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1.
Physiol Genomics ; 7(1): 45-53, 2001 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-11595791

RESUMO

Over the last few years, there has been a dramatic increase in the use of cDNA microarrays to monitor gene expression changes in biological systems. Data from these experiments are usually transformed into expression ratios between experimental samples and a common reference sample for subsequent data analysis. The accuracy of this critical transformation depends on two major parameters: the signal intensities and the normalization of the experiment vs. reference signal intensities. Here we describe and validate a new model for microarray signal intensity that has one multiplicative variation and one additive background variation. Using replicative experiments and simulated data, we found that the signal intensity is the most critical parameter that influences the performance of normalization, accuracy of ratio estimates, reproducibility, specificity, and sensitivity of microarray experiments. Therefore, we developed a statistical procedure to flag spots with weak signal intensity based on the standard deviation (delta(ij)) of background differences between a spot and the neighboring spots, i.e., a spot is considered as too weak if the signal is weaker than cdelta(ij). Our studies suggest that normalization and ratio estimates were unacceptable when this threshold (c) is small. We further showed that when a reasonable compromise of c (c = 6) is applied, normalization using trimmed mean of log ratios performed slightly better than global intensity and mean of ratios. These studies suggest that decreasing the background noise is critical to improve the quality of microarray experiments.


Assuntos
Modelos Estatísticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Teorema de Bayes , Simulação por Computador , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Camundongos , Camundongos Endogâmicos NOD/genética , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
2.
Gene ; 273(2): 275-84, 2001 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-11595174

RESUMO

We report here the cloning and characterization of a novel gene belonging to the tubby superfamily proteins (TUSP) in mouse and human. The mouse Tusp cDNA is 9120 bp in length and encodes a deduced protein of 1547 amino acids, while the human TUSP gene is 11,127 bp and encodes a deduced protein of 1544 amino acids. The human and mouse genes are 87% identical for their nucleotide sequences and 85% identical for their amino acid sequences. The protein sequences of these genes are 40-48% identical to other tubby family proteins at the C-terminal conserved 'tubby domain'. In addition, the TUSP proteins contain a tubby signature motif (FXGRVTQ), two bipartite nuclear localization signals (NLSs) at the C-terminal, two proline-rich regions, one WD40 repeat region and one suppressor of cytokines signaling domain. Transfection assay with green fluorescent protein-tagged TUSP expression constructs showed that the complete TUSP protein and the N-terminal portion of TUSP are localized in the cytoplasm but the C-terminal portion with the two NLSs produced distinct dots or spots localized in the cytoplasm. Northern blotting analysis showed that the major transcript with the complete coding sequence is expressed mainly in the brain, skeletal muscle, testis and kidney. Radiation hybrid mapping localized the mouse gene to chromosome 17q13 and the human TUSP gene to chromosome 6q25-q26 near the type 1 diabetes gene IDDM5. However, association analysis in diabetic families with a polymorphic microsatellite marker did not show any evidence for association between TUSP and type 1 diabetes. The precise biological function of the tubby superfamily genes is still unknown; the highly conserved tubby domain in different species, however, suggests that these proteins must have fundamental biological functions in a wide range of multi-cellular organisms.


Assuntos
Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Sequência de Aminoácidos , Animais , Northern Blotting , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 6/genética , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/química , DNA Complementar/genética , Diabetes Mellitus/genética , Repetições de Dinucleotídeos/genética , Feminino , Expressão Gênica , Genes/genética , Proteínas de Fluorescência Verde , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , RNA Mensageiro/genética , RNA Mensageiro/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 Tecidual
3.
J Biol Chem ; 276(44): 41357-64, 2001 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-11533054

RESUMO

The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Timo/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Dedos de Zinco , Proteína AIRE
4.
Mamm Genome ; 12(11): 830-6, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11845285

