Copyright 0 1996 by the Genetics Society of America
Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila melanogaster Lynn F. Stam and Cathy C. Laurie Department of Zoology, Duke University, Durham, North Carolina 27708 Manuscript received June 27, 1996 Accepted for publication September 9, 1996
ABSTRACT A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophilamelunogaster, 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 traitlocus 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.
A
tiple independent transformants of each construct can LCOHOL dehydrogenase (ADH) activity and probe used to avoid the complication of position effect tein levels are quantitative traits that show extensive variation in natural populations of Drosophila melavariation so that even very small differences (on the nogaster. This variation is clearly polygenic, with effects order of 5-10%) can be detected (LAURIE-AHLBERG due to genes both linkedand unlinked to the A d h locus and STAM 1987). that encodes the enzyme (reviewed by LAURIE-AHLBERG In D. melanogaster, ADH is encoded by a single gene 1985). However, the A d h locus clearly constitutes a mathat produces two distinct transcripts from alternative jor gene effect, since two allelic classes defined by an promoters, proximal and distal (Figure 1). The distal allozyme polymorphism have distributions of ADH actranscript is the predominant form in adults and late tivity withvery little overlap (LAURIE-AHLBERG et al. larvae, while the proximal transcript is the predominant 1980). Fast homozygotes generally have a 2.5-3.0-fold form in larvae up until mid-third instar (BENYAJATI et higher ADH activity levelthan Slow homozygotes, which al. 1983; SAVAKIS et al. 1986). Transcription of A d h is is partly due to a difference in catalytic efficiency and regulated by sequences immediately upstream of each partly to a difference inthe concentration of ADH propromoter in conjunction with more distant enhancer tein ( LAURIE-AHLBERG 1985). elements (POSAKONY et al. 1985; CORBIN and MANIATIS DNA sequence analysis of alleles from the two allo1989, 1990). Our transformation experiments have utizymicclasses has revealed that they differ by only a lized a genomic fragment that includesall known regusingle amino acid replacement ( W I T M A N 1983), but latory elements. It extends from a Sac1 site -5.5 kb there are many other sequence differences that show upstream of the distal promoter to a ClaI site -0.8 kb nonrandom association with the replacement polymordownstream of the polyAadditionsite (Figure 1).Adult phism (AQUADRO et al. 1986; KREITMANand AGUADE transformants with wild-type versions of this construct 1986; SIMMONS et al. 1989).These associations sugexpress ADH at levels very similar to normal flies with gested that the major expression difference between the same allele (LAURIE-AHLBERGSTAM and 1987). the classes may not be due entirely to the amino acid In a previous transformation experiment, essentially replacement. the entire difference between the allozymic classes in We have been conducting a series of experiments to ADH activity and protein levels in adults was mapped identify polymorphisms within the A d h gene that conto a HpaI/ClaI fragment, which contains all coding setribute to the expression difference between the alloquences, aswellas intronic, 3’ untranslated and 3’ zymic classes. The basic approach is to make modificaflanking sequences (LAURIE-AHLBERG and STAM1987). tions of a pair of typical Fast and Slow alleles i n vitro, This fragment contains 13 polymorphisms that differ introduce those constructs into ADH-nullflies by Pbetween consensus Fast and Slow alleles (WITMAN element transformation and then assess the effects on 1983), as shownin Figure 1. Through in vitro mutageneADH activity and protein levels in transgenic flies. Mulsis, we previously showed that the amino acid replacement causes a catalytic efficiency difference, but does Corresponding author: CathyC. Laurie, DCMB/Zoology, Box 91000, not contribute to the 1.5-fold difference in ADH proDuke University, Durham, NC 27708. E-mail:
