Los ámbitos de acción en la fase de unificación

The two-hybrid system used in this project, is based on the ability of the bait and a target protein which interact with each other, to activate the transcription of the LEU2 and lacZ reporter genes. For this reason, a bait that can activate the transcription of these reporter genes on its own is not suitable for the screen. The LEU2 reporter gene was provided by the EGY48 yeast strain, while the lacZ reporter was provided by the pJKlOS plasmid (Appendix VI). EGY48 is a high sensitivity L E U 2 reporter strain, as it contains six LexA operator binding sites to direct transcription from the

LEU2 gene. The lacZ is a lower sensitivity reporter, as the pJKlOS plasmid contains only two LexA operators.

EGY48 was transformed with the different LexA-T-Ag baits and with the pJK103 lacZ reporter plasmid. In addition, EGY48 was also transformed w ith th e p o sitiv e co n tro l plasm id pSH 17-4 w hich ex p resses a transcriptionally active LexA-Gal4 bait (Golemis and Brent, 1992), and with the n eg ativ e control plasm id pEG 202-hsR PB 7, w hich expresses a transcriptionally inactive LexA-RPB7 bait (Khazak et al., 1998).

The LexA bait vectors carry a HIS3 marker and the pJK103 plasmid has a URA3 marker, so EGY48 cells were firstly plated on glucose complete m edia lacking histidine and uracil (G lu/C M -H is-U ra) to select for transform ants. Six independent colonies were picked from each of the d ifferen t LexA -T-A g transform ations, as well as from each control transform ation, and all the transformants were assayed for their ability to activate LEU2 and lacZ transcription.

Transformants were streaked onto two Glu/CM -His-Ura plates, one also lacking leucine (-Leu) and one containing X-Gal (Figure 3.2 a and c). However, it is important to assay the transformants on galactose media too, because the B42 activation domain of the libraries used for screening is Gal- inducible and therefore, selection of interacting proteins is carried out on galactose media. Therefore, the six transformants were also streaked onto m edia supplem ented with 2% galactose and 1% raffinose (for brevity denoted Gal) as the carbon source instead of glucose (Figure 3.2 b and d). Yeast cells grow better on glucose because they can more readily metabolize it than galactose, so it should be noted that in all tests, growth and colour intensity was less in galactose compared to glucose.

The streaks were checked for growth and colour at frequent intervals for four days. The colour phenotype is visible faster than the growth phenotype, therefore, strongly activating baits usually show lacZ activity in the first 12-24 hours and LEU2 activity after two days. Inactive baits suitable to use in a two-hybrid screen should remain white on X-Gal and not have

Glucose Galactose -Leu -ve control +ve control pEG202 pM W 103 pNLexA pGilda G pGilda C X-Gal -ve control -f-ve control pEG202 pM W 103 pNLexA pGilda G pGilda C

Figure 3.2 Transactivation assays of the five constructs (day 4 results). The

four selective media are: a. Glu/CM-His-Ura-Leu, b. Gal/CM-His-Ura-Leu, c. Glu/CM-His-Ura/X-Gal and d. Gal/CM-His-Ura/X-Gal. -ve control is the LexA-RPB7 non-activating bait and 4-ve control is the LexA-Gal4 activating bait (pSH17-4 plasmid). G indicates pGilda plasmid provided by E. Golemis and C indicates pGilda plasmid purchased from Clontech.

grown on -Leu after four days (Golemis and Brent, 1997). On day 1, the pSH17-4 control had started to turn blue on X-Gal as expected, whereas the rest of the baits were white and none of them had grown on -Leu. On day 2, pSH17-4 was dark blue and had grown well on -Leu, and from the baits, pNLexA was slightly blue and had started to grow on -Leu. By day 4, pNLexA was light blue and had grown as well as the positive control. The rest of the baits showed no activity (Figure 3.2).

The results of these tests showed that the C-terminally fused T-Ag baits of pEG202, pMW103 and pGilda were all transcriptionally inactive. pEG202 and pMW103 showed slight growth on -Leu after four days, but visible only under the microscope, which should not interfere with the screen. N evertheless, this indicated that the C-terminal inducible pGilda vector expressed the m ost suitable bait for screening, at least with respect to the transactivation assay.

The N-terminal bait pNLexA showed intrinsic transactivation capacity and was therefore unsuitable for screening. In order to use this bait, a strain that is not as sensitive to LEU2 activation as EGY48 would have to be used. For this purpose, the EGY191 yeast strain {MATa, trpl, his3, ura3, leu2 :: 2LexAop-LEU2), which carries only two LexA operator binding sites and is therefore less sensitive to reporter activation, was used to transform the pNLexA bait. The transactivation assay was perform ed again (data not shown), and even though the bait still exhibited transactivation, it was less than the positive control, and so could potentially be used in a library screen.

It is im portant to note the possibility that pEG202, pMW103 and pGilda showed no transcription activation, either because they were not expressing the LexA-T-Ag baits, or because the baits failed to enter the nucleus and therefore could not bind the LexA operators. To address these possibilities, firstly the expression of the baits was tested by western blotting.