RESUMEN
OBJECTIVE: To screen and identify differentially expressed genes between a fertile patient and another infertile patient who belonged to a large Chinese pedigree affected with androgen insensitivity syndrome (AIS). METHODS: We constructed the forward and reversed subtracted libraries using genital skin fibroblasts (GSF), which were obtained from the fertile patient MJ and infertile patient ZGJ, as tester respectively. Candidate clones were screened with colony in situ hybridization, dot blot, and Southern blot analysis step by step and conformed with Northern blot analysis. The potential positive clones were sequenced and the homology of the sequences was analyzed. RESULTS: The forward and reversed subtracted libraries containing differentially expressed pattern of two GSF cell lines were constructed. Two positive clones identified by Northern blot were obtained in the reversed subtracted library. Eleven candidate clones from the two libraries that failed to hybridize with both RNA populations were obtained simultaneously, which might represent differentially expressed low abundance transcripts. Sequencing results and homology analysis demonstrated that the two positive clones were significantly homologous with the genes of autotaxin-t and calcium binding protein calcyclin (S100A6), respectively. CONCLUSIONS: Two positive clones and eleven clones showing no hybridization signals may represent differentially expressed genes between the two GSFs. This finding may be useful to elucidate the molecular mechanisms leading to phenotypic variation and preserved fertility of the AIS pedigree.
Asunto(s)
Síndrome de Resistencia Androgénica/genética , Fertilidad/genética , Perfilación de la Expresión Génica , Infertilidad Masculina/genética , Síndrome de Resistencia Androgénica/complicaciones , Northern Blotting , Fibroblastos/citología , Biblioteca de Genes , Genitales Masculinos/citología , Humanos , Técnicas In Vitro , Infertilidad Masculina/etiología , Masculino , Hibridación de Ácido Nucleico/métodos , Linaje , Reacción en Cadena de la Polimerasa , Piel/citologíaRESUMEN
Transforming growth factor-beta receptor-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TGFbetaRI and flanking intron sequences from 53 primary non-small cell lung cancer (NSCLC) tissues were examined for alterations using SSCP and direct sequencing. No somatic point mutations other than two silent mutations and a polymorphism were found in the TGFbetaRI gene. The two silent mutations located at codon 344 (AAT to AAC) and codon 406 (TTA to CTA), respectively, and the polymorphism was at the 24th base of intron 7 (G to A). To investigate whether the presence of this polymorphism is associated with NSCLC, we determined its allele distribution in all the 53 carcinomas and 89 normal controls. Interestingly, we found that the subjects with homozygous genotype A/A displayed more than 3-fold increased risk of developing NSCLC than the common wild genotype G/G. As the first report, the present study showed that TGFbetaRI gene is not a frequent site of spontaneous mutational inactivation while the detected polymorphism is frequent in the pathogenesis of NSCLC.
Asunto(s)
Receptores de Activinas Tipo I/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN de Neoplasias/genética , Neoplasias Pulmonares/genética , Polimorfismo Genético , Receptores de Factores de Crecimiento Transformadores beta/genética , Adulto , Anciano , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Neoplasias Pulmonares/fisiopatología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , Receptor Tipo I de Factor de Crecimiento Transformador betaRESUMEN
Partial sequences of 25 S ribosomal DNA for nine Candida yeast and one Kluyveromyces yeast strains were cloned and determined. We compared them with two published 25 S rDNA sequences of Saccharomyces cerevisiae and Candida albicans. An evolutionary tree of the twelve species was inferred from about 370 sites of 5' end of 25 S ribosomal DNA using the methods of neighbor-joining and bootstrap. The molecular data indicate a very close affinity between Candida kefyr CBS834 and Kluyveromyces cicerisporus CBS4857.
