Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mutagénesis Insercional , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Glándulas Sebáceas/metabolismo , Estearoil-CoA Desaturasa , Alopecia/genética , Empalme Alternativo/genética , Animales , Animales Recién Nacidos , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Exones/genética , Expresión Génica , Folículo Piloso/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , ARN Mensajero/análisis , ARN Mensajero/genética , Eliminación de Secuencia/genéticaRESUMEN
Six cloned 5' long terminal repeat (LTR) and adjoining cellular DNA regions of partially deleted feline endogenous RD-114 proviral loci were linked to the chloramphenicol acetyltransferase (CAT) gene and assayed for their ability to promote transient CAT expression. One endogenous LTR (clone CRL-3) and the LTR from an infectious RD-114 provirus, EX-LTR, were capable of actively expressing the CAT gene. DNA sequence comparison of these LTRs with an inactive endogenous LTR (CR-1) revealed extensive homology in all regions except in the 5' half of U3. The homologous portion contained transcriptional regulatory sequences including CAT, TATA, polyadenylation signal boxes and an octamer enhancer, which is rarely seen in retroviruses. Variations in the 5' half of U3 were primarily due to insertions and deletions. A major difference was in number of copies and integrity of tandemly repeated sequences. EX-LTR contained two pairs of tandem direct repeats, while the two endogenous LTRs contained different deletions of repeated sequences. DNA sequence data also revealed that the primer binding site for RD-114 loci was complementary to a glycine tRNA isotype, the use of which is distinct from any other known retrovirus. An analysis of the steady state RNA levels in T-lymphoid cell lines showed that at least three different incomplete proviral transcripts and their spliced products made up the majority of expressed RD-114 mRNA, and further demonstrated that partially deleted proviral loci have the potential to be transcriptionally vigorous in certain feline cell types.
Asunto(s)
Secuencias Repetitivas de Ácidos Nucleicos , Retroviridae/genética , Acetiltransferasas/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa , ADN Viral/genética , Genes Reguladores , Cadenas Pesadas de Inmunoglobulina/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN de Transferencia de Glicerina/genéticaRESUMEN
Identification of expressed sequence tags (ESTs) in large genomic segments is an important step in positional cloning and genomic mapping studies. A simple and efficient polymerase chain reaction (PCR)-based approach is described here to identify coding sequences in large genomic fragments of DNA cloned in vectors such as yeast artificial chromosome (YAC) vectors. The method is based on blocking of sequences such as repetitive and GC rich sequences in the genomic DNA immobilized on nylon paper discs prior to hybridization of the discs to cDNA library, and recovery of the selected cDNAs by the PCR. Single or multiple cDNA libraries can be used in the selection procedure. The procedure has been used successfully also with total yeast DNA containing a YAC.
Asunto(s)
Cromosomas Artificiales de Levadura , ADN Complementario , Reacción en Cadena de la Polimerasa/métodos , Secuencia de BasesRESUMEN
Research in hair biology has embarked in the pursuit for molecules that control hair growth. Many molecules already have been associated with the controls of hair patterning, hair maturation, and hair cycling and differentiation. Knowing how these molecules work gives us the tools for understanding and treating patients with hair disorders.
Asunto(s)
Folículo Piloso/crecimiento & desarrollo , Adulto , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Clonales , Técnicas de Cultivo , Genes Homeobox/genética , Folículo Piloso/citología , Humanos , Ratones , Ratones Transgénicos , Pigmentación , Valores de Referencia , Factores de Transcripción/fisiologíaAsunto(s)
Cromosomas Humanos Par 8 , Hipotricosis/genética , Mapeo Cromosómico , Salud de la Familia , Humanos , LinajeAsunto(s)
Alopecia/sangre , Alopecia/genética , Andrógenos/sangre , Femenino , Ligamiento Genético , Humanos , Masculino , LinajeAsunto(s)
Mapeo Cromosómico/métodos , ADN Complementario/aislamiento & purificación , Secuencia de Bases , Cromosomas Artificiales de Levadura , Cartilla de ADN , ADN Complementario/biosíntesis , Electroforesis en Gel de Campo Pulsado/métodos , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Humanos , Indicadores y Reactivos , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico/genética , Transcripción GenéticaAsunto(s)
Cromosomas Artificiales de Levadura/genética , Clonación Molecular/métodos , ADN Complementario/genética , Selección Genética , Secuencia de Bases , Cartilla de ADN , Biblioteca de Genes , Genes MHC Clase I , Genoma Humano , Humanos , Técnicas de Sonda Molecular , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
The hair follicle develops from the primitive embryonic epidermis as a result of complex epithelial-mesenchymal interactions. The full follicle, consisting of epithelial cylinders under control of a proximal lying mesenchymal papilla, grows in cycles giving rise to a new hair shaft during each cycle. The ability to cycle endows the follicle with regenerative properties. The evolution of hair follicle engineering began with the recognition in the early 1960's that hair follicles could be transplanted clinically into a foreign site and still grow a shaft typical of the donor site. Since that time, it has been found that the follicular papilla has hair follicle inducing properties and that the hair follicle houses within it epithelial stem cells that can respond to hair inductive signals. These findings have laid the foundation for isolating hair-forming cells, for expanding the cells in culture, and for forming new follicles in vivo.
