RESUMEN
The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal gamma-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Regulación de la Expresión Génica , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Tubulina (Proteína)/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Ratones , Modelos Biológicos , Células PC12 , RatasRESUMEN
The Incontinentia Pigmenti (IP) locus contains the IKBKG/NEMO/IKKgamma gene and its truncated pseudogene copy, IKBKGP/deltaNEMO. The major genetic defect in IP is a heterozygous exon4_10 IKBKG deletion (IKBKGdel) caused by a recombination between two consecutive MER67B repeats. We analyzed 91 IP females carrying the IKBKGdel, 59 of whom carrying de novo mutations (65%). In eight parents, we found two recurrent nonpathological variants of IP locus, which were also present as rare polymorphism in control population: the IKBKGPdel, corresponding to the exon4_10 deletion in the pseudogene, and the MER67Bdup, that replicates the exon4_10 region downstream of the normal IKBKG gene. Using quantitative DNA analysis and microsatellite mapping, we established that both variants might promote the generation of the pathological IKBKGdel. Indeed, in family IP-516, the exon4_10 deletion was repositioned in the same allele from the pseudogene to the gene, whereas in family IP-688, the MER67Bdup generated the pathological IKBKGdel by recombination between two direct nonadjacent MER67Bs. Moreover, we found an instance of somatic recombination in a MER67Bdup variant, creating the IKBKGdel in an IP male. Our data suggest that the IP locus undergoes recombination producing recurrent variants that might be "at risk" of generating de novo IKBKGdel by NAHR during either meiotic or mitotic division.
Asunto(s)
Cromosomas Humanos X/genética , Exones/genética , Duplicación de Gen , Quinasa I-kappa B/genética , Incontinencia Pigmentaria/genética , Eliminación de Secuencia , Secuencia de Bases , Familia , Femenino , Humanos , Quinasa I-kappa B/metabolismo , Incontinencia Pigmentaria/metabolismo , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , LinajeRESUMEN
BACKGROUND: The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members. RESULTS: During a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin. CONCLUSION: Our data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.
Asunto(s)
Genoma Humano , Familia de Multigenes , Telómero/genética , Animales , Mapeo Cromosómico , Cromosomas Humanos , Etiquetas de Secuencia Expresada , Humanos , Hibridación Fluorescente in Situ , Primates/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential modulator (NEMO), gene are the most common single cause of incontinentia pigmenti (IP) in females and anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males. The IKBKG gene, located in the Xq28 chromosomal region, encodes for the regulatory subunit of the inhibitor of kappaB (IkB) kinase (IKK) complex required for the activation of the NF-kB pathway. Therefore, the remarkably heterogeneous and often severe clinical presentation reported in IP is due to the pleiotropic role of this signaling transcription pathway. A recurrent exon 4_10 genomic rearrangement in the IKBKG gene accounts for 60 to 80% of IP-causing mutations. Besides the IKBKG rearrangement found in IP females (which is lethal in males), a total of 69 different small mutations (missense, frameshift, nonsense, and splice-site mutations) have been reported, including 13 novel ones in this work. The updated distribution of all the IP- and EDA-ID-causing mutations along the IKBKG gene highlights a secondary hotspot mutation in exon 10, which contains only 11% of the protein. Furthermore, familial inheritance analysis revealed an unexpectedly high incidence of sporadic cases (>65%). The sum of the observations can aid both in determining the molecular basis of IP and EDA-ID allelic diseases, and in genetic counseling in affected families.
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Quinasa I-kappa B/genética , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos X , ADN , Displasia Ectodérmica/genética , Femenino , Humanos , Quinasa I-kappa B/química , Incontinencia Pigmentaria/genética , Masculino , Ratones , Datos de Secuencia Molecular , Mosaicismo , Homología de Secuencia de AminoácidoRESUMEN
Two-dimensional target characterization using inverse profiling approaches with total-field phaseless data is discussed. Two different inversion schemes are compared. In the first one, the intensity-only data are exploited in a minimization scheme, thanks to a proper definition of the cost functional. Specific normalization and starting guess are introduced to avoid the need for global optimization methods. In the second scheme [J. Opt. Soc. Am. A21, 622 (2004)], one exploits the field properties and the theoretical results on the inversion of quadratic operators to derive a two-step solution strategy, wherein the (complex) scattered fields embedded in the available data are retrieved first and then a traditional inverse scattering problem is solved. In both cases, the analytical properties of the fields allow one to properly fix the measurement setup and identify the more convenient strategy to adopt. Also, indications on the number and types of sources and receivers to be used are given. Results from experimental data show the efficiency of these approaches and the tools introduced.
