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The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2.

ABSTRACT: Mutants of the necrotic (nec) gene in Drosophila melanogaster die in the late pupal stage as pharate adults, or hatch as weak, but relatively normal-looking, flies. Adults develop black melanized spots on the body and leg joints, the abdomen swells with hemolymph, and flies die within 3 or 4 days of eclosion. The TOLL-mediated immune response to fungal infections is constitutively activated in nec mutants and pleiotropic phenotypes include melanization and cellular necrosis. These changes are consistent with activation of one or more proteolytic cascades. The nec gene corresponds to Spn43Ac, one of a cluster of three putative serine proteinase inhibitors at 43A1.2, on the right arm of chromosome 2. Although serpins have been implicated in the activation of many diverse pathways, lack of an individual serpin rarely causes a detectable phenotype. Absence of Spn43Ac, however, gives a clear phenotype, which will allow a mutational analysis of critical features of the molecular structure of serpins.

SUBMITTER: Green C

PROVIDER: S-EPMC1461326 | biostudies-other | 2000 Nov

REPOSITORIES: biostudies-other

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The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2.

Green C C Levashina E E McKimmie C C Dafforn T T Reichhart J M JM Gubb D D

Genetics 20001101 3

Mutants of the necrotic (nec) gene in Drosophila melanogaster die in the late pupal stage as pharate adults, or hatch as weak, but relatively normal-looking, flies. Adults develop black melanized spots on the body and leg joints, the abdomen swells with hemolymph, and flies die within 3 or 4 days of eclosion. The TOLL-mediated immune response to fungal infections is constitutively activated in nec mutants and pleiotropic phenotypes include melanization and cellular necrosis. These changes are co ...[more]

PMID: 11063688

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Project description:BackgroundRecent analysis of the human and mouse genomes has shown that a substantial proportion of protein coding genes and cis-regulatory elements contain transposable element (TE) sequences, implicating TE domestication as a mechanism for the origin of genetic novelty. To understand the general role of TE domestication in eukaryotic genome evolution, it is important to assess the acquisition of functional TE sequences by host genomes in a variety of different species, and to understand in greater depth the population dynamics of these mutational events.ResultsUsing an in silico screen for host genes that contain TE sequences, we identified a set of 63 mature "chimeric" transcripts supported by expressed sequence tag (EST) evidence in the Drosophila melanogaster genome. We found a paucity of chimeric TEs relative to expectations derived from non-chimeric TEs, indicating that the majority (approximately 80%) of TEs that generate chimeric transcripts are deleterious and are not observed in the genome sequence. Using a pooled-PCR strategy to assay the presence of gene-TE chimeras in wild strains, we found that over half of the observed chimeric TE insertions are restricted to the sequenced strain, and approximately 15% are found at high frequencies in North American D. melanogaster populations. Estimated population frequencies of chimeric TEs did not differ significantly from non-chimeric TEs, suggesting that the distribution of fitness effects for the observed subset of chimeric TEs is indistinguishable from the general set of TEs in the genome sequence.ConclusionIn contrast to mammalian genomes, we found that fewer than 1% of Drosophila genes produce mRNAs that include bona fide TE sequences. This observation can be explained by the results of our population genomic analysis, which indicates that most potential chimeric TEs in D. melanogaster are deleterious but that a small proportion may contribute to the evolution of novel gene sequences such as nested or intercalated gene structures. Our results highlight the need to establish the fixity of putative cases of TE domestication identified using genome sequences in order to demonstrate their functional importance, and reveal that the contribution of TE domestication to genome evolution may vary drastically among animal taxa.

Project description:BACKGROUND:The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin 'suicide' inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined. RESULTS:We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities. CONCLUSIONS:In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon 'switching'. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Dataset Information

The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2.

Publications

The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2.

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