RESUMEN
Pattern recognition is at the heart of clinical dermatology and dermatopathology. Yet, while every practitioner of the art of dermatological diagnosis recognizes the supreme value of diagnostic cues provided by defined patterns of 'efflorescences', few contemplate on the biological basis of pattern formation in and of skin lesions. Vice versa, developmental and theoretical biologists, who would be best prepared to study skin lesion patterns, are lamentably slow to discover this field as a uniquely instructive testing ground for probing theoretical concepts on pattern generation in the human system. As a result, we have at best scraped the surface of understanding the biological basis of pattern formation of skin lesions, and widely open questions dominate over definitive answer. As a symmetry-breaking force, pattern formation represents one of the most fundamental principles that nature enlists for system organization. Thus, the peculiar and often characteristic arrangements that skin lesions display provide a unique opportunity to reflect upon--and to experimentally dissect--the powerful organizing principles at the crossroads of developmental, skin and theoretical biology, genetics, and clinical dermatology that underlie these--increasingly less enigmatic--phenomena. The current 'Controversies' feature offers a range of different perspectives on how pattern formation of skin lesions can be approached. With this, we hope to encourage more systematic interdisciplinary research efforts geared at unraveling the many unsolved, yet utterly fascinating mysteries of dermatological pattern formation. In short: never a dull pattern!
Asunto(s)
Enfermedades de la Piel/fisiopatología , Piel/fisiopatología , Algoritmos , Animales , Ambiente , Hormonas/fisiología , Humanos , Modelos Biológicos , Piel/metabolismo , Piel/patología , Enfermedades de la Piel/genética , Enfermedades de la Piel/patología , Pigmentación de la Piel/genética , Pigmentación de la Piel/fisiologíaRESUMEN
The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum--mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum--mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.
Asunto(s)
Complejo IV de Transporte de Electrones/biosíntesis , Hígado/enzimología , Animales , Grupo Citocromo a , Citocromos/metabolismo , Retículo Endoplásmico/enzimología , Técnicas In Vitro , Hígado/citología , Mitocondrias Hepáticas/enzimología , Ratas , Fracciones Subcelulares/enzimologíaRESUMEN
We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in lambda phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and self-annealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue.
Asunto(s)
Biblioteca de Genes , Poli A/genética , ARN/genética , Secuencia de Bases , Cromatografía , Durapatita , Amplificación de Genes , Humanos , Hidroxiapatitas , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Poli A/aislamiento & purificación , ARN/aislamiento & purificación , ARN Mensajero , ARN Ribosómico/genética , Timo/químicaRESUMEN
Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.
Asunto(s)
Clonación Molecular/métodos , ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Biblioteca de Genes , Vectores Genéticos , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The murine major histocompatibility complex (MHC) includes sequences that are responsible for haplotype-specific odor types that, in turn, influence mating preference. We report that there are several olfactory receptor genes or pseudogenes in the Class I region of the human MHC. At least one of these genes is intact, appears to encode an mRNA, and is quite homologous to a previously reported murine olfactory receptor.
Asunto(s)
Genes , Complejo Mayor de Histocompatibilidad , Proteínas de la Membrana , Seudogenes , Receptores de Superficie Celular/genética , Receptores Odorantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura , ADN Complementario/genética , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Conducta Sexual Animal/fisiología , Especificidad de la EspecieRESUMEN
A critical step in the synthesis of unsaturated fatty acids is catalysed by stearoyl-CoA desaturase (Scd). To determine the regulation of human Scd, we characterized the gene and its transcripts. Screening a human keratinocyte cDNA library and analysis of 3'-RACE (rapid amplification of cDNA ends) products from various tissues yielded a 5.2 kb cDNA encoding a 359 amino acid protein with a calculated molecular mass of 41.5 kDa. Analysis of 3'-RACE products suggested that alternative usage of polyadenylation sites generates two transcripts of 3.9 and 5.2 kb, a result consistent with Northern analysis. Southern analysis demonstrated the existance of two SCD loci in the human genome. Chromosomal mapping localized one locus to chromosome 10, and the second locus to chromosome 17. Characterization of genomic clones isolated from chromosome-specific libraries revealed that only the locus on chromosome 10 contained introns. Sequence analysis of the intron-less locus displayed multiple nucleotide insertions and deletions, as well as in-frame stop codons. Reverse transcriptase-PCR analysis performed with primers specific to the intron-less locus failed to produce a PCR product from brain, liver and skin RNA, indicating that the locus on chromosome 17 is most likely a transcriptionally inactive, fully processed pseudogene. These results suggest strongly that there is one structural SCD gene in the human genome, and that it generates two transcripts by use of alternative polyadenyation sites. Although the primary sequence and intron-exon structure of SCD is phylogenetically conserved, divergence between rodent and human is seen in the number of SCD genes and in the generation of alternative transcripts, suggesting a species-specific component of SCD regulation and function.