RESUMEN
BACKGROUND: Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect. METHODS: We carried out on linkage-candidate gene studies in a new MRX family (MRX87). All coding regions and exon-intron boundaries of ARX gene were analysed by direct sequencing. RESULTS: MRX87 patients had moderate to profound cognition impairment and a combination of minor congenital anomalies. The disease locus, MRX87, was mapped between DXS7104 and DXS1214, placing it in Xp22-p21 interval, a hot spot region for mental handicap. An in frame duplication of 24 bp (ARXdup24) in the second polyAlanine tract (polyA_II) in ARX was identified. CONCLUSION: Our study underlines the role of ARXdup24 as a critical mutational site causing mental retardation linked to Xp22. Phenotypic heterogeneity of MRX87 patients represents a new observation relevant to the functional consequences of polyAlanine expansions enriching the puzzling complexity of ARXdup24-linked diseases.
Asunto(s)
Cromosomas Humanos X , Duplicación de Gen , Ligamiento Genético , Proteínas de Homeodominio/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Péptidos/genética , Factores de Transcripción/genética , Cartilla de ADN , Humanos , Masculino , Mutación , Linaje , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The association between premature ovarian failure (POF) and the FMR1 repeat number (41> CGGn< 200) has been widely investigated. Current findings suggest that the risk estimation for POF can be calculated in the offspring of women with pre-mutated FMR1 alleles. CASE PRESENTATION: We describe the coexistence in a large Italian kindred of Fragile X syndrome and familial POF in females with ovarian dysfunctions who carried normal or expanded FMR1 alleles. Genetic analysis of the FMR1 gene in over three generations of females revealed that six carried pre-mutated alleles (61-200), of which two were also affected by POF. However a young woman, who presented a severe ovarian failure with early onset, carried normal FMR1 alleles (<40). The coexistence within the same family of two dysfunctional ovarian conditions, one FMR1-related and one not FMR1-related, suggests that the complexity of familial POF conditions is larger than expected. CONCLUSION: Our case study represents a helpful observation and will provide familial cases with heterogeneous etiology that could be further studied when candidate genes in addition to the FMR1 premutation will be available.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Insuficiencia Ovárica Primaria/genética , Adulto , Anciano , Alelos , Femenino , Síndrome del Cromosoma X Frágil/complicaciones , Genotipo , Humanos , Italia , Masculino , Persona de Mediana Edad , Linaje , Insuficiencia Ovárica Primaria/complicaciones , Expansión de Repetición de Trinucleótido , Inactivación del Cromosoma XRESUMEN
Carnitine is a molecule with well-documented pleiotropic functions whose biosynthesis involves four catalytic steps. Here, we report a detailed analysis of the expression and transcriptional control of TMLH gene, which codifies for the first enzyme of carnitine biosynthesis. TMLH maps at the extreme end of Xq28, a chromosomal region of high genomic instability. By 5' and 3' RACE, we identified and mapped two alternative 5' TMLH first exons and seven alternative 3'-splice variants, which are spread over a genomic region of about 250 kb. While the two alternative 5' exons have different expression profiles, all the 3' alternative forms are ubiquitously expressed. Reporter assays revealed that the 3'-UTRs of each TMLH isoform might influence its own expression at post-transcriptional level. In addition, we identified a highly conserved promoter region of TMLH. Functional analysis of this region showed the presence of a CpG island, whose methylation-status could control the level of TMLH transcription. Finally, by mRNA in situ hybridization, we found that TMLH expression is present at E12.5 dpc in the mouse liver, lung and brain, and is then maintained in the postnatal brain with a specific neuronal pattern. Collectively, our data highlight a tight transcriptional and post-transcriptional control of TMLH expression.
