory element (positive mode of control). According to the demand theory of gene regulation, which was first presented in qualitative form in the 1970s, the negative mode will be selected for the control of a gene whose function is in low demand in the organism's natural environment, whereas the positive mode will be selected for the control of a gene whose function is in high demand. This theory has now been further developed in a quantitative form that reveals the importance of two key parameters: cycle time C, which is the average time for a gene to complete an ON/OFF cycle, and demand D, which is the fraction of the cycle time that the gene is ON. Here we estimate nominal values for the relevant mutation rates and growth rates and apply the quantitative demand theory to the lactose and maltose operons of Escherichia coli. The results define regions of the C vs. D plot within which selection for the wild-type regulatory mechanisms is realizable, and these in turn provide the first estimates for the minimum and maximum values of demand that are required for selection of the positive and negative modes of gene control found in these systems. The ratio of mutation rate to selection coefficient is the most relevant determinant of the realizable region for selection, and the most influential parameter is the selection coefficient that reflects the reduction in growth rate when there is superfluous expression of a gene. The quantitative theory predicts the rate and extent of selection for each mode of control. It also predicts three critical values for the cycle time. The predicted maximum value for the cycle time C is consistent with the lifetime of the host. The predicted minimum value for C is consistent with the time for transit through the intestinal tract without colonization. Finally, the theory predicts an optimum value

  12. The largest subunit of RNA polymerase II as a new marker gene to study assemblages of arbuscular mycorrhizal fungi in the field.

    PubMed

    Stockinger, Herbert; Peyret-Guzzon, Marine; Koegel, Sally; Bouffaud, Marie-Lara; Redecker, Dirk

    2014-01-01

    Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.

  13. SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymerase II.

    PubMed

    Song, W; Treich, I; Qian, N; Kuchin, S; Carlson, M

    1996-01-01

    The RNA polymerase II of Saccharomyces cerevisiae exists in holoenzyme forms containing a complex, known as the mediator, associated with the carboxyl-terminal domain. The mediator includes several SRB proteins and is required for transcriptional activation. Previous work showed that a cyclin-dependent kinase-cyclin pair encoded by SSN3 and SSN8, two members of the SSN suppressor family, are identical to two SRB proteins in the mediator. Here we have identified the remaining SSN genes by cloning and genetic analysis. SSN2 and SSN5 are identical to SRB9 and SRB8, respectively, which encode additional components of the mediator. Genetic evidence implicates the SSN genes in transcriptional repression. Thus, these identities provide genetic insight into mediator and carboxyl-terminal domain function, strongly suggesting a role in mediating transcriptional repression as well as activation. We also show that SSN4 and SSN7 are the same as SIN4 and ROX3, respectively, raising the possibility that these genes also encode mediator proteins.

  14. Production of dammarane-type sapogenins in rice by expressing the dammarenediol-II synthase gene from Panax ginseng C.A. Mey.

    PubMed

    Huang, Zhiwei; Lin, Juncheng; Cheng, Zuxin; Xu, Ming; Huang, Xinying; Yang, Zhijian; Zheng, Jingui

    2015-10-01

    Ginsenosides are the main active ingredients in Chinese medicinal ginseng; 2,3-oxidosqualene is a precursor metabolite to ginsenosides that is present in rice. Because rice lacks a key rate-limiting enzyme (dammarenediol-II synthase, DS), rice cannot synthesize dammarane-type ginsenosides. In this study, the ginseng (Panax ginseng CA Mey.) DS gene (GenBank: AB265170.1) was transformed into rice using agrobacterium, and 64 rice transgenic plants were produced. The Transfer-DNA (T-DNA) insertion sites in homozygous lines of the T2 generation were determined by using high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) and differed in all tested lines. One to two copies of the T-DNA were present in each transformant, and real-time PCR and Western blotting showed that the transformed DS gene could be transcribed and highly expressed. High performance liquid chromatography (HPLC) analysis showed that the dammarane-type sapogenin 20(S)-protopanaxadiol (PPD) content was 0.35-0.59 mg/g dw and the dammarane-type sapogenin 20(S)-protopanaxatriol (PPT) content was 0.23-0.43 mg/g dw in the transgenic rice. LC/MS analysis confirmed production of PPD and PPT. These results indicate that a new "ginseng rice" germplasm containing dammarane-type sapogenins has been successfully developed by transforming the ginseng DS gene into rice.

  15. A role for selective androgen response elements in the development of the epididymis and the androgen control of the 5α reductase II gene.

    PubMed

    Kerkhofs, Stefanie; Dubois, Vanessa; De Gendt, Karel; Helsen, Christine; Clinckemalie, Liesbeth; Spans, Lien; Schuit, Frans; Boonen, Steven; Vanderschueren, Dirk; Saunders, Philippa T K; Verhoeven, Guido; Claessens, Frank

    2012-10-01

    The androgen receptor (AR) recognizes two types of DNA elements that are dimers of 5'-AGAACA-3'-like hexamers, either organized as inverted or direct repeats. We developed a mouse model [(specificity affecting AR knock-in (SPARKI)] in which the AR DNA-binding domain was mutated such that it lost binding to direct repeats but not to inverted elements. The impaired fertility of the male SPARKI mice correlates with the reduced motility of the spermatozoa, a characteristic that is developed during transit through the epididymis. Comparative transcriptome analyses revealed that the expression of 39 genes is changed in SPARKI epididymis. Remarkably, the expression of the steroid 5α-reductase type II (Srd5α2) gene, which metabolizes testosterone into the more potent dihydrotestosterone, is reduced 4-fold in SPARKI vs. wild type. The comparison of the SPARKI phenotype with that of Srd5α2-knockout mice shows, however, that the reduced Srd5α2 expression cannot explain all defects of the SPARKI epididymis. Moreover, we describe three new selective androgen response elements (AREs), which control the androgen responsiveness of the Srd5α2 gene. We conclude that the SPARKI model can be considered a knockout model for AR functioning via selective AREs and that this has a dramatic effect on sperm maturation in the epididymis.

