mutagenesis to analyze the molecular basis of the adh ... · the bamhi site of the plasmid pbsm 13-...

Copyright 0 199 1 by the Genetics Society of America

Use of in Vitro Mutagenesis to Analyze the Molecular Basis of the Difference in Adh Expression Associated With the Allozyme

Polymorphism in Drosophila melanogaster

Madhusudan Choudhary' and Cathy C. Laurie* Department of Zoology, Duke University, Durham, North Carolina 27706

Manuscript received May 14, 1991 Accepted for publication June 29, 199 1

ABSTRACT In natural populations of Drosophila melanogaster, the alcohol dehydrogenase (Adh) locus is poly-

morphic 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 HpaIIClaI 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 HpaIIClaI 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.

T HE alcohol dehydrogenase enzyme (ADH; EC 1.1.1.1) of Drosophila melanogaster is encoded

by a single gene (Adh) on chromosome arm 2L (LIN- DSLEY and GRELL 1968). Two alternative Adh transcripts are produced in a developmentally specific pattern from two tandem promoters (see Figure 1; BENYAJATI et al. 1983; SAVAKIS, ASHBURNER and WIL- LIS 1986). T h e distal transcript, which is the predominant form in adult tissues, is initiated 708 base pairs (bp) upstream of the initiation point of the proximal transcript, which is predominant in larval tissues. In addition, the distal transcript contains an extra intron of 654 bp, which separates two parts of the 5' untranslated leader sequence. T h e two processed transcripts differ in their 5' untranslated leaders, but they specify the same ADH protein. Deletion mutagenesis and P element-mediated germline transformation have been used to determine the sequences necessary for a normal pattern and level ofAdh expression. These studies show that transcription is regulated by sequences im- mediately upstream of each promoter in conjunction with distant larval- and adult-specific enhancer ele-

University, P.O. Box 1892, Houston, Texas 77251. ' Present address: Department of Ecology and Evolutionary Biology, Rice

' To whom reprint requests should be addressed.

Genetics 129: 481-488 (October, 1991)

ments (GOLDBERG, POSAKONY AND MANIATIS 1983; POSAKONY, FISCHER and MANIATIS 1985; CORBIN and MANIATIS 1989, 1990).

In natural populations throughout the world, the Adh locus is polymorphic for two allozymes, designated Slow and Fast. Several lines of evidence suggest that natural selection affects the frequencies of these alleles, a t least under some environmental conditions. Fast homozygotes, which generally have a higher activity level than Slow homozygotes, also generally have a higher tolerance to environmental alcohols in laboratory toxicity tests (see reviews by CHAMBERS 1988; GIBSON and OAKESHOTT 1982; VAN DELDEN 1982). T h e allozymes show a latitudinal cline in frequency in the same direction on each of three different continents (OAKESHOTT et al. 1982), which suggests a com- mon selective gradient. Recently, comparisons between the extent of silent nucleotide polymorphism within the melanogaster Adh gene and the extent of interspecific sequence divergence have provided evidence that there is an excess of polymorphism sur- rounding the amino acid replacement substitution that generates the allozyme polymorphism. Such an excess can be explained by postulating that the allozyme polymorphism is maintained by balancing selec-

482 M. Choudhary and C. C. Laurie

tion (KREITMAN and A G U A D ~ 1986; HUDSON, KREIT- MAN and A G U A D ~ 1987; KREITMAN and HUDSON 1991). Even though a great deal of effort has been expended on studying this polymorphism, the selective agent(s) that may cause the frequency clines and lead to a balanced polymorphism are still unknown. Additional ecological work will be necessary to understand the population genetics of the ADH allozyme polymorphism.

It is also important to understand the molecular basis of any functional differences between the two allelic classes. Fast homozygotes generally have a two- to threefold higher level of ADH activity than Slow homozygotes, which comes about through two mechanisms. Fast homozygotes have a higher concentration of ADH molecules and each of those molecules has a higher catalytic efficiency (see reviews by LAURIE- AHLBERC 1985; LAURIE and STAM 1988). DNA sequencing of several Fast and Slow alleles has shown that the two forms of the protein generally differ by a single amino acid, a threonine/lysine substitution at residue 192 (KREITMAN 1983). This amino acid replacement is clearly the cause of the catalytic efficiency difference between the allozymes, but the molecular basis of the difference in ADH concentration remains an open question.

