RESUMO
P element transposition in Drosophila melanogaster is regulated by germline-specific splicing of the P element ORF2-ORF3 intron. This regulation has been shown to depend on a cis-acting sequence located in the exon 12-31 bases from the 5' splice site. Mutations within this sequence disrupt the regulation and result in splicing of the ORF2-ORF3 intron in all tissues, indicating that the sequence is required to inhibit splicing of this intron in the soma. We now show that a trans-acting factor in a human (HeLa) cell extract can inhibit splicing of the intron, suggesting that this regulatory mechanism is conserved from flies to humans.
Assuntos
Extratos Celulares/farmacologia , Elementos de DNA Transponíveis/genética , Íntrons/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Animais , Sequência de Bases , Drosophila , Éxons/efeitos dos fármacos , Éxons/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação/genética , Peptídeos/efeitos da radiação , Splicing de RNA/genética , RNA Mensageiro/genética , Ativação Transcricional/genética , Raios UltravioletaRESUMO
P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.
Assuntos
Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Células Germinativas/fisiologia , Íntrons , Splicing de RNA , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Oligonucleotídeos/química , Reação em Cadeia da PolimeraseRESUMO
Six alpha 2-macroglobulin cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2-macroglobulin including its 27-residue signal peptide and the 3' - and part of the 5' nontranslated regions were determined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2-macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobulin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2-macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 18 h. By 24 h the levels had decreased to less than 30% of the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3-fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transcriptional processing.
Assuntos
Inflamação/genética , RNA Mensageiro/metabolismo , alfa-Macroglobulinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , DNA Recombinante , Adjuvante de Freund , Humanos , Inflamação/imunologia , Cinética , Masculino , Hibridização de Ácido Nucleico , Ratos , Ratos Endogâmicos F344 , Transcrição GênicaRESUMO
Six alpha 2-macroglobulin (alpha 2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, p alpha 2M1, carries a 4.6 kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature alpha 2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro-alpha 2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published alpha 2M amino acid sequence for all except three residues. The alpha 2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using alpha 2M cDNA as a hybridization probe.