  16. Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio.

    PubMed Central

    Wilde, A; Härtel, H; Hübschmann, T; Hoffmann, P; Shestakov, S V; Börner, T

    1995-01-01

    A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes. PMID:7780311

  17. Extended region of nodulation genes in Rhizobium meliloti 1021. II. Nucleotide sequence, transcription start sites and protein products

    SciTech Connect

    Fisher, R.F.; Swanson, J.A.; Mulligan, J.T.; Long, S.R.

    1987-10-01

    The authors have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the nod box, suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site.

  18. [Genetic counseling in osteodystrophy Albright's syndrome. Part II. GNAS1 gene and encoded protein function, genetic forecast and treatment].

    PubMed

    Janusz, Leszek; Morawska, Justyna; Wasilewska, Ewa; Sierakowski, Stanisław; Midro, Alina T

    2010-01-01

    In this article we describe localization and structure of genes from GNAS1 cluster, which mutations are responsible for characteristic group of features described mainly as Albright's syndrome. We present the capabilities of expressions of different phenotypes depending on types of molecular changes and possibilities of genetic prognosis with problems need to be solved in interdisciplinary support. PMID:21387769

  19. Heroin self-administration: II. CNS gene expression following withdrawal and cue-induced drug-seeking behavior.

    PubMed

    Kuntz, Kara L; Patel, Kruti M; Grigson, Patricia S; Freeman, Willard M; Vrana, Kent E

    2008-09-01

    In the accompanying paper, we described incubation of heroin-seeking behavior in rats following 14 days of abstinence. To gain an understanding of genomic changes that accompany this behavioral observation, we measured the expression of genes previously reported to respond to drugs of abuse. Specifically, after 1 or 14 days of abstinence, mRNA expression was measured for 11 genes in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) immediately following a single 90 min extinction session. Additionally, the role of contingency was examined in control rats that received yoked, response-independent heroin administration. Gene expression was quantified by real-time quantitative PCR. Expression of five genes (Arc, EGR1, EGR2, Fos, and Homer1b/c) was changed in the mPFC. EGR1 and EGR2 expression was increased following the 90 min extinction session in a contingency-specific manner and this increase persisted through the 14 days of abstinence. Fos expression was also increased after 1 and 14 days of abstinence, but at 14 days this increase was response-independent (i.e., it occurred in both the rats with a history of heroin self-administration and in the yoked controls). Arc expression increased following the extinction session only in rats with a history of heroin self-administration and only when tested following 1, but not 14, days of abstinence. Homer 1 b/c decreased after 14 days of enforced abstinence in rats that received non-contingent heroin. Expression of only a single gene (EGR2) was increased in the NAc. These data demonstrate that behavioral incubation is coincident with altered levels of specific transcripts and that this response is contingently-specific. Moreover, EGR1 and EGR2 are specifically upregulated in self-administering rats following extinction and this finding persists through 14 days of abstinence, suggesting that these genes are particularly associated with the incubation phenomenon. These latter observations of persistent changes

  20. Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

    PubMed Central

    Budziszewski, G J; Lewis, S P; Glover, L W; Reineke, J; Jones, G; Ziemnik, L S; Lonowski, J; Nyfeler, B; Aux, G; Zhou, Q; McElver, J; Patton, D A; Martienssen, R; Grossniklaus, U; Ma, H; Law, M; Levin, J Z

    2001-01-01

    We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype. PMID:11779813

  1. Genetic variants in IGF-I, IGF-II, IGFBP-3, and adiponectin genes and colon cancer risk in African Americans and Whites

    PubMed Central

    Keku, Temitope O.; Vidal, Adriana; Oliver, Shannon; Hoyo, Catherine; Hall, Ingrid J.; Omofoye, Seun; McDoom, Maya; Worley, Kendra; Galanko, Joseph; Sandler, Robert S.; Millikan, Robert

    2014-01-01

    Purpose Evaluating genetic susceptibility may clarify effects of known environmental factors and also identify individuals at high risk. We evaluated the association of four insulin-related pathway gene polymorphisms in insulin-like growth factor-1 (IGF-I) (CA)n repeat, insulin-like growth factor-2 (IGF-II) (rs680), insulin-like growth factor binding protein-3 (IGFBP-3) (rs2854744), and adiponectin (APM1 rs1501299) with colon cancer risk, as well as relationships with circulating IGF-I, IGF-II, IGFBP-3, and C-peptide in a population-based study. Methods Participants were African Americans (231cases, 306 controls) and Whites (297 cases, 530 controls). Consenting subjects provided blood specimens, and lifestyle/diet information. Genotyping for all genes except IGF-I was performed by the 5′-exonuclease (Taqman) assay. The IGF-I (CA)n repeat was assayed by PCR, and fragment analysis. Circulating proteins were measured by enzyme immunoassays. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. Results The IGF-I (CA)19 repeat was higher in White controls (50%) than African American controls (31%). Whites homozygous for the IGF-I (CA)19 repeat had a nearly two fold increase in risk of colon cancer (OR=1.77; 95%CI=1.15–2.73), but not African Americans (OR= 0.73, 95%CI 0.50–1.51). We observed an inverse association between the IGF-II Apa1 A-variant and colon cancer risk (OR= 0.49, 95%CI 0.28–0.88) in Whites only. Carrying the IGFBP-3 variant alleles was associated with lower IGFBP-3 protein levels, a difference most pronounced in Whites (p- trend < 0.05). Conclusions These results support an association between insulin pathway-related genes and elevated colon cancer risk in Whites but not in African Americans. PMID:22565227

  2. Cosegregation of the ND4 G11696A mutation with the LHON-associated ND4 G11778A mutation in a four generation Chinese family.

