Associations Between Restriction Site Polymorphism and Enzyme ...

Copyright 0 1990 by the Genetics Societyof America

Associations Between Restriction Site Polymorphism and Enzyme Activity Variation for Esterase6 in Drosophila melanogaster Anne Y. Game' and John G. Oakeshott C.S.I.R.O., Division of Entomology, Canberra City, ACT 2601, Australia, andResearch School of Biological Sciences, The Australian National University, Canberra City, ACT 2601, Australia Manuscript received May 16, 1990 Accepted for publication August 30, 1990 ABSTRACT Thirty-five nucleotide polymorphisms were found in a 2 1.5-kbp region including theEst6 locus among 42 isoallelic lines extracted from a single natural population of Drosophila melanogaster. The heterozygosity per nucleotide pair was estimated to be 0.010 overall, but was lower in sequences hybridizing to transcripts thanin those not hybridizing to transcripts. Eleven of 36 pairwise comparisons among the nine most common polymorphisms showed significant gametic disequilibrium. Four of these polymorphisms were also significantly associated with the major ESTG-F/ESTG-S allozyme polymorphism. Significant disequilibrium was generally restricted to polymorphisms less than 1-2 kbp apart. Previously reported measures of EST6 activity in virgin adult females proved not to be significantly associated with any of the six most common nucleotide polymorphisms located in the Est6 coding region or the 1.5 kbpimmediately 5'. However, the 11 haplotypes for fiveof these polymorphisms that lie in the 1.5-kbp 5' region could explain about halfof the previously reported in virgin adult variation among the lines for both EST6 activity and the amount of EST6 protein males. One particular polymorphism, for a RsaI site 530 bp 5' of the initiation codon, could explain 21% of the male activity variation among lines. This siteis embedded in a large palindrome and we suggest that sequences including or closetothissitemaybeinvolved in the regulation ofEST6 synthesis in the ejaculatory ductof the adult male.

A

NALYSES of restriction map variationin natural

populations are providing new information about the natureof genetic variation and insights into its relationship to thephenotype andfitness. The first issue, the natureof variation, has now been examined by restriction site analysis for many gene regions and several species (see, e.g., AQUADRO, LADO and NOON and 1988; MIYASHITAand LANGLEY1988; STEPHAN LANGLEY 1989).In Drosophila, one general finding has been the existence of substantial levels of nucleotide variation (e.g., heterozygosity >0.2% in most nuclear gene regions).Another general findinghas been the high levels of gametic disequilibrium among polymorphic sites, particularly over regions of less than about 2 kbp. Ontheotherhand, it is becoming apparent that heterozygosity and disequilibrium can vary considerably amongdifferentregions,even within species and particular chromosomes. T h e relationship between the nucleotide variation and variation in function and fitness is still, in most cases, unknown, either because the function of the particular region containing the nucleotide variation is unknown, or, if known, because the function has not been assayed for variation among those lines scored for restriction site polymorphism. Four notable

' Present address: Genetics Department, Parkville, Victoria 3052, Australia. Genetics 126: 1021-1031 (December, 1990)

T h e University of Melbourne,

exceptions are the work on the Adh (BIRLEY1984; AQUADRO et al. 1986; STRAND and MCDONALD 1989), Amy (LANGLEYet al. 1988), Ddc (ESTELLEand HODGETTS 1984a,b) and white (MIYASHITAand LANGLEY, 1988) loci of Drosophilamelanogaster. Interestingly, only a small minority of the functional variation in these systems was found to beassociated with nucleotide variation in putative cis regulatory regions. These results are somewhat surprising given the widely held view that regulatory variation is of particular importance for evolutionary change (WILSON 1976; HEDRICK and MCDONALD 1980; EDELMAN1987;SINGH and CHOUDHARY 1989). Here we report on restriction site polymorphism and its association with functional variation for the regionlocated at 69A1 onchromosome 111 of D. melanogaster that contains the structural locus (Est6) for the esterase 6 enzyme (EST6). This region is well suited to such an analysis for three reasons. First, the locations of other transcriptional units in the 2 1.5-kbp in regioncontaining Est6 havebeencharacterized detail (COLLET et al. 1990) and some information is available ontheltcation of the cis regulatory seHEALYand quences of the Est6 gene (OAKESHOTT, GAME1990). Second, high levels of functional variation have beenfoundfor EST6, not only for its structure (COOKE and OAKESHOTT 1989; OAKESHOTT

1022

A. Y . Game and J. G . Oakeshott

et al. 1990) but also, of more interest here, for two essentially independent aspects of its regulation, specifically adult male and adult female EST6 activity (GAMEand OAKESHOTT 1989). Finally, considerable evidence is now available to show that at feast some of thestructuralandregulatory variation is mainet al. 1990; tained by natural selection (OAKESHOTT RICHMOND et al. 1990, for reviews).