Asunto(s)
Empalme Alternativo , Oxigenasas de Función Mixta/genética , Regiones Promotoras Genéticas , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Encéfalo/embriología , Encéfalo/metabolismo , Carnitina/biosíntesis , Mapeo Cromosómico , Cromosomas Humanos X/genética , Islas de CpG , Cartilla de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Variación Genética , Humanos , Hibridación in Situ , Ratones , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia MolecularRESUMEN
Applicability of inverse scattering based imaging procedures can be broadened by developing new approaches exploiting only amplitude data. As a matter of fact, this can open the way to simpler and less expensive measurement set-ups. In this respect, a two-step based procedure for solving electromagnetic nonlinear inverse scattering problems from only amplitude measurements of the total field has been recently proposed [1,2]. However, in these latter both amplitude and phase of the incident field are still required. In this contribution, we show the possibility of achieving this information from the measured amplitude distribution of the incident field on the observation domain. In particular, a three steps imaging technique which exploits only amplitude measurements of the total and incident fields has been developed. The proposed procedure has been tested against benchmark experimental data available in the literature. The obtained results fully confirm the possibility of achieving faithful reconstructions of unknown targets without performing any phase measurements and any approximation on the scattering equations involved in the inverse scattering problems.
RESUMEN
A new solution approach to inverse scattering from aspect-limited phaseless measurements of the total field is introduced and discussed. In analogy with the case of measurements on closed curves [J. Opt. Soc. Am. A21, 622 (2004)], the procedure splits the problem into two different steps. In the first step, amplitude and phase of the scattered field are estimated from only amplitude information of the total field. By properly extending the concept of reduced radiated field to the case of scattered fields (as a function of both illumination and measurement variables) and taking advantage of the properties of the square amplitude distribution of the total field, criteria are given for an optimal choice of the measurement setup and a successful retrieval. Then the complex permittivity profile is reconstructed in the second step, starting from the scattered fields estimated in the previous step. Numerical examples are provided to assess the effectiveness of the whole chain in the presence of noise-corrupted data and the relevance of the representation introduced for the scattered fields.
RESUMEN
BACKGROUND: Deletions of Xq chromosome are reported for a number of familial conditions exhibiting premature ovarian failure (POF) and early menopause (EM). METHODS AND RESULTS: We describe the inheritance of an interstitial deletion of the long arm of the X chromosome associated with either POF or EM in the same family. Cytogenetic studies and heterozygosity mapping by quantitative fluorescent PCR revealed a 46,X,del(X)(q26.2-q28) karyotype in a POF female, in her EM mother, and also in her aborted fetus with severe cardiopathy. Applying a microsatellite approach, we have narrowed the extension of an identical interstitial deletion located between DXS1187 and DXS1073. These data, in line with other mapped deletions, single out the proximal Xq28 as the region most frequently involved in ovarian failure. We also propose that other factors may influence the phenotypic effect of this alteration. Indeed, skewed X inactivation has been ascertained in EM and POF to be associated with different X haplotypes. CONCLUSION: Our analysis indicates that Xq26.2-q28 deletion is responsible for gonad dysgenesis in a family with EM/POF. The dissimilar deletion penetrance may be due to epigenetic modifications of other X genes that can contribute to human reproduction, highlighting that ovarian failure should be considered as a multifactorial disease.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos X/genética , Menopausia Prematura/genética , Insuficiencia Ovárica Primaria/genética , Adulto , Mapeo Cromosómico , Femenino , Tamización de Portadores Genéticos , Genotipo , Humanos , Cariotipificación , Repeticiones de Microsatélite , Linaje , Reacción en Cadena de la Polimerasa/métodos , Inactivación del Cromosoma XRESUMEN
epsilon-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Fe(++) and 2-oxoglutarate (2OG) dependent, catalyzing the first of four enzymatic reactions of the highly conserved carnitine biosynthetic pathway. Otherwise from all the other enzymes of carnitine biosynthesis, TMLH was found to be associated to the mitochondrial fraction. We here report molecular cloning of two alternative spliced forms of TMLH, which appear ubiquitously expressed in human adult and fetal tissues. The deduced proteins are designated TMLH-a and TMLH-b, and contain 421 and 399 amino acids, respectively. They share the first N-terminal 332 amino acids, including a mitochondrial targeting signal, but diverge at the C-terminal end. TMLH-a and TMLH-b exogenous expression in COS-1 cells shows that the first 15 amino acids are necessary and sufficient for mitochondrial import. Furthermore, comparative evolutionary analysis of the C-terminal portion of TMLH-a identifies a conserved domain characterized by a key triad of residues, His242-Glu244-His389 predicted to bind 2OG end. This sequence is conserved in the TMLH enzyme from all species but is partially substituted by a unique sequence in the TMLH-b variant. Indeed, TMLH-b is not functional by itself as well as a TMLH-H389L mutant produced by site directed mutagenesis. As great interest, we found that TMLH-b and TMLH-H389L, individually co-expressed with TMLH-a in COS-1 cells, negatively affect TMLH activity. Therefore, our studies on the TMLH alternative form provide relevant novel information, first that the C-terminal region of TMLH contains the main determinants for its enzymatic activity including a key H389 residue, and second that TMLH-b could act as a crucial physiological negative regulator of TMLH.
