random mutagenesis is a powerful tool for generating enzymes , proteins , metabolic pathways , or even entire genomes with desired or improved properties . due to the technical simplicity and applicability to almost any organism , chemical or radiation mutagenesis is frequently used for the generation of genetic variability in a microorganism .
however , these methods tend to be inefficient because they can cause substantial cell damage when performed in vivo .
a novel mutagenesis technique using error - prone dna polymerase  ( pol ) , based on the disparity mutagenesis model of evolution   has been successfully employed to generate novel microorganism strains with desired traits [ 411 ] . in the disparity model , mutations occur preferentially on the lagging strand , due to the more complex , discontinuous dna replication that takes place there .
computer simulation shows that the disparity model accumulates more mutations than the parity model , in which mutations occur stochastically and evenly in both strands .
in addition , the disparity model produces greater diversity because some offspring will have mutant dna while some offspring will have nonmutated , wild - type dna .
several studies have shown that the disparity mutagenesis method often achieved more satisfactory results ( i.e. , higher mutation rate and quick attainment of the desired phenotype ) than conventional methods such as the chemical mutagen , ethyl methanesulfonate ( ems ) [ 5 , 10 ] , which is known to produce mainly g / c to a / t transitions .
however , little else is known about the spectra of mutagenic effects caused by disparity mutagenesis . 

several mutants , including the proofreading - deficient pol3 - 01 strain and several low - fidelity mutants , have been shown to elevate the mutation rate [ 1418 ] . to generate the strains with the greatest mutagenicity ,
neo - morgan laboratory ( kanagawa , japan ) has developed the plasmid ycplac33/polmkii , expressing the pol mutant allele with 2 mutations : one mutation to inactivate the proofreading activity ( d321a and e323a )   and another mutation to decrease the fidelity of replication ( l612 m ) [ 14 , 17 , 18 ] . with the recent advent of next - generation sequencing technologies , an accurate characterization of the mutant genome , relative to the parental reference strain , is now achievable .
in fact , flibotte et al . have analyzed the mutation spectra induced by various mutagens , such as ems , enu , and uv / tmp , in the whole genome of caenorhabditis elegans .
another group has also used these sequencing technologies to analyze the genetic variations between a parental and ems - mutagenized strain of yeast . in this study , we evaluate the performance of the polmkii mutator , which expresses the proofreading - deficient and low - fidelity pol in s. cerevisiae haploid strain , compared with the commonly used chemical mutagen ems .
this mutator strain possesses exogenous mutant pol supplied from a plasmid , thereby leaving the genomic one intact .
we measured the mutation rate of this mutator strain and found that the mutation frequency of polmkii was approximately 7 times higher than that of ems .
we also performed high - throughput next generation sequencing with illumina gaii to analyze the genome - wide mutation spectra produced by the 2 different mutagenesis methods and found that the mutator strain exhibited more pleiotropy and gave rise to more diverse amino acid substitution patterns .
our present study has demonstrated that a proofreading - deficient and low - fidelity polmkii mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis .
plasmid ycplac33/polmkii was constructed as follows : a 4.8  kb dna fragment containing the s. cerevisiae by2961 pol3 gene , plus the utr 1  kb upstream and 0.5  kb downstream , ( mat ura3 - 52 , his3-300 , trp1-901 , leu2 - 3 , 112 lys2 - 801 , ade2 - 2 ) was inserted into the sali - ecori site of ycplac33 , and 3 amino acid substitutions , d321a , e323a , and l612 m , were introduced into the pol3 gene using site - directed mutagenesis .
ycplac33/polmkii vector ( and ycplac33 empty vector as nonmutator control ) was introduced into s. cerevisiae by2961 strain cells using the licl method , and the transformants ( mutator strains ) were selected on synthetic complete ( sc)-agar plates without uracil .
five mutator strains were picked and independently cultivated in 1  ml sc medium at 30c for 24  h ( about 30 generations ) in order to introduce mutations into their chromosomes . to determine the mutation frequencies of the 5 mutator strains
, aliquots were spread on sc - agar plates containing l - canavanine sulfate salt ( 0.06  mg / ml ) ( sigma , st .
louis , mo , usa ) to identify can1 mutants , and incubated until resistant colonies were formed .
the mutation frequencies were calculated as the number of drug - resistant colonies divided by the number of colonies on sc - agar plate without drug .
forward mutation rates at can1 were determined by fluctuation analysis using these 5 independent cultures . in order to fix mutations , another aliquot of the mutator culture
was spread on sc - agar plates containing 5-fluoroorotic acid monohydrate ( wako ) to obtain demutatorized cells curing from ycplac33/polmkii vector .
the genomic dna was prepared from the demutatorized cells using the procedure described in the following section . 

