4. Capítulo IV Propuesta
4.5.2. Desarrollo del Marketing Educacional
This section describes the generation of constructs to allow expression of genes from the pREP3x or pREP4x vectors. The pREP vectors, which contain the inducible nmt1 (no message in thiamine) promoter allow the control of gene expression through the addition or removal of thiamine from the growth medium and are used to control the expression of the exogenous genes in S. pombe [235].
Yeast strains used in this study are auxotrophic in theS. pombegeneleu1, which encodes β-isopropylmalate dehydrogenase, a protein required for leucine biogenesis [236]. pREP3x constructs exploit this auxotropic phenotype by using the leu1 for selection. pREP3x plasmids contain the S. cerevisiae LEU2 gene, which complements for the loss of leu1 [235]. Similarly, pREP4x constructs use the S. pombe gene ura4 for selection, as these plasmids contain the ura4 gene [237]. ura4 encodes orotidine-5’-phosphate decarboxylase, which is essential for biogenesis of uracil. ura4 can be used for both positive and negative selection. Growth in the presence of 5’ fluoroorotic acid (FOA) causes cytotoxicity, whilst the absence of uracil leads to auxotrophy forura4 disruptants [238].
It should be noted that throughout this study a modified version of pREP3x was used, which contains anEcoRV site directly before theBamHI site in the pREP multiple cloning site (MCS). Table 2.17 lists all vectors used in this study to generate constructs for con- trolled gene expression and Table 2.18 lists all the vectors used to express exogenous genes throughout this study.
The following nomenclature has been used to describe the expression of genes and binding domains etc. in this study. S. pombecells transformed with pREP3x-Yfg1 and abbreviated to pYfg1 for example, implies that cells express Yfg1.
Name Description source
JD1776 pKS Stratagene
JD2320 pGADT7 BD clontech
JD2935 pREP3x-Mam2∆5 [173]
JD3374 pKS-Mam2∆tail E. McCann, PhD thesis, 2010
JD3386 pREP3x [235]
JD3453 pKS-mCherry M. Bond, PhD thesis, 2012
JD3590 pREP3x-Mam2-mCherry [173]
2.3.2.1 Creation of pREP3x-Cki constructs
These constructs were used in section 4.5.9 to elucidate the effect of constitutive expression of either Cki1, Cki2 and Cki3 in S. pombe cells. The ORFs of Cki1, Cki2 and Cki3 were amplified by PCR from an S. pombe cDNA library (created by Dr. Stephen J. Elledge [239] and kindly provided by Professor Jonathan Millar, University of Warwick) using a sense and antisense oligonucleotide, which introduced a fullBamHI site at either end of the PCR fragment (for oligonucleotides see Figure A.4). The PCR product was then digested with BamHI and cloned into BamHI digested pREP3x (JD3386) to create pREP3x-Cki1 (JD3893), pREP3x-Cki2 (JD3766) and pREP3x-Cki3 (JD3782) (Figure A.4), which enables expression of Cki1, Cki2 and Cki3 respectively inS. pombe under the control of the inducible nmt1 promoter.
2.3.2.2 Creation of pGADT7-Cki1/Cki2/Cki3 constructs
These constructs were used in section 5.2 to elucidate a possible protein-protein interaction between Cki1/ Cki2/ Cki3 with either the C-terminal tail of Mam2 or Rgs1. The pGADT7 vector (JD2320) was designed, such that when a protein of interest is fused to the GAL4 transcriptional activation domain (AD) and when co-transformed with a protein of interest expressed from pGBKT7 vector fused to the GAL4 DNA-binding domain (BD), induces transcription of the reporter genelacZ. Expression ofβ-galactosidase will only occur if these two proteins of interest interact and bind to the GAL4-responsive promoter, indicating a possible protein-protein interaction.
The ORFs of Cki1, Cki2 and Cki3 were amplified by PCR from anS. pombecDNA library (created by Dr. Stephen J. Elledge [239] and kindly provided by Professor Jonathan Millar, University of Warwick) using a sense and antisense oligonucleotide, which introduced a full BamHI site at either end of the PCR fragment (for oligonucleotides see Figure A.5). The PCR product was then digested with BamHI and cloned into the BamHI digested MCS of pGADT7 (JD2320) to create pGADT7-Cki1 (JD3770), pGADT7-Cki2 (JD3771) and pGADT7-Cki3 (JD3763) (Figure A.5), which enables expression of Cki1, Cki2 and Cki3 respectively inS. cerevisiae under the control of the T7 promoter.
2.3.2.3 Creation of pREP3x-Mam22K-mCherry
Ubiquitination typically occurs on lysine residues. There are four lysine residues in the C-terminal tail of Mam2: at position 307, 329, 345 and 346 from codon 1 from the ATG. This construct was used in Chapter 5 to elucidate the effect of removing the two lysine codons 307 and 329 on the signalling behaviour and localisation of Mam2.
Mam2 tagged mCherry was amplified on pREP3x-Mam2-mCherry (JD3590) (a plasmid where the glutamine at position 304 (Q304) relative to position 1 (ATG) has been altered
from CAA to CAg so that this residue forms the front half of a PvuII restriction site), using a mutagenic sense oligonucleotide (JO2955), initiated directly downstream of Q304 and an antisense oligonucleotide (REP2). Amplification with JO2955 introduced a muta- tion at the 307thand 329th codons of Mam2, changing the lysine codon (AAA) to arginine (AgA) by a single base mutation. The PCR product (Figure A.6, highlighted in grey) was ligated into PvuII digested pKS-Mam2∆tail. The small fragment produced from a digest with XhoI andBamHI digested pKS-Mam22K-mCherry was ligated into XhoI/BamHI di- gested pREP3x (JD3386) to produce pREP3x-Mam22K-mCherry (JD3802), which enables expression of the mutated receptor inS. pombecells under the control of the induciblenmt1 promoter (Figure A.6).
