RESUMEN
CONTEXT: Little is known about genes that contribute to polycystic ovary syndrome (PCOS). We previously found linkage and association of PCOS with the dinucleotide marker D19S884 in two independent sets of families; allele 8 of D19S884 confers increased risk. OBJECTIVE/DESIGN: The objectives of the study were: 1) use the transmission/disequilibrium test (TDT) to assess linkage and association between PCOS and D19S884 (and nearby markers) in a third set of families; and 2) test D19S884 and surrounding DNA sequence for in vitro regulatory activity in lymphoblastoid cell lines (LCLs) and granulosa cells. SETTING/SUBJECTS: We studied 98 new families with a PCOS proband, father, mother, and other available offspring. We analyzed data from these families separately and in combination with data obtained previously. INTERVENTIONS: Interventions were venipuncture. MAIN OUTCOME MEASURES: Measures were transmission frequencies and in vitro functional studies. RESULTS: The first result we found was that in the 98 new families, the TDT was significant for allele 8 of D19S884 (P = 0.043). In the total collection of 465 families, the TDT evidence is very strong (nominal P < 7 x 10(-5)). Results for all other genetic markers near D19S884 were nonsignificant after correction for multiple testing. The second result was that an approximately 800-bp fragment containing various alleles of D19S884 showed modest but reproducible promoter activity in LCLs. However, no allelic differences were detected. No activity of this fragment was detected in granulosa cells. CONCLUSIONS: This is the second independent confirmation of linkage and association of D19S884 with PCOS. We found in addition that some sequence in the region of D19S884 confers in vitro promoter activity in LCLs.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 19 , Predisposición Genética a la Enfermedad , Síndrome del Ovario Poliquístico/genética , Femenino , Genotipo , Humanos , Desequilibrio de Ligamiento , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.
Asunto(s)
Cromosomas Humanos Par 19/genética , Haplotipos/genética , Recombinación Genética , Alelos , Mapeo Cromosómico , ADN/genética , Evolución Molecular , Frecuencia de los Genes , Marcadores Genéticos , Humanos , Desequilibrio de Ligamiento , Modelos Genéticos , Polimorfismo de Nucleótido SimpleRESUMEN
This unit describes two methods of genotyping DNA sequences containing known nucleotide variations. The first protocol describes a colorimetric method for genotyping DNA samples amplified by the polymerase chain reaction (PCR) using an oligonucleotide ligation assay (OLA). The second protocol describes the ligase chain reaction (LCR), a method for simultaneously amplifying and genotyping genomic DNA samples. A Support Protocol describes the preparation of modified biotin- and digoxigenin-labeled oligonucleotide primers.
Asunto(s)
Genotipo , Reacción en Cadena de la Ligasa/métodos , Colorimetría , ADN/genética , Cartilla de ADN , Genética Médica , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
A new chemical affinity system is described for the purification of proteins. The Linx Affinity Purification System enables researchers to quickly and easily bind a protein ligand to a chromatographic matrix and use the resulting affinity resin to purify a second protein from crude mixtures. The entire process takes approximately 2 h.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas/aislamiento & purificación , Animales , Antiinfecciosos/farmacología , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Ácidos Borónicos/química , Tampones (Química) , Extractos Celulares/química , Electroforesis en Gel de Poliacrilamida , Enzimas Inmovilizadas/inmunología , Epítopos/inmunología , Concentración de Iones de Hidrógeno , Ácidos Hidroxámicos/química , Sueros Inmunes/química , Estructura Molecular , Peso Molecular , Unión Proteica/efectos de los fármacos , Proteínas/química , Proteínas/inmunología , Proteínas/metabolismo , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ribonucleasa Pancreática/inmunología , Ribonucleasa Pancreática/metabolismo , Salicilamidas/química , Sefarosa/metabolismo , EsterilizaciónRESUMEN
A library of 2302 small molecule beta-turn mimetics was screened for inhibition of the alpha 4 beta 1 integrin-CS1 splice variant binding interaction. Preliminary data revealed several active ligands, and validation with purified material culminated in the identification of some of the first small molecule ligands (1, IC50 = 5 microM, and 2, IC50 = 8 microM) to be reported for this class of integrins.
Asunto(s)
Aminoácidos/síntesis química , Ácidos Carboxílicos/síntesis química , Bases de Datos como Asunto , Integrinas/antagonistas & inhibidores , Integrinas/genética , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Receptores Mensajeros de Linfocitos/genética , Empalme Alternativo , Aminoácidos/química , Aminoácidos/farmacología , Sitios de Unión , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Diseño de Fármacos , Integrina alfa4beta1 , Integrinas/química , Ligandos , Conformación Molecular , Estructura Molecular , Receptores Mensajeros de Linfocitos/química , Receptores de Antígeno muy Tardío/antagonistas & inhibidores , Receptores de Antígeno muy Tardío/química , Receptores de Antígeno muy Tardío/genética , Reproducibilidad de los Resultados , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.
Asunto(s)
Medicina Legal/métodos , Genoma Humano , Paternidad , Reacción en Cadena de la Polimerasa/métodos , Alelos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos , Colorimetría/métodos , ADN/análisis , Cartilla de ADN , Sondas de ADN , Estudios de Factibilidad , Femenino , Marcadores Genéticos , Variación Genética , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , LinajeRESUMEN
Aspartylglycosaminuria (AGU) is a recessively inherited lysosomal storage disease that occurs with much higher frequency in Finland than elsewhere. AGU is caused by a deficiency in glycosylasparaginase (GA), which results in the accumulation of glycoasparagines in lysosomes. In the Finnish population, a single nucleotide change in the gene encoding GA is responsible for the disease. We have used the oligonucleotide ligation assay (OLA) to detect the mutation in polymerase chain reaction (PCR)-amplified DNA samples from normal, carrier, and affected individuals. Screening for AGU among 415 random Finnish DNA samples with PCR/OLA revealed five carriers of the mutant allele and demonstrated the potential of the method for use in carrier screening. PCR/OLA provides a rapid, reliable, nonisotopic method to detect the mutation responsible for AGU that can readily be applied to large population screening.
Asunto(s)
Acetilglucosamina/análogos & derivados , ADN Ligasas/metabolismo , Enfermedades por Almacenamiento Lisosomal/genética , Mutación , Oligonucleótidos/metabolismo , Acetilglucosamina/orina , Alelos , Aspartilglucosaminuria , Secuencia de Bases , Finlandia , Heterocigoto , Homocigoto , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Moldes GenéticosRESUMEN
Automated, direct cycle sequencing of purified double-stranded PCR products using Taq polymerase and fluorescently labeled dideoxynucleotide terminators provides a robust and highly reproducible method for identifying DNA sequence variations in sequence-tagged sites. We describe a simple and sensitive strategy that reliably detects the presence of DNA variations when sequencing traces from several different individuals are compared. We also demonstrate the use of this strategy to estimate allele frequencies of single nucleotide substitutions in a population. Taken together, this approach provides an automated method for conducting rapid population studies of candidate gene regions that are in linkage or association with a specific disease and for comparative evolutionary analysis of selected regions of the human genome.
