RESUMEN
We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.
Asunto(s)
Dípteros/genética , Genes de Insecto , Proteínas y Péptidos Salivales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Perfilación de la Expresión Génica , Larva , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Drosophila melanogaster was transformed with an 18 kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.
Asunto(s)
Cromosomas/genética , Dípteros/genética , Drosophila melanogaster/genética , Amplificación de Genes , Genes de Insecto , Transcripción Genética/genética , Animales , Animales Modificados Genéticamente/genética , Southern Blotting , Vectores Genéticos , Hibridación in Situ , Hibridación Fluorescente in Situ , Ensayos de Protección de NucleasasRESUMEN
The discovery of Dna sequence amplification in sciarid flies and investigations into its control and biological significance are reviewed. Results thus far show that amplification of specific salivary gland polytene chromosome bands is a general phenomenon in sciarids. It brought about as part of a final endoreplication cycle by the rising titer of ecdysterone that occurs as the Larvae approach the prepupal period. Amplification and transcription of these bands is a late, multistep effect of this hormone.The Dna puffs which form in amplified region produce mRNAs which are translated into polypeptides that appear to be involved in coccon formation. Application of molecular cloning techniques to the study of Dna amplification has allowed precise quantitation of amplification for several Dna puffs and is yielding maps of their transcription units.These techniques will ultimately help to define the origins of Dna puff replication and contribute to an understanding of the mechanism and control of the amplification phenomenon in sciaridae. Projections for future experimental approaches are presented
Asunto(s)
Animales , Femenino , Dípteros/genética , Replicación del ADN/genética , Amplificación de Genes/genética , Secuencia de Bases , Cromosomas/fisiología , Clonación Molecular , Replicación del ADN/fisiología , ADN/efectos de los fármacos , ADN/genética , ADN/fisiología , Ecdisterona/farmacología , Amplificación de Genes/fisiología , Larva , Datos de Secuencia Molecular , Glándulas Salivales , Transcripción Genética/genética , Transcripción Genética/fisiologíaRESUMEN
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