[email protected] tein level between the allozymicclasses (CHOUDHARY Genetics 1 4 4 1559-1564 (December, 1996)
1560
L. F. Stam and C. C. Laurie Proximal mRNA
sonal communication). An 8.0-kb SacI/ClaI fragment containingthe Adh regionandan 8.1-kb SalI fragment containing awild-type rosy gene are inserted into the polylinker. Plasmids were constructed using standard methods ( MANIATISet al. 1982). Adh alleles: Adh-containing genomic fragments derive from A1059 clonesprovided by M. KREITMAN, who obtained complete sequences of the transcriptional unit of (KREITMAN 1983). The Fast-typical allele each allele (A"B"CF)is a wild-type sequence (Wa-f), while the Slowtypical allele ( A S @ d ) is a chimera consisting of two flanking fragments fromWa-f (SacI/HpaI and Baa/ ClaI) and a middlefragment (HpaI/BalI) froma wild-type Slow sequence (Afs).Figure 1 shows all of the sequence differences between these two constructs, which coincide (by design) with all of the differences in the HpaI/ClaI fragment between the consensus sequencesof a random sample of five Fast and six Slow alleles analyzed by KREITMAN (1983). The eight recombinants representing all possible combinations of segments A, B and C from these two alleles were generated and analyzed by transformation. An additional construct from a previous experiment (LAURIE and STAM1994) was also analyzed: A"wl-SJBFCF, which has the same sequence as the Fast-typical allele, except that the V1 sequence in region A was changed from the Fast to the Slow form. Constructing recombinant alleles: Recombinants were generated using restriction sites ( S a d , HpaI, BamHI and C l d ) and recombinant polymerase chain reaction (PCR) (HIGUCHI 1990) with 20-base primers beginning at nucleotide sites 984,1571and 2735 (Figure1).Forexample, segment B"in the Fast-typical allele (AFBFC")was replaced by segment B 7 with the following procedure. A 984-1571 fragment from the Slow allele was amplified and annealed to a 1571-2735 fragment amplified from the Fast allele, using the overlapping complementarity of primer 1571. The 984-2735 chimeric sequence was amplified with primers 984 and2735 and cutwith BamHI and ClaZ to produce B"C", which replaced the B"C"fragment in the Fast allele, generating A"BsC". All fragments generated by PCR were completely sequenced to verify that no errors were made by the polymerase during amplification. Production andanalysis of transformant fly stocks: Transformant stocks were produced by microinjection of embryos from an ADH-null host strain, Adhfi6 cn; y, and selection of y+ offspring, as previously described (CHOUDHARY and LAURIE 1991). In some cases, additional insertions were generated by transposing an X-linked insert to the autosomes using P[ry+ A2-3](99B) (ROBERTSONet al. 1988). Stocks with a single insertwere selected by crossing a single y+ male to host strain females and analyzing a sample of progeny by Southern blotting to detect insertgenome junction fragments (as in CHOUDHARY LAURIE and 1991). The r y ' progeny from those males with a single insert were intercrossed to establish a stock. For analysis of ADH expression, r y ' males from each single insert stock were crossed to host strain females and yc male progeny were selected. A number of independent, single insert transformants (10-29) were analyzed for eachof the constructs in a randomized block design. On each of four occasions (blocks), a set of 10 maleswere sampled from a different replicate cross, aged for 7-9 days posteclosion and homogenized. ADH activity was measured spectrophotometrically, ADH-proteinwas measured by radial immunodiffusion and total protein was measured by the bicinchoninic acid procedure,as previously described (CHOUDHARY LAURIE and 1991). Unitsof activity are nanomoles NAD+ reduced per minute per milligram total proof standard tein and unitsof ADH protein are the number
1 Distal mRNA
Sac1
Sal I
-5500
43
1259984
321
816
f
BamHI
Hpal
551
SbwFam m l n o add s u b l ~ l u l ~ ( n
.:.:.:.:_:. ...... .._.. ..._..
