RESUMEN
Flight muscle Hexokinase-A (HEX-A) is the most conserved and essential hexokinase isoenzyme among Drosophila species. In this study, the Hex-A locus, encoding the HEX-A isoenzyme, has been analysed for the elements regulating its expression. By sequencing the 5' ends of Hex-A cDNA amplified by 5' RACE, we identified a transcription start site that overlapped the Initiator and downstream promoter elements. A 214 bp sequence, encompassing transcription start sites and promoter elements, was required for minimal promoter activity. DNA sequence to the 5' end of the minimal promoter element did not demonstrate any promoter activity; however, its inclusion with the basal promoter element enhanced the promoter activity. Oligonucleotide competition and site-directed mutagenesis identified the Initiator-like sequences, TCAWT, present in this region that were responsible for enhancing the promoter activity. The Hex-A locus is expressed as a single protein in Drosophila cell line, whereas in pupae, larvae and adult flies, it is expressed as two distinct types.
Asunto(s)
Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Regulación Enzimológica de la Expresión Génica , Hexoquinasa/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Isoenzimas , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Hexokinase coding DM1 and DM2 sequences were obtained from genomic DNA of a Drosophila melanogaster cell line by PCR amplification strategy. Both the sequences were found to encode an enzyme with a molecular weight of 50,000 Da. Amino acid sequence alignment of DM1 and DM2 shows approximately 45% homology with yeast and human hexokinases. The sequences also indicated the presence of conserved amino acid residues and motifs that are present in mammalian hexokinases and are involved in the binding of different substrates. Southern blot analysis suggests that the D. melanogaster genome contain a single copy of DM1 and DM2 sequences. Northern analysis indicates DM1 is expressed as more than one transcript in adult as well as in the D.Mel2 cell line. DM2 is expressed as a single transcript in adult flies. Expression levels for DM1 and DM2 encoded message were found to be similar in different stages of development as seen by RT-PCR. The biotechnological significance of these sequences in metabolic engineering of cells is discussed.
Asunto(s)
Drosophila melanogaster/enzimología , Hexoquinasa/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Drosophila melanogaster/genética , Expresión Génica , Genes de Insecto , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Chikungunya (CHIK) virus is prevalent throughout Southeast Asia and Africa. It has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic occurring in 1971. During a recent outbreak of Dengue (DEN)-like illness in eastern India, Aedes aegypti mosquitoes collected from the affected area were positive for CHIK virus. Evidence of dual infection with CHIK and DEN typel virus was also obtained. A widely circulating low-virulent CHIK virus is a possible explanation for the epidemiological pattern of the CHIK virus disease in this region.