Asunto(s)
Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/química , Secuencia de Aminoácidos , Animales , Factor Activador de Células B , Receptor del Factor Activador de Células B , Humanos , Ligandos , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Receptores del Factor de Necrosis Tumoral/genética , Distribución Tisular , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-alpha has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-beta (LT-beta) receptor (LT-betaR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-betaR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-alpha and LT-alpha1beta2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-beta and TNF-alpha receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-alpha1beta2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-alpha1beta2 expression, and HIV-1 replication. Given the critical role that LT-alpha1beta2 plays in lymphoid architecture, we speculate that LT-alpha1beta2 may be involved in HIV-associated abnormalities of the lymphoid organs.
Asunto(s)
VIH-1/crecimiento & desarrollo , Linfotoxina-alfa/metabolismo , Monocitos/virología , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antígenos CD/metabolismo , Línea Celular , Sinergismo Farmacológico , Humanos , Receptor beta de Linfotoxina , Linfotoxina-alfa/farmacología , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
The anti-common gamma chain (gammac) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Ralpha and gammac receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Ralpha, and to PHA blasts expressing both IL-4Ralpha and gammac, were used to estimate the affinity of the key interaction between gammac and the binary IL-4Ralpha.IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Ralpha.IL-4.gammac]/[IL-4Ralpha.IL-4], which we designate KR. The results show that on PHA blasts this interaction is relatively weak; KR approximately 9, implying that approximately 10% of the limiting IL-4Ralpha chain remains free of gammac even at saturating concentrations of IL-4. This quantitative treatment establishes KR as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.
Asunto(s)
Activación de Linfocitos , Receptor Cross-Talk , Receptores de Citocinas/fisiología , Receptores de Interleucina-4/fisiología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Membrana Celular/inmunología , Células Cultivadas , Humanos , Interleucina-4/farmacología , Interleucina-4/fisiología , Cinética , Ratones , Modelos Inmunológicos , Linfocitos T/efectos de los fármacosRESUMEN
During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.
Asunto(s)
Ácido Palmítico/química , Proteínas/genética , Transactivadores , Animales , Línea Celular , Colesterol/química , Proteínas Hedgehog , Humanos , Ratones , Ratones Endogámicos C3H , Mapeo Peptídico , Proteínas/química , Proteínas/metabolismo , Ratas , Transducción de SeñalRESUMEN
The lymphotoxin-alpha beta complex (LT alpha beta) is found on the surface of activated lymphocytes and binds to a specific receptor called the LT beta receptor (LT beta R). In the mouse, signaling through this pathway is important for lymph node development and splenic organization, yet the biochemical properties of murine LT alpha and LT beta are essentially unknown. Here we have used soluble receptor-Ig forms of LT beta R and TNF-R55 and mAbs specific for murine LT alpha, LT beta, and LT beta R to characterize the appearance of surface LT alpha beta complexes and LT beta R on several common murine cell lines. Cells that bound LT beta R also bound anti-LT alpha and anti-LT beta mAbs in a FACS analysis. The ability of these reagents to discriminate between surface TNF and LT was verified by analysis of surface TNF-positive, LPS-activated murine RAW 264.7 monocytic cells. Primary mouse leukocytes from spleen, thymus, lymph node, and peritoneum were activated in vitro, and CD4+ and CD8+ T cells as well as B cells expressed surface LT ligand but not the LT beta R. Conversely, elicited peritoneal monocytes/macrophages were surface LT negative yet LT beta R positive. This study shows that on mononuclear cells, surface LT complexes and receptor are expressed similarly in mice and man, and the tools described herein form the foundation for study of the functional roles of the LT system in the mouse.
