El gen de la troponina T de Drosophila melanogaster : expresión diferencial y regulación transcripcional en el músculo

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 16-04-1999In this thesis the organization of the D. melanogaster tmponin T gene has been characterized as well as some of the mechanisms controlling the expression of this protein in the dierent mude types of the organism. The complete genomic organization of the Drosophila TnT gene shows many previously unreported features, induding the first untranslated exon, an unusually large first intron, similar to intron 1 of other Drosophila mude genes, an exon 4 of only 3 nucleotides and two poliadenylation sites. This microexon is the smallest bona-fide exon so far described, placing a new lower limit on the nucleotide size required for correct splicing. Fair muscle-type specific transcripts are generated by developmentally-regulated altemative splicing. Exons 3, 4 and 5 are absent in the transcript present in jump (TDT) and flight (IFM) muscles. A total of 11 exons are present in the adult hypodermic muscles transcript, whereas the microexon is absent in the larval hypodermic rnusculature; the two hypodermic muscle specific isoforms dier only in an additional lysine residue. Post-translational regulation of the fliht musdesl tergai depressor of the trochanterspecific isoform is probably involved in flight andl or jump function. All isoforms of troponin T are phosphorylated in serine and threonine residues. To characterize the mechanism(s) of transcriptional regulation of the TnT gene during development the 'in vivo" analysis of the expression of the B-galactosidase gene under contrd of selected sequences in germ line transfectants of D. melanogaster was studied. Transgenic lines wntaining eioier the sequences 5' upstream from the initiation site or intmn 1 show, both, the sarna level and spacio-temporal pattern of expression except in the indirect flight muscles. As in other Drosophila muscle genes. elements contrciling flight muscles expression seem to be located in intron 1. These results suggest that sequences located in both regions are involved in the transcriptional regulation of this gene. By comparison between the D. melanogaster and D.vnilis TnT 5' regions upstream of the transcriptional initiation site, a group of conserved sequences that indude E-boxes, PDPl and CF-2 sites were found. In intron 1, there are also several conserved motives that seem to be grouped in two regions. These ~ 0 n s e ~ esedq uences indude thrw MEF2 sites. Moreover, "in vitro" transcribed-translated MEF-2 product binds specifically the three MEF2 sites with high affinity. These MEF-2 sites are conserved in intron 1 of several Drosophila muscle genes like the MHC and Tnl genes, therefore identing a possible comrncm feature in the transcriptional regulation of these genes