RESUMEN

Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9. To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown. We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin. By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein. Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments. We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of approximately 50 kDa. Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain. We have also shown that cochlin is N-glycosylated in its mature secreted form. Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain. Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity. To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins. We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation. These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain. It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder.

Asunto(s)

Pérdida Auditiva Sensorineural/genética , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Enfermedades Vestibulares/genética , Células 3T3 , Animales , Western Blotting , Células COS , Línea Celular , Proteínas de la Matriz Extracelular , Glicosilación , Humanos , Inmunohistoquímica , Ratones , Mutación , Mutación Missense , Plásmidos/genética , Proteínas/genética , Transfección

RESUMEN

Missense mutations in the COCH gene, which is expressed preferentially at high levels in the inner ear, cause the autosomal dominant sensorineural deafness and vestibular disorder, DFNA9 (OMIM 601369). By in situ hybridization of mouse and human inner ear sections, we find high-level expression of COCH mRNA in the fibrocytes of the spiral limbus and of the spiral ligament in the cochlea, and in the fibrocytes of the connective tissue stroma underlying the sensory epithelium of the crista ampullaris of the semicircular canals. A polyclonal antibody against the human COCH protein product, cochlin, was raised against the N-terminal 135 amino acid residues of cochlin, corresponding to the Limulus factor C-homology (cochFCH) domain; this domain harbors all five known point mutations in DFNA9. On western blots of human fetal cochlear extracts, anti-cochlin reacts with a cochlin band of the predicted full-length size as well as a smaller isoform. Immunohistochemistry performed with anti-cochlin shows staining predominantly in the regions of the fibrocytes of the spiral limbus and of the spiral ligament in mouse and in human fetal and adult tissue sections. These sites correspond to those areas that express COCH mRNA as determined by in situ hybridization, and to the regions of the inner ear which show histological abnormalities in DFNA9. The fibrocytes expressing mRNA and protein products of COCH are the very cell types which are either absent or markedly reduced and replaced by eosinophilic acellular material in temporal bone sections of individuals affected with DFNA9.

Asunto(s)

Oído Interno/metabolismo , Pérdida Auditiva Sensorineural/genética , Proteínas/metabolismo , ARN Mensajero/genética , Enfermedades Vestibulares/genética , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Oído Interno/química , Oído Interno/embriología , Proteínas de la Matriz Extracelular , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Mutación , Proteínas/genética , Proteínas/inmunología , ARN Mensajero/metabolismo , Distribución Tisular , Enfermedades Vestibulares/metabolismo , Enfermedades Vestibulares/patología

RESUMEN

We identified Eyes absent 4 (EYA4), a member of the vertebrate Eya family of transcriptional activators, as the causative gene of postlingual, progressive, autosomal dominant hearing loss at the DFNA10 locus. In two unrelated families from Belgium and the USA segregating for deafness at this locus, we found different mutations in EYA4, both of which create premature stop codons. Although EYA proteins interact with members of the SIX and DACH protein families in a conserved network that regulates early embryonic development, this finding shows that EYA4 is also important post-developmentally for continued function of the mature organ of Corti.

Asunto(s)

Sordera/genética , Pérdida Auditiva Sensorineural/genética , Transactivadores/genética , Edad de Inicio , Empalme Alternativo , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 6/genética , Cóclea/embriología , Cóclea/metabolismo , ADN/química , ADN/genética , Análisis Mutacional de ADN , Sordera/patología , Oído Interno/metabolismo , Femenino , Pérdida Auditiva Sensorineural/patología , Humanos , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos CBA , Mutación , Linaje , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