A study of restriction fragment length polymorpism in the Adh region revealed a pattern of strong non- random association among ADH activity level, ADH allozyme and several restriction site polymorphisms (AQUADRO et al., 1986). One of the strongest associations in this region occurs between the Slow/Fast substitution site and a BamHI polymorphic restriction site located about 7 kb upstream (see Figure 1). Two of the chromosomes that show a rare haplotype with respect to these two sites also show altered levels of ADH activity, as though at least part of the basis for the activity difference between the allozyme classes had been separated from the charge substitution site by recombination. These results suggested that the difference in ADH concentration between the allozyme classes might be due to linkage disequilibrium with a polymorphic regulatory site located, perhaps, in the 5’ flanking region of the gene. This possibility was investigated through P element-mediated transformation of an ADH-negative strain with chimeric Adh DNA fragments in which the 5’ flanking regions were exchanged between a pair of Slow and Fast alleles that show the typical difference in activity and ADH-protein levels. This experiment showed that both the ADH activity and protein level differences clearly map to a 2.3-kb HpaI/ClaI fragment that includes all of the Adh coding sequence and some intronic and 3’ flanking sequence, but excludes all of the 5’ flanking sequence of the distal (adult) transcriptional unit (see Figure 1 ; LAURIE-AHLBERG and STAM

1987). Although this result rejects the hypothesis of a regulatory site polymorphism in the 5’ flanking region, it does not eliminate the possibility that some site (other than the amino acid replacement) within or 3’ of the Adh transcriptional unit affects the concentration of ADH protein.

At this point, it became important to determine whether the difference in ADH concentration between allozymes is due to a corresponding difference in ADH-mRNA, as reported previously by ANDERSON and MCDONALD (1983). T o investigate this issue, we used two methods, a quantitative Northern and a quantitative RNase protection assay, both of which utilized an Adh mutant as an internal control. When applied to several alleles from each allozymic class, both methods gave the same result: the difference in ADH-protein concentration between the allozymic classes is not due to a difference in concentration of ADH-mRNA (LAURIE and STAM 1988). This leaves two possible mechanisms: either there is a difference in the translation rates of the two RNAs or there is a difference in the rates of degradation of the two proteins in vivo.

After mapping the ADH activity and protein level differences to the 2.3-kb HpaI/ClaI restriction fragment, we compared the sequences of this fragment from several Slow and Fast alleles that were cloned and sequenced by KREITMAN (1983). We analyzed Adh expression in the isochromosomal stocks from which those clones were derived and we found that all of the Fast alleles have higher ADH activity than all of the Slow alleles (LAURIE-AHLBERG and STAM 1987). Furthermore, in comparing pairs of alleles from each of four geographic locations, we found that, in each case, the Fast member of the pair had a higher concentration of ADH protein than the Slow member (LAURIE and STAM 1988). The sequence comparisons of these four pairs of alleles show that only three nucleotide substitutions distinguish all of the Fast alleles from all of the Slow alleles. These three substitutions are located in close proximity within the last coding exon (Figure 1). One is, of course, the amino acid replacement at 1490 and the others are nearby third position silent substitutions, one at 1443 and one at 1527. We previously concluded that one or more of these three substitutions is likely to be responsible for the difference in ADH level between the allozymic classes (LAURIE and STAM 1988). More recent sequence comparisons have eliminated the 1527 silent substitution as a likely candidate (LAURIE, BRIDGHAM and CHOUDHARY 1991). Here we report the results of two site-directed mutagenesis experiments designed to determine whether the 1490 and 1443 substitutions have an effect on the level of ADH protein, measured as cross-reacting material (CRM).