    PubMed

    Qu, Jia; Li, Ronghua; Zhou, Xiangtian; Tong, Yi; Yang, Li; Chen, Jie; Zhao, Fuxing; Lu, Chunjie; Qian, Yaping; Lu, Fan; Guan, Min-Xin

    2007-01-01

    We report here the characterization of a four-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON). This Chinese family exhibited a variable severity and age-at-onset of visual loss. Notably, the average age-at-onset of vision impairment changed from 26 years (generation III) to 14 years (generation IV), with the average of 18 years in this family. In addition, 30% and 50% of matrilineal relatives in generation III and IV of this family developed visual loss with a variability of severity, ranging from blindness to normal vision. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the homoplasmic ND4 G11778A mutation and 33 other variants, belonging to the Asian haplogroup D4. Of other variants, the homoplasmic G11696A mutation in the ND4 gene is of special interest as it was implicated to be associated with LHON in a large Dutch family and five Chinese pedigrees with extremely penetrance of visual loss. In fact, the G11696A mutation caused the substitution of an isoleucine for valine at amino acid position 313, located in a predicted transmembrane region of ND4. These imply that the G11696A mutation may act in synergy with the primary LHON-associated G11778A mutation in this Chinese pedigree.

  3. A survey of EMS-induced biennial Beta vulgaris mutants reveals a novel bolting locus which is unlinked to the bolting gene B.

    PubMed

    Büttner, Bianca; Abou-Elwafa, Salah F; Zhang, Wenying; Jung, Christian; Müller, Andreas E

    2010-10-01

    Beta vulgaris is a facultative perennial species which exhibits large intraspecific variation in vernalization requirement and includes cultivated biennial forms such as the sugar beet. Vernalization requirement is under the genetic control of the bolting locus B on chromosome II. Previously, ethyl methanesulfonate (EMS) mutagenesis of an annual accession had yielded several mutants which require vernalization to bolt and behave as biennials. Here, five F2 populations derived from crosses between biennial mutants and annual beets were tested for co-segregation of bolting phenotypes with genotypic markers located at the B locus. One mutant appears to be mutated at the B locus, suggesting that an EMS-induced mutation of B can be sufficient to abolish annual bolting. Co-segregation analysis in four populations indicates that the genetic control of bolting also involves previously unknown major loci not linked to B, one of which also affects bolting time and was genetically mapped to chromosome IX.

  4. Analysis of four families with the Stickler syndrome by linkage studies. Identification of a new premature stop codon in the COL2A1 gene in a family

    SciTech Connect

    Bonaventure, J.; Lasselin, C.; Toutain, A.

    1994-09-01

    The Stickler syndrome is an arthro-ophthalmopathy which associates progressive myopia with vitreal degeneration and retinal detachment. Cleft palate, cranio-facial abnormalities, deafness and osteoarthritis are often associated symptoms. Genetic heterogeneity of this autosomal dominant disease was consistent with its large clinical variability. Linkage studies have provided evidence for cosegregation of the disease with COL2A1, the gene coding for type II collagen, in about 50% of the families. Four additional families are reported here. Linkage analyses by using a VNTR located in the 3{prime} region of the gene were achieved. In three families, positive lod scores were obtained with a cumulative maximal value of 3.5 at a recombination fraction of 0. In one of these families, single strand conformation analysis of 25 exons disclosed a new mutation in exon 42. Codon for glutamic acid at position a1-803 was converted into a stop codon. The mutation was detected in DNA samples from all the affected members of the family but not in the unaffected. This result confirms that most of the Stickler syndromes linked to COL2A1 are due to premature stop codons. In a second family, an abnormal SSCP pattern of exon 34 was detected in all the affected individuals. The mutation is likely to correspond to a splicing defect in the acceptor site of intron 33. In one family the disease did not segregate with the COL2A1 locus. Further linkage studies with intragenic dimorphic sites in the COL10A1 gene and highly polymorphic markers close to the COL9A1 locus indicated that this disorder did not result from defects in these two genes.

  5. Methionine Adenosyltransferase II-dependent Histone H3K9 Methylation at the COX-2 Gene Locus*

    PubMed Central

    Kera, Yohei; Katoh, Yasutake; Ohta, Mineto; Matsumoto, Mitsuyo; Takano-Yamamoto, Teruko; Igarashi, Kazuhiko

    2013-01-01

    Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα. PMID:23539621

  6. Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice.