We have identified a novel cochlear gene, designated OTOR, from a comparative sequence analysis of over 4000 clones from a human fetal cochlear cDNA library. Northern blot analysis of human and chicken organs shows strong OTOR expression only in the cochlea; very low levels are detected in the chicken eye and spinal cord. Otor and Col2A1 are coexpressed in the cartilaginous plates of the neural and abneural limbs of the chicken cochlea, structures analogous to the mammalian spiral limbus, osseous spiral lamina, and spiral ligament, and not in any other tissues in head and body sections. The human OTOR gene localizes to chromosome 20 in bands p11.23-p12.1 and more precisely to STS marker WI-16380. We have isolated cDNAs orthologous to human OTOR in the mouse, chicken, and bullfrog. The encoded protein, designated otoraplin, has a predicted secretion signal peptide sequence and shows a high degree of cross-species conservation. Otoraplin is homologous to the protein encoded by CDRAP/MIA (cartilage-derived retinoic acid sensitive protein/melanoma inhibitory activity), which is expressed predominantly by chondrocytes, functions in cartilage development and maintenance, and has growth-inhibitory activity in melanoma cell lines.

Asunto(s)

Cóclea/metabolismo , Secuencia Conservada/genética , Mapeo Físico de Cromosoma , Proteínas/genética , Animales , Northern Blotting , Pollos , Cromosomas Humanos Par 20/genética , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Proteínas de la Matriz Extracelular , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias , Especificidad de Órganos , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/genética , ARN Mensajero/biosíntesis , Rana catesbeiana , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

Mutations in myosin VI (Myo6) cause deafness and vestibular dysfunction in Snell's waltzer mice. Mutations in two other unconventional myosins cause deafness in both humans and mice, making myosin VI an attractive candidate for human deafness. In this report, we refined the map position of human myosin VI (MYO6) by radiation hybrid mapping and characterized the genomic structure of myosin VI. Human myosin VI is composed of 32 coding exons, spanning a genomic region of approximately 70 kb. Exon 30, containing a putative CKII site, was found to be alternatively spliced and appears only in fetal and adult human brain. D6S280 and D6S284 flank the myosin VI gene and were used to screen hearing impaired sib pairs for concordance with the polymorphic markers. No disease-associated mutations were identified in twenty-five families screened for myosin VI mutations by SSCP analysis. Three coding single nucleotide polymorphisms (cSNPs) were identified in myosin VI that did not alter the amino acid sequence. Myosin VI mutations may be rare in the human deaf population or alternatively, may be found in a population not yet examined. The determination of the MYO6 genomic structure will enable screening of individuals with non-syndromic deafness, Usher's syndrome, or retinopathies associated with human chromosome 6q for mutations in this unconventional myosin.

Asunto(s)

Genes/genética , Cadenas Pesadas de Miosina/genética , Adulto , Secuencia de Bases , Encéfalo/embriología , Encéfalo/metabolismo , ADN/química , ADN/genética , Análisis Mutacional de ADN , Sordera/genética , Exones , Salud de la Familia , Feto , Frecuencia de los Genes , Humanos , Intrones , Mutación , Mutación Puntual , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple

RESUMEN

We report the genetic analysis of one large Belgian and two small Dutch families with autosomal dominant non-syndromic progressive sensorineural hearing loss associated with vestibular dysfunction. Linkage studies in the Belgian family mapped the disease to the DFNA9 locus on chromosome 14. Mutation analysis of the COCH gene, which is responsible for DFNA9, revealed a missense mutation changing a highly conserved residue. One of the patients, who had an earlier age of onset in comparison with most of the affected family members, was shown to be homozygous for the mutation. After the mutation was found in the Belgian family, we discovered that the same missense mutation was also present in two Dutch families with similar cochleo-vestibular symptoms. In all three families with hearing loss and imbalance problems, >25% of the patients showed additional symptoms, including episodes of vertigo, tinnitus, aural fullness and hearing loss. Clinically, these symptoms are consistent with the criteria for Menière's disease. The importance of genetic factors in Menière's disease has been suggested on many occasions, but this study is the first report of a mutation in a gene leading to the symptoms of Menière's disease in a significant portion of the carriers. The COCH gene may be one of the genetic factors contributing to Menière's disease and the possibility of a COCH mutation should be considered in patients with Menière's disease symptoms.