RESUMO

We cloned a novel mouse gene that encodes a protein with homology to the mitochondria solute carrier proteins (Mscp). The major full-length Mscp transcript contains 4112 bp of cDNA and a deduced protein of 338 amino acids. The Mscp protein shares 50%, 40%, and 39% sequence identity with the C. elegans hypothetical protein T26089 and the yeast mitochondria carrier proteins MRS3 and MRS4, respectively. It also showed homology with the uncoupling proteins (UCP1, UCP2, and UCP3; 22%, 24%, and 29% identity, respectively). The protein has six transmembrane domains and three mitochondria energy-transfer protein signature motifs, which are conserved among all the members of mitochondria carrier protein family. Northern analysis indicated that the Mscp gene is highly expressed in the spleen. Using cDNA microarray and Northern analysis, we have shown a significant decrease of the splenic Mscp mRNA levels around 4-5 weeks of age in several mouse strains including C57BL/6J, nonobese diabetic (NOD), and several NOD-congenic mice. These results suggest that the Mscp gene is decreased during splenic lymphocyte maturation in these mice.


Assuntos
Proteínas de Transporte/genética , RNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas de Transporte da Membrana Mitocondrial , Dados de Sequência Molecular , RNA/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Tempo
5.
Autoimmunity ; 31(1): 47-53, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10593569

RESUMO

We have recently cloned the murine autoimmune regulator (Aire) gene, the homologue of human AIRE responsible for the autoimmune polyglandular syndrome type 1 (APS1) or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). Here, we report the genomic sequence (18,413 bp) for the entire Aire gene and its 5' flanking region, which contains putative regulatory sequences. Comparison of the genomic and cDNA sequences indicates that the Aire gene is composed of 14 exons and the coding sequence shares high similarities between mouse and human. The sizes of the homologous introns in the two species are conserved; however, the introns do not share significant sequence homologies except the sequences near the splice donor and acceptor sites. Sequence analyses of the 5' regulatory region and the complete coding region in three mouse strains (B6, NOD and SJL) did not reveal any sequence variation, suggesting sequence conservation between different inbred mouse strains. Using one of the six microsatellite markers identified by genomic sequencing and a B6 x Cast backcross mapping panel, we mapped the mouse Aire gene to chromosome 10, a syntenic region containing the Cdl18 and Pfkl genes on human chromosome 21q22.


Assuntos
Mapeamento Cromossômico , Análise de Sequência de DNA , Fatores de Transcrição/genética , Animais , Sequência de Bases , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Poliendocrinopatias Autoimunes/genética , RNA/isolamento & purificação , Proteína AIRE
6.
J Autoimmun ; 13(3): 307-13, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10550218

RESUMO

AIRE, the gene responsible for the autoimmune polyglandular syndrome type 1 (APS1) or APECED, may act as a transcription factor according to its predicated protein structure. Here we demonstrate the low expression level of the mouse Aire gene as it is undetectable by Northern blot analyses. However, RT-PCR analyses revealed expression of Aire in mouse thymus, ovary, lung, testis, kidney and adrenal gland. Barely detectable level of RT-PCR product was also found in thyroid gland and heart but no amplification was detected in pancreas, spleen and liver. Competitive RT-PCR assays demonstrated highest expression level of Aire mRNA in thymus. In addition to the complete cDNA (Aire or Aire-1a), we identified 11 alternative splicing forms (designated as Aire-1b, Aire-1c, Aire-1d, Aire-2a, Aire-2b, Aire-2c, Aire-2d, Aire-3a, Aire-3b, Aire-3c and Aire-3d). These forms result from combinations of four alternative splicing units (exon 10, exon 11, 12 bp in exon 6 and 3 bp in exon 8). The relative abundance of these splicing forms was also determined.


Assuntos
Fatores de Transcrição/genética , Processamento Alternativo , Animais , Sequência de Bases , Éxons , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/metabolismo , Distribuição Tecidual , Fatores de Transcrição/biossíntese , Proteína AIRE
7.
Am J Med Genet ; 84(5): 454-9, 1999 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-10360399

RESUMO

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disorder characterized by abnormal facial expression and urinary abnormalities. Previously, we mapped the gene to a genomic interval of approximately 1 cM on chromosome region 10q23-24, using families from Columbia. Here we demonstrate genetic homogeneity of the syndrome through homozygosity mapping in American patients with Irish heritage. We established a physical map and identified novel polymorphic markers in the UFS critical region. Haplotype analysis using the new markers mapped the UFS gene within one YAC clone of 1,410 kb. We also determined the precise location of the gene encoding for glutamate oxaloacetate transaminase (GOT1) within the new UFS critical region and determined its genomic structure. However, mutation analysis excluded GOT1 as a candidate for the UFS gene.