Asunto(s)
Empalme Alternativo , Poli A/genética , Estearoil-CoA Desaturasa/genética , Secuencias Repetidas en Tándem , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 17/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/análisis , ADN Complementario/genética , Genoma Humano , Humanos , Intrones/genética , Queratinocitos/metabolismo , Datos de Secuencia Molecular , Seudogenes/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia , Piel/citologíaRESUMEN
It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC.
Asunto(s)
Cromosomas Humanos Par 6 , Antígenos HLA-A/genética , Hemocromatosis/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Línea Celular , Cromosomas Artificiales de Levadura , ADN Complementario , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Hibridación de Ácido Nucleico , Mapeo Restrictivo , Análisis de SecuenciaRESUMEN
A search for new genes was performed in a 220-kb region around the tumor necrosis factor gene cluster in the human central major histocompatibility complex region using a cDNA hybridization and selection method. In addition to the seven known genes in this region, we identified a new gene that is preferentially expressed in spleen. We also identified two pseudogenes that have high degrees of homology to cytokeratin and cyclophillin, respectively. Expressed sequences for a human homologue of the mouse B144 gene were also found in the current analysis. RT-PCR analysis showed that B144 is expressed in spleen, in thymus, and prominently in the macrophage cell line, U937. We also independently identified the BAT1 gene to be the well-conserved homologue of a previously described rat liver nuclear protein.
Asunto(s)
Complejo Mayor de Histocompatibilidad/genética , Familia de Multigenes/genética , Factores de Transcripción , Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/genética , Células CHO , Cricetinae , ADN Complementario/genética , Biblioteca de Genes , Genes MHC Clase I , Genoma Humano , Humanos , Péptidos y Proteínas de Señalización Intracelular , Queratinas/genética , Linfotoxina-alfa/genética , Linfotoxina beta , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/genética , Ratas , Receptores Inmunológicos/genética , Factor de Transcripción ReIBRESUMEN
We have developed a novel efficient approach, termed differential subtraction display, for the identification of differentially expressed genes. Several critical parameters for the reproducibility and enhanced sensitivity of display, as well as steps to reduce the number of false positive cDNA species, have been defined. These include- (a) use of standardized oligo(dT)-primed cDNA pools rather than total RNA as the starting material for differential display, (b) critical role of optimal cDNA input for each distinct class of primers, (c) phenomena of primer dominance and interference, and (d) design of a novel set of enhanced specificity anchor primers. Introduction of an efficient subtractive hybridization step prior to cloning of cDNA species enriches the bona fide cDNA species that are either exclusively present in one sample (+/-) or show altered expression (up-/down-regulation) in RNA samples from two different tissues or cell types. This approach, in comparison to differential display, has several advantages in terms of reproducibility and enhanced sensitivity of display coupled to the cloning of enriched bona fide cDNA species corresponding to differentially expressed RNAs.
Asunto(s)
ADN Complementario/aislamiento & purificación , Expresión Génica , Animales , Biotinilación , Northern Blotting , Clonación Molecular/métodos , Cartilla de ADN , ADN Complementario/síntesis química , ADN Complementario/química , Regulación de la Expresión Génica , Ratones , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN/química , ARN/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We have refined the position of asebia locus by genotyping DNA from more than 600 backcross mice derived from asebia mouse and a genetically unrelated strain. One of the candidate genes in the locus is stearoyl-CoA desaturase (SCD). Previously two members of this gene family, namely SCD1 and SCD2, have been described. We have found, for the first time, that these SCD genes are expressed in skin. Moreover, we have identified a third species of SCD in the mouse skin. The most prominent SCD species is SCD1 in the mouse skin. The implications of this gene family to skin are discussed.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Piel/enzimología , Estearoil-CoA Desaturasa/biosíntesis , Estearoil-CoA Desaturasa/genética , Animales , Ratones , Mutación , Análisis de Secuencia de ADNRESUMEN
A cDNA library for 6S-9S poly(A)-containing RNA from rat liver was constructed in E. coli. Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3'-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.