Asunto(s)

Enfermedad de Meniere/genética , Proteínas/genética , Cromosomas Humanos Par 14/genética , ADN/química , ADN/genética , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular , Salud de la Familia , Femenino , Ligamiento Genético , Pérdida Auditiva Sensorineural/genética , Humanos , Escala de Lod , Masculino , Enfermedad de Meniere/epidemiología , Enfermedad de Meniere/patología , Repeticiones de Microsatélite , Mutación , Linaje , Prevalencia

Asunto(s)

Sordera/genética , Audición/genética , Sordera/patología , Humanos

RESUMEN

We analysed a Dutch family with autosomal dominant non-syndromic progressive sensorineural hearing loss and mapped the underlying gene defect by genetic linkage analysis to a 11.0 cM region overlapping the DFNA9 interval on chromosome 14q12-q13. Clinically, the Dutch family differs from the original DFNA9 family by a later age at onset and a more clearly established vestibular impairment. A gene that is highly and specifically expressed in the human fetal cochlea and vestibule, COCH (previously described as Coch5B2 ), was mapped to the DFNA9 critical region. Sequence analysis revealed a 208C-->T mutation in the COCH gene, resulting in a Pro51Ser substitution in the predicted protein in all affected individuals of the family but not in unaffected family members and 200 control individuals. The same mutation was also identified in three apparently unrelated families with a similar phenotype, suggesting the presence of a Dutch founder mutation. The function of COCH is unknown but several characteristics of the protein point to a structural role in the extracellular matrix. The mutant serine at position 51 is situated between cysteines and possibly interferes with proper COCH protein folding or its interaction with extracellular matrix proteins.

Asunto(s)

Genes Dominantes/genética , Pérdida Auditiva Sensorineural/genética , Proteínas/genética , Enfermedades Vestibulares/genética , Edad de Inicio , Sustitución de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , ADN/química , ADN/genética , Análisis Mutacional de ADN , Sordera/genética , Proteínas de la Matriz Extracelular , Femenino , Ligamiento Genético , Pérdida Auditiva Sensorineural/complicaciones , Pérdida Auditiva Sensorineural/patología , Humanos , Masculino , Repeticiones de Microsatélite , Linaje , Mutación Puntual , Prolina/genética , Serina/genética , Enfermedades Vestibulares/complicaciones , Enfermedades Vestibulares/patología

RESUMEN

To identify candidate genes for human hearing disorders and to understand better human hearing at the molecular level, we constructed a human cochlear cDNA library. An aliquot of the unsubtracted cochlear library was contributed to the IMAGE Consortium at Lawrence Livermore National Laboratory for the generation of expressed sequence tags (ESTs) by the Merck/WashU EST project. Over 4000 ESTs were developed from the cochlear cDNA library and deposited in the GenBank EST database. Sequence clustering shows that the majority of clones are in low copy numbers, demonstrating the high complexity of the library. The sequences of 1388 cochlear ESTs (33%) match 517 known human genes. Among these are genes previously shown to cause both syndromic and non-syndromic hearing loss. A number of the cochlear ESTs show high homology to non-human genes, suggesting new gene family members or human homologs of animal genes. We also report the chromosomal map positions of 437 cochlear ESTs. These provide positional candidate genes for 18 different non-syndromic hearing disorders. A Human Cochlear EST Database web site (http://www.bwh.partners. org/pathology ) has been created to provide access to the cochlear clone data for gene discovery investigations.

Asunto(s)

Cóclea/metabolismo , Sordera/genética , Etiquetas de Secuencia Expresada , Expresión Génica , Animales , Mapeo Cromosómico , Cromosomas Humanos/genética , ADN Complementario/genética , Humanos

RESUMEN

DFNA9 is an autosomal dominant, nonsyndromic, progressive sensorineural hearing loss with vestibular pathology. Here we report three missense mutations in human COCH (previously described as Coch5b2), a novel cochlear gene, in three unrelated kindreds with DFNA9. All three residues mutated in DFNA9 are conserved in mouse and chicken Coch, and are found in a region containing four conserved cysteines with homology to a domain in factor C, a lipopolysaccharide-binding coagulation factor in Limulus polyphemus. COCH message, found at high levels in human cochlear and vestibular organs, occurs in the chicken inner ear in the regions of the auditory and vestibular nerve fibres, the neural and abneural limbs adjacent to the cochlear sensory epithelium and the stroma of the crista ampullaris of the vestibular labyrinth. These areas correspond to human inner ear structures which show histopathological findings of acidophilic ground substance in DFNA9 patients.

