RESUMO
With the advance of sequencing technology, the number of sequenced plant genomes has been rapidly increasing. However, understanding of the gene function in these sequenced genomes lags far behind; as a result, many coding plant sequences in public databases are annotated as proteins with domains of unknown function (DUF). Function of a protein family DUF810 in rice is not known. In this study, we analysed seven members of OsDU810 (OsDUF810.1-OsDUF810.7) family with three distinct motifs in rice Nipponbare. By phylogenetic analysis, OsDUF810 proteins fall into three major groups (I, II, III). Expression patterns of the seven corresponding OsDUF810 protein-encoding genes in 15 different rice tissues vary. Under drought, salt, cold and heat stress conditions and ABA treatment, the expression of OsDUF810.7 significantly increases. Overexpression of this protein in E. coli lead to a significant enhancement of catalase (CAT) and peroxidase (POD) activities, and improved bacterial resistance to salt and drought.
Assuntos
Oryza/genética , Filogenia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Secas , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Cloreto de Sódio/toxicidadeRESUMO
The genomic instability of persons with Bloom's syndrome (BS) features particularly an increased number of sister-chromatid exchanges (SCEs). The primary cause of the genomic instability is mutation at BLM, which encodes a DNA helicase of the RecQ family. BLM interacts with Topoisomerase IIIalpha (Topo IIIalpha), and both BLM and Topo IIIalpha localize to the nuclear organelles referred to as the promyelocytic leukemia protein (PML) nuclear bodies. In this study we show, by analysis of cells that express various deletion constructs of green fluorescent protein (GFP)-tagged BLM, that the first 133 amino acids of BLM are necessary and sufficient for interaction between Topo IIIalpha and BLM. The Topo IIIalpha-interaction domain of BLM is not required for BLM's localization to the PML nuclear bodies; in contrast, Topo IIIalpha is recruited to the PML nuclear bodies via its interaction with BLM. Expression of a full-length BLM (amino acids 1-1417) in BS cells can correct their high SCEs to normal levels, whereas expression of a BLM fragment that lacks the Topo IIIalpha interaction domain (amino acids 133-1417) results in intermediate SCE levels. The deficiency of amino acids 133-1417 in the reduction of SCEs was not explained by a defect in DNA helicase activity, because immunoprecipitated 133-1417 protein had 4-fold higher activity than GFP-BLM. The data implicate the BLM-Topo IIIalpha complex in the regulation of recombination in somatic cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Síndrome de Bloom/enzimologia , Síndrome de Bloom/genética , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sítios de Ligação , Síndrome de Bloom/metabolismo , Linhagem Celular Transformada , DNA Helicases/química , DNA Helicases/genética , DNA Topoisomerases Tipo I/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Fenótipo , Estrutura Terciária de Proteína , RecQ Helicases , Troca de Cromátide Irmã , Células Tumorais CultivadasRESUMO
A human cDNA and gene encoding for human ERMAP, a putative erythroid transmembrane adhesion/receptor protein, is reported. The predicted protein is made up of 475 amino acids and shares high homology with the murine ERMAP (73% identity and 14% conservative changes). Human Ermap is highly expressed in erythroid tissues and the protein localizes to the plasma membrane, particularly in sites of cell contact, and "cytoplasmic bodies." The extracellular segment contains one IgV fold that shares high homology with the butyrophilin family of milk proteins, autoantigens, and avian blood group antigens. In the intracellular region, there is a conserved B30.2 domain that is encoded by a single exon and is highly homologous with a similar domain in a diverse group of proteins, including butyrophilin, pyrin, and MID 1. The human Ermap gene is composed of 11 exons spanning 19 kb on chromosome 1p34.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Antígenos de Grupos Sanguíneos , Butirofilinas , Compartimento Celular , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Células Precursoras Eritroides , Humanos , Dados de Sequência Molecular , Família Multigênica , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
Ermap (erythroid membrane-associated protein), a gene coding for a novel transmembrane protein produced exclusively in erythroid cells, is described. It is mapped to murine Chromosome 4, 57 cM distal to the centromere. The initial cDNA clone was isolated from a day 9 murine embryonic erythroid cell cDNA library. The predicted peptide sequence suggests that ERMAP is a transmembrane protein with two extracellular immunoglobulin folds, as well as a highly conserved B30.2 domain and several phosphorylation consensus sequences in the cytoplasmic region. ERMAP shares a high homology throughout the entire peptide with butyrophilin, a glycoprotein essential for milk lipid droplet formation and release. A GFP-ERMAP fusion protein was localized to the plasma membrane and cytoplasmic vesicles in transiently transfected 293T cells. Northern blot analysis and in-situ hybridization demonstrated that Ermap expression was restricted to fetal and adult erythroid tissues. ERMAP is likely a novel adhesion/receptor molecule specific for erythroid cells.
