RESUMO
Randomly amplified polymorphic DNA (RAPD) analysis is a DNA polymorphism assay commonly used for fingerprinting genomes. After optimizing the reaction conditions, samples of Escherichia coli H10407 DNA were assayed to determine the influence of osmotic and/or oligotrophic stress on variations in RAPD banding patterns. Genetic rearrangements or DNA topology variations could be detected as changes in agarose gel electrophoresis banding profiles. A new amplicon generated using DNA extracted from bacteria prestarved by an osmotic stress and resuscitated in rich medium was observed. Enrichment improved recovery of mutator cells and allowed them to be detected in samples, suggesting that DNA modifications, such as stress-induced alterations and supercoiling phenomena, should be taken into consideration before beginning RAPD analyses.
Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Rearranjo Gênico , Genes Bacterianos , Sequência de Bases , Impressões Digitais de DNA , Primers do DNA/genética , Variação Genética , Pressão Osmótica , Fenótipo , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
In the past 10 years, much attention has been focused on transcription preinitiation complex formation as a target for regulating gene expression, and other targets such as transcription termination complex assemblage have been less intensively investigated. We established the existence of poly(A) site choice and fusion splicing of two adjacent genes, galactose-1-phosphate uridylyltransferase (GALT) and interleukin-11 receptor alpha-chain (IL-11Ralpha), in normal human cells. This 16-kilobase (kb) transcription unit contains two promoters (the first one is constitutive, and the second one, 8 kb downstream, is highly regulated) and two cleavage/polyadenylation signals separated by 12 kb. The promoter from the GALT gene yields two mRNAs, a 1.4-kb mRNA encoding GALT and a 3-kb fusion mRNA when the first poly(A) site is spliced out and the second poly(A) is used. The 3-kb mRNA codes for a fusion protein of unknown function, containing part of the GALT protein and the entire IL-11Ralpha protein. The GALT promoter/IL-11Ralpha poly(A) transcript results from leaky termination and alternative splicing. This feature of RNA polymerase (pol) II transcription, which contrasts with efficient RNA pol I and pol III termination, may be involved, together with chromosome rearrangements, in the generation of fusion proteins with multiple domains and would have major evolutionary implications in terms of natural processes to generate novel proteins with common motifs. Our results, together with accumulation of genomic informations, will stimulate new considerations and experiments in gene expression studies.
Assuntos
Cromossomos Humanos Par 9 , Splicing de RNA , Receptores de Interleucina/genética , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Animais , Células COS , Mapeamento Cromossômico , Evolução Molecular , Humanos , Subunidade alfa de Receptor de Interleucina-11 , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores de Interleucina-11 , Transcrição GênicaRESUMO
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder displaying a large spectrum of symptoms. Linkage studies have shown two loci, TSC1 in 9q34 and TSC2 in 16p13.3, to be involved in the disease. The TSC2 gene, composed of 41 exons, has been isolated and is shown to encode a protein, tuberin, from a 5.5-kb transcript. Mutation screening for both clinical diagnosis and identification of functional domains within the tuberin is in progress. In this study we identify a 33-bp in-frame deletion (1462del33) in the mRNA which segregates in two unrelated French families with severe TSC phenotypes. The corresponding 11 amino acids deletion (aa 482-492) is shown to result from two different splice site mutations at exon 14 and, when compared with the position of two previously described missense mutations, indicates a novel functionally important region of the protein.
Assuntos
Splicing de RNA/genética , Proteínas Repressoras/genética , Deleção de Sequência/genética , Esclerose Tuberosa/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , Éxons/genética , Feminino , França , Heterogeneidade Genética , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de TumorRESUMO
LCN1 gene encodes the tear lipocalin; the lipocalins are a large and growing family of proteins characterized by their ability to bind small hydrophobic molecules. We report here the location of a dinucleotide repeat microsatellite marker (D9S1826) close to LCN1 gene. Using the CEPH reference families, the position of LCN1 is located within the 9q34 genetic map between D9S23 and D9S158.
