Γενετική και μοριακή ανάλυση μιας φυλοσύνδετης θερμοευαίσθητης μετάλλαξης που επηρεάζει την εκκόλαψη του ακμαίου ατόμου στη Drosophila melanogaster

Abstract

In the present study we describe the genetic, molecular and developmental properties of an X-linked temperature sensitive mutation in Drosophila melanogaster. The mutant flies emerged at 19oC have incised wings, smaller and rough eyes. For this reason we have named the mutation wiser (wings scalloped-eyes rough). At 29oC the mutation is lethal. The lethality occurs at the stage of late pupae, just before or during the eclosion of imago. The mutation occurred from a dysgenic cross with the 23.5 hobo MRF strain and is due to a P element insertion at the locus 7E. Viability studies showed that the mutation causes the death of the mutants also at the stage of 3rd instar larvae at 29oC and acts as semi-lethal. The P insertion is located 490bp upstream the predicted coding region of the gene CG32711. The gene is transcribed in two mRNA, not in one as predicted in Flybase. The two transcripts have shown quantitative differences among the developmental stages that where examined in both the mutant strain wiser and the wild-type Canton-s both in 19oC and 29oC. The longer transcript derives from alternative splicing in 260nt of the first intron. The predicted translated region is identical to both transcripts. Western analysis revealed that the gene is translated in two protein isoforms of 8 kD and 9 kD probably due to post-translational modification. The pattern expression of the two isoforms in the wild-type strain is the following: at the stages of 3rd instar larvae and 1st instar pupae the isoform of 8 kD, at the stages of 2nd and 3rd instar pupae both isoforms and in adults just the protein isoform of 9 kD is expressed. The mutant phenotype and properties are due to the abnormal expression of the protein isoform of 9 kD during the development of the mutants. RNA in situ hybridization, immunohistochemistry and ectoping expression of the predicted translated region of the gene CG32711 revealed that the gene plays a functional role in gliogenesis, tracheal and hemocyte development, wing, eye and leg formation and is essential for the survival of the individuals. Ectoping expression of the predicted translated region of the gene CG32711 revealed that the translated protein is functional and when expressed under control of the regulatory region of the gene apterous rescues the mutant flies at 29oC. The function of the gene CG32711 is also dosage-sensitive due to the fact that many individuals carrying phenotype abnormalities in the wings, thorax, eyes and legs were developed by the ectoping expression. Two other lethal mutations PL26 and PL28 are located at the 5´ regulatory region of the gene CG32711. The differences concerning the pattern expression and the properties of these two mutations in relation to those of the wiser mutation, revealed that the three mutations affect the same gene but in a different way.