Criterios que determinan la intervención del Banco Central en el

The low sensitivity EGY191 yeast strain (two LexA operators) was used for both pNLexA screens. The mouse embryonic library was screened by direct transformation and the HeLa library was screened by interaction mating. Both libraries are constructed in the pJG4-5 vector, which expresses cDNAs as translational fusions to the SV40 NLS, the B42 activation domain and the hemagglutinin (HA) epitope tag (Appendix IV).

Mouse day 19 embryo library (for detailed protocol see Methods 2.2.2.3) An individual, characterized colony of EGY191, carrying the pNLexA bait plasm id and the pJK103 la c Z reporter plasm id, was used for the transformation of the library. Transformations were carried out using library DNA, except one which was carried out with the empty pJG4-5 library vector as control, to estim ate the number of cD NA -independent false positive colonies. A total of 3x10^ library transformants were obtained, and to fully represent each independent transformant, 3x10^ cells were plated on media lacking leucine to test for LEU2 activation.

A fter four days, 270 colonies had grown. However, a comparable number of colonies carrying the empty vector (244) bad also grown, meaning th at there was a very high background of non-specific colonies. Nevertheless, it was decided to test the library colonies for any potential interactors that may exhibit LEU2 and lacZ activation above background. All 270 colonies were therefore picked and streaked onto the 4 selective plates to assay their LEU 2 and lacZ phenotype (see 2.2.2.3). The Lex A-T-Ag bait transformed with the pJG4-5 empty vector was used as a negative control (LexA-T-Ag/B42), and the LexA-RPB7/B42-RPB4 interaction was used as a positive control (Figure 4.2).

Glucose Galactose a. b. 201 -Leu 270 -ve +ve c. d. X-Gal

Figure 4.2 pNLexA mouse clones 201-270 on the 4 selective plates. The selective media are: a. Glu/CM-His-Ura-Trp-Leu, b. Gal/CM-His-Ura-Trp- Leu, c. Glu/CM-His-Ura-Trp/X-Gal and d. Gal/CM-His-Ura-Trp/X-Gal. The -ve controls are LexA-T-Ag/B42 clones and the +ve controls are LexA- RPB7/B42-RPB4 clones.

All of the 270 colonies grew as well on glucose as on galactose. Similarly, the colonies that turned blue, did so on both Glu/X-Gal and Gal/X- Gal media. This result indicated that the LEU2+AacZ-^ phenotype of these colonies was not galactose-dependent, and therefore not due to the presence of the library plasmid, but due to trans activation of the reporters by the LexA-T-Ag bait. Therefore, none of the 270 colonies picked were pursued any further. Interestingly, the negative control Lex A-T-Ag baits did not activate the reporters, which suggests that the increase in intrinsic transactivation was somehow triggered by the library transformation.

HeLa library (for detailed protocol see Methods 2.2.2.4)

The screening of the HeLa library was performed by interaction mating (Bendixen et al., 1994 ; Finley Jr. and Brent, 1994). In a standard screening as described above, the library is transformed into a haploid yeast strain carrying the bait and the la c Z reporter. In the interaction m ating screening, one haploid strain is transformed with the library, and another haploid strain of the opposite mating type (a or a ) is transformed with the bait and the lacZ

reporter. The two strains are mated to produce a diploid strain that carries the bait plasmid, the library plasmid and the LEU2 and lacZ reporters. Any two haploid strains can be used for interaction mating, as long as their mating types and nutritional requirements (auxotrophic markers) can be combined.

The strain carrying the pNLexA bait and the lacZ reporter was again EGY191. The strain transformed with the HeLa library was RFY206 (MATa, tr p lA : : h is G his3A 200 ura3-52 lys2A201 leu2-3). RFY206 was also transformed with the empty library pJG4-5 vector as a control.

A characterized colony of EGY191 was used to setup the culture for the mating. Two matings of EGY191 were setup, one with an aliquot of the pretransformed library strain, and one with an aliquot of the pretransformed control strain. The mating efficiency for pNLexA was estimated (see Methods

2 .2 .2 4 ) to be 25%, and 1x10^ transformants were obtained. In order to fully represent the transformants, a total of 1x10^ cells had to be plated. However, since 1x10^ cells is the maximum number of cells that can be plated onto a dish without cross-feeding between colonies being a problem, and the mating efficiency was 25%, forty dishes had to be plated to screen 1x10^ cells. Instead it was decided to start with a small scale screen first to get an indication of the number of colonies growing, and subsequently plate more cells if necessary. Therefore, 1x10^ cells were plated on media lacking leucine to test for LEU2 activation.

After four days a total of 504 colonies had grown. Similar to the mouse screen, a high num ber of control colonies had also grown (486). A representative group of 96 colonies (colonies that had appeared on the second, third and fourth day) were picked. When streaked on the 4 selective plates, none of the 96 colonies exhibited a Gal-dependent LEU2+/lacZ'^

phenotype. It was highly unlikely that any potential interactors would exist in the remaining 408 colonies, and therefore the screen was not pursued any further.