OAKESHOTT et al. ( 1 987) andCOLLET et al. (1990). The full restriction map of the region was compiled by GAME(1 989). Also Indicated on Figure 1 is the location of a second esterase gene in the 2 1.5 kbp region. The coding region of this gene, EstP, begins 197 bp 3' of the Est6 stop codon, The two genes presumably reflect a tandem duplication event, although they now show only64% sequence similarity and are maximally transcribed in different life stages (adults et al. 1990). for Est6, late larvae for EstP; COLLET

MATERIALS AND METHODS

RESULTS

The analysis is based on 42 third chromosome isoallelic lines extracted from a population at Coffs Harbour, Australia. The extraction procedures were described by COOKE, RICHMOND and OAKEsHOTT (1987) who also reported that six EST6 allozymes segregate among the 42 lines. These ailozymes cover four major electrophoretic mobility classes, ESTG-vF (otherwise denoted ESTG-I), ESTG-F'(or EST62), EST6-F and ESTG-S, with the latter two, more common classes each including two minor mobility variants (EST6-4 and EST6-5 within EST6-F and EST6-8 and EST6-9 within EST6-S). The 42 lines havealso been assayed for variation in EST6 1989). The activity in 4-5activity (GAMEand OAKESHOTT day-old virgins of each sex shows heritable threefold variation across the 42 lines. Males havefrom three tonine times more EST6 activity than females, depending on the line. The activity variation among lines is largely independent of the allozymic differences and onlyweakly correlated between the two sexes. The basis of the variation in females has not been investigated but in males is due almost entirely to variation in the number of EST6 protein molecules. In the present experiments, genomic DNA was extracted from adult flies from each line using a modification of the and DOVER(1982). Single procedure of COEN,THODAY digests of the DNA from each line withnine hexanucleotide restriction enzymes (BamHI, BglI, BglII EcoRI, EcoRV, HindIII, PstI, PuuII and XbaI) and six tetranucleotide restriction enzymes(DdeI, HaeIII, Hinfl, Rsal, Sau3A and TuqI) were then fractionated by electrophoresis and transferred to nitrocellulose membranes by standard techniques. Membranes were prehybridized in 5 X SSPE (0.9 M NaCI, 50 mM sodium phosphate, pH 7.0, 5 mM EDTA), 5 X Denhardt's solution (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1 % Ficoll), 0.1 ?6 SDS, and 500 ,ug/ ml sonicated denatured salmon sperm DNA at 65" for 4-8 hr. Hybridizations were then carried outunder similar conditions except that only 250 pg/ml carrier DNAwas used and incubations were for 14-18 hr. Double-stranded DNA probes were made by nick translation (RIGBYet al. 1977). After hybridization, membranes were subjected to three 5-min washes at room temperature in 0.1 X SSC, 0.1 % SDS followed by two 30-min washes at 65" in 0.5 X SSC, 0.1% SDS. They were then exposed to Kodak RP X-ray film at -70" for 3-15 days. Figure 1 shows the four clones used to probe the 2 1.5kbp region screened. Genomic DNA digested with the six tetranucleotide enzymes and PstI was probed with two plasmid subclones, HR-1.15 and RH-2.75. These cover a region from 1.15 kbp 5' ofEst6, throughits I .69-kbp coding region (including its 51-bp intron), to 1.06 kbp 3' of the gene. Genomic DNA digested with the othereight hexanucleotide enzymes was probed with two XEMBL4 bacteriophage clones, A801 and h201, which together span the 21.5-kbp region. All four clones used as probes had been isolated from the Dm145 stock of D. melanogaster as described in

