RESUMO
A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.
Assuntos
Perfilação da Expressão Gênica , Marcadores Genéticos , Neoplasias de Cabeça e Pescoço/genética , RNA Mensageiro/genética , Neoplasias da Glândula Tireoide/genética , Transcrição Gênica , Processamento Alternativo , Etiquetas de Sequências Expressas , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Laringe/metabolismo , Boca/metabolismo , Faringe/metabolismo , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismoRESUMO
The peroxisomal methanol metabolism of Hansenula polymorpha depends on a group of genes that are coordinately regulated. Methanol oxidase (Mox) plays a key role in this pathway and its synthesis has been shown to be regulated at the transcriptional level. MOX expression is strongly repressed on glucose and activated on glycerol or methanol. In this study we have identified two MOX transcripts that are differentially expressed along MOX derepression. The first one, named l-MOX (for longer MOX), starts at position -425, is only weakly and transiently transcribed and is not translated into the Mox protein. The other is the true MOX mRNA, which initiates around position -25. Using a strain bearing multiple copies of MOX(Q1N) and a reporter gene fused to the MOX promoter, regulation of the two transcripts was investigated. Initiation of the true MOX correlates with repression of l-MOX and conditions that are repressive for MOX transcription, such as the inhibition of mitochondrial activity, lead to higher levels of l-MOX expression. This effect was first observed in a mox mutant (Q1N-M8) unable to grow on nonfermentable carbon sources. No function was detected for l-MOX, but its regulation follows a pattern similar to that of catalase, which is essential for methanol metabolism. This suggests that, l-MOX, although precisely regulated, seems to be a remnant of the evolution of the methanol metabolism network.
