2.7.1 Recombination mediated cassette exchange overview
The recombination mediated cassette exchange (RMCE) system was previously developed in the Carr lab and provides an efficient, site-specific method for gene tagging and gene replacement (Watson 2008). The method relies on creation of a ‘base strain’
whereby two incompatible Cre-recombinase recognition sites, loxP and loxM3, which are integrated into the genome along with the ura4 selectable marker creating a ‘cassette’ at a region of interest. The required tag or gene sequence can then be introduced into the genome via introduction of a Cre-expression plasmid containing an equivalent cassette.
In this thesis two classes of base strain were used, gene replacement and C-terminal tagging.
In the case of a gene replacement base strain, the loxP recognition site is positioned upstream of the gene promoter region, and the ura4 and loxM3 are integrated directly downstream of the stop codon (as shown for PCNA in Figure 4-9). C-terminal tagging base strains involve integration of the loxP-ura4-loxM3 cassette downstream of a gene open reading frame, removing the stop codon in the process.
2.7.2 Creating pcn1 gene replacement base strain
RMCE was used to introduce a copy of the pcn1 (PCNA) gene fused to the photoactivatable fluorophore mEos3.1. pcn1 strains were created using the strategy designed for the replacement of an essential gene (Watson, 2008). A pcn1 “base strain”
was constructed by inserting the Cre recognition sequence, loxP, upstream of the pcn1 start codon and ura4+-loxM3 directly downstream of the pcn1 stop codon. To insert the loxP site, Phusion DNA Polymerase (New England Biolabs) was used to amplify the loxP-ura4+-loxP cassette from plasmid template pAW41(Watson, 2008) using primers P1 and P2. The resulting PCR fragment consisted of ura4 flanked by loxP sites with 80bp stretches of sequence homologous to the pcn1 locus 927bp upstream of the pcn1 start codon.
This PCR fragment was used to transform S. pombe strain AW310. Following transformation, cells were directly plated onto EMM supplemented with amino acids adenine and leucine plates and grown at 30 °C for 4–5 days until colonies appeared.
Transformants were re-streaked onto fresh plates.
In order to remove the ura4 marker and leave only one loxP site, cells were transformed with the Cre-recombinase expressing plasmid pAW5(Watson, 2008), and were plated onto EMM plates supplemented with uracil (EMM+U) and grown at 30°C. Cassette exchange was then performed using successful transformants. Cells that were leucine and uracil auxotrophic were retained and the insertion of the loxP site was confirmed by DNA sequencing.
Cells containing the loxP site upstream of the start codon were subject to a second integration designed to introduce the ura4 marker and loxM3 site downstream of the stop codon was then performed. The ura4-loxM3 cassette from plasmid template pAW12 (Watson, 2008) was amplified using primers P3 and P4. The resulting PCR fragment was flanked with 80bp stretches homologous to sequences directly downstream of the pcn1 stop codon and was transformed into cells. After transformation, cells were directly plated onto EMM+L+A plates and grown at 30 °C for 4–5 days to allow colony growth.
Transformants were re-streaked onto fresh EMM+L+A plates. The resulting pcn1 base strain TJE47 (h- loxP:pcn1:ura4:loxM3, leu1-32, ura4-D18) was confirmed by PCR and sequencing.
2.7.3 Creating pAW8-mEos3.1-pcn1 plasmid
To create the mEos3.1-pcn1 construct the pcn1 ORF and 927bp of upstream sequence was amplified from wild type S. pombe genomic DNA using primers P5 and P6. The product was then cloned into SphI/SpeI restricted plasmid pGEM5fz to create pGEM5fz-pcn1 and the insert confirmed by sequencing.
In order to N-terminally tag Pcn1 with the mEos3.1 protein, a BamHI restriction site was introduced directly upstream of the pcn1 start codon by single site mutational PCR using primers P7 and P8. An mEos3.1 coding sequence was commercially synthesised that was codon-optimised for S. pombe (Forsburg, 1994), and encoded a C-terminal poly-threonine-glycine-serine (TGS) linker (Genscript). This sequence was flanked by BamHI
restriction sites and was subcloned into BamHI restricted pGEM5fz-pcn1 to create pGEM5fz-mEos3.1-pcn1. The mEos3.1-pcn1 construct was then finally subcloned from pGEM5fz- mEos3.1-pcn1 into the Cre-expression plasmid pAW8 (Watson et al 2008) to create pAW8- mEos3.1-pcn1.
2.7.4 Creating mEos3.1-pcn1 his3::pcn1+ strain
It has been previously reported that pcn1 can be N-terminally tagged in S. pombe, however this can cause sensitivity to HU (Meister et al 2007). This was also observed in this study as an mEos3.1 N-terminally tagged pcn1 strain was created by cassette exchanging the pAW8- mEos3.1-pcn1 into the pcn1 base strain. This construct was not stable inside cells and was selected against over time resulting in a mixed population of tagged and untagged cells. Therefore, a strain containing one mEos3.1 tagged pcn1 and one wild type copy of pcn1 was created. This approach has been previously shown to result in PCNA homotrimers that contain both tagged and un-tagged subunits.
To introduce a wild type copy of the pcn1 gene we used a his3 targeting plasmid (Matsuyama, 2008). The promoter-pcn1 fragment from plasmid pAW8-pcn1 was PCR amplified by primers P9 and P10. The resulting fragment and plasmid pHIS3K were restricted with SphI and SalI enzymes before the fragment was ligated into the vector to create plasmid pHIS3-pcn1. Plasmid pHIS3-pcn1 was linearised with NotI and transformed into the pcn1 base strain (TJE47). Cells were grown on YEA plates before being replica plated onto YEA+Hygromycin (Melford) to select for successful transformants. Integration was further confirmed by patching cells on EMM+U+L plates to check for histidine auxotrophy. Cells which were Hygromycin resistant and histidine auxotrophic generated strain TJE177. The mEos3.1-pcn1 construct was introduced into strain TJE177 by RMCE using the plasmid pAW8-mEos3.1-pcn1. The resulting strain was TJE211.
2.7.5 Creating C-terminally tagged strains using RMCE
C-terminal tagging RMCE base strains were used to tag Mcm4 and Cdc20 with mEos3.1 for experiments in Chapter 4 and 5 respectively. In both cases, the base strains had been previously created in the Carr lab, and were gifts from Izumi Miyabe. Integration of the mEos3.1 fluorescent tag into both strains was achieved by transformation with plasmid
pAW8-mEos3.1 and RMCE to create the final strains mcm4-mEos3.1 (TJE148) and
Table 2-6. Table of strains used in this study.
Strains starting with AW were gifts from Adam Watson, strains marked with YDP were gifts from Yasukazu Daigaku and strains marked with * were created by the Murray lab
Number Sequence
Table 2-7. Primers used in the creation of TJE211