RESUMEN
CONTEXT: Beckwith-Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer. OBJECTIVE: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS. METHODS: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease. RESULTS: Both qPCR and RPA clearly demonstrated a marked decrease (86-93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter. CONCLUSIONS: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.
Asunto(s)
Síndrome de Beckwith-Wiedemann/genética , Metilación de ADN , Inhibidores Enzimáticos/metabolismo , Silenciador del Gen/fisiología , Impresión Genómica/genética , Proteínas de la Membrana , Proteínas Nucleares/genética , Síndrome de Beckwith-Wiedemann/enzimología , Línea Celular , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Fibroblastos/química , Regulación de la Expresión Génica/genética , Humanos , Canales de Potasio con Entrada de Voltaje , ARN Largo no Codificante , ARN no Traducido/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
We have identified a number of transcribed sequences within a 400-kb interval on chromosome 11p15.1--> p14. Six genes and 13 novel transcripts including ESTs, cDNAs and exons have been identified and assigned to this region. Comparison of mRNA sequence with genomic sequence has enabled us to determine the exon/intron structure of four of the genes (NUCB2, PIK3C2A, RPS13 and OR7E14P).
Asunto(s)
Cromosomas Humanos Par 11/genética , Genes , Mapeo Físico de Cromosoma , Transcripción Genética/genética , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Exones/genética , Etiquetas de Secuencia Expresada , Orden Génico/genética , Humanos , Hibridación Fluorescente in Situ , Intrones/genética , Familia de Multigenes/genética , Seudogenes/genética , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
In an effort to control the variability of transgene expression in plants, we used Cre-lox mediated recombination to insert a gus reporter gene precisely and reproducibly into different target loci. Each integrant line chosen for analysis harbors a single copy of the transgene at the designated target site. At any given target site, nearly half of the insertions give a full spatial pattern of transgene expression. The absolute level of expression, however, showed target site dependency that varied up to 10-fold. This substantiates the view that the chromosome position can affect the level of gene expression. An unexpected finding was that nearly half of the insertions at any given target site failed to give a full spatial pattern of transgene expression. These partial patterns of expression appear to be attributable to gene silencing, as low gus expression correlates with DNA methylation and low transcription. The methylation is specific for the newly integrated DNA. Methylation changes are not found outside of the newly inserted DNA. Both the full and the partial expression states are meiotically heritable. The silencing of the introduced transgenes may be a stochastic event that occurs during transformation.
Asunto(s)
Silenciador del Gen , Genes de Plantas , Transgenes , Alelos , Metilación de ADN , ADN de Plantas/genética , Expresión Génica , Genes Reporteros , Genoma de Planta , Glucuronidasa/genética , Meiosis , Modelos Genéticos , Mutagénesis Insercional , Plantas Tóxicas , ARN Mensajero/genética , ARN de Planta/genética , Recombinación Genética , Distribución Tisular , Nicotiana/genética , Transformación GenéticaRESUMEN
Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.
Asunto(s)
Síndrome de Beckwith-Wiedemann/genética , Cromosomas Humanos Par 15 , ADN sin Sentido/genética , Fosfatos de Dinucleósidos/análisis , Impresión Genómica , Proteínas de la Membrana , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , ADN/genética , Metilación de ADN , Femenino , Humanos , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Síndrome de QT Prolongado/genética , Linfocitos/fisiología , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The APETALA3 floral homeotic gene is required for petal and stamen development in Arabidopsis. APETALA3 transcripts are first detected in a meristematic region that will give rise to the petal and stamen primordia, and expression is maintained in this region during subsequent development of these organs. To dissect how the APETALA3 gene is expressed in this spatially and temporally restricted domain, various APETALA3 promoter fragments were fused to the uidA reporter gene encoding beta-glucuronidase and assayed for the resulting patterns of expression in transgenic Arabidopsis plants. Based on these promoter analyses, we defined cis-acting elements required for distinct phases of APETALA3 expression, as well as for petal-specific and stamen-specific expression. By crossing the petal-specific construct into different mutant backgrounds, we have shown that several floral genes, including APETALA3, PISTILLATA, UNUSUAL FLORAL ORGANS, and APETALA1, encode trans-acting factors required for second-whorl-specific APETALA3 expression. We have also shown that the products of the APETALA1, APETALA3, PISTILLATA and AGAMOUS genes bind to several conserved sequence motifs within the APETALA3 promoter. We present a model whereby spatially and temporally restricted APETALA3 transcription is controlled via interactions between proteins binding to different domains of the APETALA3 promoter.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Proteínas de Dominio MADS , Regiones Promotoras Genéticas/genética , Secuencia de Bases , ADN de Plantas/metabolismo , Genes de Plantas/genética , Genes Reporteros , Glucuronidasa/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Unión Proteica , Homología de Secuencia de Ácido Nucleico , Transactivadores/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
