RESUMEN
The Anopheles merus (Diptera, Nematocera, Culicoidea) alpha-amylase gene (AmerAmy, GenBank Accession Number U01210) was amplified with its own or with the Zabrotes subfasciatusalpha-amylase signal peptide (ZsAmerAmy, GenBank Accession Number AY270183) by PCR, using designed primers. The AmerAmy gene was sequenced from its promotor to the TGA codon. As a positive control, the Z. subfasciatusalpha-amylase gene with its own signal peptide (ZsAmy, GenBank Accession Number AF255722) was also amplified by PCR. These three sequences were inserted into the baculovirus genome using the Bac-to-Bac trade mark system. Recombinant baculovirus preparations were used to infect Sf9 Spodoptera frugiperda insect cells. The A. merusalpha-amylase was successfully expressed as an active enzyme detected mainly in cell culture supernatants.
Asunto(s)
Anopheles/genética , Baculoviridae/genética , Escarabajos/genética , Expresión Génica , Spodoptera/enzimología , Transformación Genética , alfa-Amilasas/genética , Animales , Secuencia de Bases , Técnicas de Cultivo de Célula , Cromatografía , Cartilla de ADN , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , Spodoptera/genética , alfa-Amilasas/metabolismoRESUMEN
The synchronous development of a sibling group of Rhynchosciara larvae enables us to follow the relationship between the local transcription and extrareplication of C3 and C8 DNA puffs. Although DNA amplification at these two loci takes place during the last cycle of DNA duplication in salivary gland chromosomes, a different timing of puff expression was observed for the two regions analysed. C3 puff transcription is a late event in relation to the C8 counterpart. We present evidence that this might be a consequence of the different firing of DNA amplification in both loci. No signs of DNA rearrangements were detected with probes that extend the previously analysed regions.
Asunto(s)
Replicación del ADN/fisiología , Dípteros/genética , Amplificación de Genes/fisiología , Transcripción Genética/fisiología , Animales , Northern Blotting , Southern Blotting , ADN/fisiología , Sondas de ADN , Drosophila/genética , Biblioteca de Genes , Larva , Glándulas SalivalesRESUMEN
1. Fourth-instar larvae of Rhynchosciara americana were injected with the insect molting hormone, ecdysterone, giving final hemolymph concentrations from 4.46 to 223 microM. 2. Induction of the DNA puff, B2b, in the proximal (S1) region of the salivary glands of Rhynchosciara americana by 22.6 microM ecdysterone, was accompanied by the production of an mRNA and a polypeptide with the same characteristics as B2b products produced during normal development. This mRNA and polypeptide were restricted to the proximal region of the gland, as is the B2b puff. 3. Synthesis of other poly(A)+RNAs was also stimulated in S1 by ecdysterone, and other puffs that appear during normal development were induced. However, rRNA production in S1 goes through a pattern of inhibition, followed by recovery when B2b is puffed, and subsequent inhibition. 4. Low molecular weight RNA, with a peak in the region of 4S, is stimulated after ecdysterone administration.
Asunto(s)
ADN/efectos de los fármacos , Dípteros/genética , Ecdisterona/farmacología , Poli A/biosíntesis , ARN/biosíntesis , Animales , Femenino , Larva/fisiología , Glándulas SalivalesRESUMEN
1. Fourth-instar larvae of Rhynchosciara americana were injected with the insect molting hormone, ecdysterone, giving final hemolymph concentrations from 4.46 to 223µM. 2. Induction of the DNA puff, B2b, in the proximal (S1) region of the salivary glands of Rhynchosciara americana by 22.6 µM ecdyesterone, was accompanied by the production of an mRNA and a polypeptide with the same characteristics as B2b products produced during normal development. This mRNA and polypeptide were restricted to the proximal region of the gland, as is the B2b puff. 3. Synthesis of other poly (A) +RNAs was also stimulated in S1 by ecdysterone, and other puffs that appear during normal development were induced. However, rRNA production in S1 goes through a pattern of inhibition, followed by recovery when B2b is puffed, and subsequent inhibition. 4. Low molecular weight RNA, with a peak in the region of 4S, is stimulated after ecdysterone administration
Asunto(s)
Animales , Femenino , Dípteros , Ecdisterona/farmacología , Glándulas Salivales/fisiología , Poli A/biosíntesis , ARN/biosíntesis , Ecdisterona/administración & dosificación , Larva/fisiologíaRESUMEN
Among the anurans of the family Ceratophrydidae two polyploid species have been found: Odontophrynus americanus (4n = 44) and Ceratophrys dorsata (8n = 104). Comparative measurements in the diploid O. cultripes and the tetraploid O. americanus indicated a reduced activity or a partial inactivation of the genes which control the enzyme lactate dehydrogenase and the hemoglobin in the tetraploid.