Targeted mutagenesis of the Sap47 gene of Drosophila: flies lacking the synapse associated protein of 47 kDa are viable and fertile.
BACKGROUND
Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila. Sequence comparison identifies homologous proteins in numerous species including C. elegans, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila.


RESULTS
Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels.


CONCLUSIONS
The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila.
Funk
Natalja
N
Theodor Boveri-Institut für Biowissenschaften, Lehrstuhl für Genetik und Neurobiologie, Am Hubland D-97074 Würzburg, Germany. nfunk@biozentrum.uni-wuerzburg.de
Becker
Sonja
S
Huber
Saskia
S
Brunner
Marion
M
Buchner
Erich
E
D017398:Alternative Splicing; D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D005091:Exons; D005298:Fertility / Q000235:genetics*; D005328:Fetal Viability / Q000235:genetics*; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D011995:Recombination, Genetic; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D019076:Transgenes
D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C097026:Sap47 protein, Drosophila