    PubMed

    Itoh, Toshiaki; Kuwahara, Tomomi; Suzuki, Takayoshi; Hayashi, Makoto; Ohnishi, Yoshinari

    2003-08-01

    Transgenic mouse assays have revealed that the mouse intestine, despite its resistance to carcinogenesis, is sensitive to the mutagenicity of some heterocyclic amines (HCAs). Little is known, however, about the level and localization of that sensitivity. We assessed the mutagenicity of four orally administered (20 mg/kg per day for 5 days) HCAs-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hydrochloride, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate-in the intestine of male MutaMice. Two weeks after the last administration, we isolated epithelium from the small intestine, cecum, and colon and analyzed lacZ and cII transgene mutations. PhIP increased the lacZ mutant frequency (MF) in all the samples, and in the small intestine, cII and lacZ MFs were comparable. In the cII gene, G:C to T:A and G:C to C:G transversions were characteristic PhIP-induced mutations (which has also been reported for the rat colon, where PhIP is carcinogenic). In the small intestine, PhIP increased the cII MF to four-fold that of the control, but IQ, MeIQ, and Trp-P-2 did not have a significant mutagenic effect. In the cecum, cII MFs induced by IQ and MeIQ were 1.9 and 2.7 times those in the control, respectively. The MF induced by MeIQ in the colon was 3.1 times the control value. Mutagenic potency was in the order PhIP>MeIQ>IQ; Trp-P-2 did not significantly increase the MF in any tissue. The cecum was the most susceptible organ to HCA mutagenicity. PMID:12948818

  7. A comparison of batch effect removal methods for enhancement of prediction performance using MAQC-II microarray gene expression data.

    PubMed

    Luo, J; Schumacher, M; Scherer, A; Sanoudou, D; Megherbi, D; Davison, T; Shi, T; Tong, W; Shi, L; Hong, H; Zhao, C; Elloumi, F; Shi, W; Thomas, R; Lin, S; Tillinghast, G; Liu, G; Zhou, Y; Herman, D; Li, Y; Deng, Y; Fang, H; Bushel, P; Woods, M; Zhang, J

    2010-08-01

    Batch effects are the systematic non-biological differences between batches (groups) of samples in microarray experiments due to various causes such as differences in sample preparation and hybridization protocols. Previous work focused mainly on the development of methods for effective batch effects removal. However, their impact on cross-batch prediction performance, which is one of the most important goals in microarray-based applications, has not been addressed. This paper uses a broad selection of data sets from the Microarray Quality Control Phase II (MAQC-II) effort, generated on three microarray platforms with different causes of batch effects to assess the efficacy of their removal. Two data sets from cross-tissue and cross-platform experiments are also included. Of the 120 cases studied using Support vector machines (SVM) and K nearest neighbors (KNN) as classifiers and Matthews correlation coefficient (MCC) as performance metric, we find that Ratio-G, Ratio-A, EJLR, mean-centering and standardization methods perform better or equivalent to no batch effect removal in 89, 85, 83, 79 and 75% of the cases, respectively, suggesting that the application of these methods is generally advisable and ratio-based methods are preferred.

  8. An Atypical psbA Gene Encodes a Sentinel D1 Protein to Form a Physiologically Relevant Inactive Photosystem II Complex in Cyanobacteria*

    PubMed Central

    Wegener, Kimberly M.; Nagarajan, Aparna; Pakrasi, Himadri B.

    2015-01-01

    Photosystem II, a large membrane-bound enzyme complex in cyanobacteria and chloroplasts, mediates light-induced oxidation of water to molecular oxygen. The D1 protein of PSII, encoded by the psbA gene, provides multiple ligands for cofactors crucial to this enzymatic reaction. Cyanobacteria contain multiple psbA genes that respond to various physiological cues and environmental factors. Certain unicellular cyanobacterial cells, such as Cyanothece sp. ATCC 51142, are capable of nitrogen fixation, a highly oxygen-sensitive process, by separating oxygen evolution from nitrogen fixation using a day-night cycle. We have shown that c-psbA4, one of the five psbA orthologs in this cyanobacterium, is exclusively expressed during nighttime. Remarkably, the corresponding D1 isoform has replacements of a number of amino acids that are essential ligands for the catalytic Mn4CaO5 metal center for water oxidation by PSII. At least 30 cyanobacterial strains, most of which are known to have nitrogen fixing abilities, have similar psbA orthologs. We expressed the c-psbA4 gene from Cyanothece 51142 in a 4E-3 mutant strain of the model non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803, which lacks any psbA gene. The resultant strain could not grow photoautotrophically. Moreover, these Synechocystis 6803 cells were incapable of PSII-mediated oxygen evolution. Based on our findings, we have named this physiologically relevant, unusual D1 isoform sentinel D1. Sentinel D1 represents a new class of D1 protein that, when incorporated in a PSII complex, ensures that PSII cannot mediate water oxidation, thus allowing oxygen-sensitive processes such as nitrogen fixation to occur in cyanobacterial cells. PMID:25525275

  9. Genetic variants within obesity-related genes are associated with tumor recurrence in patients with stages II/III colon cancer

    PubMed Central

    Sebio, Ana; Gerger, Armin; Matsusaka, Satoshi; Yang, Dongyun; Zhang, Wu; Stremitzer, Stefan; Stintizing, Sebastian; Sunakawa, Yu; Yamauchi, Shinichi; Ning, Yan; Fujimoto, Yoshiya; Ueno, Masashi; Lenz, Heinz-Josef

