RESUMO
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.
Assuntos
Replicação do DNA/fisiologia , Dípteros/genética , Amplificação de Genes/fisiologia , Transcrição Gênica/fisiologia , Animais , Northern Blotting , Southern Blotting , DNA/fisiologia , Sondas de DNA , Drosophila/genética , Biblioteca Gênica , Larva , Glândulas SalivaresRESUMO
We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.
Assuntos
Replicação do DNA , Dípteros/genética , Amplificação de Genes , Animais , Cromossomos/ultraestrutura , Clonagem Molecular , Dípteros/ultraestrutura , Larva , Sequências Repetitivas de Ácido Nucleico , Replicon , Saccharomyces cerevisiae/genética , Glândulas Salivares/ultraestruturaRESUMO
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.
Assuntos
DNA/efeitos dos fármacos , Dípteros/genética , Ecdisterona/farmacologia , Poli A/biossíntese , RNA/biossíntese , Animais , Feminino , Larva/fisiologia , Glândulas SalivaresRESUMO
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.
Assuntos
Anopheles/genética , Baculoviridae/genética , Besouros/genética , Expressão Gênica , Spodoptera/enzimologia , Transformação Genética , alfa-Amilases/genética , Animais , Sequência de Bases , Técnicas de Cultura de Células , Cromatografia , Primers do DNA , Eletroforese em Gel de Ágar , Dados de Sequência Molecular , Spodoptera/genética , alfa-Amilases/metabolismoRESUMO
Late in the fourth larval instar, several regions of the Rhynchosciara americana salivary gland chromosomes undergo "DNA puffing." We have constructed a library of cloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands during the period of development when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puff sites C3 and C8 and encode 1.25-kilobase and 1.95-kilobase RNA molecules, respectively. We estimate from the quantitation of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.
Assuntos
Cromossomos/ultraestrutura , Dípteros/genética , Amplificação de Genes , Glândulas Salivares/ultraestrutura , Animais , DNA , Dípteros/crescimento & desenvolvimento , Larva , Hibridização de Ácido NucleicoRESUMO
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.
Assuntos
Animais , DNA , Anfíbios/genética , Hemoglobinas/análise , L-Lactato Desidrogenase/análise , Miocárdio/enzimologia , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/química , Genes Reguladores , PoliploidiaRESUMO
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