…..Y2H INTERACTORS 88
The following section will describe the results of the processes used to identify plausible LQTS candidate genes.
74 3.1 YEAST TWO-HYBRID ANALYSIS
3.1.1 Generation of Y2H bait construct
Following successful generation of the pGBKT7-KCNE2 bait construct, the construct was sequenced to establish whether the reading frame had been maintained and also to determine the integrity of the reading frame. Sequence analysis confirmed that the pGBKT7-KCNE2 insert was in the correct reading frame. The insert sequence was preserved throughout the multiple rounds of PCR amplification that had been used to create the fragment (Section 2.2.2) (Appendix IV).
3.1.2 Assessment of the AH109 bait strain
3.1.2.1 Phenotypic assessment
The bait construct was successfully transformed into the AH109 S.cerevisiae yeast strain and was able to grow on SD-Trp selection plates. Growth on SD-Leu and SD-Ada plates was however inhibited, thus confirming that the pGKBT7-KCNE2 construct did not autonomously activate transcription of the host reporter genes (Section 2.13.1). Conversely, moderate growth was observed when colonies were streaked onto SD-His plates. The transformed yeast cells showed growth on SD-Ura plates, indicating that the phenotype of the S.cerevisiae AH109 strain was not altered. Additionally, this growth indicated that the phenotype of the yeast remained conserved following transformation with the pGBKT7-KCNE2 bait construct.
3.1.2.2 Toxicity Test
In order to establish whether the pGBKT7-KCNE2 bait construct was toxic to the yeast strain AH109, a growth curve was generated (Section 2.12.2) in which growth of AH109 transformed with the bait construct was compared to that of AH109 transformed with a non-recombinant pGBKT7 plasmid. When comparing the slopes of the linearized test curves of the AH109 strain transformed with the bait construct and the AH109 transformed with the non-recombinant plasmid, there was no significant difference (Figure 3.1). This indicates that these transformants were able to grow at similar rates. It was therefore determined that the pGBKT7-KCNE2 construct was not toxic to the yeast.
75 Figure 3.1: Linear growth curve of the yeast strain AH109 transformed with either pGBKT7-KCNE2 bait construct (purple squares and purple line) or a non-recombinant pGBKT7 plasmid (green diamonds and green line). The growth rates of the two yeast transformants were compared to each other by measuring absorbance at 600nm every two hours in order to determine whether the bait construct had a toxic effect on the AH109 strain. This was determined by calculating the slope of each curve. It was concluded that the growth of the host strain was not affected by the bait construct as the slopes were similar and comparable.
3.1.2.3 Mating efficiency of AH109 transformed with bait construct
Small scale mating efficiency testing was performed (Section 2.12.3) in order to determine whether the transformation of pGBKT7-KCNE2 had affected the ability of the AH109 to mate.
The mating efficiency results ultimately showed that the bait construct did not affect the mating efficiency of the AH109 yeast strain. The observed mating efficiency was calculated at 10.2% (Appendix III), which is higher than the minimum 2% mating efficiency specified by the manufacturer of the MATCHMAKER Y2H system (BD Biosciences, clontech, USA) (Table 3.1).
Table 3.1: Effect of the pGBKT7-KCNE2 bait construct on AH109 mating efficiency
Mating Mating efficiency
pGBKT7-KCNE2 (AH109) × pACT2 (Y187) 10.2%
pGBKT7-53 (AH109) × pACT2 (Y187) 2.15%
-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0 5 10 15 20 25 30 Log Ab sor b an ce (600 n m) Time (Hours) pGBKT7 pGBKT7-KCNE2 Linear (pGBKT7) Linear (pGBKT7-KCNE2)
76 3.1.3 Y2H screen of pre-transformed cardiac cDNA library
3.1.3.1 Bait culture titre
The titre of the bait culture was established by counting the cells with a Haemocytometric counting chamber (Section 2.13.3). The average of the total amount of cells counted on both sides of the chamber was 603 and following calculations (Appendix III) the bait culture titre amounted to 6.03×109 colony forming units (cfu)/ml, with the commercial transformed bait titre
estimated at 5×107.
3.1.3.2 Library titre and library mating efficiency
The library titre and mating efficiency was established as described in section 2.13.5 and after the four day incubation period, approximately 2984 cfu were counted on the 1:10000 dilution SD-Leu plate. Following the necessary calculations (Appendix II) a library titre of 2.9×108 was established.
The number of offspring S.cerevisiae cells that were present on the SD-Trp, SD-Leu and SD-Trp-Leu media plates was counted (Table 3.2) and the library mating efficiency calculated to 2.1%, which is slightly higher than the manufacturer’s recommended minimum value of 2% (Appendix III).
Table 3.2: Library mating efficiency as established by progeny colonies on growth selection media
Library mating: pGBKT7-KCNE2 × pACT2
Mating culture dilution 1:10 1:100 1:1000 1:10000
SD-Trp * * * *
SD-Leu * * 725 87
SD-Trp-Leu 220 45 15 0
Mating efficiency (%) 2.1%
* Too many colonies to count. Abbreviations: SD, single dropout; -Trp, without Tryptophan; -Leu, without Leucine; -Trp-Leu, without Tryptophan and Leucine.
77 Further analysis included determining the number of pre-transformed cardiac cDNA clones screened, which was calculated to be 3.066×105 independent clones with a final resuspension volume of 14.6ml (Appendix III).
3.1.3.3 Y2H screen of pre-transformed cardiac cDNA library
The diploid yeast colonies identified through procedures explained in section 2.13.6, were exposed to a cascade of nutritional selection with increasing stringency in order to enhance the chances of identifying true interactors.
The screen ultimately yielded 721 clones that were able to grow on TDO plates containing 10mM 3-AT (Section 2.13.6.2) (Appendix I), thus indicating that they were capable of activating the HIS3 reporter gene. For the second stage, the 721 clones were transferred to QDO plates containing 10mM 3-AT (Section 2.13.6.2) and after four days, 427 clones were selected based on their ability to activate both the HIS3 and ADE2 reporter genes as determined by the growth on the nutritional selection plates (Appendix I).
Subsequently, the ability of the 427 clones to activate the colourimetric reporter gene, MEL1 (Section 2.13.7) was evaluated. After the two day incubation period, it was determined that 379 clones were able to activate the MEL1 reporter gene. This was established by plating the 427 clones onto QDO plates containing 10mM 3-AT as well as 20mg/ml x-α-galactosidase solution (Appendix I).
Table 3.3: Grouping of primary and secondary clones based on the x-α-galactosidase colour production and intensity
Group x-α-galactosidase Colour