Generation of the cmd RIP mutant strains

7. Genomatix software: This is used for promoter analysis of Genomic DNA sequence

4.2 Results

4.2.2 Generation of the cmd RIP mutant strains

The plasmid construct Ptcu-1::cmd::5xGly::V5::gfp was transformed into the N. crassa strain 51-IV-8a (rid-1::nat; mus-51::hph; mat a; Table 2.1) by electroporation (described in materials and methods) and the initial heterokaryotic transformants were selected on Vogel’s sorbose agar medium (with VM containing 0.5% L-proline in lieu of NH4NO3) supplemented with basta (400 μg/ml) and pantothenate (0.01 mg/ml).

Integration of the Ptcu-1::cmd::5xGly::V5::gfp construct at the pan-2 locus in the LG VI of the transformed strains was PCR verified using primers Ptcu-CaM-RIP-FP-3 and GFP- RV (Table 4.1). PCRs were performed using custom oliogonucleotide primers (IDT, USA), Phusion High-Fidelity PCR Kit (Thermo Scientific, USA) according to the manufacturer’s protocol. The initial heterokaryotic transformants designated as 26-35- IV-8 of genotype [(rid-1::nat; mus-51::hph mat a) + (∆pan-2::bar::Ptcu- 1::cmd::5xGly::V5::gfp; mat a)], crossed with the wild-type; mat A strain. Crosses were harvested after about 20-25 days, ascospores were germinated by heat shock (described in materials and methods) and five homokaryotic strains of genotype rid-1::nat; ∆pan- 2::bar::Ptcu-1::cmd::V5::gfp; mat a (850, 851, 852, 853 and 854; Table 2.1) were isolated. The homokaryotic strains were isolated based on their resistance to basta (400 µg/ml) to verify the presence of the bar selectable marker, and also screened for nourseothricin (50 µg/ml) for the nat selectable marker on the Vogel’s sorbose agar medium supplemented with pantothenate (0.01 mg/ml). These homokaryotic strains contained the cmd gene under the Ptcu-1 in the pan-2 locus, and its expression is regulated by the addition of BCS (induction) and CuSO4 (repression). The 850 and 853 strains were crossed with the wild-type; mat A strain (Table 4.2) and isolated Δpan-2::bar::Ptcu- 1::cmd::v5::gfp; mat A (2, 4, 12, 13, 15, 16, 17, 24, 28, 30, 31, 34 and 41; Table 2.1) as well as pan-2::bar::Ptcu-1::cmd^RIP::V5::gfp; mat A (RIP-26 and RIP-38; Table 2.1) progenies; and further used for generation of other cmd^RIP mutants and for meiotic silencing assay. Furthermore, I isolated the strains RIP-35 and RIP-50 (Table 2.1; Table 4.2) from the cross of the strains 24 with 850; strains RIP-3, RIP-6 and RIP-9 (Table 2.1; Table 4.2) from the cross of the strains RIP-26 with 850; and the strain RIP-55 from the cross of the strains RIP-38 with 853 (Table 2.1; Table 4.2) based on their initial slow growth phenotype (data not shown). Thus, I screened a total of 208 progenies from these crosses and isolated eight strains (Table 4.3) based on their initial slow growth phenotype (data not shown). All these eight strains contain the cmd gene in the endogenous locus

in the LG V and its tagged ectopy copy at the pan-2 locus in the LG VI. A 1241 bp fragment of the cmd endogenous allele was PCR amplified using primer pairs CaM-RIP- FP-1 and CaM-RIP-RP and a 1272 bp fragment of the tagged cmd construct in pan-2 locus was PCR amplified using primer pairs Ptcu-CaM-RIP-FP-3 and GFP-RV from the eight strains; PCR purified products were sequenced for confirmation of cmd^RIPmutant (SciGenom Labs, Cochin, India and Eurofins Genomics India Pvt. Ltd. Bangalore, India), using the same pairs of primers used for the PCR amplification and the common primer CaM-RIP-FP-2 for sequencing of middle region of both endogenous and the ectopic copies of the cmd gene (Figure 4.1; Figure 4.3; Table 4.1). The sequences of the eight cmd^RIPmutant alleles were aligned with the wild-type cmd gene sequence to identify the mutations and deposited in GenBank with accession numbers KX009461-71 ( Table 4.3; Laxmi and Tamuli 2016). Analysis of the sequence revealed that the cmd^RIP mutants contained both conventional (G:C to A:T) as well as non-conventional RIP- induced mutations in the cmd allele (Figure 4.4; Figure 4.5; Table 4.3; Kim and Nelson 2005). However, only one mutant, the pan-2::bar::Ptcu-1::cmd^RIP::V5::gfp; mat A (RIP- 26) contained nonsynonymous substitutions in both the endogenous and ectopic cmd gene (Laxmi and Tamuli 2016). The pan-2::bar::Ptcu-1::cmd^RIP::V5::gfp; mat A (RIP- 26) mutant strain contained D57Y (GAT–TAT codon change) and Q148H (CAG–CAC codon change) mutations in the endogenous copy and M1W (ATG–TGG codon change) and E8K (GAG–AAG codon change) mutations in the ectopic copy of the cmd (Figure 4.6). Therefore, the pan-2::bar::Ptcu-1::cmd^RIP::V5::gfp; mat A (RIP-26) mutant strain has been used for further studies. In addition, I found that the CaM::V5::GFP fusion protein was expressed in the N. crassa strains (Figure 4.7; Laxmi and Tamuli 2016).

Table 4.2: Crosses to generate the Ptcu-1::cmd::V5::gfp; mat A and cmd^RIP mutant strains

Sl.

no.

Cross Strains

1. rid-1::nat; pan-2::bar::Ptcu- 1::cmd::V5::gfp; mat a (850) X Wild-type; mat A