GEPHE SUMMARY Print
Entry Status
Published
GepheID
GP00001962
Main curator
Courtier
PHENOTYPIC CHANGE
Trait Category
Trait State in Taxon A
Drosophila melanogaster - slow allele - lower enzyme activity
Trait State in Taxon B
Drosophila melanogaster - fast allele - higher enzyme activity
Ancestral State
Taxon A
Taxonomic Status
Taxon A
Common Name
fruit fly
Synonyms
Sophophora melanogaster; fruit fly; Drosophila melanogaster Meigen, 1830; Sophophora melanogaster (Meigen, 1830); Drosophila melangaster
Rank
species
Lineage
Show more ... Brachycera; Muscomorpha; Eremoneura; Cyclorrhapha; Schizophora; Acalyptratae; Ephydroidea; Drosophilidae; Drosophilinae; Drosophilini; Drosophila; Sophophora; melanogaster group; melanogaster subgroup
NCBI Taxonomy ID
is Taxon A an Infraspecies?
No
Taxon B
Common Name
fruit fly
Synonyms
Sophophora melanogaster; fruit fly; Drosophila melanogaster Meigen, 1830; Sophophora melanogaster (Meigen, 1830); Drosophila melangaster
Rank
species
Lineage
Show more ... Brachycera; Muscomorpha; Eremoneura; Cyclorrhapha; Schizophora; Acalyptratae; Ephydroidea; Drosophilidae; Drosophilinae; Drosophilini; Drosophila; Sophophora; melanogaster group; melanogaster subgroup
NCBI Taxonomy ID
is Taxon B an Infraspecies?
No
GENOTYPIC CHANGE
Mutation #1
Presumptive Null
No
Molecular Type
Aberration Type
SNP
SNP Coding Change
Nonsynonymous
Molecular Details of the Mutation
Thr192Lys. Historical note : this discovery is actually based on progressive mapping started before 1980; and on early population genetics work by Kreitman 1983 (PMID: 6410283). There are three other (noncoding) substitutions in Adh coding regions. In vitro tests showed that none of the other three noncoding substitutions contributed a detectable effect. The K192T substitution is thus sufficient to explain the entirety of the activity difference from the coding region.
Experimental Evidence
Taxon A Taxon B Position
Codon - - -
Amino-acid - - -
Authors
Choudhary M; Laurie CC
Abstract
In natural populations of Drosophila melanogaster, the alcohol dehydrogenase (Adh) locus is polymorphic for two allozymes, designated Slow and Fast. Fast homozygotes generally have a two- to threefold higher ADH activity level than Slow homozygotes for two reasons: they have a higher concentration of ADH protein and the Fast protein has a higher catalytic efficiency. DNA sequencing studies have shown that the two allozymes generally differ by only a single amino acid at residue 192, which must therefore be the cause of the catalytic efficiency difference. A previous P element-transformation experiment mapped the difference in ADH protein level to a 2.3-kb HpaI/ClaI restriction fragment; which contains all of the Adh coding sequences but excludes all of the 5' flanking region of the distal transcriptional unit. Here we report the results of a site-directed in vitro mutagenesis experiment designed to investigate the effects of the amino acid replacement. This replacement has the expected effect on catalytic efficiency, but there is no detectable effect on the concentration of ADH protein estimated immunologically. This result shows that the average difference in ADH protein level between the allozymic classes is due to linkage disequilibrium between the amino acid replacement and one or more other polymorphisms within the HpaI/ClaI fragment. Sequence analysis of several Fast and Slow alleles suggested that the other polymorphism might be a silent substitution at nucleotide 1443, but another in vitro mutagenesis experiment reported here shows that this is not the case. Therefore, the molecular basis of the difference in ADH protein concentration between the allozymic classes remains an open question.
Mutation #2
Presumptive Null
No
Molecular Type
Aberration Type
Indel Size
10-99 bp
Molecular Details of the Mutation
Indel in 5'UTR intron. Slow alleles nearly always have the 29 bp version of delta1 and Fast alleles predominantly have the 34bp version.
Experimental Evidence
Authors
Laurie CC; Stam LF
Abstract
Several lines of evidence indicate that natural selection controls the frequencies of an allozyme polymorphism at the alcohol dehydrogenase (Adh) locus in Drosophila melanogaster. However, because of associations among sequence polymorphisms in the Adh region, it is not clear whether selection acts directly (or solely) on the allozymic site. This problem has been approached by using in vitro mutagenesis to distinguish among the effects on Adh expression of individual polymorphisms. This study shows that a polymorphism within the first Adh intron (delta 1) has a significant effect on the level of ADH protein. Like the allozyme, delta 1 shows a geographic cline in frequency, indicating that it may also be a target of natural selection. These results suggest that multisite selection models may be required to understand the evolutionary dynamics of individual loci.
Mutation #3
Presumptive Null
No
Molecular Type
Aberration Type
Molecular Details of the Mutation
mutation in 3'UTR region. Two substitutions occur in the pair of fast and slow alleles from Loehlin et al 2019: a poly-A tract of length 14 in slow and length 15 in fast, and a complex substitution of sequence CA in slow and G- in fast. Authors attempted to separate the effects of the A(14)/A(15) from the CA/G- with a construct that was recombined between the two sites. Activity of this construct was not significantly different from either neighboring construct; indicating that they did not have sufficient power to determine the causative site(s). In spite of this; Stam and Laurie also mapped an activity difference with a similar effect size of 1.1-fold to a region containing the 3’-UTR. Their fast and slow haplotypes carry a different suite of substitutions in this region; but the C/G nucleotide substitution is shared in both studies. Additional mapping work would be required to determine if this one substitution or if multiple substitutions are responsible for the activity difference from the 3'-UTR.
Experimental Evidence
Authors
Stam LF; Laurie CC
Abstract
A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce "superalleles" with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect.
Mutation #4
Presumptive Null
No
Molecular Type
Aberration Type
SNP
Molecular Details of the Mutation
C/T SNP in 5' flanking region upstream of the 5'UTR is responsible for the change in Adh activity. Adh-slow: TCACCGATT; Adh-fast: TCATGCATT (where TCAC is the end of the 5' flanking region and CGATT the beginning of the 5'UTR). The causing mutation seems to be a 3-bp multi-nucleotide mutation.
Experimental Evidence
Authors
Loehlin DW; Ames JR; Vaccaro K; Carroll SB
Abstract
The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Copyright © 2019 the Author(s). Published by PNAS.
Additional References
Mutation #5
Presumptive Null
No
Molecular Type
Aberration Type
SNP
Molecular Details of the Mutation
C/G SNP in 5'UTR region
Experimental Evidence
Authors
Loehlin DW; Ames JR; Vaccaro K; Carroll SB
Abstract
The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Copyright © 2019 the Author(s). Published by PNAS.
Additional References
Mutation #6
Presumptive Null
No
Molecular Type
Aberration Type
Insertion Size
10-99 bp
Molecular Details of the Mutation
delta-2 indel in 5'UTR region. 37-bp insertion in the fast allele.
Experimental Evidence
Authors
Loehlin DW; Ames JR; Vaccaro K; Carroll SB
Abstract
The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Copyright © 2019 the Author(s). Published by PNAS.
Additional References
COMMENTS
@SeveralMutationsWithEffect @Fitness - All six higher-activity variants appear to be derived; suggesting a history of directional selection. The three causative substitutions in the 5′ UTR occur within 100 bp. - Entry validated by David Loehlin - http://flybase.org/reports/FBal0000310 - http://flybase.org/reports/FBal0000314
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