Asunto(s)
Linfocitos/química , Linfocitos/metabolismo , Linfotoxina-alfa/química , Proteínas de la Membrana/química , Factor de Necrosis Tumoral alfa/química , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Linfocitos B/química , Línea Celular , Citometría de Flujo , Humanos , Hibridomas , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Linfocitos/inmunología , Linfoma de Células T , Linfotoxina-alfa/inmunología , Linfotoxina beta , Macrófagos , Proteínas de la Membrana/inmunología , Ratones , Ratas , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Especificidad de la Especie , Linfocitos T/química , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized. When recombinant mLT alpha was produced by either of several methods, the protein had a very low specific activity relative to that of human LT alpha in the conventional WEHI 164 cytotoxicity bioassay. The weak activity observed was inhibited by a soluble murine TNF-R55 Ig fusion protein (mTNF-R55-Ig), but not by mLT beta R-Ig. Coexpression of both mLT alpha and a soluble version of mLT beta in insect cells led to an LT alpha beta form that was cytotoxic in the WEHI 164 assay via the LT beta R. To determine whether natural mLT alpha-like forms with cytotoxic activity comparable to that of secreted human LT alpha were secreted from primary spleen cells, splenic lymphocytes were activated in various ways, and their supernatants were analyzed for cytotoxic activity. Using specific Abs to distinguish between mTNF and mLT, a TNF component was readily detected; however, there was no evidence for a secreted mLT alpha cytotoxic activity using this assay. Combined, these observations suggest that secreted mLT alpha may not play a role in the mouse via interactions with TNF-R55, and the ramifications of this hypothesis are discussed.
Asunto(s)
Citotoxicidad Inmunológica , Linfotoxina-alfa/química , Proteínas de la Membrana/química , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Pruebas Inmunológicas de Citotoxicidad , Humanos , Activación de Linfocitos , Linfotoxina-alfa/metabolismo , Linfotoxina-alfa/toxicidad , Linfotoxina beta , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/toxicidad , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores del Factor de Necrosis Tumoral/fisiología , Proteínas Recombinantes/metabolismo , Solubilidad , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have applied exon amplification, GRAIL2 exon prediction and EST database searching to a 2 Mb segment of chromosome 4p16.3. Experimental and computational methods of identifying exons were comparable in efficiency and apparent false positive rate, but were complementary in gene identification, revealing distinct overlapping sets of expressed sequences. EST searching was most powerful when we considered only those ESTs that show evidence of splicing relative to the genomic sequence. The combination of the three gene finding methods produced a transcription map of 30 loci in this segment of 4p16.3 that includes known human genes, homologs of loci identified in rodents and several anonymous transcripts, including a putative novel DNA polymerase and a gene related to Drosophila ash1. While most of the genes in the region have been found, our data suggest that even with the entire DNA sequence available, complete saturation of the transcript map will require additional, focused experimental effort.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 4/genética , Análisis de Secuencia de ADN/métodos , Transcripción Genética , Exones/genética , HumanosRESUMEN
Huntingtin expression was examined by Western blot and immunoprecipitation studies of lymphoblastoid cell lines from Huntington's disease (HD) homozygotes, heterozygotes, and a phenotypically normal individual with a t(4p16.3;12p13.3) breakpoint in the HD gene. The latter produced a reduced level of normal huntingtin without evidence of an altered protein, indicating that simple loss of huntingtin activity does not cause HD. In juvenile onset HD heterozygotes, NH2- and COOH-terminal antisera revealed reduced relative expression from the mutant allele. Pulse-chase studies indicated that huntingtin is a stable protein whose differential allelic expression is not due to destabilization of the mutant isoform. No stable breakdown products specific to mutant huntingtin were detected in either HD homozygotes or heterozygotes. These data are consistent with HD involving either a gain of function or a dominant negative loss of function that operates within severe constraints and suggest that in either case the pathogenic process is usually saturated by the amount of abnormal huntingtin produced from a single mutant allele.
