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1.
Curr Protoc Hum Genet ; Chapter 2: Unit 2.2, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-18428266

RESUMEN

These protocols construct a representative small-insert genomic DNA library in a phagemid vector. First, size-selected DNA fragments are ligated into a phagemid vector. In the second protocol, the resulting small-insert phagemid library is propagated in a bacterial strain combining mutations at the dut and ung loci, which permit incorporation of uracil in place of thymidine during DNA replication. Infection of the phagemid library with M13 helper phage permits recovery of this library as single-stranded DNA (ssDNA). Finally, this uracil-substituted ssDNA is used as a template for primer extension using an oligonucleotide whose sequence corresponds to the STR class of interest [e.g., (GATA)10] as primer. The products of this primer-extension reaction are transformed into an E. coli strain maintaining wild-type genes at the dut and ung loci. Under these conditions, uracil-substituted ssDNA will be restricted from growing by the host-encoded uracil-N-glycosylase, while the primer-extended products are capable of replicating.


Asunto(s)
Biblioteca Genómica , Repeticiones de Microsatélite , Colifagos/genética , ADN/genética , ADN/aislamiento & purificación , Marcadores Genéticos , Técnicas Genéticas , Vectores Genéticos , Genética Médica , Humanos
2.
Genome Res ; 7(7): 716-24, 1997 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9253600

RESUMEN

The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.


Asunto(s)
Mapeo Cromosómico , Genoma Humano , Repeticiones de Microsatélite/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Cromosomas Artificiales de Levadura , Humanos , Ratones
3.
Diabetes ; 46(6): 1081-6, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9166684

RESUMEN

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) located on chromosome 12q. We have identified four novel HNF-1alpha missense mutations in MODY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in MODY3. We observed no HNF-1alpha mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.


Asunto(s)
Cromosomas Humanos Par 12/genética , Proteínas de Unión al ADN , Diabetes Mellitus Tipo 2/genética , Mutación/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Análisis Mutacional de ADN , Cartilla de ADN/química , Familia , Ligamiento Genético , Haplotipos , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple
4.
Genomics ; 40(1): 147-50, 1997 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-9070932

RESUMEN

During the recent cloning of the mouse Lyst gene we developed both a high-resolution genetic map and a complete YAC and BAC contig of the Lyst critical region on mouse Chromosome 13. We also report the mapping of the human homologue of the mouse Lyst gene (LYST) to 1q43. These data are consistent with LYST being the gene for the human Chediak-Higashi Syndrome and strengthen the synteny relationship between MMU13 and human 1q43.


Asunto(s)
Mapeo Cromosómico , Proteínas/genética , Animales , Secuencia de Bases , Cromosomas Humanos Par 1 , ADN Complementario , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Proteínas de Transporte Vesicular
5.
Proc Natl Acad Sci U S A ; 94(26): 14837-42, 1997 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-9405700

RESUMEN

Hearing is one of the last sensory modalities to be subjected to genetic analysis in Drosophila melanogaster. We describe a behavioral assay for auditory function involving courtship among groups of males triggered by the pulse component of the courtship song. In a mutagenesis screen for mutations that disrupt the auditory response, we have recovered 15 mutations that either reduce or abolish this response. Mutant audiograms indicate that seven mutants reduced the amplitude of the response at all intensities. Another seven abolished the response altogether. The other mutant, 5L3, responded only at high sound intensities, indicating that the threshold was shifted in this mutant. Six mutants were characterized in greater detail. 5L3 had a general courtship defect; courtship of females by 5L3 males also was affected strongly. 5P1 males courted females normally but had reduced success at copulation. 5P1 and 5N18 showed a significant decrement in olfactory response, indicating that the defects in these mutations are not specific to the auditory pathway. Two other mutants, 5M8 and 5N30, produced amotile sperm although in 5N30 this phenotype was genetically separable from the auditory phenotype. Finally, a new adult circling behavior phenotype, the pirouette phenotype, associated with massive neurodegeneration in the brain, was discovered in two mutants, 5G10 and 5N18. This study provides the basis for a genetic and molecular dissection of auditory mechanosensation and auditory behavior.