t m
I
-ivz
I Tffi
* ; E 1527 1557
+
Bell
C/a I
1571
1971
2671 2735
$1 1
1693
T/C /vc
FIGURE1.-The structure of the Adh gene and sequence differences between the Fast- and Slowtypical alleles. Solid blocks represent coding sequences, open blocks represent introns and stippled blocks represent 5' and 3' untranslated sequences (BENYAJATI et al. 1983). Numbering of nucleotide sites is from the consensus sequence of KREITMAN (1983), except for theSacI site, which is based on restriction fragment length. The proximal ml2NAis the primary form in larval stages while the distal mRNA is the primary form in adults (BENYAJATI et al. 1983). The sequenceused for transformation extends from a SacI site -5.5 kb upstream to a ClaI site 2.7 kb downstream of the site of distal transcript initiation. This sequence consists of an upstream SacI/HpaI fragment, which is common to all alleles, and a downstream HpaI/ClaI fragment, which is composed of three regions (A, B and C) that vary in sequence among alleles.
and LAURIE 1991). In addition, we showed that an intronic length polymorphism ( V l ) causes part, but not all, of the ADH protein level difference (LAURIE and STAM1994). Here we report a molecular mapping experiment to localize other polymorphisms that contribute to this protein level difference. This experiment dissects the HpuI/CZuI fragment into three regions (A, B and C) shown in Figure 1. Each of the eight possible combinations among the three regions were constructed by making recombinants between a pair of Fast- and SZowtypical alleles. The Fusttypical allele used in this experiment (AFLfCF)is a wildtype sequence (from allele Waf) with a HpuI/CluI fragment that happens to have the consensus sequence of a random sample of five Fast alleles (KREITMAN 1983). The Slowtypical allele (A'E'C') has a HpuI/CluI fragment that was engineered to have the consensus sequence of six random Slow alleles. Both alleles have a common upstream fragment (SucI/HpuI) from the Waf allele. The effects of each region wereassessed by analyzing variation in ADH expression in multiple independent transformants of each of the eight construct types.
ry+
MATERIALSAND METHODS
Plasmid constructions: Theplasmidsusedfor P-element transformation all have the samebasic structure (see and STAM1987). The vector Figure 1 of LAURIE-AHLBERG pPLAl consists of a defective P-element containingpolya perlinker, which was inserted into PUG9 (J. POSAKONY,
Adh Variation in Drosophila
1561
tein level, contains foursites that differ between consensus Fast and Slow alleles. One of these polymorphisms Means of ADH activity and ADH protein ( V l ) is a complexsequencelength polymorphism within the first intron of the distal transcript (KREITMAN ADH ADH Adh allele N" activity' protein' 1983), which is the only detectable RNA found in the adult male transformants of the typeanalyzed here Transformant types A 1"BFCF 18 596 13.3 (LAURIE and STAM1994). We have analyzed this polyA /BP-c\ morphism previously by in vitro mutagenesis and found 10.929 519 AFB"Cb 11 355 14.4 that it has a significant effect on ADH protein level A1:B.\c.5 17 313 12.4 (LAURIE and STAM1994). A construct used in the previA.SB6CF 11 442 9.6 ous experiment is identical to the Fast-typical allele, A .TB1's;( 428 28 9.2 except that the VI sequence was changed to the Slow A .SB s (,.I' 272 21 10.6 typical form [ i.e., AFplS)B"C"]. Transformants with this A SB5 c s 19 248 9.6 A1"p1 - 5 )B FC F construct do not differ significantly in ADH protein 10 465 9.9 level from the A"B"C" transformants, which indicates Wild-type stocks that V1 alone can account for the effect of region A. Wa-f 14.5 652 274 9.9 Wa-s Region B contains six closely linked sites that differ between consensus Fast and Slow alleles. One is the "N is thenumber of independent, single-insert transamino acid replacement that causes the allozymic difformant lines analyzed. 'Units are given in MATERIALS AND METHODS. Means are ference, while the others are third position silent substiover Nlines with four observations per line fortransformants tutions. We havepreviouslyanalyzed the amino acid and over four observations for wildtype stocks. The variance replacement at nucleotide 1490 and a silent substituamong lines within a transformant type is estimated by the tion at 1443by in uitromutagenesis. Although the amino corresponding mean square from ANOVA. This mean square acid replacement significantly affects ADH activity due is 9843 for ADH activity and 7.11 for ADH protein (with 146 d.f. in both cases). Standard errors of the means are shown to a change in catalytic efficiency,neither of these subin Figure 2. stitutions has a detectable effect on the level of ADH protein (CHOUDHARY and LAURIE 1991). Therefore, it (Hochi-R inbred) fly-equivalents per milligram total prois very likely that the region B effect is due to one or tein. The data were analyzed by factorial analysis of varimore of the other four silent substitutions. ance after excluding four lines (from a total of 168) that Region C contains three polymorphisms in the 3' are outlierssatisfying a statistical rejection rule (SNEDECOR untranslated sequence of the transcript that differ beand COCHRAN1967, p. 322). tween Fast and Slow consensus alleles. One is a length polymorphism in a run of As, while the other two are RESULTS single base substitutions. Anyof these three polymorphisms could be responsible for the effect of Region C. Transformants with the Fast-typical allele have 2 . 4 fold more ADH activityand 1.4-fold more ADH protein than those with the Slowtypical allele (Table l ) ,which DISCUSSION is very similar to the average for completely wild-type Slow and Fast flies (LAURIE and STAM1988). For examSeveral of the polymorphisms analyzed here show ple, in this experiment, the wild-type stocks Wa-f and significant linkage disequilibrium in large surveys of Wa-s showed an activity ratio of 2.4 and anADH protein natural populations. A strong association between the ratio of 1.5. The following analysis will focus on ADH V1 and amino acid replacement polymorphisms has protein level rather than activity, since only one of the been detected in several geographic locations (KREITsequence differences between the twotypical alleles MAN and AGUADE1986; AGUADE1988, LAURIE et al. causes an amino acid replacement that could affect ac1991; BERRY and KREITMAN 1993; BENASSIet al. 1993), tivity without affecting ADH protein level. which suggests that V1 plays an important role in causEach of the three regions has a highly significant ing the world-wide difference in ADH protein level beeffect on the level of ADH protein in adult males ( P < tween the allozymic classes. Twoof the silent polymorO.OOOl), but the direction of effect differs between B phisms in region B (sites 1518 and 1527) are in strong and the other two regions (Table 1 and Figure 2). Allinkage disequilibrium with each other and with the leles with A" have 32% more ADH protein than those amino acid replacement polymorphism in several difwith A', while alleles with C" have 14% more than C". ferentpopulations (e.g., SIMMONS et al. 1989). The In contrast, alleles with B" have 10% less ADH protein length polymorphism in region C has severalallelic than those with By.In addition, regions A and C show states in natural populations (from 10 to 16 A s ) and a significant epistatic interaction (P< 0.002), such that shows linkage disequilibrium with the amino acid polythe difference between A" and As is greater on a C" morphism such that longer runs are associated with the than on a C y background (Figure 2a). Fast amino acid ( P = 0.058 in a chi-square contingency Region A, which has the largest effect on ADH protest using data from KREITMAN and AGUADE1986). TABLE 1
1562
L. F. Stam and C. C. Laurie
I a. Region A
15
-I-
7
... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... XFF
:
:
XFS
M ... ... ... ... .. ... .. ... ... .. ... .. ... .. ... .. ... .. ... .. ... .. ... ...
