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  1. Jun 2019
  2. May 2019
    1. All data were presented as mean ± standard deviation (SD)/standard error of mean (SEM) fromthree independent experiments. Statistical analysis was performed using Student's t-test and one-way ANOVA followed by a post hoc Tukey testwherever applicable. Results were analyzed and illustrated by SPSS statistical software package (SPSS for Windows,version 16). Comparisons are done within and between the test groups (i.e.,parentalcells and profilin-stable cells). Asterisk (*) symbol indicates statistical difference between parentaland profilin-stable cells, whereas Number (#) and Dollar ($) signsindicatestatistical difference within parentaland profilin-stable cells, respectively. Significance of results was determined as p ≤ 0.01 and p ≤ 0.05(*indicates p ≤0.05, **indicates p ≤0.01, *** indicates p ≤0.001 and **** indicates p ≤0.0001
    2. Statistical analysis
    1. was fused to the 5’ region of the nat1gene. The 3’ region of the nat1gene was in turn fused to the 5’ region of UBC11, the gene that is downstream of RPA43in the genome. These two fragments were transformed into the NOY222 rpa34strain using yeast transformation kit (Clontech) and selected on nourseothricin(NAT) 200 μg/mL to identify strains carrying mutant RPA43generated by homologous recombination (Fig. 2.4) containing YPD. Colony PCR was performed using primers that amplify the merged region of RPA43fragmentand natgene to verify the site of insertion
    2. To conduct yeast transformation competent cells were made from relevant haploid yeast strain using Frozen EZ Yeast Transformation II Kit from Zymo Research Corporation, according to the manufacturer’s instructions.To conduct plasmid shuffling in the NOY222, NOY222rpa34Δ and NOY222rpa34ΔRPA43S/322/323/325/A strainsto generate native and S/A mutant versions of the RPA190gene, the competent cells were transformed with either pRS314RPA190 or pRS314 RPA190S1413/1415/1417/A harbouring wild type a mutant versions of RPA190 generespectively,and transformants were selected by growing them on yeast nitrogen base containing leucine, G418 (200 g/mL) and canavanine (6 g/mL) but lacking uracil. The resulting strains have chromosomal deletions of RPA34 and RPA190 and harbour either wild type or mutant versions of RPA190on pRS314 plasmids
    3. genomic mutation on the A43 protein coding gene RPA43was inserted in the wild type BY4741 and NOY222 rpa34strains. Plasmid pGP5 RPA43, gifted by Dr.Herbert Tschochner, and a plasmid harbouring the nourseothricin N-acetyltransferase (nat1) gene(Goldstein and McCusker, 1999)were used for a PCR based site directedmutagenesis to create a DNA fragment to generate a genomic mutant of RPA43 (Fig


    4. with 0.01% NP 40 in water, andwere diluted serially and plated on rich medium (YPD). RPA34in the genome was replaced with kanMx4that provides resistance toG418 and RPA190gene deletion was done by replacing it with URA3gene that supports growth of the strain in the absence of uracil in the medium. pNOY20, a shuffle plasmid harbours a LEU gene which supports yeast growth in the absence of uracil. Therefore germinated spores were streaked on a selection mediumlacking leucine and uracil and containing 200 g/mLG418to select the strain containing double mutation. The genotype of the strain was confirmed by growing the straindifferent combinations of media such as SC-Ura, SC-Leu, SC-Trp, SC-Met and SC-His. The procedure to generate RPA190 genome deletion in the background of rpa34Δ(NOY222 rpa34) was conducted by a colleague, Ms. Sitalakshmi Thampatty, in thelaboratory
    5. TheS. cerevisiaestrains used were BY4741 rpa34and NOY222 (Table 2.1) (Gerberet al., 2008).NOY222 harbours agenomic deletion of RPA190 with the RPA190encoded on pNOY20, a shuffle plasmid,as the genomic deletion is lethal. BY4741 rpa34(MATa), and NOY222(MATα)were mated by mixing these two strains and patched on the YPD agar. The resulting diploids were sporulated on yeast sporulating medium at 25°Cfor 2-3 weeks.Thespores were treated with 5U zymolyase and sonicated briefly in a bath sonicator to release the spores. The free spores were washed
    6. Generation of yeast mutants