Amount of restriction site variation: A total of 80 restriction sites (55 six-base sites and 25 four-base sites) have been scored in the 21.5-kbp region among the 42 Coffs Harbour lines (Figure 1). Thirty-one of these sites are polymorphic (Table 1, Figure 2). The frequency of the rarer form at each of the polymorphic sites ranges from 2% to 34%. Four polymorphic insertionldeletion differences (0.5-4.9 kbpin length) are also found and their frequencies range from 2% to 5%. The estimate of heterozygosity per nucleotide eair (6 k overthe 21.5 kbpscreened is 0.00960.0003 SE; EWENS,SPIELMAN and HARRIS1981 Eq. 12; HUDSON I982 Eq. 19). The overall proportion of polymorphic nucleotide sites is 0.045 f 0.001 $ 4 SE; HUDSON1982 Eqs. 7 and 11). However, estimates of these two parameters vary among regions hybridizing to transcripts (56 sites from -4.9 to -1.6 kbp and 0 to +12.3 kbp; see Figure 1) and not hybridizing to transcripts (24 sites from -1.6 to 0 kbp and+12.3 to +16.6 kbp). Fortheformer regions 8 is 0.0079 k 0.0003 and is 0.035 +- 0.001 andforthelatter is 0.0138 f 0.0006 and is 0.071 +- 0.003. Thus the regions hybridizing to transcripts are only about half as variable as regions not hybridizingto transcripts. A more detailed breakdown of heterozygosity values for regions around and including Est6 is given in Figure 3. The Est6 structural region has the lowest heterozygosity; about one third of that for the region of highest heterozygosity, the 1.15-kbpregion immediately 5' of Est6. The latter region is known from germ-line transformation analysis to include all promoter elements required for wild-type EST6 expresHEALY and GAME1990). The codsion (OAKESHOTT, ing region of EstP, like that for Est6, shows relatively low heterozygosity. The heterozygosities for the regions 3' are about double those of the Est4 and EstP codingregions. The same pattern of differences among regions is evident for the proportion of polymorphic sites i n each of the regions (data not shown); the values of'p range from 0.019f 0.002 for the Est6 structuralregionto0.065 0.004 forthe1.15-kbp region immediately 5' of Est6. Gametic disequilibrium: Only 25 of the 593 pairwise comparisons among all nucleotide pofymorphisms (including insertion variants) show statistically

6

1023

Est6 Restriction Site Variation

4 j

3

- 2 ;

0

-1

+1

4

j12

e

+4

+5

1

r7

4

4

+S

+10

+11

+12 +14 i +13 1

+15

+16

Wi1.15 0

i

RK2.75

1 In01

M l l l.26

'4 ++

j

ji

1.68 1.25 1.63

ii ' j

1.39

1.25 1.92

3.20

-

J

0.60 1.16

FIGURE1 .-The 2 1 .bkbp region aroundEst6 and EstP (left andright hatched regions, respectively) showing the 35 polymorphisms (above the line) and 49 monomorphic restriction sites scored (below). A coordinate line (in kbp) with zero at the initiation codon of Est6 is also shown. Locations of all sites scored are taken from the full restriction map of the region in GAME(1989). The positions given for the insertions represent aproximate locations only and are the midpoint of the smallest restriction fragment to which they could be localized. The four clones used as probes to detect the restriction variation are shown immediately below the coordinate line. At bottom are the sizes and orientations of transcripts which COLLET et al. (1990) found to hybridize to the region. Vertical lines between transcripts mark the ends of the fragments used in the Northern analyses to detect the transcripts. A = HaeIII, B = BamHI, D = DdeI, El = EcoRI, EV = EcoRV, F = Hinfl, GI = BglI, GI1 = BglII, H = HindIII, P = PstI, R = RsaI, S = SauJA, T = TaqI, V = PvuI1, X = XbaI.