Adh Expression in Drosophila 483

MATERIALS AND METHODS

Plasmid constructions: The plasmids used for P-element transformation all have the same basic structure (see Figure 1 of LAURIE-AHLBERG and STAM 1987). The vector pPLA1 consists of a defective P element containing a polylinker, which was inserted into pUC9 (J. POSAKONY, personal com- munication). An 8.6-kb SacI/ClaI fragment containing the Adh gene (Figure 1) and an 8.1-kb Sal1 fragment containing a wild-type ry gene are inserted within the polylinker. The Adh fragments originate from the Wa-s and Wa-fX1059 clones of KREITMAN (1983). The Wa-s and Wa-f fly stocks from which these clones derive show typical levels of Adh expression within their respective allozymic classes (LAURIE- AHLBERG and STAM 1987; LAURIE and STAM 1988). Num- bering of nucleotides begins with the distal transcript initiation point and is based on KREITMAN'S (1983) consensus sequence. Plasmids were constructed using standard methods (MANIATIS, FRITSCH and SAMBROOK 1982).

Plasmids that differ from the wild-type Wa-s by a single nucleotide substitution were constructed in the following way. The plasmid pAWa-s (containing the 8.6-kbSacI/ClaI Wa-s fragment inserted within pPLAl) was digested with BamHI and BglII. This plasmid has a unique BamHI site at 1257 within the Adh fragment and a unique BglII site approximately 20 nucleotides into the polylinker from the ClaI site. The 1.4-kb BamHI/BglII fragment was inserted at the BamHI site of the plasmid pBSM 13- (Stratagene, Inc.). This pBS clone was used in the in vitro mutagenesis procedure described below. The mutant fragment was then re- moved from the pBS clone by digestion with Bum HI and Clal and was used to replace the corresponding BamHI/ ClaI fragment in the wild-type pAWa-s plasmid. The final step was to insert the 8.1-kb ry Sal1 fragment into the XhoI site of this plasmid. The final constructions used for P- element transformation are referred to as pAWasla (wild- type), pAWasl490Cla (amino acid replacement) and pAWas1443Gla (silent substitution at 1443). The Wa-f plasmids were constructed by the same procedure and are referred to as pAWaEa, pAWafl490A2a, pAWafl443C2a. In each of these plasmids, the ry gene is in the same transcriptional orientation as Adh.

Site-specific mutagenesis: Oligonucleotide-directed, site- specific mutagenesis was performed according to a protocol provided by Amersham Inc. (code RPN.1523), which is based on a procedure described by TAYLOR, OTT and ECK- STEIN (1985). The Adh fragment to be mutagenized was cloned into the phagemid pBSMl3-, which produces single stranded template when the host JM109 is coinfected with helper phage R408. A mutagenic oligonucleotide is an- nealed to the single stranded template and then extended by Klenow polymerase in the presence of a thionucleotide (dCTPaS) and T4 DNA ligase to generate a mutant heter- oduplex. Selective removal of the nonmutant strand is ac- complished by restriction with NciI, which cannot cleave the newly synthesized phosphorothioate DNA but does nick the template strand. Nicking is followed by digestion with exonuclease 111 or T7 exonuclease V. The mutant strand is then used as a template to generate a mutant homoduplex using DNA polymerase I and T4 DNA ligase. The mutagenic oligonucleotides were each 19 nucleotides long, with the mismatch at position 9, 10 or 11 and with two G or c nucleotides at each terminus. The BamHIIClaI fragment containing the mutation was completely sequenced to be sure that no other changes had occurred. P element transformation and stock construction. Mi-

croinjection of embryos was performed essentially as described by GOLDBERG, POSAKONY and MANIATIS (1983).

Embryos from the ADH-null host stock Ad@' cn; ry506

(provided by J. POSAKONY) were injected with 5 mM KCI/ 0.1 M sodium phosphate, pH 6.8, containing the wings- clipped helper P element plasmid, pr25.7WC (KARESS and RUBIN 1984), at 150 pg/ml and one of the pAWa plasmids described above at 600 pg/ml. Insert-containing chromosomes were extracted into the genetic background of the host strain as described previously (LAURIE-AHLBERG and STAM 1987). Stocks having just a single insertion were identified by digesting the fly DNA with BamHI, EcoRI and XhoI and probing Southern blots with a 1.4-kb BamHI/ClaI probe that contains part of the Adh gene and 800 bp of 3' flanking sequence. This probe detects a single constant fragment from the resident Adh gene and a junction fragment of unique size from each transposon. All of the transformant stocks were checked by starch gel electrophoresis to determine whether they express the correct ADH allozyme (LAURIE-AHLBERG and WEIR 1979). In addition, two each of the transformants containing the 1443 silent site mutants were checked by sequencing of polymerase chain reaction (PCR)-amplified Adh DNA from the transformed fly stocks to verify the nucleotide at 1443. PCR methods are described in LAURIE, BRIDGHAM and CHOUDHARY