    2014-01-01

    Objective Obesity is an established risk factor for colorectal cancer (CRC) incidence and it is also linked to CRC recurrence and survival. Polymorphisms located in obesity-related genes are associated with increased risk of developing several cancer types including colorectal cancer. We evaluated whether SNPs in obesity-related genes may predict tumor recurrence in colon cancer patients. Methods Genotypes were obtained from germline DNA from 207 patients with stage II or III colon cancer at the Norris Comprehensive Cancer Center. Nine polymorphisms in eight obesity-related genes (PPAR, LEP, NFKB, CD36, DRG1, NGAL, REGIA and DSCR1) were evaluated. The primary endpoint of the study was 3-year recurrence rate. Positive associations were also tested in an independent Japanese cohort of 350 stage III CRC patients. Results In univariate analysis, for PPAR rs1801282, patients with a CC genotype had significantly lower recurrence probability (29± 4% standard error, SE) compared to patients with a CG genotype (48% ± 8% SE), HR: 1.77; 95%CI, 1.01-3.10; p=0.040. For DSCR1 rs6517239, patients with an AA genotype had higher recurrence probability than patients carrying at least one allele G (37% ± 4% SE vs 15% ± 6% SE), HR: 0.51, 95% CI, 0.27-0.94; p=0.027. This association was stronger in the patients bearing a left-sided tumor (HR: 0.34; 95%CI, 0.13-0.88; p=0.018). In the Japanese cohort no associations were found. Conclusion This hypothesis generating study suggests a potential influence of polymorphisms within obesity-related genes in the recurrence probability of colon cancer. These interesting results should be further evaluated. PMID:25379721

  10. Type II and type IV topoisomerase mutations in clinical isolates of Morganella morganii harbouring the qnrD gene

    PubMed Central

    2014-01-01

    Introduction The aim of this study was to show the emergence of the qnrD gene among fluoroquinolone-resistant Morganella morganii isolate. The occurrence of mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC,parE) genes was also investigated in this strain. Methodology 95 clinical Enterobacteria were screened for harbouring the qnrD gene. The clinical isolate of M. morganii was recovered from urine from a patient hospitalized in the urology unit at Fattouma Bourguiba Hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method. Quinolone susceptibility was studied with microbroth dilution technique. The investigations of plasmid mediated quinolone resistance (PMQR) and topoisomerases mutations were performed by polymerase chain reaction and nucleotide sequencing. Results This isolate showed high level of resistance to quinolones. The MIC with microbroth dilution technique was 512 μg/ml for norfloxacin, 256 μg/ml for ofloxacin and ciprofloxacin and 64μg/ml for levofloxacin. This strain was found to harbour the quinolone resistance determinant qnrD. In addition, this strain harboured two new gyrB mutations (S463A, S464Y) and one parC mutation (S80I). Conclusions This is the first report in Tunisia of qnrD determinant and tow new gyrB muations in M. morganii. The nosocomial infection due to this proteeae invites further study of its epidemiologic evolution. PMID:25106550

  11. Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure.

    PubMed

    Herdegen, M; Babik, W; Radwan, J

    2014-11-01

    Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre-eminent system for the study of selective pressures that arise from host-pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population-genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.

  12. Mechanisms of Nrf2/Keap1-Dependent Phase II Cytoprotective and Detoxifying Gene Expression and Potential Cellular Targets of Chemopreventive Isothiocyanates

    PubMed Central

    Das, Biswa Nath; Kim, Young-Woo

    2013-01-01

    Isothiocyanates (ITCs) are abundantly found in cruciferous vegetables. Epidemiological studies suggest that chronic consumption of cruciferous vegetables can lower the overall risk of cancer. Natural ITCs are key chemopreventive ingredients of cruciferous vegetables, and one of the prime chemopreventive mechanisms of natural isothiocyanates is the induction of Nrf2/ARE-dependent gene expression that plays a critical role in cellular defense against electrophiles and reactive oxygen species. In the present review, we first discuss the underlying mechanisms how natural ITCs affect the intracellular signaling kinase cascades to regulate the Keap1/Nrf2 activities, thereby inducing phase II cytoprotective and detoxifying enzymes. We also discuss the potential cellular protein targets to which natural ITCs are directly conjugated and how these events aid in the chemopreventive effects of natural ITCs. Finally, we discuss the posttranslational modifications of Keap1 and nucleocytoplasmic trafficking of Nrf2 in response to electrophiles and oxidants. PMID:23781297

  13. A group II intron in the Neurospora mitochondrial coI gene: nucleotide sequence and implications for splicing and molecular evolution.

    PubMed Central

    Field, D J; Sommerfield, A; Saville, B J; Collins, R A

    1989-01-01

    The temperature-sensitive Neurospora nuclear mutant cyt18-1 is deficient in splicing many Group I mitochondrial introns when grown at its non-permissive temperature; however, splicing of intron 1 in the coI gene of the Adiopodoume (formerly called North Africa) strain is unaffected (R.A. Collins and A.M. Lambowitz, J. Mol. Biol. 184: 413-428, 1985). Here we show that coI intron 1 is a typical Group II intron, the only one identified to date in Neurospora. The differential effect of the cyt18-1 mutation suggests that splicing of certain introns could be regulated independently of others by nuclear-encoded proteins. The intron contains a long open reading frame (ORF) resembling that of the Neurospora Mauriceville mitochondrial plasmid. The intron and plasmid ORFs share unusual features of codon usage that suggest both evolved outside of the Neurospora mitochondrial genetic system. Images PMID:2531370

  14. Efficient one-pot synthesis of trans-Pt(II)(salicylaldimine)(4-picoline)Cl complexes: effective agents for enhanced expression of p53 tumor suppressor genes.