Assuntos
Moléculas de Adesão Celular/genética , Eritrócitos/metabolismo , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Antígenos de Grupos Sanguíneos , Northern Blotting , Butirofilinas , Adesão Celular , Linhagem Celular , Núcleo Celular/química , Mapeamento Cromossômico , Clonagem Molecular , Citoplasma/química , DNA Complementar/química , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Eritrócitos/citologia , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Humanos , Hibridização In Situ , Células K562 , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Muridae , RNA/genética , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The gene BLM, mutated in Bloom syndrome (BS), encodes the nuclear protein BLM, which when absent, as it is from most BS cells, results in genomic instability. A manifestation of this instability is an excessive rate of sister-chromatid exchange (SCE). Here we describe the effects on this abnormal cellular phenotype of stable transfection of normal BLM cDNAs into two types of BS cells, SV40-transformed fibroblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Clones of BLM-transfected fibroblasts produced normal amounts of BLM by western blot analysis and displayed a normal nuclear localization of the protein by immunofluorescence microscopy. They had a mean of 24 SCEs/46 chromosomes, in contrast to the mean of 69 SCEs in controls transfected only with the vector. BLM-transfected fibroblast clones that expressed highest levels of the BLM protein had lowest levels of SCE. The lymphoblastoid cells transfected with BLM had SCE frequencies of 22 and 42 in two separate experiments in which two different selectable markers were used, in contrast to 57 and 58 in vector-transfected cells; in this type cell, however, the BLM protein was below the level detectable by western blot analysis. These experiments prove that BLM cDNA encodes a functional protein capable of restoring to or toward normal the uniquely characteristic high-SCE phenotype of BS cells.
Assuntos
Adenosina Trifosfatases/genética , Síndrome de Bloom/genética , DNA Helicases/genética , Troca de Cromátide Irmã/genética , Adenosina Trifosfatases/metabolismo , Western Blotting , Células Cultivadas , DNA Helicases/metabolismo , Fibroblastos , Imunofluorescência , Herpesvirus Humano 4/genética , Humanos , Linfócitos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , RecQ Helicases , Vírus 40 dos Símios/genética , Transfecção , Transformação GenéticaRESUMO
Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Síndrome de Bloom/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Adenosina Trifosfatases/isolamento & purificação , Síndrome de Bloom/metabolismo , Pré-Escolar , DNA Helicases/isolamento & purificação , DNA Complementar/metabolismo , Feminino , Regulação da Expressão Gênica , Teste de Complementação Genética , Humanos , Masculino , Mutação , Fenótipo , RecQ Helicases , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiaeRESUMO
The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells. The Bloom syndrome gene BLM encodes a RecQ DNA helicase, whose absence from the cell results in genomic instability epitomized by high levels of sister-chromatid exchange (SCE) and cancer predisposition. We show here that BLM co-localizes with PML to the NB. In cells from persons with Bloom syndrome the localization of PML is unperturbed, whereas in APL cells carrying the PML-RARalpha oncoprotein, both PML and BLM are delocalized from the NB into microspeckled nuclear regions. Treatment with retinoic acid (RA) induces the relocalization of both proteins to the NB. In primary PML-/- cells, BLM fails to accumulate in the NB. Strikingly, in PML-/- cells the frequency of SCEs is increased relative to PML+/+ cells. These data demonstrate that BLM is a constituent of the NB and that PML is required for its accumulation in these nuclear domains and for the normal function of BLM. Thus, our findings suggest a role for BLM in APL pathogenesis and implicate the PML NB in the maintenance of genomic stability.