s. cerevisiae by2961 strain cells were suspended in 0.1  m phosphate - buffered saline ( pbs ) ( ph 7.0 ) containing 1.5 , 2.0 , 2.5 , or 3.0% ethyl methanesulfonate ( ems ) and were incubated at 30c for 1  h to introduce chromosomal mutations . the cells were washed 3 times with 5% sodium thiosulfate , suspended in sterilized water , and spread on sc - agar plates containing l - canavanine sulfate salt ( 0.06  mg / ml ) ( sigma ) to identify can1 mutants .
another aliquot of the ems - treated cell suspension was spread on a ypd - agar plate to isolate single clones .
the genomic dna was prepared from 5 single clones derived from the cells treated with 1.5% ems using the procedure described in the following section .
the genomic dna from s. cerevisiae was extracted using the dneasy blood and tissue kit ( qiagen , valencia , ca , usa ) .
briefly , 3  g of genomic dna was fragmented to an average length of 200  bp by using the covaris s2 system ( covaris , woburn , ma , usa ) .
the fragmented dna was repaired , a single  a  nucleotide was ligated to the 3 end , illumina index pe adapters ( illumina , san diego , ca , usa ) were ligated to the fragments , and the sample was size selected for a 300  bp product using e - gel sizeselect 2% ( invitrogen , grand island , ny , usa ) .
the size - selected product was amplified by 18 cycles of pcr with the primers inpe1.0 , inpe2.0 , and the index primer containing 6-nt barcodes ( illumina ) .
the final product was validated using the agilent bioanalyzer 2100 ( agilent , santa clara , ca , usa ) .
the 11 barcoded libraries ( the parental strain by2961 , 5 colonies from the mutator strain , and 5 colonies from the ems - treated strain ) were used for cluster generation in several multiplexed flow cell lanes in the illumina genome analyzer ii system .
ninety - one cycles of multiplexed paired - end sequencing was performed , running phi x 174 genomic dna as a control in a separate lane of the flow cell . after the sequencing reactions were complete , illumina analysis pipeline ( casava 1.6.0 ) was used to carry out image analysis , base calling , and quality score calibration .
the quality of each sequencing library was assessed by evaluating the quality score chart and the nucleotide distribution plot using fastx - toolkit ( http://hannonlab.cshl.edu/fastx_toolkit/ ) .
once the raw sequence data were curated , the reads of each sample were aligned to the s288c reference genome ( http://www.yeastgenome.org/ ) using the bwa software ( ver .
0.5.1 ) with default parameters . to avoid false positives and mutations from repetitive regions , we removed repetitive reads from the alignment files .
0.1.9 )   to produce the lists of mutations . to identify mutations that were produced by mutagenesis , we applied the following filtering criteria to the lists of mutations : the coverage at the mismatch positions should be at least 10 ; the variant is not present in the sequenced parental strain ; indels meet a snp quality threshold of 50 and substitutions meet a snp quality threshold of 20 ( samtools assigns snp quality , which is the phred - scaled probability that the consensus is identical to the reference ) ; samples meet a mapping quality of 30 ( samtools assigns mapping quality , which is the phred - scaled probability that the read alignment is wrong ) ; the percentage of reads showing the variant allele exceeds 90% . a variant must pass this filter to be considered a mutation .
the lists of mutations were then annotated using cova ( comparison of variants and functional annotation ) ( http://sourceforge.net/projects/cova ) .
cova was specifically designed to annotate the large number of identified mutants using the genbank annotation files .
in this study , we evaluated the performance of the polmkii mutator , compared with that of the commonly used chemical mutagen , ems . to assess ems efficiency ,
the lethality and mutation frequencies of the canavanine resistant colonies are shown in table 1 . at an ems concentration of 1.5% ,
the mutation frequency was approximately 18-fold higher than that in the control ( untreated ) strain . above 2.0% ems ,
based on this result , we decided to use cells treated with 1.5% ems for whole - genome sequencing . to assess the effectiveness of the mutator
, we transformed the haploid by2961 strain with a yeast expression plasmid , ycplac33/polmkii , expressing the pol mutant allele containing both the mutation to inactivate the proofreading activity ( d321a and e323a ) and the mutation to decrease the fidelity of replication ( l612 m ) .
