RESUMEN
We describe the molecular analysis of a large three generation Palestinian family segregating for monilethrix. Previous reports have shown that mutations in type-II hair cortex keratin genes, hHb1 and hHb6, are associated with monilethrix. Genetic linkage analysis performed on this family using markers flanking the hHb6 gene exhibited strong evidence for linkage. Sequence analysis revealed a nucleotide substitution of G --> T at nucleotide 1230 resulting in a glutamic acid to aspartic acid amino acid substitution at codon 410, identical to that reported in a French family. The family in our study provides further evidence that mutations of the hHb6 gene are responsible for monilethrix.
Asunto(s)
Enfermedad de Darier/genética , Cabello/anomalías , Biología Molecular , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Árabes , Secuencia de Bases , Análisis Mutacional de ADN , Enfermedad de Darier/diagnóstico , Familia , Ligamiento Genético , Marcadores Genéticos , Humanos , Israel/etnología , Masculino , Biología Molecular/métodos , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA.
Asunto(s)
Alpharetrovirus/enzimología , Ácido Aspártico Endopeptidasas/genética , Proteínas de Fusión gag-pol/genética , Productos del Gen gag/genética , Mutación Puntual , Alpharetrovirus/fisiología , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Escherichia coli/genética , Proteínas de Fusión gag-pol/metabolismo , Productos del Gen gag/metabolismo , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Virión/enzimología , Replicación ViralRESUMEN
The apterous (ap) gene in Drosophila melanogaster encodes a homeodomain transcription factor. It is required for the development of the wings and of a subset of embryonic muscles. The gene has been implicated in the juvenile hormone (JH) system because mutations in ap lead to JH deficiency, and are associated with defective histolysis of the larval fat body, arrested vitellogenesis, sterility, and aberrant sexual behavior, all of which are dependent on JH. We describe here the use of hemizygotes and germ-line clones, of X-ray- and hybrid dysgenesis-induced lethal ap alleles to determine the primary role of the gene during development. We find that ap lethality is polyphasic, but occurs primarily at the larval and pupal stages. The lethal phenotype is not associated with any overt morphological abnormality, suggesting that death occurs from a systemic malfunction. Strong interallelic complementation for the wing phenotype was found between some ap mutations induced by X-rays or by hybrid-dysgenesis. By Northern blot analysis, we demonstrate an increase in ap expression in pupae and adults as compared to embryos and larvae, suggesting that it is developmentally regulated. Finally, primer extension is used to determine the transcription start site of the gene.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Proteínas de Homeodominio , Hormonas Juveniles/genética , Factores de Transcripción/genética , Alelos , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Regulación de la Expresión Génica , Prueba de Complementación Genética , Proteínas con Homeodominio LIM , Masculino , Datos de Secuencia Molecular , MutaciónRESUMEN
Chick embryo fibroblasts grew normally in the presence of 1 X 10(-3) to 10 X 10(-3) M alpha-difluoromethylornithine (DFMO). This drug did not interfere with protein and DNA synthesis of normal fibroblasts and of cells transformed by Rous sarcoma virus. The morphological appearance of normal and transformed cells was not altered by DFMO, as determined by scanning electron microscopy. Flow microfluorometric analyses also confirmed the notion that normal or transformed cells were not blocked by DFMO in the G1 phase of the cell cycle. As expected, DFMO reduced cellular putrescine levels. This diamine, however, was replaced by the analogue cadaverine (1,5-diaminopentane), which accumulated mainly in the transformed cells. The increase in cellular cadaverine levels was also demonstrated during the infection of chick embryo fibroblasts with a temperature-sensitive mutant of Rous sarcoma virus under permissive conditions. Under restrictive conditions (42 degrees C), less cadaverine accumulated in the infected cells. These findings suggest that diamines and polyamines are necessary for the transformation process and that blocking one pathway by DFMO leads to the activation of an alternative biosynthetic pathway.