Asunto(s)
Percepción Auditiva/fisiología , Conducta Animal , Drosophila melanogaster/fisiología , Mutación , Animales , Genes de Insecto
6.
Nat Genet ; 14(3): 307-11, 1996 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8896560

RESUMEN

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by hypopigmentation, severe immunologic deficiency with neutropenia and lack of natural killer (NK) cells, a bleeding tendency and neurologic abnormalities. Most patients die in childhood. The CHS hallmark is the occurrence of giant inclusion bodies and organelles in a variety of cell types, and protein sorting defects into these organelles. Similar abnormalities occur in the beige mouse, the proposed model for human CHS. Two groups have recently reported the identification of the beige gene, however the two cDNAs were not at all similar. Here we describe the sequence of a human cDNA homologous to mouse beige, identify pathologic mutations and clarify the discrepancies of the previous reports. Analysis of the CHS polypeptide demonstrates that its modular architecture is similar to the yeast vacuolar sorting protein, VPS15.


Asunto(s)
Síndrome de Chediak-Higashi/genética , Análisis Mutacional de ADN , Proteínas/genética , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Femenino , Homocigoto , Humanos , Lactante , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas/química , Homología de Secuencia de Aminoácido , Proteína de Clasificación Vacuolar VPS15 , Proteínas de Transporte Vesicular
7.
Hum Mol Genet ; 5(7): 1047-50, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8817345

RESUMEN

We report a novel locus responsible for postlingual progressive sensorineural hearing loss (designated DFNA9) that maps to chromosome 14q12-13. A large kindred with autosomal dominant transmission of non-syndromic hearing loss was clinically studied. Hearing in affected individuals deteriorated at approximately 20 years of age and progressed to anacusis in the fifth decade. A random genome-wide search using polymorphic short tandem repeats demonstrated linkage with D14S121 (maximum two point LOD score = 6.19, theta = 0). Haplotype analysis of recombination events defined a 9 cM disease interval, between D14S252 and D14S49.


Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , Genes Dominantes/genética , Pérdida Auditiva Sensorineural/genética , Adulto , Edad de Inicio , Línea Celular , Femenino , Haplotipos , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Escala de Lod , Linfocitos , Masculino , Persona de Mediana Edad , Linaje
8.
Nat Genet ; 13(3): 303-8, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8673129

RESUMEN

The beige mutation is a murine autosomal recessive disorder, resulting in hypopigmentation, bleeding and immune cell dysfunction. The gene defective in beige is thought to be a homologue of the gene for the human disorder Chediak-Higashi syndrome. We have identified the murine beige gene by in vitro complementation and positional cloning, and confirmed its identification by defining mutations in two independent mutant alleles. The sequence of the beige gene message shows strong nucleotide homology to multiple human ESTs, one or more of which may be associated with the Chediak-Higashi syndrome gene. The amino acid sequence of the Beige protein revealed a novel protein with significant amino acid homology to orphan proteins identified in Saccharomyces cerevisiae, Caenorhabditis elegans and humans.


Asunto(s)
Síndrome de Chediak-Higashi/genética , Mutación , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Clonación Molecular/métodos , Prueba de Complementación Genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos , Ratones Mutantes , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular
9.
Cell ; 85(2): 281-90, 1996 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-8612280

RESUMEN

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.


Asunto(s)
Obesidad/genética , Proteínas/química , Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Animales , Secuencia de Bases , Química Encefálica/fisiología , Mapeo Cromosómico , Clonación Molecular , Exones/genética , Expresión Génica/fisiología , Variación Genética , Hibridación in Situ , Resistencia a la Insulina/genética , Ratones , Ratones Obesos , Datos de Secuencia Molecular , Mutación/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido
10.
Cell ; 84(3): 491-5, 1996 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-8608603

RESUMEN

OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.