the mutator strain harboring the ycplac33/polmkii plasmid will be referred to from here on as  mutator .
as summarized in table 2 , the mutation frequency of the mutator was approximately 132-fold higher than in the cells containing the empty vector .
the forward mutation rate at the can1 ( arginine permease ) locus was calculated to be 7.9  10/cell division .
these results show that the plasmid - generated mutated pol protein effectively competes with the endogenous wild - type pol protein that is produced from the chromosome , and the semidominant negative expression of mutated pol was effective in introducing mutations .
these results also demonstrate that the mutation frequency of the mutator was approximately 7 times higher than that of ems . to analyze the genome - wide mutation spectra of the 2 different mutagenesis methods
, we implemented a parallel sequencing approach with the illumina solexa technology ( gaii instrument ) .
we sequenced the parental haploid strain by2961 , each of the 5 clones from the mutator strains , and each of the 5 clones from the ems - treated strains under nonselective conditions .
sequencing reads were aligned to the s288c reference genome using the bwa software . to avoid false positives due to mutations from repetitive regions ,
reads mapped to multiple locations were discarded , and only uniquely mapped reads were used for subsequent analysis . in the current study , the average genomic coverage ranged from 32 to 87 ( table 3 ) . on average , 94.18% of the s288c reference genome was covered with at least 1 uniquely mapped read at each base .
subsequently , we analyzed the data for 2 kinds of mutational events : single nucleotide variants ( snvs ) and small insertions and deletions ( indels ) .
we compared the average number of mutations between mutator strains and ems - mutagenized strains ( figure 1 ) .
mutator and ems produced few deletions ( 1.6 versus 2.8 per strain , resp . ) , as well as few insertions ( 0.2 versus 0.6 per strain , resp . ) .
an average of 1.14  10 nucleotide sites fulfilled our criteria of read depth ( 10 ) , with an average base - substitutional mutation rate estimate of ems : 4.87 ( se = 1.34 )  10 per site , mutator : 2.09  ( 0.55 )  10 per site per cell division ( about 30 generations ) .
the rate we calculated for the mutator is 100-fold higher than the previously reported spontaneous mutation rate , 3.3  ( 0.8 )  10 , based on 454 analyses of 4 mutation - accumulation ( ma)-lines . the 2 mutagens generate mutations that are distributed similarly across the various gene features although the mutator did produce more snvs within exons than did ems ( figure 2 ) .  
the mutation spectra are shown in figure 3(a ) . in the genome - wide profile
, we found that the mutator primarily induced transversions ( 72% ) while ems primarily induced transitions ( 97% ) , well in accord with the known mutagenic specificity of ems .
similarly , the mutator primarily induced transversions ( 69% ) in the nonsynonymous substitutions in exons ( figure 3(b ) ) , similar to what has been seen in pol3 - 01 study using ura3 reporter gene .
ems treatment was also in agreement with the genome - wide spectra , induced transitions with a prevalence of 98% .
the mutation spectra of a given mutagenesis method influences the repertoire of changed amino acids at the protein level , and we were able to evaluate the amino acid substitution patterns generated by our 2 protocols ( table 4 ) .
initially , we classified mutations into those that preserved the corresponding amino acid , changed the amino acid , or generated a stop codon .
a clear difference was seen between mutator and ems . of the total mutations , the mutator changed the amino acid in approximately 85% , whereas ems changed the amino acid in approximately 61% .
the mutator also generated more stop codons than ems ( 7% versus 2% , resp . ) . while mutator generated more changes to the first or second nucleotide of the codon , ems generated changes in all 3 positions in approximately equal proportions .  
amino acid changes were classified into conservative and nonconservative substitutions , where a conservative substitution changed the encoded amino acid to a similar amino acid according to the criteria of the blosum62 matrix . of the amino acid changes ,
for the comparison of random mutagenesis methods , wong et al .   proposed a useful structure indicator that takes into account gly and pro substitutions as well as stop codons . in our study , the mutator produced an equivalent number of gly / pro and stop codon substitutions , whereas ems generated only stop codon substitutions .
in this study , we evaluated the performance of a novel mutagenesis technique using error - prone proofreading - deficient and low - fidelity dna polymerase  by determining the mutation rate of the strain harboring the enzyme .