significant disequilibrium (Table 2). However, eleven of the significant disequilibrium values lie among the 36 pairwise comparisons of the nine most common polymorphisms (variant frequencies between10% and 90%; and the three 5' insertions, which may all have arisen from the same ancestral insertion event, being classified as a single polymorphism). Polymorphisms involved in significant associations are scattered across the 2 1.5-kbp region but most significant associations involve pairs of closely linked polymorphisms. This can be seen in Figure 4, which plots the significance values against the distance between the two sites. The average probability value from the significance tests is only below 5 % for associations between sites less thanabout1kbpapart. T h e total of 35 polymorphisms found generate 30 haplotypes, ranging in frequencies from 2% to 14% (Table 1). T h e estimate of haplotype diversity is 0.973, with a SD of 0.031 (NEI 1978; NEI and TAJIMA 1981 Eq. 7). Four of the nine most common nucleotide polymorphisms individually show significant associations with the major EST6-F us. EST6-S allozyme difference (Table 2). Three of these associations involve polymorphisms less than 2 kbp from the nucleotide polymorphism coding for the ESTG-F/ESTG-S difference (an A/G polymorphism at +0.772 producing an Asn/Asp amino acid difference; COOKEand OAKESHOTT 1989). Strong disequilibrium with the allozymes is also evident if all sixEST6 allozymes are considered, rather than just the two major EST6-F and EST6-S forms. For example, only one of the 30 nucleotide haplotypes is found in more than one allozyme (haplotype 19 in EST6-2 and EST6-9; Table 1). When variation across all 21.5 kbp is considered, comparable levels of haplotype diversity and nucleo-

tide heterozygosity arefound within thedifferent allozymes. Thus, the six EST6-4 lines represent six haplotypes and a heterozygosity of 0.010; the three EST6-5 lines yield three haplotypes and a 0 of 0.010; the 22 EST6-8 lines give 15 haplotypes and 6 of 0.008; and the eight EST6-9 lines five haplotypes and ?i of 0.006. (The other two allozymes, EST6-1 and EST62, were too rare for meaningful analysis.) This suggests that all four of these electrophoretically defined Est6 alleles have existed long enough to accumulate similar levels of nucleotide variation in the 2 1.5-kbp region including the gene. However, these overall results for the whole 21.5 kbp conceal differences among the allozymes for variation within the coding region of Est6. In particular, Table 3 shows that the EST6-8 allozyme completely lacks nucleotide polymorphism or haplotype diversity within this region. This corroborates both the findings of LABATEet al. (1989), who found no EST6 thermostability variation within 28 EST6-8 lines, and (1 989),who found those of COOKEand OAKESHOTT no nucleotide differences in two independent EST68 lines for which they and COLLETet al. (1990) obtained full sequence data. We therefore concur with these authors' conclusions that Est6-8 is a relatively recent allele (which has nevertheless risen to a relatively high frequency). Our finding that EST6-8 resembles the other allozymes in nucleotide heterozygosity and haplotype diversity for the larger 2 1.5-kbp region therefore emphasizes the evidence presented above that recombinationhas broken upmost gametic disequilibrium over distances greater than 1-2 kbp. Following TEMPLETON, BOERWINKLE and SING (1987) and TEMPLETON et al. (1988), an attempt was made to arrange the 30 haplotypes in a cladogram

1024

A. Y. Game and J. G . Oakeshott

TABLE 1 Distribution of the 35 polymorphisms across the 42 lines Haplotypes Variant

XbaI-4.3 BglI-3.6 XbaI-3.5 Ins(a)- 1.4 Ins(b)-1.4 Ins(c)-1.4 DdeI-0.90

1

2

3

4

5

6

7

8

9

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+

-

-

-

-

-

+ +

-

-

+

-

-

-

-

-

+

+ +

+

+ +

-

+ +

Hinfl-0.80

Hinfl-0.68 Tuql-0.63 Rsal-0.53 Sau3A-0.20 RsaI+O.20 TaqI+0.80 BarnHI+2.3 HaeIII+2.4 BgllI+3.1 Ins(d)+3.3 DdeI+4.3 EcoRI+4.3 Dde1+4.7 EcoRI+7.0 BglI+8.1 EcoRI+9.9 EcoRV+10.8 EcoRV+l 1.3 EcoR1+11.8 BglII+II.9 EcoRV+l3.0 EcoR+I13.1 BumHI+13.4 BglII+14.7 HindIII+14.8 BglII+15.7 EcoRI+16.6

