RESUMEN
Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.
Asunto(s)
Endorribonucleasas/metabolismo , Complejos Multienzimáticos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Helicasas , Procesamiento Postranscripcional del ARN , ARN Bacteriano/metabolismo , ARN Ribosómico/metabolismo , Cromatografía de Afinidad , Endorribonucleasas/inmunología , Endorribonucleasas/aislamiento & purificación , Escherichia coli/enzimología , Complejos Multienzimáticos/inmunología , Complejos Multienzimáticos/aislamiento & purificación , Oligopéptidos , Péptidos , Polirribonucleótido Nucleotidiltransferasa/inmunología , Polirribonucleótido Nucleotidiltransferasa/aislamiento & purificación , Precursores del ARN/metabolismo , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 5S/metabolismo , ARN de Transferencia/metabolismo , Especificidad por SustratoRESUMEN
The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Ojo/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Núcleo Celular/química , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Drosophila/química , Drosophila/genética , Expresión Génica/genética , Expresión Génica/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto/genética , Proteínas de Homeodominio/análisis , Datos de Secuencia Molecular , Mutación/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Fenotipo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
We report here a method for the in vivo dissection of the regulatory region of a gene in the Drosophila genome. Our system includes (i) the reporter genes lacZ and white to detect transcriptional enhancer and silencer activities in a target gene, (ii) an efficient way to induce integration of gypsy elements in the genome, and (iii) unidirectional blocking of regulatory activities by the gypsy element, which is dependent on the su(Hw) protein. The optomotor-blind (omb) gene was analyzed. In the omb(P1) line, a P[lacW] construct is inserted about 1.4 kb upstream of the omb transcription start site. The lacZ reporter gene within P[lacW] exhibits the same expression pattern as omb. The white reporter gene is expressed in a "bipolar" pattern. We induced high frequency gypsy mobilization in omb(P1) and identified two lines (D11 and D13-1) with altered eye pigmentation pattern, which is dependent on su(Hw) activity. A gypsy element was found inserted in the first intron of omb in D13-1 and in P[lacW] in D11. These results indicate that it is the blocking of regulatory activities by gypsy that caused the changes in the white reporter gene expression. The effect of these gypsy insertions on the expression patterns allowed us to predict several aspects of the organization of the regulatory elements in the omb locus.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila/genética , Genes de Insecto , Genes Reguladores , Proteínas del Tejido Nervioso/genética , Retroelementos/genética , Proteínas de Dominio T Box , AnimalesRESUMEN
The sequences of a 1.5 kb long stretch of the 5' flanking region of the gene for the alpha 3 isoform of the catalytic subunit of human Na(+)-K+ ATPase (located on chromosome 19) and of more than a 2 kb stretch of the 5' flanking region of the gene for the alpha 2 isoform (located on chromosome 1) have been determined. Transcription start sites for the gene for the alpha 3 isoform have been mapped at positions -152 and -155 relative to the translation initiation codon by primer extension analysis and S1-nuclease mapping of mRNA from human brain. The 5' flanking region of the gene for isoform alpha 3 contains a CCAAT box on the noncoding chain and six putative Sp1 binding sites. Absence of a conventional TATA box and a high GC content are other features of the region. The 5' upstream region of the gene for the alpha 2 isoform contains potential TATA and CCAAT boxes and one potential Sp1 binding site. Upstream of the putative TATA box there is an octanucleotide repeat, GGGGGAGA, which is also found in several eukaryotic genes in analogous positions. Pairwise comparison of the putative 5' regulatory regions of the genes coding for the different isoforms of the Na(+)-K(+)-ATPase catalytic subunit shows the existence of conserved elements, as well as of oligonucleotide blocks with very different structures. It is suggested that the differences in the primary structure of the 5' upstream regions may provide the basis for tissue-specific expression of the Na(+)-K(+)-ATPase isoforms.
Asunto(s)
Secuencias Reguladoras de Ácidos Nucleicos , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Humanos , Isoenzimas , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Biosíntesis de Proteínas , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The existence of a chromosome gene family containing at least one gene and one pseudogene was shown for the Na+,K+-ATPase beta-subunit. A partial structure of the beta 1-gene was determined, the coding part of which was completely homologous to cDNA of the Na+,K+-ATPase beta I-subunit from HeLa cells. The region encoding the putative protein transmembrane domain was shown to be bordered by two introns. The structure of a pseudogene (beta psi) was determined. This pseudogene is processed and contains multiple stop codons. Its homology to the beta I-subunit cDNA from HeLa cells is about 88%.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , Secuencia de Bases , Células HeLa , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Familia de Multigenes , Seudogenes , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , PorcinosRESUMEN
The primary structure of the putative regulatory region of a gene of the Na+,K+-ATPase multigene family in the human genome has been determined. This region includes the first exon with all of the untranslatable sequence of mRNA and a dozen nucleotides, coding for the first four amino acids of the hypothetic precursor of the alpha+-subunit. The entire region comprises over 1400 bp. The possible role of specific nucleotide blocks within this region in comparison with other genes is discussed.