    PubMed

    Rahman, Faiz-Ur; Ali, Amjad; Guo, Rong; Wang, Wei-Kun; Wang, Hui; Li, Zhan-Ting; Lin, Yuejian; Zhang, Dan-Wei

    2015-06-01

    A series of trans-Pt(II)(salicylaldimine)(4-picoline)Cl complexes were synthesized in 78-87% yield using a one-pot procedure from commercially available precursors. The structures of these complexes were characterized by (1)H, (19)F and (13)C NMR spectroscopy, HRMS (ESI) as well as single crystal X-ray analysis. Bioactivity investigations including bio-assay, time- and dose-dependent, cell cycle progression study, caspase 3 and 9 apoptosis marker assay and DNA interaction using pBR322 plasmid DNA by gel electrophoresis were performed. The results indicated that the complexes showed promising in vitro cytotoxicity in MCF-7 and A549 cancer cell lines. Moreover these complexes enhanced the expression of p53 tumor suppressor gene family members such as p63 and p73.

  15. CO2 induced seawater acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae.

    PubMed

    Stumpp, M; Dupont, S; Thorndyke, M C; Melzner, F

    2011-11-01

    Extensive use of fossil fuels is leading to increasing CO(2) concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO(2). As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO(2) 41 Pa e.g. 399 μatm) and CO(2) acidified seawater with pH of 7.7 (pCO(2) 134 Pa e.g. 1318 μatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10% reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0.518). Significant differences in gene expression patterns (ANOSIM R=0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO(2) treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO(2) effect. We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes

  16. Linkage between the variegate porphyria (VP) and the alpha-1-antitrypsin (PI) genes on human chromosome 14.

    PubMed

    Bissbort, S; Hitzeroth, H W; du Wentzel, D P; Van den Berg, C W; Senff, H; Wienker, T F; Bender, K

    1988-07-01

    From family studies close linkage between the gene locus for variegate porphyria (VP) and the alpha-1-antitrypsin (PI) gene became evident. The maximal lod score from male meioses was 4.33 at theta = 0.04 and from both sexes combined 3.56 at theta = 0.12. Three pedigrees were triple informative regarding loci VP, PI, and IGHC (immunoglobulin heavy chain cluster, Gm polymorphism). In two of the respective meioses recombinations were observed, and in both cases the co-segregating VP and PI alleles were separated from the Gm haplotypes. These findings argue in favour of gene order either VP:PI:IGHC or PI:VP:IGHC.

  17. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  18. A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

    PubMed

    Spitzer, Franz; Vahjen, Wilfried; Pieper, Robert; Martinez-Vallespin, Beatriz; Zentek, Jürgen

    2014-12-01

    This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions.

  19. Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois.

    PubMed

    Mona, S; Crestanello, B; Bankhead-Dronnet, S; Pecchioli, E; Ingrosso, S; D'Amelio, S; Rossi, L; Meneguz, P G; Bertorelle, G

    2008-09-01

    The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen-mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d(n)/d(s) ratio and the presence of trans-species polymorphisms suggest that a strong long-term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D-loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000-30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift.

  20. A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

    PubMed

    Spitzer, Franz; Vahjen, Wilfried; Pieper, Robert; Martinez-Vallespin, Beatriz; Zentek, Jürgen

    2014-12-01

    This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions. PMID:25313936

  1. Association between Single Nucleotide Polymorphisms of the Major Histocompatibility Complex Class II Gene and Newcastle Disease Virus Titre and Body Weight in Leung Hang Khao Chickens

    PubMed Central

    Molee, A.; Kongroi, K.; Kuadsantia, P.; Poompramun, C.; Likitdecharote, B.

    2016-01-01

    The aim of the present study was to investigate the effect of single nucleotide polymorphisms in the major histocompatibility complex (MHC) class II gene on resistance to Newcastle disease virus and body weight of the Thai indigenous chicken, Leung Hang Khao (Gallus gallus domesticus). Blood samples were collected for single nucleotide polymorphism analysis from 485 chickens. Polymerase chain reaction sequencing was used to classify single nucleotide polymorphisms of class II MHC. Body weights were measured at the ages of 3, 4, 5, and 7 months. Titres of Newcastle disease virus at 2 weeks to 7 months were determined and the correlation between body weight and titre was analysed. The association between single nucleotide polymorphisms and body weight and titre were analysed by a generalized linear model. Seven single nucleotide polymorphisms were identified: C125T, A126T, C209G, C242T, A243T, C244T, and A254T. Significant correlations between log titre and body weight were found at 2 and 4 weeks. Associations between single nucleotide polymorphisms and titre were found for C209G and A254T, and between all single nucleotide polymorphisms (except A243T) and body weight. The results showed that class II MHC is associated with both titre of Newcastle disease virus and body weight in Leung Hang Khao chickens. This is of concern because improved growth traits are the main goal of breeding selection. Moreover, the results suggested that MHC has a pleiotropic effect on the titre and growth performance. This mechanism should be investigated in a future study. PMID:26732325

  2. Type II hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine-436 in the C1 inhibitor gene

    SciTech Connect

    Levy, N.J.; Ramesh, N.; Daviss, A.E. III ); Cicardi, M. ); Harrison, R.A. )

    1990-01-01

    Identical single-base changes in the C1 inhibitor gene that may result in dysfunctional inhibitor proteins are described in two different families with type II hereditary angioneurotic edema. Initially, a restriction fragment length polymorphism was defined that resulted from loss of a Pst I site within exon VIII, which encodes the region containing the reactive center. Exon VIII from the normal and abnormal allelles was amplified by the polymerase chain reaction. Amplified DNA product was cloned into plasmid pUC18; clones representing normal and mutant allelles were distinguished by the presence and absence, respectively of the Pst I restriction site. DNA sequence analysis revealed a G {yields} A mutation in the codon for alanine-436, which would result in replacement with a threonine residue. This position is nine amino acid residues amino-terminal to the reactive-center arginylthreonine peptide bond. In contrast, previously defined mutations in type II hereditary angioneurotic edema result in replacement of the reactive-center arginine.