Human chromosomal band 11p15.5 has been shown to contain genes involved in the development of several pediatric and adult tumors and in Beckwith-Wiedemann syndrome (BWS). Overlapping P1 artificial chromosome clones from this region have been used as templates for genomic sequencing in an effort to identify candidate genes for these disorders. PowerBLAST identified several matches with expressed sequence tags (ESTs) from fetal brain and liver cDNA libraries. Northern blot analysis indicated that two of the genes identified by these ESTs encode transcripts of 1-1.5 kb with predominant expression in fetal and adult liver and kidney. With RT-PCR and RACE, full-length transcripts were isolated for these two genes, with the largest open reading frames encoding putative proteins of 253 and 424 amino acids. Database comparison of the predicted amino acid sequence of the larger transcript indicated homology to integral membrane organic cation transporters; hence, we designate this gene ORCTL2 (organic cation transporter-like 2). An expressed sequence polymorphism provided evidence that the ORCTL2 gene exhibits "leaky" imprinting in both human fetal kidney and human fetal liver. The mouse orthologue (Orctl2) was identified, and a similar polymorphism was used to demonstrate maternal-specific expression of this gene in fetal liver from interspecific F1 mice. The predicted protein of the smaller gene showed no significant similarity in the database. Northern and RACE analyses suggest that this gene may have multiple transcription start sites. Determination of the genomic structure in humans indicated that the 5'-end of this transcript overlaps in divergent orientation with the first two exons of ORCTL2, suggesting a possible role for antisense regulation of one gene by the other. We, therefore, provisionally name this second transcript ORCTL2S (ORCTL2-antisense). The expression patterns of these genes and the imprinted expression of ORCTL2 are suggestive of a possible role in the development of Wilms tumor (WT) and hepatoblastoma. Although SSCP analysis of 62 WT samples and 10 BWS patients did not result in the identification of any mutations in ORCTL2 or ORCTL2S, it will be important to examine their expression pattern in tumors and BWS patients, since epigenetic alteration at these loci may play a role in the etiology of these diseases.
Asunto(s)
Síndrome de Beckwith-Wiedemann/genética , Cromosomas Humanos Par 11 , Genes Sobrepuestos , Impresión Genómica , Proteínas de la Membrana , Transcripción Genética , Tumor de Wilms/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Análisis Mutacional de ADN , ADN Complementario , Humanos , Riñón/metabolismo , Hígado/metabolismo , Ratones , Datos de Secuencia MolecularRESUMEN
Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration. The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14-15.1 between D11S899 and D11S861. In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu-PCR hybridization. The YAC contig is approximately 3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1 )-MYOD1-D11S902D11S921-D11S 1890-TEL. Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921. To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu-PCR products generated from the YACs, and PAC end probes. A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping. Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced. PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes (BIR, SUR1) mapped previously to this region, and a previously characterized but unmapped gene NEFA (DNA binding/EF hand/acidic amino-acid-rich). GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality. These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts. Based on their map location, these loci represent candidate disease loci for USH1C. The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 11/genética , Genes , Pérdida Auditiva Conductiva/genética , Bacteriófago P1/genética , Cromosomas Artificiales de Levadura , Genoma Humano , Haplotipos , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Two models have been proposed to explain the coordinated development of the four whorls of floral organs. The spatial model predicts that positional information defines the four whorls simultaneously, and that individual organs develop independently of surrounding tissues. The sequential model suggests that inductive events between the outer and inner whorl primordia are required for appropriate organogenesis. To test these models we have genetically ablated second and third whorl floral organ primordia to determine if organ identity, number or position are perturbed in the first or fourth whorls. We used diphtheria toxin to specifically ablate floral cells early in development in Nicotiana tabacum and in Arabidopsis thaliana. Second and third whorl expression of the diphtheria toxin A chain coding sequence (DTA) was conferred by the Arabidopsis APETALA3 (AP3) promoter. Both Nicotiana and Arabidopsis flowers that express the AP3-DTA construct lack petals and stamens; it appears that the second and third whorl cells expressing this construct arrest early in floral development. These results show that first and fourth whorl development is normal and can proceed without information from adjacent second and third whorl primordia. We propose that positional information specifies the establishment of all four whorls of organs prior to the expression of AP3 in the floral meristem.
Asunto(s)
Comunicación Celular , Genes de Plantas , Meristema/fisiología , Modelos Biológicos , Secuencia de Aminoácidos , Arabidopsis/genética , Toxina Diftérica/farmacología , Meristema/efectos de los fármacos , Datos de Secuencia Molecular , Plantas Tóxicas , Nicotiana/genéticaRESUMEN
Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.