Asunto(s)
Variación Genética , Mitocondrias/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Células COS , Catálisis , Dominio Catalítico , Chlorocebus aethiops , ADN Complementario/genética , Perfilación de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Mitocondrias/enzimología , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Transporte de Proteínas , Alineación de SecuenciaAsunto(s)
Proteínas Portadoras , Incontinencia Pigmentaria/genética , Proteínas/genética , Femenino , Humanos , Quinasa I-kappa B , Lactante , TurquíaRESUMEN
Incontinentia Pigmenti (IP) is an X-linked genodermatosis that is lethal for males and present in females with abnormal skin pigmentation and high variable clinical signs, including retinal detachment, anodontia, alopecia, nail dystrophy and nervous system defects. The NF-kappaB essential modulator (NEMO) gene, responsible for IP, encodes the regulatory subunit of the IkappaB kinase (IKK) complex required for nuclear factor kappaB (NF-kappaB) activation. We analyzed the NEMO gene in 122 IP patients and identified mutations in 83 (36 familiar and 47 sporadic cases). The recurrent NEMO exon 4-10 deletion that is the major cause of the disease was present in 73 females (59.8%). In addition 10 point alterations (8.2% of females) were identified: three frameshift, three nonsense, three missense and one in-frame deletion of a single amino acid. We measured the effects of these NEMO point-mutations on NF-kappaB signaling in nemo(-/-) deficient murine pre-B cells. A mutation in the N-terminal domain, required for IKK assembly, reduced but did not abolish NF-kappaB activation following lipopolysaccharide stimulation. Mutations that disrupt the C-terminal domain, required for the recruitment of upstream factors, showed lower or no NF-kappaB activation. A phenotype score based on clinical features of our IP patients was applied for summarizing disease severity. The score did not correlate with mutation type or domain affected indicating that other factors influence the severity of IP. Such a factor is likely to be X-inactivation. Indeed, 64% of our patients have extremely skewed X-inactivation pattern (>/=80 : 20). Overall IP pathogenesis thus depends on a combination of X-inactivation and protein domain that recruit upstream factors and activate NF-kappaB.
Asunto(s)
Proteínas Portadoras/genética , Incontinencia Pigmentaria/genética , Mutación/genética , FN-kappa B/metabolismo , Fenotipo , Animales , Secuencia de Bases , Western Blotting , Proteínas Portadoras/metabolismo , Células Cultivadas , Compensación de Dosificación (Genética) , Exones/genética , Componentes del Gen , Prueba de Complementación Genética , Humanos , Quinasa I-kappa B , Inmunoprecipitación , Lipopolisacáridos , Ratones , Mutagénesis Sitio-Dirigida , Linaje , Plásmidos/genética , Transducción de Señal/genética , TransfecciónRESUMEN
In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes. Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors. Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies: RG-SNAREs and RD-SNAREs. The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters.