Asunto(s)

Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/fisiopatología , Mutación Missense , Proteínas/genética , Vestíbulo del Laberinto/fisiopatología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Secuencia Conservada , Cartilla de ADN/genética , Proteínas de la Matriz Extracelular , Femenino , Genes Dominantes , Pérdida Auditiva Sensorineural/patología , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Vestíbulo del Laberinto/patología

RESUMEN

We constructed and screened a human fetal cochlear cDNA library to identify genes involved in hearing and deafness. From this library we isolated a cDNA corresponding to the highly conserved ancient gene antiquitin (ATQ1). The plant homolog of ATQ1 is thought to be involved in regulating turgor pressure, a function that also would be essential for cells of the mammalian cochlea. Northern blots of 13 human fetal tissues show antiquitin to be highly expressed in cochlea, ovary, eye, heart, and kidney. Using RT-PCR of rat cochlear hair cell-specific cDNA libraries, we detect antiquitin expression in outer hair cells, but not in inner or vestibular type 1 hair cells, suggesting that antiquitin is not expressed ubiquitously in the cochlea. Human ATQ1 was mapped to human chromosome region 5q31 using fluorescence in situ hybridization, and mouse ATQ1 was mapped to mouse chromosome 18 by single-strand conformation polymorphism mapping of interspecific backcross progeny DNAs. Four human antiquitin-like sequences, possibly pseudogenes, were also identified and mapped.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Cromosomas Humanos Par 5 , Oído Interno/fisiología , Proteínas/genética , Proteínas/metabolismo , Aldehído Deshidrogenasa , Animales , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/genética , Mapeo Cromosómico , Cóclea/fisiología , Secuencia Conservada , ADN Complementario , Feto/metabolismo , Forminas , Biblioteca de Genes , Cabello/fisiología , Humanos , L-Aminoadipato-Semialdehído Deshidrogenasa , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ratas , Análisis de Secuencia , Distribución Tisular

RESUMEN

Previously we identified a partial human cDNA for a novel cochlear transcript, hCoch-5B2 (HGMW-approved symbol D14S564E), using subtractive hybridization techniques. Herein we report isolation and characterization of both human and mouse (D12H14S564E) cDNAs for Coch-5B2. Full-length Coch5B2 deduced amino acid sequences reveal a very high degree of conservation in the coding region (89% nucleotide and 94% amino acid identity and a potential signal peptide and two regions of extensive homology to the collagen-binding type A domains of von Willebrand factor, also present in other secreted proteins, including extracellular matrix components. High levels of hCoch-5B2 expression are seen only in human fetal inner ear structures, cochlea, and vestibule, among a large panel of human fetal and adult tissues. Coch-5B2 expression in the mouse is more widespread than in the human, with message detected in mouse adult spleen, cerebrum, cerebellum/medulla, and thymus. In both species very low level expression is detected in total eye. More specifically, mouse retina shows a higher level of mCoch-5B2 message than sclera and choroid. We have mapped hCoch-5B2 to human 14q11.2-q13 by somatic cell hybrid analysis and FISH and, more precisely, using radiation hybrids to a region of markers linked to DFNA9, a nonsyndromic autosomal dominant sensorineural hearing loss with vestibular defects. Furthermore, we detect hCoch-5B2 on three overlapping YACs, two of which also contain one of the markers linked to DFNA9. mCoch-5B2 was genetically mapped in the mouse to chromosome 12, in a region of homologous synteny with human 14q11.2-q13, which contains the asp1 (audiogenic seizure prone) locus in the mouse.