Assuntos
Anormalidades Múltiplas/genética , Aspartato Aminotransferases/genética , Cromossomos Humanos Par 10/genética , Face/anormalidades , Mapeamento Físico do Cromossomo , Sistema Urinário/anormalidades , Sequência de Bases , Análise Mutacional de DNA , Éxons , Genes Recessivos , Haplótipos , Homozigoto , Humanos , Íntrons , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Síndrome
8.
Diabetes ; 48(1): 219-22, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9892248

RESUMO

A new viral sequence (IDDMK(1,2)22) similar to the human endogenous retrovirus (HERV)-K10/K18 subfamilies has recently been isolated from the culture supernatants of leukocyte-infiltrated islets from two patients who died at the onset of type 1 diabetes. It was claimed that this endogenous retrovirus is expressed in patients with type 1 diabetes but not in healthy control subjects, suggesting an important role of the retrovirus in beta-cell-specific autoimmunity that results in type 1 diabetes. However, despite exhaustive attempts involving identical and expanded methods of detection, we did not observe the IDDMK(1,2)22 viral sequence in genomic DNA, lymphocyte, or plasma RNA in any subject. Therefore, we believe that the viral sequence is not derived from an endogenous retrovirus and that a role for the retrovirus in the pathogenesis of type 1 diabetes must be reconsidered.


Assuntos
DNA Viral/análise , Diabetes Mellitus Tipo 1/virologia , RNA Mensageiro/análise , Retroviridae/imunologia , Retroviridae/isolamento & purificação , Superantígenos/análise , Adolescente , Adulto , Criança , Diabetes Mellitus Tipo 1/genética , Feminino , Genoma Viral , Humanos , Masculino , Proteínas de Membrana
9.
Yi Chuan Xue Bao ; 26(5): 439-46, 1999.
Artigo em Chinês | MEDLINE | ID: mdl-10665219

RESUMO

X-linked, early onset Pelizaeus-Merzbacher disease (PMD) and part of X-linked spastic paraplegia are caused by mutation of proteolipid protein. M6b (U45955) partially cloned by Olinsky was considered as a member of PLP gene family. One novel fragment about 300 bp partially overlapped but differed in 5'part with U45955 was obtained by nested PCR. Assembly of the novel sequence with U45955 make a 1.642kb cDNA sequence with an open reading frame encoding 265 amino acids, which was verified by sequence of PCR products from brain cDNA library. The cDNA (termed M6ba) and its deduced peptide sequence showed significant similarity to murine M6b gene and protein (91.2% and 93.4% respectively). Northern blot, PCR amplification in cDNA library and EST analysis indicated that human M6b gene has at least three splicing forms. M6ba also showed significant similarity to PLP gene, they encode strongly hydrophobic protein and all their hydrophobic region are highly conserved. Gene structure analysis showed that the coding region of M6ba was composed of seven exons.


Assuntos
DNA Complementar/genética , Proteolipídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Splicing de RNA
10.
Chin Med J (Engl) ; 107(4): 257-9, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8088191

RESUMO

A successful method for constructing human chromosome 8q24.1 band specific probe pool by microdissection and microcloning is described in this paper. The probe pool was proved to be band-specific by fluorescence in situ hybridization. Through Southern blot analysis, 34 single copy DNA segments were obtained and four of them were sequenced. It is possible to provide useful landmarks for constructing human chromosome 8q24.1 physical map and STSs map.


Assuntos
Cromossomos Humanos Par 8 , Análise de Sequência de DNA , Sequência de Bases , Southern Blotting , DNA/genética , Humanos , Hibridização In Situ , Dados de Sequência Molecular
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