+ +

+ + + + + +

+ + + + +

-

-

-

+ + -

+ + +

Haplotypefre0.02 quency EST6 allozyme

8

+ + +

+

-

-

-

-

+

-

+

-

+ + + + + + + + + + +

+ + + + + + + n + + +

+

-

+ +

-

+ +

-

-

+ + -

+ +

+ +

-

-

-

+ + +

+ + +

+ + -

+ + +

-

-

-

0.05

0.02 0.05 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

1

4

8

+ + + +

+ + + + +

-

-

s

-

+ + +

+

-

+

+ + + + + +

-

+ +

-

-

-

+ -

+ +

+ -

+ -

+ + + + +

+ + + + + + +

+ + + + +

8

4

8

-

-

-

+ +

+ -

-

+ + + 8

-

-

-

+ +

+ +

+ +

-

-

-

-

-

-

-

-

-

+ + -

-

+ +

-

-

+

-

+

+

+ + + -

+

+

-

+ + + + -

+ -

+ -

+ +

+ -

-

-

-

+ + +

+ +

+ +

4

16

+ +

-

-

15

+ +

-

+ -

13

+ +

+ + + +

+

1412

+ +

+ + + +

+ +

11

+ + + + +

-

-

+ +

-

+ + +

+ + +

-

+

-

-

-

+ +

10

8

+ +

8

-

+

+ +

+ + + + +

-

+

+

+ -

-

+ + + + + -

+

-

-

+ +

+ + + + + +

ns

+

+ + + + + + + + + + + -

-

-

+ + -

+ + +

+ + +

+ + +

5

4

8

8

+ + +

-

-

+

- + + + + +

+ + +

+ + + + + + +

-

+ + + + + + + + +

-

-

-

+ -

+ + -

-

+ + + + + + 9

Polymorphisms are listed with respect to their positions on the restriction mapin Figure 1. T h e presence of a restriction site or insertion is indicated by and the absence by -. Two sites in a total of three lines could not be scored (“ns”). Variant frequency refers to the less common form for each polymorphism, either presence or absence. Haplotype frequency indicates the frequency of the haplotype in the sample. The EST6 allozyme status of each haplotype is also shown; for haplotype 19, one line is EST6-2 and the other two lines are EST69.

+

(results not shown). However, only 11 of the 30 haplotypes, representing 19 of the 42 lines, couldbe placed in such a cladogram, without invoking some recombination. This compares with an analysis of a 13-kbp regionaround Adh in this species, in which 25 of 29 haplotypes (representing 41 of 49 lines) could be placed in a single cladogramwithoutapparent recombination (TEMPLETON, BOERWINKLE and SING 1987; TEMPLETON et al. 1988). The reason for the difference between the two studies might reflect the greater length of the Est6 region scored,which might, in turn, allow for more recombination. Associations with activity variation: Since only a minority of the Est6 haplotypes could be incorporated

into a cladogram without recombination,the cladistic approach which TEMPLETON, BOERWINKLE and SING (1 987)and TEMPLETON et al. (1988) developed to test for associations between nucleotide and activity variation is not appropriate for our data. The alternative approach used below has four principal elements. First, we restrict our attentiontothenine most common polymorphisms (variant frequenciesbetween 10% and go%), since any effects of rarer polymorphisms are more likely to be confounded by genetic background differences. Second, given the gametic disequilibrium among several of the polymorphisms, we analyse the activity associations of the haplotypes before dissecting them into the contributions of indi-

1025

Est6 Restriction Site Variation

Haplotypes

17

18

19

20

21

22

23

24

26

+ + -

-

+ + + + + + + + + +

-

+ + + + + + -

+

-

+ + +

27

28

29

30

Variant frequency

0.05 0.05 0.02 0.05 0.05 0.02 0.19 0.02

0.02 0.24 0.26 0.17

0.02 0.26 0.02 0.05 0.02 0.02

0.05 0.34 0.05 0.10 0.07 0.02 0.33 0.33 0.05 0.02

0.05 0.07 0.02 0.02 0.05 0.02 0.07

0.05 9

vidual polymorphisms. Third, kcause thecause of its association with activity may vary with the region in which the nucleotide variation occurs, separate analyses are conducted for the variation in three regions, namely the region 5‘ to Est6 (todetectregulatory polymorphisms), the coding region of Est6 (to detect structural polymorphisms) and the region 3’ to Est6 (for which no causal associations with EST6 activity are expected). Finally, in order to examine further the causes of any associations detected, they were then compared with the corresponding associations with EST6 amount (only possible in males) and with EST6 allozymes. Regulatory polymorphisms should show similar associations with activity and amount and these associations should be independentof EST6 allozyme status (which GAMEand OAKESHOTT (1989) showed