we also analyzed the spectra of mutations across the entire s. cerevisiae genome and then assessed the diversity of mutation types at the amino acid level .
proofreading - deficient pol mutants , such as pol3 - 01 strain , and several low - fidelity pol mutants , such as l612 m , have been shown to present a mutator phenotype and to elevate the mutation rate [ 1418 ] .
we generated a by2961 strain expressing a polmkii mutator , pol mutant allele containing a combination of mutations to inactivate the proofreading activity ( d321a and e323a ) and to decrease the fidelity of replication ( l612 m ) .
this mutant allele acts as a strong mutator , as evidenced by the high frequency of spontaneous mutations ( 131-fold over control , compared to 18-fold for ems strains ) .
reported the forward can1 mutation rates of pol mutants as 1.5  10 in l612 m , and 5.6  10 in pol3 - 01 .
these mutant strains were constructed by integrating the pol3 - 01 or pol3-l612 m allele into the chromosomal pol3 gene by targeted integration , thereby disrupting the endogenous pol3 gene .
in contrast , our mutator plasmid expressing the pol mutant allele produced a mutation rate of 7.9  10 , which shows a high mutation rate as well as chromosomal integration .
the use of the polmkii mutator plasmid allows the continued expression of the endogenous wild - type pol3 and provides for an efficient restoration of the wild - type mutation rate by curing the yeast strains of the mutator plasmid .
once the desired trait(s ) has been selected , curing the cells from the mutator plasmid can stabilize the newly obtained phenotype .
in general , all random mutagenesis methods developed to date are biased toward transition mutations , although efforts have been made to overcome this . while transition bias was observed in ems , we actually observed transversion bias with the mutator ( figure 3(a ) ) . because of this
the mutator was also biased toward transversions in the nonsynonymous substitutions ( figure 3(b ) ) . for ems ,
the spectrum of mutation events we observed is similar to what has been reported by others . at the protein level
, the amino acid substitution pattern differed between the mutator and ems ( table 4 ) .
mutations generated by the mutator resulted in amino acid substitutions more often than did mutations generated by ems ( 85% versus 61% , resp . ) .
most of the substitutions made by the mutator were nonconservative , whereas only half of the substitutions made by ems were nonconservative .
in addition , the mutator generated more structure - disturbing amino acid changes ( gly / pro ) .
the transversion bias of non - synonymous substitutions by the mutator generates more diverse amino acid substitution patterns than does the transition bias of ems .
although the average base - substitution mutation rate of ems was approximately 100 times higher than that of the mutator , the mutation frequency of the mutator was approximately 7 times higher than that of ems .
this gap between a higher apparent mutation frequency and fewer mutations may be explained by the higher proportion of amino acid changes and the diversity of amino acid substitutions by the mutator .
the disparity mutagenesis technique has been successfully applied to not only eukaryotic microorganisms such as s. cerevisiae [ 5 , 79 ] , s. pombe , and ashbya gossypii , but also to prokaryotic microorganisms such as escherichia coli   and bradyrhizobium japonicum .
we believe that this novel mutagenesis technique has the potential to be applied to a wide variety of microorganisms . our present study has demonstrated that a proofreading - deficient and low - fidelity polmkii mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis .
it has been suggested that organisms may accelerate evolution by decreasing the fidelity of the proofreading activity of pol in nature ; therefore , this mutator may also be useful for studying the acceleration of evolution .
 a novel mutagenesis technique using error - prone dna polymerase  ( pol ) , the disparity mutagenesis model of evolution , has been successfully employed to generate novel microorganism strains with desired traits . 
 however , little else is known about the spectra of mutagenic effects caused by disparity mutagenesis . 
 we evaluated and compared the performance of the polmkii mutator , which expresses the proofreading - deficient and low - fidelity pol , in saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate ( ems ) . 
 this mutator strain possesses exogenous mutant pol supplied from a plasmid , tthereby leaving the genomic one intact . 
 we measured the mutation rate achieved by each mutagen and performed high - throughput next generation sequencing to analyze the genome - wide mutation spectra produced by the 2 mutagenesis methods . 
 the mutation frequency of the mutator was approximately 7 times higher than that of ems . 
 our analysis confirmed the strong g / c to a / t transition bias of ems , whereas we found that the mutator mainly produces transversions , giving rise to more diverse amino acid substitution patterns . 
 our present study demonstrated that the polmkii mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion