RESUMO
In Drosophila melanogaster structural genes are located close to moderately reiterated sequences. One of the clones obtained contains the DNA related to intercalary heterochromatin of D. melanogaster. These are individual differences in the distribution of genetic material in polytenic chromosomes of different stocks of D. melanogaster. The techniques that allow isolation of DNA fragments containing structural genes at the beginning, in the middle, or the end of the coding strand have been elaborated.
Assuntos
DNA/isolamento & purificação , Genes , Ligação Genética , Animais , Sequência de Bases , Colífagos , Drosophila melanogaster , Engenharia Genética , Heterocromatina/química , Métodos , Camundongos , Hibridização de Ácido Nucleico , RNA MensageiroAssuntos
Núcleo Celular/metabolismo , RNA Mensageiro , RNA Neoplásico , Animais , Sequência de Bases , Carcinoma de Ehrlich/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Hidroxiapatitas , Camundongos , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Renaturação de Ácido Nucleico , Pâncreas/enzimologia , Radioisótopos de Fósforo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Ribonucleases , Ribonucleotídeos/análise , Temperatura , Transcrição GênicaRESUMO
A method for the isolation of the DNA fragments containing structural genes at the beginning, in the middle and at the end of the coding strand is described. It involves mechanical or enzymatic shearing, exonuclease treatment, hybridization with mRNA and subsequent retention of DNA-mRNA complex on poly(U)-Sepharose. Hybridization of the DNA fragments with mRNA takes place with high specificity and isolated material is enriched in structural genes. Applications of this method for gene isolation are discussed.
Assuntos
DNA/isolamento & purificação , Genes , Animais , Carcinoma de Ehrlich/análise , Cromatografia de Afinidade , Enzimas de Restrição do DNA , Exonucleases , Fígado/análise , Métodos , Camundongos , Peso Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/isolamento & purificaçãoRESUMO
In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using 32P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions ("jumping") of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with "synthetic" karyotoypes characterized by different combinations of D. virilis homologous chromosomes and "hybrid" chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition ("jumping") took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such "synthetic" stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.
Assuntos
Cromossomos/fisiologia , DNA/genética , Drosophila/genética , Animais , Clonagem Molecular , Cruzamentos Genéticos , Elementos de DNA Transponíveis , DNA Recombinante/metabolismo , Drosophila melanogaster/genética , Feminino , Hibridização Genética , Masculino , Hibridização de Ácido Nucleico , Poli A/genética , RNA Mensageiro/genética , Especificidade da EspécieRESUMO
The esterase S gene (estS) of Drosophila virilis is specifically expressed in the ejaculatory bulbs of males. Its sequencing shows similarities between estS product and other esterases of different origin. The transcription of estS in ejaculatory bulbs is at least 2 orders of magnitude higher than in other tissues of males. Two promoters, P1 (distal) and P2 (proximal), and two different transcripts were identified. The promoter P2 is used much more efficiently, and in a stringent, tissue-specific manner. The transcription from P1 takes place in different tissues and stages of development of D. virilis. However, the mRNA transcribed from P1 seems to be inactive in translation as there are three open-reading frames (ORF) between P1 and P2, which may block the translation in P1 initiated mRNA. Insertion of sequence containing the three ORFs into the 5' untranslated region of the CAT gene strongly inhibited expression of CAT. Point mutations destroying the three ORFs completely eliminate the inhibitory effect. Hence tissue-specific expression of the estS gene may depend on control at the level of transcription, promoter selection and translation.
Assuntos
Hidrolases de Éster Carboxílico/genética , Proteínas de Drosophila , Drosophila/genética , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Carboxilesterase , Linhagem Celular , Galinhas , Cloranfenicol O-Acetiltransferase/genética , Códon , Feminino , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta , Mutação Puntual , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Transcrição Gênica , TransfecçãoRESUMO
We have cloned the gene for the esterase S isozymes complex from the genome of Drosophila virilis in pBR322. Esterase S is an enzyme which is specifically synthesized in the ejaculatory bulbs of D. virilis adult males. The gene for the esterase S isozyme complex (estS) has been localized in band 2G5e of chromosome II. Poly(A)+ RNA prepared from ejaculatory bulbs actively hybridizes with this band. A cloned 15-kb fragment of D. virilis DNA (pVE9) also hybridizes with band 2G5e. The area encoding the poly(A)+ RNA is located in the middle part of the cloned fragment whose ends are not transcribed in vivo. Only one poly(A)+ RNA which is 1.9 kb long and complementary to pVE9 DNA can be revealed in the cytoplasm. The mRNA preselected by hybridization to pVE9 DNA was microinjected into the cytoplasm of Xenopus laevis oocytes. In other experiments, the pVE9 DNA itself was microinjected into oocyte nuclei. In both cases, esterase S is synthesized in the oocytes, and the major part of the protein is transported from the oocytes and accumulated in the incubation medium.