2.3.1 Yeast-two-hybrid screens l-III
The first three library screens (I-III) were performed using the full length Mirror protein as bait. Due to the activation of reporter genes by this construct in Y190 cells, the HF7c strain was used in these experiments. Library scale transformations were carried out using the pAS2-1-Mirror FL construct and amplified library DNA (see chapter 6). A small aliquot of the transformations were diluted and plated on selective medium lacking tryptophan and leucine, but not histidine. The colonies from these plates were counted and the transformation efficiency per pg of library DNA calculated. The total number of library clones screened was calculated by multiplying the transformation efficiency with the amount of library DNA used in pg (formula from the MATCHMAKER Yeast-Two- Hybrid manual). In the first two screens the transformation efficiency was not high enough to ensure that all the clones in the library had been screened, so three screens were performed in total. The rest of the transformation was plated on selective medium lacking histidine and containing 1.5mM 3AT. The plates were incubated at 30°C for up to 14 days and growing colonies picked and re-streaked on selective medium. A summary of the numbers of clones screened and the numbers of HISS positive colonies picked form screens I-III can be found in table 2.2.
2.3.2 Tirtation o f HIS + clones on 3AT
All the HIS3 positive colonies picked in screens I-III were tested for activation of the lacZ reporter using the |3-galactosidase filter assay, but they were all negative. This is probably due to the very weak promoter used for the lacZ gene in this strain. In order to try to select for true positives, a more stringent test for activation of the H1S3 reporter was
Chapter 2
employed. The colonies were streaked onto selective medium containing increasing amounts of 3AT to titer the activity of the HIS3 gene product, so that only colonies that expressed high levels of the reporter could be picked. Titering growth on 3AT in this way has been used to identify true yeast-two-hybrid interactors even in the absence of lacZ
activation (Alberts et a l 1998). Table 2.2 below contains the results of screens I-in.
Table 2.2 Summary of results form screen I-III. Screen Library clones
screened
HIS3-^
colonies
Total colonies tested on 3AT
Colonies growing on 3AT
2mM 5mM lOmM
I 250 000 65 45 29 21 11
II 500 000 70 48 35 17 10
III 4 000 000 83 50 37 24 16
Table 2.2. The number of library clones screened is calculated by multiplying the transformation efficiency with the amount of library DNA (in |xg) used in the transform ations. HIS3+ colonies were streaked onto plates with increasing concentrations of 3 AT and scored as growing if large colonies had appeared within 7 days.
2.3.3 Sequencing o f putative yeast-two hybrid interactors form screens I-III.
In order to investigate what types of proteins had been selected during screens I-III, plasmid DNA was isolated from most of the colonies that grew on lOmM 3AT. The plasmids were then used as templates for PCR reactions to amplify the cDNA inserts. The PCR products were sequenced at the 5 ’ end and the sequence used to search the
Drosophila genome database for matches. Due to the failure of some inserts to amplify or
sequence well, the final number of sequenced clones were 8 for screen I, 7 for screen II and 10 for screen III. The results of the sequencing are presented in table 2.3.
Chapter 2
Table 2.3 Sequencing results from screen I-III
Clone CG number Protein name . domains or homologies to other proteins I 5a CG10642 Klp64D IKinesin-like protein at 64D)
I8a/b CG8286 Chaperone J-domain, Tetratricopeptide repeat (TPR)
110a none P element sequence
116b CGI 1604 Zn-finger C2HC, NLS 123a CG4027 Act5C lActin at 5 0
136a CGI 8642 Bem46. Esterase. Hydrolase
142a CG1135 FHA domain (homologous to nucleolar protein Ms/Hu) 150b CG12262 Acyl-CoA dehydrogenase domains
113a CG2103 Polypeptide N-acetylgalactosaminyltransferase 11 9b CG9184 rhodopsin C-terminal tail
1112a CG1135 FHA domain (homologous to nucleolar protein Ms/Hu) 1113a none Mitochondrial Cytochrome C oxidase 1
1131b CG1467 Svxl6 ISvntaxin 161
1148a CGI 1584 C-terminal homology to mucins/phosphoproteoglycans 1148c CG12919 TNF (Tumor Necrosis Factor) family motif
111 Id CG7558 Arp66B lActin-related protein 66B1 111 6a CG2102 Castor fZn-finger transcription factor) 111 11a CG13389 RpS13 IRibosomal protein)
111 12b CG8448 Several polypeptides predicted, all contain DNAJ domain. 111 13c CG7558 Arp66B (Actin-related protein 66B)
111 14c CGI 846 inositol-3,4,-bisphosphate 4-phosphatase 111 17d CG6163 No homologues or motifs
111 27a CG1135 FHA domain (homologous to nucleolar protein Ms/Hu) 111 39a CG5740 C-terminal coiled-coil region (homo, non-muscle myosins) 11141a CG13067 No homologues or motifs
111 50a CG4954 elF3-S8 (translation initiation factor)
T able 2.3. Each partial cDNA sequence was used to search the entire Drosophila
genome sequence using the BDGP Fly-BLAST server to identify the predicted gene product (CG numbers). Information about the predicted protein sequences was taken from GadFly where identified domains and homologues can be found for each gene.
Chapter 2
As the sequencing of selected clones form screens I-III shows, most of the different yeast colonies selected contained different cDNA clones. Two genes were represented more that once, CGI 135 which was found three times, once in each screen, and Arp66B which was found twice in screen III.