Asunto(s)
Alelos , Enfermedad de Huntington/genética , Mutación , Adulto , Edad de Inicio , Western Blotting , Niño , Humanos , Proteína Huntingtina , Enfermedad de Huntington/epidemiología , Enfermedad de Huntington/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Pruebas de Precipitina , Valores de Referencia , Translocación GenéticaRESUMEN
The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells. The signaling function of this receptor-ligand pair is not precisely known but is believed to be involved in the development of the peripheral lymphoid organs. To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired. The LT-beta molecule was engineered into a secreted form and co-expressed with LT-alpha using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-alpha3, LT-alpha2/beta1, and LT-alpha1/beta2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-alpha3-like components in the LT-alpha1/beta2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors. Heteromeric LT complexes were prepared with this mutant LT- alpha form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors. A LT-alpha3 based cytotoxic activity was used to show that the LT-alpha1/beta2 form cannot readily scramble into a mixture of forms following various treatments and storage periods. This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analysis of the specificity of the LT-bet a and TNF receptors for the various LT forms.
Asunto(s)
Linfotoxina-alfa/química , Proteínas de la Membrana/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bioensayo , Cromatografía Líquida de Alta Presión , Citotoxinas/química , Cartilla de ADN/química , Humanos , Linfotoxina beta , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Peso Molecular , Nucleopoliedrovirus , Proteínas Recombinantes , Solubilidad , SpodopteraRESUMEN
BACKGROUND: An expanded CAG trinucleotide repeat is the genetic trigger of neuronal degeneration in Huntington's disease (HD), but its mode of action has yet to be discovered. The sequence of the HD gene places the CAG repeat near the 5' end in a region where it may be translated as a variable polyglutamine segment in the protein product, huntingtin. MATERIALS AND METHODS: Antisera directed at amino acid stretches predicted by the DNA sequence upstream and downstream of the CAG repeat were used in Western blot and immunohistochemical analyses to examine huntingtin expression from the normal and the HD allele in lymphoblastoid cells and postmortem brain tissue. RESULTS: CAG repeat segments of both normal and expanded HD alleles are indeed translated, as part of a discrete approximately 350-kD protein that is found primarily in the cytosol. The difference in the length of the N-terminal polyglutamine segment is sufficient to distinguish normal and HD huntingtin in a Western blot assay. CONCLUSIONS: The HD mutation does not eliminate expression of the HD gene but instead produces an altered protein with an expanded polyglutamine stretch near the N terminus. Thus, HD pathogenesis is probably triggered by an effect at the level of huntingtin protein.
Asunto(s)
Enfermedad de Huntington/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alelos , Secuencia de Aminoácidos , Anticuerpos , Encéfalo/inmunología , Encéfalo/patología , Humanos , Enfermedad de Huntington/inmunología , Inmunohistoquímica , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos/inmunología , Células Tumorales CultivadasRESUMEN
Huntington's disease (HD) is an autosomal dominant disorder characterized by involuntary movements, dementia, and progressive, global, but regionally accentuated, brain atrophy. The disease affects the striatum most severely. An expansion of a trinucleotide repeat on chromosome 4p16.3 within the coding region of a gene termed IT15 has been identified as the mutation causing HD. The normal function of IT15 and the mechanisms by which the presence of the mutation causes HD are unknown. Although IT15 expression has been detected in the brain, as well as in other organ tissues, by Northern blot and in situ hybridization, it is not known whether a preferential regional or cellular expression of IT15 exists within the central nervous system of normal, affected, and presymptomatic individuals. Using quantitative in situ hybridization methods, we examined extensively the regional and cellular expression of IT15. In controls, IT15 expression was observed in all brain regions examined with the highest levels seen in cerebellum, hippocampus, cerebral cortex, substantia nigra pars compacta, and pontine nuclei. Expression in the striatum was intermediate and expression in the globus pallidus was low. IT15 was expressed predominantly in neurons; a low but significant level of expression was seen in glial cells. Analysis of grain counts per square micrometer in neurons showed that the regional differences in the level of mRNA expression were related to density and size of neurons in a given region and not primarily to differences in levels of mRNA expression in individual cells after correction for cell size. Neurons susceptible to degeneration in HD did not selectively express high levels of IT15 mRNA. In HD brains (grades 2-4), the distribution and levels of IT15 mRNA were comparable with controls in all areas except in neostriatum where the intensity of labeling was significantly reduced. Presymptomatic HD brains had a striatal expression similar to controls and surviving striatal neurons in more advanced HD had an expression of IT15 within normal limits. It is apparent from these results that the presence of expanded trinucleotide repeats in HD does not result in the absence of IT15 mRNA expression or in altered patterns or levels of expression. The lack of correlation between the levels of IT15 mRNA expression and susceptibility to degeneration in HD strongly suggests that the mutant gene acts in concert with other factors to cause the distinctive pattern of neurodegeneration in HD.