  3. Angiotensin II blockade upregulates the expression of Klotho, the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy

    PubMed Central

    Yoon, Hye Eun; Ghee, Jung Yeon; Piao, ShangGuo; Song, Ji-Hyun; Han, Dong He; Kim, Sol; Ohashi, Naro; Kobori, Hiroyuki; Kuro-o, Makoto; Yang, Chul Woo

    2011-01-01

    Background. The Klotho gene plays a role in suppressing ageing-related disorders. It is suggested that activation of renin–angiotensin system (RAS) or oxidative stress suppresses Klotho in the kidney. This study evaluated the association between Klotho expression and RAS in cyclosporine (CsA)-induced renal injury. Methods. Chronic CsA nephropathy was induced by administering CsA (30 mg/kg) to mice on a low-salt diet (LSD) for 4 weeks. A normal-salt diet (NSD) was used as the control. Reverse transcription–polymerase chain reaction, western blot and immunohistochemistry were performed for Klotho and intrarenal RAS activity was measured using immunohistochemistry for angiotensinogen and renin. Oxidative stress was measured with urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Results. CsA treatment decreased Klotho mRNA and protein in mouse kidney in a dose-dependent and time-dependent manner, but a concurrent treatment with losartan, an angiotensin II type 1 (AT1) receptor blocker, reversed the decrease in Klotho expression with histological improvement. This finding was more marked in the LSD than the NSD. Klotho expression was correlated with angiotensinogen and renin expression, tubulointerstitial fibrosis score and urinary 8-OHdG excretion. Conclusions. Angiotensin II may play a pivotal role in regulating Klotho expression in CsA-induced renal injury. AT1 receptor blocker may inhibit the ageing process by decreasing oxidative stress caused by CsA. PMID:20813770

  4. A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains.

    PubMed Central

    Goppelt, A; Stelzer, G; Lottspeich, F; Meisterernst, M

    1996-01-01

    Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II. Images PMID:8670811

  5. Isolation and gene disruption of the Tox5 gene encoding trichodiene synthase in Gibberella pulicaris.

    PubMed

    Hohn, T M; Desjardins, A E

    1992-01-01

    The trichodiene synthase gene (Tox5) was isolated from Gibberella pulicaris, and its nucleotide sequence was determined. Tox5 was disrupted through transformation with a plasmid carrying a doubly truncated copy of the coding region and a selectable marker for resistance to hygromycin B (Hygr). Analysis of 82 transformants for their ability to produce the trichothecene, 4,15-diacetoxyscirpenol (DAS), resulted in the identification of five DAS- strains. Southern hybridization analysis of DAS- Hygr transformants indicated that the plasmid integrated at the Tox5 locus. The disrupted Tox5 gene was shown to be mitotically stable. Analysis of nine tetrads revealed either the cosegregation of the disrupter plasmid and the DAS- phenotype or the loss of the disrupter plasmid. These results demonstrate the feasibility of using gene disruption in G. pulicaris and suggest a general method for obtaining Tox5- mutants in other trichothecene-producing fungi. PMID:1421511

  6. Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

    PubMed Central

    2010-01-01

    Background Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida. Results GSII sequences were isolated from four species of green algae (Trebouxiophyceae), and additional green algal (Chlorophyceae and Prasinophytae) and streptophyte (Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta) sequences were obtained from public databases. In Bayesian and maximum likelihood analyses, eubacterial (GSIIB) and eukaryotic (GSIIE) GSII sequences formed distinct clades. Both GSIIB and GSIIE were found in chlorophytes and early-diverging streptophytes. The GSIIB enzymes from these groups formed a well-supported sister clade with the γ-Proteobacteria, providing evidence that GSIIB in the Chloroplastida arose by horizontal gene transfer (HGT). Bayesian relaxed molecular clock analyses suggest that GSIIB and GSIIE coexisted for an extended period of time but it is unclear whether the proposed HGT happened prior to or after the divergence of the primary endosymbiotic lineages (the Archaeplastida). However, GSIIB genes have not been identified in glaucophytes or red algae, favoring the hypothesis that GSIIB was gained after the divergence of the primary endosymbiotic lineages. Duplicate copies of the GSIIB gene were present in Chlamydomonas reinhardtii, Volvox carteri f. nagariensis, and Physcomitrella patens. Both GSIIB proteins in C. reinhardtii and V. carteri f. nagariensis had N-terminal transit sequences, indicating they are targeted to the chloroplast or mitochondrion. In contrast, GSIIB proteins of P. patens lacked transit sequences, suggesting a cytosolic

  7. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    SciTech Connect

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  8. Analysis of Gln223Agr polymorphism of Leptin Receptor Gene in type II diabetic mellitus subjects among Malaysians.

    PubMed

    Etemad, Ali; Ramachandran, Vasudevan; Pishva, Seyyed Reza; Heidari, Farzad; Aziz, Ahmad Fazli Abdul; Yusof, Ahmad Khairuddin Mohamed; Pei, Chong Pei; Ismail, Patimah

    2013-09-18

    Leptin is known as the adipose peptide hormone. It plays an important role in the regulation of body fat and inhibits food intake by its action. Moreover, it is believed that leptin level deductions might be the cause of obesity and may play an important role in the development of Type 2 Diabetes Mellitus (T2DM), as well as in cardiovascular diseases (CVD). The Leptin Receptor (LEPR) gene and its polymorphisms have not been extensively studied in relation to the T2DM and its complications in various populations. In this study, we have determined the association of Gln223Agr loci of LEPR gene in three ethnic groups of Malaysia, namely: Malays, Chinese and Indians. A total of 284 T2DM subjects and 281 healthy individuals were recruited based on International Diabetes Federation (IDF) criteria. Genomic DNA was extracted from the buccal specimens of the subjects. The commercial polymerase chain reaction (PCR) method was carried out by proper restriction enzyme MSP I to both amplify and digest the Gln223Agr polymorphism. The p-value among the three studied races was 0.057, 0.011 and 0.095, respectively. The values such as age, WHR, FPG, HbA1C, LDL, HDL, Chol and Family History were significantly different among the subjects with Gln223Agr polymorphism of LEPR (p < 0.05).

