All 981 (3*327) possible point mutations were represented in the library, and 99.4% of the 53,301 (327*326/2) possible pairs of sites were jointly mutated, most of them in alleles that contained additional mutations. In addition to the SNPs, 43.6% of variants also contained short deletions (median length 1 nt) or insertions (median length 1 nt). We generated two independent mutant libraries, which contained on average 3 and 10 single nucleotide polymorphisms (SNPs) per allele, respectively. Our mutagenesis approach ensures uniform coverage of mutations among positions 7-333, encompassing 98% of the gene, and prevents bias towards specific types of mutations ( Figs. U3 basepairs to the primary rRNA transcript (pre-rRNA) and this interaction is required for pre-rRNA cleavage and 18S rRNA biogenesis. We used “doped” oligonucleotides to synthesize ~130,000 randomly mutated variants of the 333-nucleotide Saccharomyces cerevisiae gene SNR17A, which encodes the U3 small nucleolar RNA (snoRNA). However, previous studies focused on relatively small networks of interactions, and the comprehensive pattern of epistasis has not yet been determined for any gene. Although genome-wide studies have revealed a network of intergenic epistasis ( 6), it has been suggested that interactions within genes may be even more common ( 7– 11). Epistasis plays a major role in evolution it determines the accessibility of mutational pathways ( 3), thereby influencing the rate of adaptation and the diversity and robustness of genetic variants ( 4, 5). This phenomenon, known as epistasis, explains synthetic lethal interactions, where a combination of two individually viable mutations causes death, and compensatory interactions, where the fitness cost of a mutation is reduced by a second mutation ( 1, 2). The effect of a mutation on phenotype may depend on the presence of additional mutations.
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