-L
XSF
xss
Fxs
sxs
b. Region B
FXF
SXF
It is not clear why many polymorphisms in the Adh gene show extensive linkage disequilibrium, but a contributing factor may be the relatively recent origin of the Fast amino acid mutation followed by a rapid increase in frequency due to selection. Several studies have shown that the Fast allozyme class has less haplotype diversity than the Slow class, indicating a recent origin (AQUADRO et 01. 1986; KREITMAN and AGUADE 1986). Repeatable geographic clines provide strong evidence for differential selection on the allozymic classes (OAKESHOTT et al. 1982; DAVIDet al. 1989;JIANG et al. 1989; P-H et al. 1992), but the targets of selection are not clearly defined. BERRYand KREITMAN (1993) found that both the amino acid and V1 polymorphisms show amarkedlatitudinal cline in North America, whereas most other polymorphisms in Adh that were analyzed in their study do not show significant clinai variation. This observation suggests that both the amino acid and V1 polymorphisms may be targets of selection. The other polymorphisms that contribute to the difference in ADH levels could also be targets of selection, but most of the candidate polymorphisms identified here have not been included in clinal studies. The results of this study, combined with those of previous analyses, showthat several typesof polymorphisms distributed throughout the Adh gene contribute to a “major gene” effect associated with the allozyme polymorphism in natural populations. One of these effects is due to the amino acid replacement, which causes an approximately twofold difference in catalytic efficiency ( C H O U D H A RLAURIE Y ~ ~ ~1991). The ADH protein level difference is caused by a length polymorphism in an intron, atleast one (probably silent) substitution in the coding sequenceand atleast one substitution or length difference in the 3‘ untranslated region. In addition, a previous study has shown that the upstream fragment (SacI/HpaI) from Wa-fdiffers from the corresponding fragment from a Slow allele ( Wa-s) by one or moresubstitutions that cause an 8% ADH protein level difference (LAURIE-AHLBERG and STAM1987). However, it is possible that this upstream effect is unique to the Wuf/Wa-s comparison and is not necessarily characteristic of Fast and Slow alleles. The findings of this study have important implications for interpreting quantitative trait locus mapping experiments because they show that detectionof a “major gene” effect is not necessarily due topolymorphism at a single site. Because of extensive linkage disequilibrium among polymorphisms in the Adh region, the effects of several different polymorphisms combine (in some cases synergistically) to produce a “superallele”
2 I c. Region C
15
-I
T
... ... ... ... ... ...
... ... ... ... ... ... ... ... ... FFX
FSX
SFX
ssx
Transposon Type FIGURE2.-The mean level of ADH protein in adultmales of transformant lines containing one of 8 different Adh alleles. All alleles have the same upstream region, but they vary in genotype of the A, B and C regions shown in Figure 1. The three-letter label for each transformant type describes (in order) the sequence of the A, B and C regions. For example, “XSF” for X = F represents the allele AFBsCF.The mean values are taken over a number of independent transformant lines, with four replicate samples per line. These values and
the sample sizes are given in Table 1. The errorbars represent the standard error of the mean (calculated from the common mean square for lines within transformant type and the number of lines of each type). Units of ADH protein are given in MATERIALS AND METHODS.
Drosophila
in
Variation
Adh
effect. Similar situations may occur inother systems. For example, association between certain polymorphisms in apolipoprotein genes and traits associated with cardiovascular disease suggest physiologically significant effects (DUNNING et al. 1992). However, linkage disequilibrium occurs within these loci (SINGand MOLL1990), so in this case (like A&) multiple polymorphisms may combine to produce the observed effects. An important issue in the evolution of morphology and other traits concerns the relative contributions of mutations with major us. minor effect (CHARLESWORTH et al. 1982; LANDE 1983; COYNEand LANDE 1985). One approach to this problem is mapping the quantitative trait loci that cause differences between species or selected lines and estimating the magnitude of their effects. In recent years, several studies have reported evidence thatsmall regions of the genomemay contribute a relatively large effect (DOEBLEY and STEC1991; TANKSLEY 1993; BRADSHAW et al. 1995; LONGet al. 1995; LIU et al. 1996). In addition to recognizing that closely linked genes may be involved in causing localized effects, it is important to realize that even if a single locus is responsible, the effect may be due to multiple substitutions. Moreover, when dealing with species differences, multiple substitutions may have occurred sequentially, rather than being due simultaneous to fixation of multiple linked polymorphisms. Thus, it is clear that mutations with minor effects may combine to produce alleles that segregate with major effect on the trait. Although ADH protein level may not be typical ofother quantitative traits, it provides a well studied example that demonstrates the possible complexity of “major gene” effects. We thank SAMANTHA GASSONand MERYL CARTER for technical assisCUNNINGHAM and JANIS ANTONOVICS tance. JIANJUN LIU, CLIFFORD provided helpful comments on the manuscript. This work was s u p ported by National Science Foundation grant DEB-9207215.
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Communicating editor: M. J. SIMMONS