Asunto(s)
Proteínas Portadoras/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Mutación Puntual , Proteínas/metabolismo , Receptores de Superficie Celular , Receptores de Citocinas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Leptina , Ratones , Ratones Endogámicos , Ratones Obesos , Datos de Secuencia Molecular , Obesidad/genética , Obesidad/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa , Receptores de Leptina , Homología de Secuencia de Aminoácido , Transducción de Señal
11.
Genomics ; 32(1): 15-20, 1996 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-8786107

RESUMEN

Two thousand nine hundred and thirty-one tri- and tetranucleotide short tandem repeat polymorphisms (STRPs) developed by the Cooperative Human Linkage Center were assigned to chromosomes using the NIGMS somatic cell hybrid mapping panel 2 and an efficient pooling strategy. Approximately 82% of all STRPs tested were assigned by this method, with 96.7% accuracy. Many of the single chromosome cell lines contained portions of additional chromosomes, confirming previous reports. The cell lines for chromosomes 6, 14, and 20 contained extensive portions of other chromosomes. Five previously unreported chromosomal contaminants were identified and are reported. A new pooling strategy was designed to minimize ambiguous assignments.


Asunto(s)
Mapeo Cromosómico/métodos , Marcadores Genéticos , Repeticiones de Microsatélite , Repeticiones de Trinucleótidos , Animales , Secuencia de Bases , Línea Celular , Bandeo Cromosómico , Cromosomas Humanos/genética , Cricetinae , Femenino , Humanos , Células Híbridas , Masculino , Ratones
12.
Genomics ; 32(1): 75-85, 1996 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-8786123

RESUMEN

The expansion of a (CAG/CTG)n triplet repeat has been found to be associated with at least seven genetic diseases, suggesting that this mechanism of disease may be fairly common. To accelerate the discovery of new loci containing (CAG/CTG)n triplet expansions, we have isolated numerous genomic clones containing this class of repeats. We have developed 338 sequence-tagged sites (STSs) containing (CAG/CTG)n repeat sequences. Two hundred ninety-nine STSs were unambiguously assigned to chromosomes, and 89 of the total were assigned to YACs. The 141 STSs that were developed based on (CAG/CTG)n repeats of at least seven units were genotyped on four reference CEPH individuals to estimate their polymorphic quality.


Asunto(s)
Mutación , Repeticiones de Trinucleótidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos/genética , Clonación Molecular , Femenino , Enfermedades Genéticas Congénitas/genética , Genotipo , Humanos , Masculino , Repeticiones de Minisatélite , Polimorfismo Genético , Lugares Marcados de Secuencia
13.
Neuron ; 16(1): 77-87, 1996 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-8562093

RESUMEN

Periventricular heterotopia (PH) involves dramatic malformations of the human cerebral cortex. Here we show that PH is closely linked to markers in distal Xq28 (maximal two-point lod score = 4.77 for F8C at theta = 0; maximal multipoint lod score = 5.37), so that affected females are obligatory mosaics for the mutation; that PH is lethal to at least some affected males; that PH malformations consist of well-differentiated cortical neurons filling the adult subependymal zone; and that individuals with PH are at high risk for epilepsy, though they have no other neurological or external stigmata. The PH gene may represent an important epilepsy susceptibility locus in addition to playing a key role in normal cortical development.


Asunto(s)
Encefalopatías/genética , Corteza Cerebral , Coristoma/genética , Epilepsia/genética , Cromosoma X , Aborto Habitual/genética , Adulto , Encefalopatías/patología , Coristoma/patología , Epilepsia/patología , Epilepsia Generalizada/genética , Epilepsia Generalizada/patología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/patología , Femenino , Muerte Fetal/genética , Genes Dominantes , Genes Letales , Humanos , Recién Nacido , Escala de Lod , Imagen por Resonancia Magnética , Masculino , Linaje , Embarazo
14.
Mamm Genome ; 6(10): 714-24, 1995 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8563170

RESUMEN

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.


