Limits...
The Evolutionary Potential of Phenotypic Mutations.

Yanagida H, Gispan A, Kadouri N, Rozen S, Sharon M, Barkai N, Tawfik DS - PLoS Genet. (2015)

Bottom Line: Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals.Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location.Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT
Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids ß-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to +1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3'-UTR. Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

No MeSH data available.


Related in: MedlinePlus

Laboratory evolution for peroxisomal localization is driven by genetic mutations at the same location where phenotypic mutations occur.(A) The C-terminal region of K. waltii IDP2 including the 3’-UTR (100 bp around the stop codon) was randomly mutated by error-prone PCR at an average of ~1 mutation per gene. The mutated genes were cloned and selected by plasmid complementation of the ΔIdp3 growth in YP medium with petroselinate. After 200 hours of culture, a burst in growth rate was observed. (B) Six out of seven randomly sequenced clones from the 300 hours culture had a single base deletion just before the stop codon (g1-g3), all resulting in an in-frame PTS1 signal (-AKL*). (C) The three deletion sites were modified by silent mutations of the wild-type base into G. The resulting mutants were tested by plasmid complementation of the ΔIdp3 strain for growth on petroselinate. Two silent mutations (g2 and g3) resulted in growth inhibition, and the triple mutant (K.wal IDP2+g1+2+3) showed essentially no growth, thus indicating that the translational slippage leading to dual localization of K. waltii IDP2 occurs within this segment. It should be noted that we used an adapted ΔIdp3 strain whereby the growth of ΔIdp3 complemented with K.wal IDP2 increased (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526572&req=5

pgen.1005445.g004: Laboratory evolution for peroxisomal localization is driven by genetic mutations at the same location where phenotypic mutations occur.(A) The C-terminal region of K. waltii IDP2 including the 3’-UTR (100 bp around the stop codon) was randomly mutated by error-prone PCR at an average of ~1 mutation per gene. The mutated genes were cloned and selected by plasmid complementation of the ΔIdp3 growth in YP medium with petroselinate. After 200 hours of culture, a burst in growth rate was observed. (B) Six out of seven randomly sequenced clones from the 300 hours culture had a single base deletion just before the stop codon (g1-g3), all resulting in an in-frame PTS1 signal (-AKL*). (C) The three deletion sites were modified by silent mutations of the wild-type base into G. The resulting mutants were tested by plasmid complementation of the ΔIdp3 strain for growth on petroselinate. Two silent mutations (g2 and g3) resulted in growth inhibition, and the triple mutant (K.wal IDP2+g1+2+3) showed essentially no growth, thus indicating that the translational slippage leading to dual localization of K. waltii IDP2 occurs within this segment. It should be noted that we used an adapted ΔIdp3 strain whereby the growth of ΔIdp3 complemented with K.wal IDP2 increased (see Materials and Methods).

Mentions: In fact, we began our exploration with the latter—namely, we sought to identify hotspots for genetic, single base deletions that may occur upstream to K. waltii IDP2’s stop-codon and result in its cryptic PTS1 becoming in-frame (K. waltii IDP2 was the most poorly bypassed pre-duplication IDP2; and, as mentioned above, has no >3 bases repeats in its C-terminus; Fig 1B). We randomly mutated the segment of 100 bases around K. waltii IDP2’s stop-codon, transformed the mutated gene library to the S. cerevisiae ΔIdp3 strain and selected the transformed yeast cells for growth on petroselinate. After 200 hours, the culture’s growth rate dramatically increased (Fig 4A). The selected pool was analyzed by sequencing seven randomly chosen clones. We identified 3 different single base deletions that all occurred within a stretch comprising 3 repeats of 3 bases each just before the stop codon (AAATCCCAAA; Fig 4B). To examine whether the phenotypic frameshifts occur within the very same stretch, we applied the same test applied to validate the 6T repeat as the site of ribosomal slippage in A. gossyppii IDP2. Namely, we introduced silent mutations at each of the 3 deletion sites (AAA TCC CAA A; in bold, the sites of silent mutations; Fig 4B) and examined whether the frequency of slippage, as reflected by the rate of growth on petroselinate, would be reduced. Indeed, silent mutations in the two deletion sites that are closer to the stop-codon showed a marked inhibition of growth, and the triple mutant showed effectively no growth (Fig 4C). It therefore appears that the phenotypic mutations leading to cryptic peroxisomal localization in the cytosolic IDP2s are readily ‘immortalized’ via genetic deletion mutations that occur within the very same site.


