As the main aim for identifying proteins that interact with Mirror was to discover more about how Mirror acts as a transcription factor in Drosophila, the primary concern when analysing the interactors from the yeast-two-hybrid screen was to establish if an in vivo interaction would be possible. There were three main criteria that were thought to be important for investigating the biological relevance of the putative interactions. Firstly, it was reasoned that if the protein interacts with Mirror in vivo, it must be co-expressed with Mirror in the same cells and at the same time. Therefore, a comparison of the expression patterns between the putative interactors and m irror was undertaken. In addition to confirming the likelihood of a physical interaction, studying the expression patterns of putative interactors could also provide hints as to the nature of the interaction. The transcription factor Mirror might be expected to have both general interaction partners such as co-factors and regulators, and specific interaction partners such as other transcription factors or signalling molecules. These different types of interactors would be predicted to have different expression patterns, the former to be ubiquitously expressed and the latter to overlap with Mirror at specific sites. It was also thought that a comparison of the phenotypes of mutant alleles with the mirror phenotype might aid the establishment
Chapter 3
of a functional interaction. Unfortunately, this type of analysis would only be possible for two of the putative interactors, as most of the proteins were previously unknown and lacked any characterised alleles. Finally, an important way of ascertaining if there was an in vivo association between the proteins would be to demonstrate a genetic interaction. This could either be an interaction between alleles of mirror and the gene in question or it could be the effect of loss of the putative interactor on an over-expression phenotype of Mirror. The types of genetic interaction observed might also imply the relationship between the two proteins, ie if the putative interactor is a regulator of Mirror function or a necessary cofactor. The results of the above investigations would then be used to determine which of the putative interactors would be studied in detail.
3.1.1 Selected clones fo r further analysis
The main parameter for selecting putative interactors for further analysis was that the interaction seemed to be a true yeast-two-hybrid interaction. Therefore, some of the proteins selected that gave strong or variable background activation of reporter genes in the absence of Mirror were not selected. In addition, some of the yeast-two-hybrid interactors that had known or predicted functions that did not seem compatible with a role as a transcription factor binding partner were also not analysed further. Table 3.2 below lists the yeast-two-hybrid clones that were selected for further analysis in Drosophila,
including information on any known domains or homologies to proteins from other species. The table also lists the exact portion of the protein that was selected in the yeast- two-hybrid screens and which is the putative Mirror interaction domain. Of the proteins of known function. Castor and Dichaete will be introduced in separate sections below and CHDl is discussed in detail in chapter 5.
Some of the previously uncharacterised proteins contain recognised domains or motifs or share limited homology with proteins from other species. The novel protein
Chapter 3
CG I 135 which contains a forkhead associated (FHA) domain is discussed in detail in chapter 4. CG30337 and CG5740 both have regions that are predicted to form coiled-coils, which are protein interaction domains. CG30337 consists of mainly coiled-coil regions and is moderately homologous to a novel mouse protein termed Rab6 interaction protein 2 (unpublished submission to GenBank). CG5740 contains an N-terminal coiled-coil region which shows weak homology to myosins. CG1962 also contains an N-terminal coiled-coil region, which is specifically homologous to an auto-antigen associated with paraneoplastic cerebellar degeneration in humans (Fathallah-Shaykh et al. 1991). C G I3367 shows limited homology to mouse and human cDNAs which are highly expressed during eye development (unpublished submission to GenBank). The novel gene CG12919 contains a tumour necrosis factor-like (TNF) motif. The TNF motif is a trimeric motif found in all TNF ligands and is involved in the interaction with the TNF receptor via cystein-rich motifs (Bodmer et al. 2002). The presence of a TNF-like motif indicates that this protein may be involved in extracellular signalling. Finally, CG8448 contains a DNAJ domain. DNAJ domains were first studied in E. coli molecular chaperones and are believed to function as co-chaperones with Hsp70-type proteins (Haiti 1996). Mammalian relatives of DNAJ (Mrj) have been found to have a role in placental development (Hunter et al. 1999) and as chaperones for neuronal proteins (Chuang et al. 2002).
Chapter 3
Table 3.1 Putative interactors chosen for further analysis. Gene Location Function/known domains/
homologies
Total aa Interaction domain
Dichaete 70D3 HMG domain transcription factor 384 324-384
Castor 83C1 Zn-finger transcription factor 799 428-799
CG13367 1B13 No motifs. Limited homology mouse and human cDNA
488 75-488
CHDl 23C4 Chromo-Helicase-DNAbinding
domain protein
1883 1481-1883
CG1962 38EI-E2 N-terminal Homology to paraneoplastic cerebellar degeneration antigen, cdr2
790 525-790
CG10395 41D1 No motifs or homologies 173 1-173
CG30337 45F1-F3 N-terminal coiled-coil motif 1456 917-1456 CGI2919 46F1 Tumour necrosis factor (TNF)- like
motif
325 117-325
CG8448 53B4-C1 Several polypeptides predicted, all contain DNAJ domain.
CG8448- RC 259
69-256
CG1135 64A4 Forkhead associated (FHA) domain 578 169-578
CG3987 88E2 No motifs or homologies 404 213-404
CG5740 94A2 N-terminal coiled-coil region, weak homology to myosins
1483 1026-1483
Table 3.1 Information on location of genes, known functions, domains, homologies and total length in amino acids from Gad-Fly and Flybase. Interaction domain indicates amino acids present in the cDNA clone isolated from the yeast-two-hybrid screens.