Iproof polymerase

Total RNA was extracted from bat cell lysates using RNeasy Mini Spin Column (QIAgen). cDNA was PCR amplified with primers, 5′-GTCACCAGAGGGTCATAAA-3′ and 5′-CCACTTCCTCTGCCATCAAA-3′. The PCR mixture (25 μl) contained cDNA, PCR buffer, 200 μM (each) dNTPs, and 1.0 U Iproof Polymerase (Bio-Rad, Hercules, CA, USA). The mixtures were amplified for 40 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 72 s and a final extension at 72 °C for 10 min in an automated thermal cycler (Applied Biosystems, Foster City, CA, USA). RT-PCR products were gel purified using QIAquick gel extraction kit (Qiagen), and sequenced with an ABI Prism 3700 DNA Analyzer (Applied Biosystems). The sequences obtained were compared with sequences of DPP4 genes in GenBank database. Phylogenetic tree construction was performed based on an amino acid alignment of partial DPP4 sequences (corresponding to residue 229–346 of hDPP4) using the Neighbor-Joining method with JTT model by MEGA 6.0, with bootstrap values calculated from 1000 trees.

A library of genomic DNA from JI:522 was prepared in λEMBL4 (Martin et al., 1991) and screened with pJAM1494. DNA inserts from positive plaques were subcloned as EcoRI fragments into pBluescript. DNA was also isolated from inc I¹ : del and inc I² : del lines digested with EcoRI, size‐fractionated and cloned into λNM1149. Inserts were screened with pJAM1494 and the positive EcoRI inserts were subcloned into pGEM‐T (Promega) and sequenced.
Amplification of inc I, delila and Delila‐like alleles from genomic DNA was performed with iProof polymerase (Bio‐Rad) and gene‐specific primers (Supporting Information Table S1). Sequences have been submitted to GenBank with the following accession numbers: del²³, genomic DNA, MW027119: inc I¹, genomic DNA, MW027120; Incolorata I, cDNA, MW027121; WDR1, cDNA, MW027122.

For each reaction, we mixed 0.5 μl of iProof polymerase (Bio-Rad, 1725301), 1 μl of 10 mM dNTP (NEB, N0447L), 1 μl of 100% DMSO, template (genomic DNA), 1 μl of 10 μM mixed primers (forward and reverse) at a molar ratio of 1:1, 10 μl of 5× iProof buffer, and nuclease-free water with a total volume of 50 μl. The polymerase was added by the end of the first step of PCR (“hot start” at 98°C, 3-min step).
PCR program: $$\begin{array}{c}98{}^{\circ }\mathrm{C}3\min \mathrm{polymerase}\mathrm{added}\\ \begin{array}{c}98{}^{\circ }\mathrm{C}30\mathrm{s}\\ 69{}^{\circ }\mathrm{C}45{\mathrm{s}}^{\ast }\end{array}\}10\mathrm{cycles}\\ 72{}^{\circ }\mathrm{C}1\min \end{array}$$
*temperature decreased by 1°C per cycle. $$\begin{array}{c}98{}^{\circ }\mathrm{C}30\mathrm{s}\\ \begin{array}{c}59{}^{\circ }\mathrm{C}45\mathrm{s}\\ 72{}^{\circ }\mathrm{C}1\min \\ 72{}^{\circ }\mathrm{C}5\min \end{array}\}18\mathrm{cycles}\\ 4{}^{\circ }\mathrm{C}\mathrm{\infty }\end{array}$$
Used primers:
Forward:
TGACTGGAGTTCAGACGTGtgctcttccgatctNNNNN(N)_1–4GCTAGCGCCGCCACCATG
Reverse:
CACTCTTTCCCTACACGACgctcttccgatctNNNNN(N)_1–4GCAGCGGAGCCAGCAGAAC

All DNA manipulation procedures followed standard molecular cloning techniques. Primers were synthesized and purified by Sigma-Aldrich and are all listed in Table S10. Phusion polymerase, restriction enzymes, and T4 DNA ligase were obtained from New England Biolabs (NEB). iProof polymerase was obtained from Bio-Rad. Where indicated, plasmids were constructed using the ligation-independent cloning method In-Fusion (TaKaRa), following the manufacturer’s recommendations. All plasmids used within this study can be found in Table S9, and cloning experiments are summarized in Fig S11. PCR-amplified inserts were routinely subjected to DNA sequencing (Macrogen).

Table S9. Plasmids used in this work.

Table S10 Oligonucleotides used in this study.

The diploid isolates were transformed using a lithium acetate procedure (Gietz & Woods, 2002) with a cassette that targeted the terminal region of the highly expressed PGK1 gene (Figure S2) and contained: (1) either mCherry or GFP, and (2) antibiotic resistance through KanMX (Wach, Brachat, Pohlmann, & Philippsen, 1994), NatMX or HphMX (Goldstein & McCusker, 1999). Plasmids pFA6a‐GFP‐KanMX6 (Longtine et al., 1998) and pBS34‐mCherry‐KanMX6 (Hailey, Davis, & Muller, 2002) were digested with NotI and used as template for PCR (Yeast Resource Center, University of Washington). For some strains, the antibiotic resistance in fluorescently labeled yeast was subsequently switched via transformation with NatMX or HphMX (Table S1). All polymerase chain reactions were performed with iProof polymerase (Bio‐Rad) using the manufacturer's recommendations for cycling conditions using the primers listed in Table S2; DMSO was added to 3% to reaction mixtures. Strains with killer toxin virus K2 (29‐06) (Pieczynska et al., 2013) (generously provided by D. Wloch‐Salamon) were used for assays with toxin activity. K2 is active in the acidic pH range of 2.5–5.0 at temperatures between 20 and 25°C (Lukša, Serva, & Servienė, 2016).