ABSTRACT
In vertebrates, tissue inhibitors of metalloproteinases (TIMPs) play key roles in extracellular matrix (ECM) homeostasis and growth control. Deletion of the recently cloned Timp gene of Drosophila results in a subviable phenotype. Adult flies display inflated wings similar to integrin mutants, suffer from a bloated gut and progressive dissolution of internal tissues, and die prematurely. Our results demonstrate that the Timp gene product controls selective aspects of ECM function in Drosophila, and suggest that it is involved in cell adhesion/cell signaling pathways. Hence, Drosophila Timp mutants may prove useful as a model system for a wide variety of pathological conditions related to ECM dysregulation.
Subject(s)
Drosophila/genetics , Integrins/genetics , Tissue Inhibitor of Metalloproteinases/genetics , Abdomen/pathology , Animals , Blotting, Northern , Cell Adhesion , Crosses, Genetic , DNA Transposable Elements/genetics , Extracellular Matrix/metabolism , Female , Gene Deletion , Genotype , Immunohistochemistry , In Situ Hybridization , Male , Models, Genetic , Mutagenesis, Site-Directed , Phenotype , Protein Binding , Signal Transduction , Synapsins/genetics , Time Factors , Tissue Inhibitor of Metalloproteinases/physiology , Wings, Animal/physiologyABSTRACT
Vertebrate tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix metalloproteinases and are thus involved in a wide variety of developmental and physiological processes. By identifying cDNAs of a transcript detected within an intron of the Drosophila synapsin gene we have cloned the Drosophila TIMP gene (Timp), which represents the first invertebrate member of the TIMP gene family. Sequence analysis revealed an open reading frame of 210 amino acids with 35% identity to human TIMPs and a conserved exon-intron structure. Analysis of sequence data from the Sanger Centre demonstrated that the human TIMP3 gene is encoded within intron V of the human synapsin-III gene, indicating that the nested organization of TIMP and synapsin genes may be a general feature conserved in evolution. We therefore speculate that the human TIMP4 gene will be located in intron V of the human synapsin-II gene on chromosome 3p25, and we present preliminary evidence that a human synapsin-IV gene is located near the TIMP2 gene on chromosome 17q23-q25.
Subject(s)
Invertebrates/genetics , Tissue Inhibitor of Metalloproteinases/genetics , Amino Acid Sequence , Animals , Chromosomes, Human, Pair 17/genetics , Conserved Sequence , DNA, Complementary/chemistry , DNA, Complementary/genetics , Drosophila/chemistry , Drosophila/enzymology , Drosophila/genetics , Exons , Genes, Insect/genetics , Humans , Introns , Invertebrates/enzymology , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Synapsins/geneticsABSTRACT
Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.