The Evolutionary Potential of Phenotypic Mutations.

Yanagida H, Gispan A, Kadouri N, Rozen S, Sharon M, Barkai N, Tawfik DS - PLoS Genet. (2015)

Laboratory evolution for peroxisomal localization is driven by genetic mutations at the same location where phenotypic mutations occur.(A) The C-terminal region of K. waltii IDP2 including the 3’-UTR (100 bp around the stop codon) was randomly mutated by error-prone PCR at an average of ~1 mutation per gene. The mutated genes were cloned and selected by plasmid complementation of the ΔIdp3 growth in YP medium with petroselinate. After 200 hours of culture, a burst in growth rate was observed. (B) Six out of seven randomly sequenced clones from the 300 hours culture had a single base deletion just before the stop codon (g1-g3), all resulting in an in-frame PTS1 signal (-AKL*). (C) The three deletion sites were modified by silent mutations of the wild-type base into G. The resulting mutants were tested by plasmid complementation of the ΔIdp3 strain for growth on petroselinate. Two silent mutations (g2 and g3) resulted in growth inhibition, and the triple mutant (K.wal IDP2+g1+2+3) showed essentially no growth, thus indicating that the translational slippage leading to dual localization of K. waltii IDP2 occurs within this segment. It should be noted that we used an adapted ΔIdp3 strain whereby the growth of ΔIdp3 complemented with K.wal IDP2 increased (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526572&req=5

pgen.1005445.g004: Laboratory evolution for peroxisomal localization is driven by genetic mutations at the same location where phenotypic mutations occur.(A) The C-terminal region of K. waltii IDP2 including the 3’-UTR (100 bp around the stop codon) was randomly mutated by error-prone PCR at an average of ~1 mutation per gene. The mutated genes were cloned and selected by plasmid complementation of the ΔIdp3 growth in YP medium with petroselinate. After 200 hours of culture, a burst in growth rate was observed. (B) Six out of seven randomly sequenced clones from the 300 hours culture had a single base deletion just before the stop codon (g1-g3), all resulting in an in-frame PTS1 signal (-AKL*). (C) The three deletion sites were modified by silent mutations of the wild-type base into G. The resulting mutants were tested by plasmid complementation of the ΔIdp3 strain for growth on petroselinate. Two silent mutations (g2 and g3) resulted in growth inhibition, and the triple mutant (K.wal IDP2+g1+2+3) showed essentially no growth, thus indicating that the translational slippage leading to dual localization of K. waltii IDP2 occurs within this segment. It should be noted that we used an adapted ΔIdp3 strain whereby the growth of ΔIdp3 complemented with K.wal IDP2 increased (see Materials and Methods).
Mentions: In fact, we began our exploration with the latter—namely, we sought to identify hotspots for genetic, single base deletions that may occur upstream to K. waltii IDP2’s stop-codon and result in its cryptic PTS1 becoming in-frame (K. waltii IDP2 was the most poorly bypassed pre-duplication IDP2; and, as mentioned above, has no >3 bases repeats in its C-terminus; Fig 1B). We randomly mutated the segment of 100 bases around K. waltii IDP2’s stop-codon, transformed the mutated gene library to the S. cerevisiae ΔIdp3 strain and selected the transformed yeast cells for growth on petroselinate. After 200 hours, the culture’s growth rate dramatically increased (Fig 4A). The selected pool was analyzed by sequencing seven randomly chosen clones. We identified 3 different single base deletions that all occurred within a stretch comprising 3 repeats of 3 bases each just before the stop codon (AAATCCCAAA; Fig 4B). To examine whether the phenotypic frameshifts occur within the very same stretch, we applied the same test applied to validate the 6T repeat as the site of ribosomal slippage in A. gossyppii IDP2. Namely, we introduced silent mutations at each of the 3 deletion sites (AAA TCC CAA A; in bold, the sites of silent mutations; Fig 4B) and examined whether the frequency of slippage, as reflected by the rate of growth on petroselinate, would be reduced. Indeed, silent mutations in the two deletion sites that are closer to the stop-codon showed a marked inhibition of growth, and the triple mutant showed effectively no growth (Fig 4C). It therefore appears that the phenotypic mutations leading to cryptic peroxisomal localization in the cytosolic IDP2s are readily ‘immortalized’ via genetic deletion mutations that occur within the very same site.

Bottom Line: Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals.Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location.Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT
Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids ß-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to +1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3'-UTR. Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

No MeSH data available.


Related in: MedlinePlus