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BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.
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Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.
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Alquil e Aril Transferases/genética , Plaquetas/citologia , Divisão Celular/genética , Mutação , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Cromossomos Artificiais de Levedura , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Fenótipo , Prenilação de ProteínaAssuntos
Mapeamento Cromossômico , Camundongos/genética , Proteína-Lisina 6-Oxidase/genética , Animais , Sondas de DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Ligação Genética , Marcadores Genéticos , Haplótipos/genética , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
Chediak-Higashi syndrome is an autosomal recessive, immune deficiency disorder of human (CHS) and mouse (beige, bg) that is characterized by abnormal intracellular protein transport to, and from, the lysosome. Recent reports have described the identification of homologous genes that are mutated in human CHS and bg mice. Here we report the sequences of two major mRNA isoforms of the CHS gene in human and mouse. These isoforms differ both in size and in sequence at the 3' end of their coding domains, with the smaller isoform (approximately 5.8 kb) arising from incomplete splicing and reading through an intron. These mRNAs also differ in tissue distribution of transcription and in predicted biological properties. Novel mutations were identified within the region of the coding domain common to both isoforms in three CHS patients: C-->T transitions that generated stop codons (R50X and Q1029X) were found in two patients, and a novel frameshift mutation (deletion of nucleotides 3073 and 3074 of the coding domain) was found in a third. Northern blots of lymphoblastoid mRNA from CHS patients revealed loss of the largest transcript (approximately 13.5 kb) in two of seven CHS patients, while the small mRNA was undiminished in abundance. These results suggest that the small isoform alone cannot complement Chediak-Higashi syndrome.
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Processamento Alternativo , Síndrome de Chediak-Higashi/genética , Mutação , Proteínas/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Análise Mutacional de DNA , DNA Complementar , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isomerismo , Camundongos , Dados de Sequência Molecular , Proteínas/metabolismo , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Proteínas de Transporte VesicularRESUMO
Current knowledge of genes that regulate pattern formation and differentiation processes during mammalian embryonic development is limited. In an effort to isolate developmentally relevant genes, 20 novel, end-sequenced cDNAs selected from a Day 10.5 postcoitum mouse embryo library were genetically mapped in intersubspecific backcross mice. Eleven of 20 cDNA clones mapped to three mouse autosomes (Chr 5, 11, and 14), a result that was unlikely (P < 0.03) if the distribution of genes expressed in embryos is random within the mouse genome. Several clones were candidates for mouse developmental mutations by virtue of genetic colocalization and concordance of embryonic expression patterns with the distribution of defects in mutant mice: Estm11 was a candidate for the mouse mutation wabbler-lethal (wl), since Estm11 mapped in the vicinity of wl on mouse Chr 14 and was expressed in those regions of embryonic brain that exhibit axonal degeneration in wl. End-sequence analysis, genetic mapping, and in situ hybridization appeared to be an effective combination of methods for identification and characterization of genes with potential regulatory functions during mammalian embryogenesis.
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Mapeamento Cromossômico , DNA Complementar , Família Multigênica , Animais , Sequência de Bases , Embrião de Mamíferos , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
BACKGROUND: Chediak-Higashi syndrome (CHS) is a systemic disorder of human and mouse (beige, bg) that is characterized by aberrant intracellular protein kinesis and lysosomal trafficking. Affected individuals exhibit a severe primary immune deficiency that principally affects the function of granulocytes and cytolytic lymphocytes and partial oculocutaneous albinism, platelet dysfunction, and neurodegeneration. Chediak-Higashi syndrome is inherited as an autosomal recessive Mendelian trait in human and mouse and maps on proximal mouse Chromosome 13. METHODS: To clone positionally the defective gene in CHS, we have generated a large number of backcross mice who segregate for beige. Genomic DNA from these mice was genotyped for 26 genetic markers known to map on proximal mouse Chromosome 13. RESULTS: By segregation analysis, bg was localized to a 0.24 centiMorgan interval and was shown to cosegregate with 6 genetic markers (Nid, Estm9, D13Mit56, D13Mit162, D13Mit237, and D13Mit240). Two of these loci, Nid and Estm9, are genes and represent candidates for bg. Nidogen (Nid) encodes an extracellular matrix protein that is a component of basement membranes. Estm9 is a sequence that is transcribed ubiquitously in mouse embryos and encodes a protein of unknown function. Mutation analysis of Nid and Estm9 was undertaken in 6 bg alleles; no differences were observed between bg and coisogenic controls by analysis of Northern blots, Southern blots, or by quantitative reverse transcription and polymerase chain reaction. CONCLUSIONS: These studies indicate that a genomic rearrangement affecting Nid or Estm9 does not underlie bg. The bg locus has been localized on mouse Chromosome 13 with sufficient precision to enable rapid cloning of the bg non-recombinant interval and eventual identification of the gene for Chediak-Higashi syndrome among sequences within the interval.
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Síndrome de Chediak-Higashi/genética , Mapeamento Cromossômico/métodos , Clonagem Molecular/métodos , Proteínas/genética , Animais , Cruzamentos Genéticos , Feminino , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Polimorfismo Genético , Proteínas de Transporte VesicularRESUMO
Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.