Assuntos
Adenosina Trifosfatases/genética , Núcleo Celular/metabolismo , DNA Helicases/genética , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Síndrome de Bloom/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Códon de Terminação , DNA Helicases/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Células HL-60 , Células HeLa , Homozigoto , Humanos , Queratinócitos/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , RecQ Helicases , Troca de Cromátide Irmã , Transfecção , Tretinoína/farmacologia , Proteínas Supressoras de Tumor , Células U937RESUMO
The gene for Bloom syndrome (BLM) has been mapped to human chromosome 15 band q26.1 by homozygosity mapping. Further refinement of the location of BLM has relied upon linkage-disequilibrium mapping and somatic intragenic recombination. In combination with these mapping approaches and to identify novel DNA markers and probes for the BLM candidate region, a contiguous representation of the 2-Mb region that contains the BLM gene was generated and is presented here. YAC and P1 clones from the region have been identified and ordered by using previously available genetic markers in the region along with newly developed sequence-tagged sites from radiation-reduced hybrids, polymorphic dinucleotide repeat loci, and end sequences of YACs and P1s. A long-range restriction map of the 2-Mb region that allowed estimation of the distance between polymorphic microsatellite loci is also reported. This map and the DNA markers derived from it were instrumental in the recent identification of the BLM gene.
Assuntos
Adenosina Trifosfatases/genética , Bacteriófago P1/genética , Síndrome de Bloom/genética , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 15/genética , DNA Helicases/genética , Genes Recessivos , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Animais , Sequência de Bases , Células CHO , Hidrolases de Éster Carboxílico/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 15/ultraestrutura , Consanguinidade , Cricetinae , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Desequilíbrio de Ligação , Repetições de Microssatélites , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-fes , Proto-Oncogenes , RecQ HelicasesRESUMO
The Bloom's syndrome (BS) gene, BLM, plays an important role in the maintenance of genomic stability in somatic cells. A candidate for BLM was identified by direct selection of a cDNA derived from a 250 kb segment of the genome to which BLM had been assigned by somatic crossover point mapping. In this novel mapping method, cells were used from persons with BS that had undergone intragenic recombination within BLM. cDNA analysis of the candidate gene identified a 4437 bp cDNA that encodes a 1417 amino acid peptide with homology to the RecQ helicases, a subfamily of DExH box-containing DNA and RNA helicases. The presence of chain-terminating mutations in the candidate gene in persons with BS proved that it was BLM.
Assuntos
Adenosina Trifosfatases/genética , Síndrome de Bloom/genética , DNA Helicases/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas/fisiologia , Clonagem Molecular , DNA/análise , Humanos , Dados de Sequência Molecular , Mutação/genética , RNA/fisiologia , RecQ Helicases , Homologia de Sequência do Ácido NucleicoRESUMO
We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG.
Assuntos
Antígenos CD , Antígenos de Grupos Sanguíneos/genética , Moléculas de Adesão Celular/genética , Genes , Antígeno 12E7 , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Western Blotting , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/fisiologia , Reações Cruzadas , Epitopos/imunologia , Fibroblastos/metabolismo , Ligação Genética , Testes de Hemaglutinação , Células-Tronco Hematopoéticas/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Coelhos , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The pseudoautosomal boundaries are the interface between pseudoautosomal and sex chromosome-specific DNA sequences. We have isolated a gene, PBDX, from the human pseudoautosomal boundary region of Xp. The three exons at the 5' end of PBDX are situated in the pseudoautosomal region immediately downstream of MIC2, whereas the other seven exons are in the X-specific region. Hence, PBDX is inherited in two modes: its 5' end is pseudoautosomally inherited and its 3' end is X-linked. The predicted amino acid sequence of the 540 bp coding region is 48% homologous to 12E7, the product of MIC2. By virtue of its position, PBDX becomes an excellent candidate for the XG blood group gene.