2.3.2.4 Creation of pREP3x-Mam24K-mCherry
To abolish ubiquitination of the C-terminal tail of Mam2, all four lysine residues (at codon positions 307, 329, 345 and 346 from codon 1 from the ATG) were either mutated or removed to elucidate their influence on the localisation and signalling behaviour of Mam2 (Chapter 5).
pREP3x-Mam2∆5 (JD2935) (where the last 5 amino acid residues of Mam2 have been removed, which include lysine residues 345 and 346 and this plasmid has also been altered at Q304 from CAA to CAg so that this residue forms the front half of a PvuII restriction site) was amplified with sense (JO2955) and antisense (REP2) oligonucleotide to create a PCR product that changed the 307th and 329th lysine (AAA) codons to arginines (AgA). The PCR product (Figure A.7, highlighted in grey) was ligated into PvuII digested pKS- Mam2∆tail to create pKS-Mam24K. A second PCR amplification was performed on pKS- Mam24Kwith sense (REP1) and antisense (JO3038) oligonucleotides. This produces a DNA
fragment (Figure A.7, boxed by a red dotted line) that includes anXbaI site upstream of the Mam2 ATG codon and excludes the Stop codon, such that when the fragment is digested with XbaI and ligated into XbaI/ PvuII digested pKS-mCherry it creates a vector that lacks any of the lysine residues in the C-terminal tail of Mam2 and is tagged with mCherry (pKS-Mam24K-mCherry).
Finally, the small fragment produced when digesting pKS-Mam24K-mCherry withXhoI andBamHI was ligated intoXhoI/BamHI digested pREP3x (JD3386) to produce pREP3x- Mam24K-mCherry (JD3804), which enables expression of the mutated receptor inS. pombe under the control of the induciblenmt1 promoter (Figure A.7).
Name Description Source JD557 pREP41x [98] JD636 pREP41x-Sxa2 [98] JD1170 pGBKT7 BD Clontech JD1627 pREP3x-Mam2 [184] JD2261 pREP3x-GFP [222] JD2320 pGADT7 BD Clontech JD2332 pREP3x-Gpa1 [82] JD2388 pREP4x-Rgs1 [82] JD2555 pREP3x-Rgs1 [87] JD2673 pREP3x-Gpa1G223S [82] JD2880 pREP3x-Mam2∆tail [173] JD2885 pREP3x-Mam2-GFP [184] JD2999 pREP3x-Mam2∆5-GFP [173] JD3001 pREP3x-Mam2∆13-GFP [173] JD3003 pREP3x-Mam2∆21-GFP [173] JD3007 pREP3x-Mam2∆37-GFP [173] JD3180 pGADT7-Rgs1 [173] JD3182 pGBKT7-Rgs1 [173] JD3221 pGBKT7-Mam2tail [173]
JD3280 pGBKT7-Map3tail E. McCann, PhD thesis, 2010
JD3284 pGBKT7-STE2tail E. McCann, PhD thesis, 2010
JD3317 pREP3x-Mam2∆tail-GFP [173]
JD3386 pREP3x [235]
JD3514 pREP3x-mCherry [228]
JD3590 pREP3x-Mam2-mCherry [173]
JD2621 pREP3x-Mam2∆tail-mCherry [173]
JD3681 pREP-RBD M. Bond, University of Warwick
JD3763 pGADT7-Cki3 This study
JD3766 pREP3x-Cki2 This study
JD3770 pGADT7-Cki1 This study
JD3771 pGADT7-Cki2 This study
JD3782 pREP3x-Cki3 This study
JD3893 pREP3x-Cki1 This study
JD3802 pREP3x-Mam22K-mCherry This study
JD3804 pREP3x-Mam24K-mCherry This study
Table 2.18: Inducible plasmidsThe name, description and source of the plasmids used in this study.
Extending the mathematical model
Mam2 Mam2 Rgs1 Gpa1 GDP Gpa1GTP P-factor Gap1 Ras1 GDP Byr2 Byr1 Spk1 Ste6 Transcriptional response Pmp1 Spk1 Previously described Described in this chapter P-factor Ras1 GTP P P P PFigure 3.1: The currentS. pombemodel. P-factor binds the GPCR Mam2 promoting GDP/GTP exchange on the G protein Gpa1. GTP-bound Gpa1 activates Ras1 via the GEF Ste6, transducing signalling via sequential phosphorylation of a MAPK cascade (Byr2, Byr1 and Spk1), resulting in activation of Ste11 (not included), which induces transcription of mating-responsive genes. There are various points of regulation through the pathway (highlighted with red text) including: Rgs1, which catalyses the hydrolysis of Gpa1. Gap1, which hydrolyses Ras1 and Pmp1 a phosphatase that regulates the action of Spk1 (Figure adapted form [50]). The red dashed box highlights components described in the previous models and the purple box contains components that are incorporated into the model in
this chapter.
3.1
Chapter aim
This chapter extends the previously discussed mathematical models of theS. pombemating- response pathway to include all known components, including known points of adaptation (signal regulation) (Figure 3.1).