Asunto(s)
Secuencias de Aminoácidos , Proteínas de Transporte Vesicular/química , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos/genética , Sustitución de Aminoácidos/genética , Animales , Secuencia de Consenso/genética , Secuencia Conservada/genética , Células Eucariotas/química , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas SNARE , Homología de Secuencia de Aminoácido , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Proteínas de Transporte Vesicular/genéticaRESUMEN
A new approach for quantitative electromagnetic imaging of scatterers located in free space from phaseless data is proposed and discussed. The procedure splits the problem into two steps. In the first one, we solve a phase-retrieval problem for the total field, thus estimating the amplitude and phase of the scattered field. Careful analysis of properties and possible representations of both scattered and incident fields allow us to introduce a criterion for an optimal choice of the measurement setup and a successful retrieval. Then the complex permittivity profile is reconstructed in the second step by use of the estimated scattered field. Numerical examples are provided to check the whole chain in the presence of noise-corrupted data.
RESUMEN
We recently published the genomic characterization of the STAT5A and STAT5B paralogous genes that are located head to head in the 17q21 chromosome and share large regions of sequence identity. We here demonstrate by transient in vitro transfection that STAT5A and STAT5B promoters are able to direct comparable levels of transcription. The expression of basal promoters is enhanced after Sp1 up-regulation in HeLa and SL2 cells while DNA methylation associated to the recruitment of MeCP2 methyl CpG binding protein down-regulates STAT5A and B promoters by interfering with Sp1-induced transcription. In addition, cross-species sequence comparison identified a bi-directional negative cis-acting regulatory element located in the STAT5 intergenic region.
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Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas de la Leche , Regiones Promotoras Genéticas , Transactivadores/genética , Transcripción Genética , Animales , Sitios de Unión , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Ratones , Factor de Transcripción STAT5 , Análisis de Secuencia de ADN , Transactivadores/metabolismo , Proteínas Supresoras de TumorRESUMEN
The synaptobrevin-like 1 (SYBL1) gene is ubiquitously expressed and codes for an unusual member of the v-SNAREs molecules implicated in cellular exocytosis. This X-linked gene has the peculiarity of also being present on the Y chromosome in a transcriptional inactive status. Moreover, although ubiquitous, the function of SYBL1 is prominent in specific tissues, such as brain. As a first insight into the molecular mechanisms controlling SYBL1 expression, in this report we describe the extent and role of SYBL1 upstream regions and characterize the binding of trans-acting factors. In vivo foot-printing experiments identify three protected regions. Band shift and transient reporter gene assays indicate a strong role of two of these evolutionary conserved regions in regulating SYBL1 transcription. Because one site is the classical CAAT box, we characterized the binding to the other site of the mammalian homologues of the selenocysteine tRNA gene transcription activating factor (Staf) family, zinc-finger transcription factors, and their role in regulating SYBL1 expression. The results reported here clarify that a Staf-zinc finger family factor, together with the CAAT factor, is the major nuclear protein bound to the SYBL1 promoter region and is responsible for its regulation in HeLa cells, thus identifying the basic control of SYBL1 transcription. In vivo binding of Staf proteins to the SYBL1 promoter is confirmed by chromatin immunoprecipitation assays. Our results identify a fourth mRNA promoter stimulated by a member of the Staf-zinc finger family, the function of which on mRNA polymerase II promoters is still very poorly understood.
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Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/genética , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Secuencia Conservada , ADN/química , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas/genética , Proteínas R-SNARE , Relación Estructura-Actividad , Transactivadores/química , Transactivadores/genética , TransfecciónRESUMEN
Otosclerosis is the single most common cause of hearing impairment among adult caucasians. Little is known about its aetiology and its molecular aspects. Until now, genetic linkage in otosclerosis has been demonstrated in an Indian family and a Belgian family, showing the presence of two otosclerosis loci, OTSC1 and OTSC2, respectively. Linkage analysis has never been applied to Italian otosclerotic families. We have collected four multigenerational Italian otosclerotic families that show dominant transmission for the pathology. Here, we report a detailed audiological analysis of these families and a genetic linkage study on the OTSC1 and OTSC2 loci. Statistical analysis revealed the absence of linkage between the disease in our families and the OTSC1 and OTSC2 loci. These data strongly suggest the presence of one or more additional loci for otosclerosis, which still need to be defined.