Asunto(s)
Encéfalo/metabolismo , Enfermedad de Huntington/genética , Biosíntesis de Proteínas , Anciano , Análisis de Varianza , Humanos , Proteína Huntingtina , Hibridación in Situ , Persona de Mediana Edad , Proteínas del Tejido Nervioso , Neuronas/metabolismo , Proteínas Nucleares , Proteínas/análisis , ARN Mensajero/biosíntesis , Valores de ReferenciaRESUMEN
To examine the expression of the gene which causes Huntington's disease (HD), IT15, during development, in situ hybridization of radiolabeled riboprobes was performed in human fetal (gestational ages 20-23 weeks) and adult brain. Optical densities of autoradiographs were determined in various brain regions and compared to cell density in those regions. IT15 expression was found in all regions of the fetal and adult brain, and there was a high degree of correlation of autoradiographic signal with cell number in all regions but germinal matrix in fetal brain and white matter in adult brain. These two regions are notable for their significant proportion of glial cells, and suggest that IT15 expression is predominantly neuronal. There was no preponderance of IT15 expression in striatal compartments in fetal brain as demonstrated by acetylcholinesterase activity, nor was there differential expression of IT15 in brain regions known to be particularly affected in HD. IT15 gene expression is present by 20 weeks gestation in human brain, and at that stage of development exhibits a pattern of distribution which is similar to adult brain. If a developmentally-regulated role for IT15 exists in the pathogenesis of HD, it must occur prior to 20 weeks gestation.
Asunto(s)
Encéfalo/embriología , Feto/fisiología , Expresión Génica , Enfermedad de Huntington/genética , Anciano , Envejecimiento/fisiología , Encéfalo/fisiología , Desarrollo Embrionario y Fetal , Humanos , Hibridación in Situ , ARN Mensajero/metabolismoRESUMEN
The mouse homologs of the Huntington's disease (HD) gene and 17 other human Chromosome (Chr) 4 loci (including six previously unmapped) were localized by use of an interspecific cross. All loci mapped in a continuous linkage group on mouse Chr 5, distal to En2 and I16, whose human counterparts are located on Chr 7. The relative order of the loci on human Chr 4 and mouse Chr 5 was maintained, except for a break between D5H4S115E and Idua/rd, with relocation of the latter to the opposite end of the map. The mouse HD homolog (Hdh) mapped within a cluster of seven genes that were completely linked in our data set. In human these loci span a approximately 1.8 Mb stretch of human 4p16.3 that has been entirely cloned. To date, there is no phenotypic correspondence between human and mouse mutations mapping to this region of synteny conservation.