was not related to EST6 amount).On the other hand, coding region polymorphisms affecting the structure of the EST6 protein may be associated with EST6 allozyme status. We consider first those five of the most common polymorphisms that lie in the 1.5 kbp region immediately 5’ to the coding region of Est6. Four of these five polymorphisms (DdeI(-0.90), TuqI(-0.63), RsaI(-0.53), Suu3A(-0.20)) actually lie in the 1.15 kbp known to contain the Est6 promoter; the fifth polymorphism, for the insertions at about -1.4 kbp, lies outside this region but is included with*the other four in this analysis because of the possibility that transcription from the inserted material could interfere with Est6 transcription ( c j STRANDand McDONALD1989). The five polymorphisms form 11

A. Y. Game and J. G . Oakeshott

1026 0 Restriction sites insertions

0.1

0

1

NUCLEOTIDE POLYMORPHISM

5

FIGURE2.-Frequency

spectrum for restriction map variants in the21.5-kbp Est6 region. The frequency of the least frequent variant at each site is shown.

"

Est 6 Est P

GENOMIC REGION AROUND EST6

FIGURE3.-Mean

haplotypes among the 42 lines and these haplotypes differ significantly in male activity and male amount, but not in female activity (Table 4). The mean activity and mean amount of enzyme in males each vary over almost a twofold range among thevarious haplotypes, and these haplotypes explain 58% of the variation in activity and 44% of the variation in amount among lines. The most common haplotype, whichlacksall four restriction sites and the insertions, is relatively low forboth male activity and amount. T h e most extreme male activity and amount values are for relatively rare haplotypes. The correlation between male activity and male amount across the 5' haplotypes is highly significant ( T = 0.94, d.f. = 9, P < 0.001), further suggesting that variation in this region is important in regulating the number of EST6 molecules produced. RsaI(-0.53) is the only one of the five most common polymorphisms 5' to Est6 that individually shows a significant effect on male EST6 activity (explaining 21 % of the variance among lines) and none show a significant effect on female activity (Table 5). T h e presence of the RsaI(-0.53) site is associated with higher male activity than its absence (2358 f 176 us. 1881 f 60). It is also associated with higher male EST6 amounts (1865 f 180 us. 1587 f 60; P < 0.05 on a one-tailed test). Further analyses of variance and covariance (not shown) indicated that the difference in amounts could explain 73% of the activity difference associated with the RsuI(-0.53) polymorphism, whereas the ESTG-F/ESTG-S electrophoretic difference could only explain 38% of the activity difference associated with this polymorphism. These results suggest that the RsaI(-0.53) polymorphism may mark an element regulating transcription,and, since the effect of this polymorphism on female activity is insignificant, the element may control transcription in a sexspecific manner. The one common restriction site polymorphism within the Est6 structural region, TaqI(+0.80), shows

values for the heterozygosity per nucleotide pair (+SE) for regions around and including Est6 and E S P . The values are calculated for restriction sites only (i.e., excluding insertions) and the numbers in parentheses are the numbers of sites scored within each region.

a significant association (explaining 10% of variance among lines) with male activity but not female activity (Table 5). Absence of the site is associated with higher male activity (2245 f 157 us. 1921 f 74), and higher amount (1810 f 136 us. 1607 f 74), althoughthe latter difference is not statistically significant (Table 5 ) . Most of the male activity difference associated with the TaqI(+0.80) polymorphism could be explained either by the difference in male amount (90%)or by association with ESTG-F/ESTG-S electrophoretic class (97%).One interpretation of these results is that the TaqI(+0.80) polymorphism produces structural a change in theprotein which is associated with the electrophoretic difference and which sufficiently destabilizes theprotein to cause adifference inits amount. Some primary effect on the structure of the protein would certainly be possible, given the evidence from 13 sequenced isolates of Est6 (COOKEand OAKESHOTT 1989), that a nucleotide substitution within + TCGG) this TuqI recognitionsequence(TCGA produces an amino acid substitution (Thr + Ala, at residue 247). On the other hand, it seems unlikely that aneffect due toa structural changewould be sexlimited. Given this anomaly, and the fact that it is a weak effect anyway, the statistical significance of the TuqI(+0.80) association with male activity may reflect no more than a chance result among a large number of associations calculated. Finally we considerthe three common polymorphisms 3' of the Est6 codingregion, EcoRI(+4.3), EcoRV(+10.8) and EcoRV(+ 1 1.3). The six haplotypes for these three polymorphisms do not differ significantly in male or female EST6 activity or male EST6 amount (Table 4). However, both EcoRV(+10.8) and EcoRV(+l 1.3) individually show significant effects on female activity (explaining 16% and 13% of variance

