Cloning, genomic organization and expression pattern of a novel Drosophila gene, the disco-interacting protein 2 (dip2), and its murine homolog
Introduction
To fully understand the signal transduction network that regulates nervous system development, identification of genes encoding the individual components of such network is essential. A forward genetic approach, in which genes that play a role in a given pathway are identified through mutagenesis, has been fruitful in the dissection of a variety of biological processes, in particular those that govern nervous system development. Recent analyses of genomic databases have substantiated the previously inferred DNA sequence conservation between Drosophila and mouse. Therefore, genes initially identified in Drosophila through classical genetic approaches such as mutagenesis have provided an excellent entry point for analyzing their function in a more complex murine model system. Here we report the first step in such an approach.
Drosophila disconnected (disco) gene is required for proper neuronal connections in both the larval and adult visual system of Drosophila (Steller et al., 1987, Campos et al., 1995). The disco gene codes for a protein with two C2H2 type zinc-finger domains, a feature shared by many transcription factors (Heilig et al., 1991, Pieler and Bellefroid, 1994). In embryos carrying apparent null mutant alleles of disco, in which the highly conserved Cysteins of the zinc finger motifs have been substituted, expression of Disco protein is seen in all tissues that normally express disco except in the optic lobe region (Lee et al., 1991). Upregulation of the disco gene in transgenic flies under the control of a heat shock promoter is sufficient to restore disco expression in the optic lobe primordium (Lee et al., 1999). No vertebrate homolog of the disco gene has been identified so far. Currently, the only vertebrate gene that shows some degree of sequence similarity is the human and mouse basonuclin genes (Tseng and Green, 1992). The sequence conservation between these genes is restricted to the three pairs of zinc fingers of basonuclin and the single pair of disco.
Dissecting the disco autoregulatory pathway is an important step towards understanding the role of disco in visual system connectivity. To address the molecular mechanism of tissue-specific autoregulation of disco, we focused our study initially on identifying proteins that interact with disco. The expectation is that some of these proteins may represent molecules that regulate tissue-specific disco function. To that end, we used the yeast interaction trap technique (Gyuris et al., 1993). Using this assay system we have identified a novel gene, the disco-interacting protein 2 (dip2). Here, we report isolation and initial characterization of the Drosophila and the mouse dip2 homologs.
Section snippets
Yeast interaction trap screening and cloning of the Drosophila dip2 gene
A Drosophila melanogaster 0–12 h embryonic DNA complementary to RNA (cDNA) library constructed in the galactose-inducible yeast expression vector pJG4–5, was kindly provided by Dr Roger Brent. The library was screened according to Gyuris et al. (1993). The baits used for the interaction trap assay were constructed by cloning 5′ (corresponding to 186 N-terminal amino acids) or 3′ (corresponding to 153 C-terminal amino acids) or full coding region of disco cDNA in-frame with the lexA coding
Cloning of the Drosophila dip2 gene
We have used the N-terminal portion of the disco gene in the interaction trap screen. A Disco bait was constructed where the cysteine residue at position 127 was replaced with a Serine residue. The Cys127 to Ser substitution abolished the transcription activation property of the N-terminal Disco protein, thus allowing us to use this portion of the gene as a bait in a yeast interaction trap screening. The construct was subsequently sequenced to confirm the presence of the mutation.
About 2.5×106
Conclusion
Using the yeast two hybrid system, we have identified a novel Drosophila gene that interacts with the transcription factor disco in vitro and in yeast.
We have isolated the mouse homolog of the dip2 gene. The amino acid sequence of the predicted mouse protein is 60% identical to that of Ddip2.
The Ddip2 gene is located on left arm of chromosome 3 at band 61B, while the mouse homolog is located on mouse chromosome 10.
The dip2 gene is evolutionarily conserved. dip2 homologs exist in organisms as
Acknowledgements
We thank Dr Roger Brent for Yeast Interaction trap constructs and yeast strains. Our heartfelt thanks to Julie Ruston from the Rudnicki laboratory who provided some of the mouse embryos used in this work. We gratefully acknowledge the kind support of Dr Heiner Westphal (National Institutes of Health). This work was supported by an operating grant from NSERC (National Science and Engineering Research Council of Canada) to A.R.C. B.K was supported by NSERC and The Hospital for Sick Children
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