RESUMEN
Normal silkworms (Bombyx mori) have opaque larval skin due to uric acid accumulation in the epidermis while a mutant, og, is translucent owing to a deficiency in xanthine dehydrogenase (XDH), which synthesizes uric acid. Molybdenum cofactor (MoCo) sulfurase is responsible for XDH activation in various organisms. A silkworm MoCo sulfurase gene was cloned and found to be on the og locus, whose mutant alleles, og(k) and og(t), show premature stop codons, proving that og is the MoCo sulfurase gene. It was observed that a miniature inverted-repeat transposable element (MITE), named Organdy, when inserted in an og(t) mutant allele exon, causes unstable splicing of a downstream intron leading to incomplete open reading frames.
Asunto(s)
Bombyx/enzimología , Bombyx/genética , Sulfurtransferasas/genética , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis , Secuencia de Bases , Bombyx/crecimiento & desarrollo , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Genes , Larva , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Fenómenos Fisiológicos de la Piel , Sulfurtransferasas/química , Sulfurtransferasas/metabolismo , Xantina Deshidrogenasa/genéticaRESUMEN
Kynurenine 3-monooxygenase (KMO, EC 1.14.13.9), which catalyzes the oxidation of kynurenine to 3-hydroxykynurenine, is involved in the synthesis of ommochrome pigments in insects. A silkworm mutant, white egg 1 ( w-1), has been shown to be deficient in this enzyme activity. The mutant is characterized morphologically by its white eyes and the fact that the females lay white eggs. To analyze the relationship between the KMO gene and the mutation, we first determined the entire sequence of a full-length 2.0-kb cDNA and examined its expression pattern in the wild type. The cDNA sequence contains one ORF encoding a polypeptide of 456 amino acids, and transcripts were detected in the larval Malpighian tubules and the pupal ovaries, but not in other tissues. Southern analysis and nucleotide sequencing showed that the KMO gene is present in a single copy and consists of ten exons distributed over a 16-kb region. Comparison of the transcripts between the wild type and mutant silkworms showed that the wild type expressed a single transcript, whereas the mutant exhibited markedly reduced amounts of two transcripts with sizes of 2.0 kb and 1.8 kb. Nucleotide sequence analysis of these mutant transcripts indicated that sequences corresponding to the ninth and tenth exons were missing. Inverse PCR and Southern analysis of the mutant gene demonstrated that the corresponding genomic region was deleted in the w-1 mutant.
Asunto(s)
Bombyx/genética , Oxigenasas de Función Mixta/genética , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , Expresión Génica , Quinurenina 3-Monooxigenasa , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de AminoácidoRESUMEN
We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.
Asunto(s)
Bombyx/genética , Elementos Transponibles de ADN/genética , Vectores Genéticos/genética , Mutación de Línea Germinal/genética , Transformación Genética/genética , Actinas/genética , Envejecimiento/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Bombyx/embriología , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Cruzamientos Genéticos , Femenino , Proteínas Fluorescentes Verdes , Larva/metabolismo , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Masculino , Mariposas Nocturnas/enzimología , Mariposas Nocturnas/genética , Mutagénesis Sitio-Dirigida/genética , Regiones Promotoras Genéticas/genética , Pupa/metabolismo , Recombinación Genética/genética , Análisis de Secuencia de ADN , Secuencias Repetidas Terminales/genética , Transgenes/genética , Transposasas/genética , Transposasas/metabolismoRESUMEN
Xanthine dehydrogenase (XDH) is a molybdoenzyme which catalyses oxidation of xanthine and hypoxanthine to uric acid. We isolated genomic clones of silkworm (Bombyx mori) XDH genes (BmXDH1 and BmXDH2). The BmXDH2 gene is located upstream from the BmXDH1 gene and they show a tandemly duplicated structure. Both BmXDH genes were expressed in the fat body and Malpighian tubules, whereas only the BmXDH1 gene was expressed in the midgut. Phylogenetic analysis indicates that BmXDH gene duplication occurred after the divergence of the silkworm and dipteran species. Intron insertion site comparison shows that some introns were lost during insect XDH gene evolution.
Asunto(s)
Bombyx/enzimología , Genes Duplicados , Genes de Insecto , Secuencias Repetidas en Tándem , Xantina Deshidrogenasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Bombyx/genética , ADN Complementario , Expresión Génica , Humanos , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Xantina Deshidrogenasa/fisiologíaRESUMEN
Seedlings of dwarf pea grown under red light for 9 d were homogenized in 0.1 M phosphate buffer (pH 6.8) and a "water-soluble extract" was obtained by centrifugation, dialysis and lyophilization. The extract contained a proteinaceous substance (or substances) which interfered with the GA3 response of dwarf peas, probably due to complex formation with the hormone.