1027

Est6 Restriction Site Variation TABLE 2

D’ values (LEWONTIN1964) for gametic disequilibrium among polymorphic sites where the variant frequencies are between 10%and 90%and between these sites and EST6-F vs. EST6-S allozyme status DdeI(-0.90)

Ins(-l.4) DdeI(-0.90) TagI(-0.63) RsaI(-0.53) Sau3A(-0.20) TaqI(+0.80) EcoRI(+4.3) EcoRV(+10.8) EcoRV(+ 11.3)

0.04 0.01 0.19

TaqI(-0.63)RsaI(-0.53)

0.21 0.84***

Sau3A(-0.20) TaqI(+O.AO) EcoR1(+4.3)

0.66** 0.73***

0.65** 1.0*** 0.81***

0.24 -0.49* -0.19 -0.38* -0.23

-0.09 -0.24 0.12 0.45 -0.03 -0.08 0.31

EcoRV(+lO.R) EcoRV(+11.3)

1.0**

0.06 0.25 0.32 0.14 -0.05 -0.02

1 .o -0.25 -0. I O

-0.14 -0.18 -0.58* l.O***

ESTG-F/ESTG-S

0

0.48* 0.16 0.36* 0.20 -l.O*** -0.73*** 0 0.13

The 5’ insertions (lns(-l.4)) are considered as one polymorphism and their position represents an approximate location only (see Figure 1).

Other significant associations involving less common nucleotide polymorphisms: 5% significance: XbaI(-3.5) and Ins(c)(-1.4), D’ = 1; XbaI(-3.5) and BamH1(+2.3), D’ = -1; Ins(c)(-1.4) and BarnHI(+2.3), D’ = -1; HinfI(-0.68) and EcoRI(+11.8), D’ = -1; TagI(-0.63) and EcoRV(+l3.0), D‘ = -1; Rsal(-0.53) and EcoRI(+7.0), D’ = 0.66; Sau3A(-0.20) and EcoRV(+13.0), D’ = -1; RsaI(+0.20) and DdeI(+4.7), D’ = 1; EcoRI(+4.3) and EcoRI(+16.6), D’ = 1; EcoRI(+9.9) and BglII(+15.7), D’ = I ; BglII(+I 1.9) andBarnH1(+13.4),D’ = 1. 1 % significance: Sau3A(-0.20) and EcoRI(+7.0), D‘ = 0.70; TaqI(+0.80) and EcoR1(+16.6), D’ = 0.55; EcoRI(+7.0) and EcoRV(+IS.O), D‘ = -1. 0.1% significance: XbaI(-4.3) and Ins(b)(-1.4). D’ = -1. * P < 0.05, ** P < 0.01, *** P < 0.001. 08r

0 I-

%

0.3

4

.. DISTANCE APART (kbp)

FIGURE4.-Plot of gametic disequilibrium against distance for all sites where the variant frequencies are between 7% and 93% (preferred over the range 5-95% used elsewhere herein to increase the data available for comparison). Gametic disequilibrium is given as the probability value from x’ tests. Average values and their standard errors are shown over 1-kbp regions for sites 0 to 6 kbp apart and 2-kbp regions for sites 6 to 18 kbp apart. TABLE 3

Comparison of EST6-8 with other allozymes for estimates of heterozygosity per nucleotide pair(2SE) in different regions of the 21.5 kbp scored EST6-8

Region

(22 lines)

-1.15 kbp to 0 kbp Est6 coding region Other regions

0.014 & 0.001 0

0.008 f 0.001

Others (20 lines)

0.014 f 0.001 0.006 f 0.001 0.007 f 0.001

among lines respectively). In both cases, presence of the site is associated with higher activity (409 f 22 us. 350 & 11 for EcoRV(+10.8); 387 If: 14 vs. 335 k 14 for EcoRV(+l 1.3)). In neither case could much of the activity variance duetothe polymorphism beexplained by electrophoretic class (