Asunto(s)
Mapeo Cromosómico , Enfermedad de Huntington/genética , Animales , Secuencia de Bases , Cromosomas Humanos Par 4 , Cruzamientos Genéticos , ADN Complementario , Ligamiento Genético , Marcadores Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Datos de Secuencia MolecularRESUMEN
The incurable neurodegenerative disorder, Huntington's disease (HD), is caused by an expanded, unstable CAG repeat encoding a stretch of polyglutamine in a 4p16.3 gene (HD) of unknown function. Near the CAG repeat is a polyproline-encoding CCG repeat that shows more limited allelic variation. The mouse homologue, Hdh, has been mapped to chromosome 5, in a region devoid of mutations causing any comparable phenotype. We have isolated overlapping cDNAs from the Hdh gene and compared their sequences with the human transcript. The consensus mouse coding sequence is 86% identical to the human at the DNA level and 91% identical at the protein level. Despite the overall high level of conservation, Hdh possesses an imperfect CAG repeat encoding only seven consecutive glutamines, compared to the 13-36 residues that are normal in man. Although no evidence for polymorphic variation of the CAG repeat was seen, a nearby CCG repeat differed in length by one unit between several strains of laboratory mouse and Mus spretus. The absence of a long CAG repeat in the mouse is consistent with the lack of a spontaneous mouse model of HD. The information presented concerning the sequence of the mouse gene should facilitate attempts to create such a model.
Asunto(s)
Enfermedad de Huntington/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Humanos , Ratones , Datos de Secuencia Molecular , Polimorfismo Genético , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Instability of a CAG repeat in 4p16.3 has been found in Huntington's disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.
Asunto(s)
Enfermedad de Huntington/genética , Secuencias Repetitivas de Ácidos Nucleicos , Adolescente , Adulto , ADN/sangre , ADN/genética , Enfermedades en Gemelos/genética , Femenino , Variación Genética , Edad Gestacional , Humanos , Enfermedad de Huntington/embriología , Masculino , Mosaicismo , Oligodesoxirribonucleótidos/genética , Espermatozoides/química , Distribución Tisular , Gemelos MonocigóticosRESUMEN
Huntington's disease is an inherited disorder in which selective neuronal loss in the brain leads to a characteristic choreic movement disorder. The successful mapping of the Huntington's disease gene to chromosome 4 set off a torrent of similar studies in other inherited disorders as investigators attempted to locate and isolate human disease genes with this new approach. Although it took a decade-long quest since the initial mapping of the genetic defect, the gene causing Huntington's disease has recently been isolated. Discovery of the mutational mechanism causing Huntington's disease has explained some of the peculiarities of inheritance of this intriguing disorder and creates hope for a better understanding of the cause of neuronal cell death that could eventually lead to a treatment.
Asunto(s)
Enfermedad de Huntington/genética , Biología Molecular , Secuencia de Aminoácidos , Cromosomas Humanos Par 4 , ADN/genética , Marcadores Genéticos , Humanos , Enfermedad de Huntington/diagnóstico , Datos de Secuencia Molecular , Mutación , Nucleótidos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
Huntington's disease (HD) chromosomes contain an expanded unstable (CAG)n repeat in chromosome 4p16.3. We have examined nine families with potential de novo expression of the disease. With one exception, all of the affected individuals had 42 or more repeat units, well above the normal range. In four families, elderly unaffected relatives inherited the same chromosome as that containing the expanded repeat in the proband, but had repeat lengths of 34-38 units, spanning the gap between the normal and HD distributions. Thus, mutation to HD is usually associated with an expansion from an already large repeat.
Asunto(s)
Enfermedad de Huntington/genética , Secuencias Repetitivas de Ácidos Nucleicos , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 4 , Femenino , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
We have previously used exon amplification to identify the ADD1 gene in cosmid Y24 from the Huntington's disease (HD) region of 4p16.3. The same technique has now yielded a second gene from this cosmid. This gene appears to encode a novel member of a superfamily of transporter proteins that includes active and passive transporters in a number of species. The predicted protein of 455 amino acids displays sequence similarity with the E. coli tetracycline resistance efflux protein encoded by cloning vector pBR322, and with a number of related transporters. This gene should open a route to isolating additional mammalian members of this growing superfamily.