RESUMO
Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.
Assuntos
Asma/genética , Genes Reporter/genética , Proteínas Imediatamente Precoces , Lipoxigenase/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Transcrição Gênica/genética , Alelos , Sequência de Bases , Códon de Iniciação , Primers do DNA , Proteínas de Ligação a DNA/genética , Hipersensibilidade a Drogas/genética , Proteína 1 de Resposta de Crescimento Precoce , Éxons , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Recombinação Genética , Proteínas Oncogênicas de Retroviridae/genética , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Our data demonstrate the presence of a naturally occurring family of alleles in the core promoter of the 5-LO gene, which is characterized by the deletion or addition of consensus Sp1 (-GGGCGG) and Egr-1 (-GCGGGGGCG-) binding motifs. Each of the variant alleles can bind Sp1 and Egr-1 protein, as indicated by EMSA and supershift analysis with nuclear extracts. In addition, preliminary data from CAT reporter assays indicate that these alleles are less effective than the wild-type allele in initiating 5-LO gene expression. Whether patients harboring the various alleles identified herein have different capacities to transcribe the 5-LO gene and the importance of such potential regulation to the clinical expression of 5-LO have yet to be determined.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Mutação/genética , Regiões 5' não Traduzidas/genética , Adulto , Alelos , Asma/enzimologia , Asma/genética , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Feminino , Genes Reporter , Haplótipos , Humanos , Masculino , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genéticaRESUMO
Multiple highly polymorphic markers have been used to construct a genetic map of the q12-q13.1 region of chromosome 20 and to map the location of the maturity-onset diabetes of the young (MODY) locus. The genetic map encompasses 23 cM and includes 11 loci with PIC values > .50, seven of which have PICs > .70. New dinucleotide repeat polymorphisms associated with the D20S17, PPGB, and ADA loci have been identified and mapped. The dinucleotide repeat polymorphisms have increased the PIC of the ADA locus to .89 and, with an additional RFLP at the D20S17 locus, the PIC of the D20S17 locus to .88. The order of the D20S17 and ADA loci determined genetically (cen-ADA-D20S17-qter) was confirmed by multicolor fluorescence in situ hybridization. The previously unmapped PPGB marker is closely linked to D20S17, with a two-point lod score of 50.53 at theta = .005. These markers and dinucleotide repeat markers associated with the D20S43, D20S46, D20S55, D20S75, and PLC1 loci and RFLPs at the D20S16, D20S17, D20S22, and D20S33 have been used to map the MODY locus on chromosome 20 to a 13-cM (sex averaged) interval encompassing ADA, D20S17, PPGB, D20S16, and D20S75 on the long arm of chromosome 20 and to create a genetic framework for additional genetic and physical mapping studies of the region. With these multiple highly polymorphic loci, any MODY family of appropriate size can be tested for the chromosome 20 linkage.