  9. The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis

    PubMed Central

    Mesoraca, Alvaro; Di Natale, Manuela; Cima, Antonella; Di Giacomo, Gianluca; Sarti, Monica; Barone, Maria Antonietta; Bizzoco, Domenico; Cignini, Pietro; Mobili, Luisa; DʼEmidio, Laura; Giorlandino, Claudio

    2010-01-01

    Objective: The aim of the study is to evaluate the role of Denaturing High Performance Liquid Chromatography (DHPLC) in the second level screening of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Methods: A 9-month prospective study, between June 2008 and March 2009 at Artemisia Fetal Medical Centre, included 3829 samples of amniotic fluid collected from women undergoing mid-trimester amniocentesis. The genetic diagnosis of CF was based on research of the main mutations of the CFTR gene on fetal DNA extracted from the amniocytes, (first level screening) using different commercial diagnostic systems. A second level screening using DHPLC, on the amniotic fluid and on a blood sample from the couple, was offered in case of fetuses heterozygous at first level screening. Results: Of 3829 fetuses, 134 were found to be positive, 129 heterozygous and 5 affected. Of the 129 couples, following appropriate genetic counselling, 53 requested a second level screening. Through the use of DHPLC, 44 couples were found to be negative, and in nine couples, nine rare mutations were identified. Conclusions: The first level screening can be useful to evidence up to 75% of the CF mutations. The second level screening can identify a further 10% of mutant alleles. DHPLC was found to be a reliable and specific method for the rapid identification of the rare CFTR mutations which were not revealed in initial first level screening. PMID:22439061

  10. Analysis of Gln223Agr polymorphism of Leptin Receptor Gene in type II diabetic mellitus subjects among Malaysians.

    PubMed

    Etemad, Ali; Ramachandran, Vasudevan; Pishva, Seyyed Reza; Heidari, Farzad; Aziz, Ahmad Fazli Abdul; Yusof, Ahmad Khairuddin Mohamed; Pei, Chong Pei; Ismail, Patimah

    2013-01-01

    Leptin is known as the adipose peptide hormone. It plays an important role in the regulation of body fat and inhibits food intake by its action. Moreover, it is believed that leptin level deductions might be the cause of obesity and may play an important role in the development of Type 2 Diabetes Mellitus (T2DM), as well as in cardiovascular diseases (CVD). The Leptin Receptor (LEPR) gene and its polymorphisms have not been extensively studied in relation to the T2DM and its complications in various populations. In this study, we have determined the association of Gln223Agr loci of LEPR gene in three ethnic groups of Malaysia, namely: Malays, Chinese and Indians. A total of 284 T2DM subjects and 281 healthy individuals were recruited based on International Diabetes Federation (IDF) criteria. Genomic DNA was extracted from the buccal specimens of the subjects. The commercial polymerase chain reaction (PCR) method was carried out by proper restriction enzyme MSP I to both amplify and digest the Gln223Agr polymorphism. The p-value among the three studied races was 0.057, 0.011 and 0.095, respectively. The values such as age, WHR, FPG, HbA1C, LDL, HDL, Chol and Family History were significantly different among the subjects with Gln223Agr polymorphism of LEPR (p < 0.05). PMID:24051404

  11. Analysis of Gln223Agr Polymorphism of Leptin Receptor Gene in Type II Diabetic Mellitus Subjects among Malaysians

    PubMed Central

    Etemad, Ali; Ramachandran, Vasudevan; Pishva, Seyyed Reza; Heidari, Farzad; Aziz, Ahmad Fazli Abdul; Yusof, Ahmad Khairuddin Mohamed; Pei, Chong Pei; Ismail, Patimah

    2013-01-01

    Leptin is known as the adipose peptide hormone. It plays an important role in the regulation of body fat and inhibits food intake by its action. Moreover, it is believed that leptin level deductions might be the cause of obesity and may play an important role in the development of Type 2 Diabetes Mellitus (T2DM), as well as in cardiovascular diseases (CVD). The Leptin Receptor (LEPR) gene and its polymorphisms have not been extensively studied in relation to the T2DM and its complications in various populations. In this study, we have determined the association of Gln223Agr loci of LEPR gene in three ethnic groups of Malaysia, namely: Malays, Chinese and Indians. A total of 284 T2DM subjects and 281 healthy individuals were recruited based on International Diabetes Federation (IDF) criteria. Genomic DNA was extracted from the buccal specimens of the subjects. The commercial polymerase chain reaction (PCR) method was carried out by proper restriction enzyme MSP I to both amplify and digest the Gln223Agr polymorphism. The p-value among the three studied races was 0.057, 0.011 and 0.095, respectively. The values such as age, WHR, FPG, HbA1C, LDL, HDL, Chol and Family History were significantly different among the subjects with Gln223Agr polymorphism of LEPR (p < 0.05). PMID:24051404

  12. A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid.

    PubMed

    Mruk, Iwona; Kaczor