Asunto(s)

Cromosomas Humanos Par 14 , Sordera/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Conducto Coclear/metabolismo , ADN Complementario , Proteínas de la Matriz Extracelular , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

RESUMEN

We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with 32P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than with brain cDNA probes revealed some previously characterized genes, including mitochondrial sequences, collagen type I alpha-2 (COL1A2), collagen type II alpha-1 (COL2A1), collagen type III alpha-1 (COL3A1), spermidine/spermine N1-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, showing differential expression of this novel gene in the cochlea.

Asunto(s)

Cóclea/química , ADN Complementario/genética , Proteínas Fetales/genética , Regulación del Desarrollo de la Expresión Génica , Genes , Audición/genética , Proteínas del Tejido Nervioso/genética , Avidina , Biotina , Cóclea/embriología , Proteínas Fetales/biosíntesis , Biblioteca de Genes , Edad Gestacional , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Hibridación de Ácido Nucleico , ARN Mensajero/genética

RESUMEN

The expression and localization of COL2A1 mRNA and protein was examined in human fetal cochlea to study the role of this gene in hearing and to begin to understand the pathogenesis of mutations in COL2A1 in hearing disorders. Northern blot analysis revealed COL2A1 expression in fetal membranous cochlea to be markedly greater than that in fetal skin, kidney, cartilage, eye and brain. In situ hybridization revealed COL2A1 expression in marrow cells, osteoblasts, fibroblasts and some osteocytes, in addition to chondrocytes in otic capsule. In the membranous cochlea, connective tissue elements (spiral ligament, spiral limbus and modiolar connective tissue), neuronal cells, secretory cells (stria vascularis) and organ of Corti cells (sensory hair cells) were found to express COL2A1. Immunohistochemistry was performed to assess distribution of type II collagen and correlation with COL2A1 mRNA in these morphologically and functionally diverse cell populations. In otic capsule, only cartilage was found to stain positively, and in membranous cochlea, only connective tissue structures including spiral ligament, spiral limbus, tectorial and basilar membranes, modiolar and spiral lamina cartilage contained type II collagen. Nonconnective tissue cells, marrow cells and osteoblasts did not contain immunohistochemically identifiable protein. Absence of type II collagen in a subset of cochlear cells may reflect potentially either inability to detect low levels of protein in these cells or posttranscriptional regulation.

Asunto(s)

Cóclea/metabolismo , Colágeno/biosíntesis , Regulación del Desarrollo de la Expresión Génica/genética , Trastornos de la Audición/genética , ARN Mensajero/biosíntesis , Autorradiografía , Northern Blotting , Encéfalo/embriología , Encéfalo/metabolismo , Cartílago/citología , Cartílago/embriología , Cartílago/metabolismo , Diferenciación Celular/genética , Cóclea/embriología , Colágeno/genética , Ojo/embriología , Ojo/metabolismo , Genes , Humanos , Inmunohistoquímica , Hibridación in Situ , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Mutación/genética , ARN Mensajero/genética , Piel/citología , Piel/embriología , Piel/metabolismo , Transcripción Genética/genética

RESUMEN

The myc family of proto-oncogenes consists of several members that possess regions of sequence homology and some have known similarities in structure and function. We have isolated an 8.8-kb EcoRI fragment from a human genomic library by hybridization to a 28-base oligonucleotide probe derived from a region of the second exon of MYC, which is highly conserved in the myc gene family. Sequence analysis of this myc-like (MYCLK1) DNA fragment has revealed the existence of a region with 85% homology to the 28-base oligonucleotide probe. An open reading frame of 207 nucleotides containing the region of homology was found. We have mapped MYCLK1 to human chromosome 7 at band p15 by chromosome in situ hybridization; this site is distinct from the map location of previously characterized myc genes. Whether MYCLK1 represents a new functional member of the myc family of proto-oncogenes remains to be determined.

