Abstract
Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3' of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.
Highlights
The kermit gene was first described in Xenopus [1] and it was later shown that it shares 72% identity with mammalian GIPC proteins [2]
Characterization of the anomalous mRNA (LpinK-w) expressed in the KG00562 mutant fly Northern blot of mRNA from both heterozygous and homozygous KG00562 mutant flies probed with a fragment derived from the first exon of DmLpinK and kermit revealed that an mRNA of about 2.3 kb was not detected in CS wild-type flies
The open reading frame of LpinK-w encodes a 770-amino acid sequence that corresponds to the white protein lacking the first 23 amino acids, which maintains the ATP Binding Cassette (ABC) Transporter Complex domain required for its activity (Figure 2B)
Summary
The kermit gene was first described in Xenopus [1] and it was later shown that it shares 72% identity with mammalian GIPC proteins [2]. The isoforms encoded by DmLpin are homologous of lipins [8], which constitute a novel family of Mg2+-dependent phosphatidate phosphatase (PAP1) enzymes [9]. These enzymes catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol. The reaction proceeded for 15 min presents a high lethality rate that was not rescued in the at 70°C in 20 mM Tris-HCl, pH 8, 50 mM KCl, 10 mM DTT, wild-type background, characterizing a gain of function 0.5 mM dNTP, and 200 U Superscript II-RT (Invitrogen). We demonstrated that neither mM LpinB/Kermit first exon-specific primer (GSP-RACE-3’; of these alterations is related to the lethality phenotype Table 1), 0.2 mM Abridged Universal Amplification Primer of the KG00562 fly line. We found that the (AUAP), 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM overexpression of the kermit gene in Malpighian tubules MgCl2, and 0.1 U Taq DNA polymerase (Fermentas, USA). seems to be associated with the gain of function observed The 3’-end was obtained with 37 cycles of denaturation in the KG00562 fly line
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