Asunto(s)
Bovinos/genética , Biblioteca Genómica , Repeticiones de Microsatélite/genética , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Datos de Secuencia Molecular
15.
Hum Mol Genet ; 4(10): 1829-36, 1995 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8595403

RESUMEN

Genetic markers based upon PCR amplification of short tandem repeat-containing sequence tagged sites (STSs) have become the standard for genetic mapping. We have completed a survey based on the direct isolation of representative members of each of the 10 trinucleotide repeat classes to determine their relative abundance, repeat size distribution, and general utility as genetic markers. Trinucleotide repeats, depending on the repeat class, are one to two orders of magnitude less frequent than (AC)n repeats. The average size of trinucleotide repeats sequenced was less than 15 repeat units in length, and only three of the STSs developed for this study demonstrated more than 25 repeats units. The (AAT)n class of repeats are the most abundant and also the most frequently polymorphic. Other classes of trinucleotide repeat classes observed to be frequently polymorphic include (AAC)n, (ACT)n, (ATC)n and (AAG)n; however, the relative abundance of these classes is less than that observed for the (AAT)n class of repeats. Based upon this initial survey, we have initiated saturation cloning of the (AAT)n class of repeats. At the time of submission of this manuscript, we have developed, as part of the Cooperative Human Linkage Center (CHLC), more than 415 new high heterozygosity (AAT)n genetic markers (more than two alleles in four individuals) and 200 new low heterozygosity (AAT)n STSs from this larger screening effort combined with the initial survey.


Asunto(s)
Mapeo Cromosómico , Genoma Humano , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Cromosomas Humanos , Cartilla de ADN , Marcadores Genéticos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Lugares Marcados de Secuencia , Terminología como Asunto
17.
Genomics ; 27(3): 389-98, 1995 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-7558018

RESUMEN

We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a subunit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear.


Asunto(s)
ADN Complementario/genética , Órgano Espiral/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Anticuerpos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN/genética , Cobayas , Inmunohistoquímica , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Quinasas Asociadas a Fase-S , Distribución Tisular , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo
18.
Hum Mol Genet ; 4(3): 449-52, 1995 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-7795601

RESUMEN

For the purpose of initial screening of the human genome in linkage mapping, two overlapping sets of high quality short tandem repeat polymorphisms (STRPs) which span the autosomes have been assembled. The higher density set contains a total of 363 markers with an average heterozygosity of 79% and an average sex-equal genetic distance between markers of 10.5 cM. The lower density set, which is a subset of the other, contains 156 markers with an average heterozygosity of 80% and an average spacing of 26.5 cM. Tri- and tetranucleotide STRPs comprised 47 and 63%, respectively, of the markers within the higher and lower density sets. Markers within the screening sets were selected to have maximum quality, where quality was defined as a blend of high informativeness, strong amplification under standard PCR conditions, low amplification background, and ease in scoring. The screening sets along with combinations of STRPs which can be amplified and electrophoresed simultaneously are available electronically through anonymous ftp.


Asunto(s)
Ligamiento Genético , Marcadores Genéticos , Genoma Humano , Secuencias Repetitivas de Ácidos Nucleicos/genética , Cromosomas Humanos Par 14/genética , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo Genético
19.
Genomics ; 25(1): 1-8, 1995 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-7774906

RESUMEN

In this report, we describe the successful application of a rapid and efficient procedure, based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs). This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome. Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis. Several key steps have been designed to achieve optimal subtraction and to obtain preferential amplification and recovery of the target sequences. Methods for efficient construction of small insert libraries and rapid, nonradioactive screening have also been integrated into the protocol. Using a 750-kb YAC as a target, we identified a minimum of 14 unique microsatellite containing clones, leading to the development of 12 polymorphic STSs (sequence-tagged sites). These new markers will facilitate the genetic localization of targeted locus and allow the accurate ordering by STS content mapping of a cloned contig spanning the interval. In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.


Asunto(s)
Cromosomas Artificiales de Levadura , ADN de Hongos/análisis , ADN Satélite/análisis , Marcadores Genéticos , Técnicas Genéticas , Reacción en Cadena de la Polimerasa/métodos , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN de Hongos/genética , ADN Satélite/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Mapeo Restrictivo , Lugares Marcados de Secuencia
20.
Nucleic Acids Res ; 22(20): 4148-53, 1994 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-7937140

RESUMEN

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3'-terminal exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' exons in addition to a great reduction of non-specific background. As a test for the system, 3' exon trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.


Asunto(s)
Clonación Molecular/métodos , Exones , Globinas/genética , Secuencia de Bases , Línea Celular , Cromosomas Humanos Par 16 , Cósmidos , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Poli A/metabolismo , Reacción en Cadena de la Polimerasa , Empalme del ARN , Transfección
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