Asunto(s)

Cromosomas Humanos Par 7 , Familia de Multigenes , Secuencia de Bases , Genes myc , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Ácido Nucleico

RESUMEN

We have characterized the expression of MYCL2, an intronless X-linked gene related to MYCL1. RNase protection analysis of a panel of human normal and tumor tissues has revealed that MYCL2 is expressed almost exclusively in human adult normal testis; much lower levels of transcript were detected in one human lung adenocarcinoma. No MYCL2 transcript was found in human testis RNA obtained from second trimester fetuses. This observation suggests a germ cell rather than somatic cell origin of the transcript and possible developmental regulation of MYCL2. Northern blot analysis of poly(A)+ RNA from adult human normal testis with an antisense riboprobe revealed a transcript of approximately 4.8-kb, which is in agreement with the size predicted from the MYCL2 nucleotide sequence. Antisense transcripts were found spanning regions of MYCL2 corresponding to all three exons of MYCL1. No sizable open reading frame was seen for the MYCL2 antisense transcripts suggesting that they may represent either regulatory sequences or an intron of a gene encoded by the complementary strand. RNase protection assays and the 5' RACE protocol (Rapid Amplification of cDNA Ends) were used to address the localization of the transcription start site of the MYCL2 sense transcript and different putative promoters and transcription regulatory elements have been identified.

Asunto(s)

Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , Testículo/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN/genética , Femenino , Ligamiento Genético , Humanos , Masculino , Datos de Secuencia Molecular , Placenta/metabolismo , Embarazo , ARN/análisis , ARN sin Sentido/análisis , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Células Tumorales Cultivadas , Cromosoma X

RESUMEN

Polygalacturonase is extractable from ripe tomatoes in two isoenzyme forms, polygalacturonase 1 and 2. These isoenzymes have previously been shown to have substantially different properties although their polypeptides appear similar. Green fruit contain a heat-stable, non-dialysable factor capable of the conversion of polygalacturonase 2 in vitro into another isoenzyme which, on the basis of heat stability, molecular weight and density in caesium chloride, is equivalent to polygalacturonase 1. The amount of this factor extractable from tomato tissue increases during ripening.

Asunto(s)

Glicósido Hidrolasas/aislamiento & purificación , Isoenzimas/aislamiento & purificación , Poligalacturonasa/aislamiento & purificación , Verduras , Fenómenos Químicos , Química , Calor , Peso Molecular

RESUMEN

1. Polygalacturonase activity is not detectable in mature green tomato fruits but appears as fruits begin to change colour and continues to increase during the ripening period. There is a sequential appearance of two isoenzymes, polygalacturonase 1 and 2, during ripening. These isoenzymes have been purified and their properties compared. Polygalacturonase 1 has a Mr of 100,000, is 50% inactivated at 78 degrees C and has a density of 1.343 g cm-3 in caesium chloride. Polygalacturonase 2 has a Mr of 42,000, is 50% inactivated at 57 degrees C and has a density of 1.300 g cm-3 in caesium chloride. 2. Fruits from isogenic lines homozygous for the 'Neverripe' (Nr) mutation do not ripen normally and contain reduced amounts of polygalacturonase. Only polygalacturonase 1 is produced in Nr fruit. Tomatoes from isogenic lines homozygous for the 'ripening inhibitor' (rin) mutation do not ripen normally and produce very little detectable polygalacturonase. 3. Although polygalacturonases 1 and 2 have different properties they both give rise to a single polypeptide on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl-sulphate (Mr = 46,000). A comparison of the major fragments produced by limited proteolysis of polygalacturonase 1 and 2 with chymotrypsin suggests that the polypeptides from the two isoenzymes are similar. The same conclusion was reached from a comparison of polygalacturonase 1 and 2 by radioimmunoassay, using antibody prepared against polygalacturonase 2 and 125I-labelled polygalacturonase 2. 4. The results from radioimmunoassay of extracts from green and ripening fruits suggest that the increase in polygalacturonase activity during ripening is due to net synthesis of protein.

Asunto(s)

Glicósido Hidrolasas/metabolismo , Isoenzimas/metabolismo , Desarrollo de la Planta , Poligalacturonasa/metabolismo , Centrifugación por Gradiente de Densidad , Calor , Peso Molecular , Mutación , Plantas/enzimología , Plantas/genética , Radioinmunoensayo