A2-3-induced transposon mobilization. Certain second chromosome insertions were mobilized to new locations on the third chromosome by crossing in a source of transposase, the insertion called P[ry+ A2-3](99B) (ROBERTSON et al. 1988). The target chromosome came from the Ad@' cn; 9'"' host stock and those third chromosomes containing a new insertion were extracted into the host stock genetic background using stocks with second and third chromosome balancers already in that background. Hence, the genetic background of the A2-3-induced insertions should be the same as those produced by embryo injection. Single insert stocks were identified by the Southern blotting procedure described above and checked for ADH allozyme by starch gel electrophoresis.

Protein assays: ADH activity was measure by the spectro- photometric method of MARONI (1978) using isopropanol as substrate. ADH units are r.anomoles NAD+ reduced per minute per mg total protein. Total protein was determined by the Fohn phenol procedure (LOWRY et al. 1951). ADH protein or cross-reacting material (CRM) was estimated by radial immunodiffusion (MANCINI, CARBONARA and HERE- MANS 1965). This procedure was tested with purified ADH- F and ADH-S (LEE 1982; CHAMBERS, FLETCHER and AYALA 1984) to verify that there is no difference between allozymes in the extent of antibody-antigen reaction (ie., equal quan- tities of ADH-protein gave equal immunodiffusion diame- ters). Each diffusion plate contained a dilution series of a standard extract of flies from a reference stock, Hochi-R, to ensure linearity and to provide a standard for comparison among plates. ADH-CRM units are given in terms of Hochi- R fly equivalents per milligram of total protein.

Transformant line surveys: In each experiment, adult males were sampled from each of the single insert transformant stocks and crossed to females from the A d p ' cn; rySo6 host stock during each of two time blocks. Within each block, four separate crosses of 5 pairs each were set up in 8- dram vials. From the pooled progeny of those four crosses, two sets of ten 6-8-day-old males were homogenized for the assay of ADH activity and CRM level. This makes a total of four observations from each stock. The ANOVA model contained the following main effects and all of their interactions: block, replicate within block, transposon type and line within transposon type. In devising F tests, all effects were considered random except for transposon type. The

(1991).

484 M . Choudhary and C. C. Laurie

GLM procedure of the SAS statistical software package (SAS Institute, Inc.) was used to perform the unbalanced ANO- VAS. Satterthwaite’s approximate F test was used for testing significance of transposon type (STEEL and TORRIE 1980). A linear combination of mean squares was used to estimate the error variance used in calculation of the least significant differences between transposon type classes reported in Figures 3 and 5.

RESULTS

Experimental plan: In each of the two experiments described below, four Adh DNA fragments are compared, two wild-type and two mutant. The two wild- type fragments derive from the Wa-f and Wa-s alleles of KREITMAN (1983), which were cloned from stocks showing typical Adh expression within their respective allozymic classes. Each of the two wild-type fragments was subjected to oligonucleotide-directed mutagenesis at a particular site (nucleotide 1490 in one experiment and 1443 in the other) to produce two mutant fragments. Each Adh fragment was inserted within a defective P element vector, which also contains a wild- type ry gene to provide an eye color marker for detecting transformants. Germline transformants of each of the four types were produced by embryo injection. Subsequently, some additional stocks containing insertions at new sites were produced by A2- 3-induced transposon mobilization. Each stock that was analyzed for ADH activity and CRM level had just a single transposon insertion on one of the two autosomes. Analyses of variance (ANOVA) were performed to test for differences among transformants of the same transposon type that were produced by embryo injection at different times and/or by A2-3 mobilization. There were no significant differences among transformants produced by the different methods or at different times.

The Adh fragment consists of an 8.6-kb SacI/ClaI fragment, which includes the Adh transcriptional unit along with about 0.8 kb of 3’ flanking DNA and about 5.9 kb of 5’ flanking DNA (Figure 1). GOLDBERG, POSAKONY and MANIATIS (1983) previously demon- strated that this amount of 5‘ flanking DNA is sufficient to provide for a normal tissue and developmental specificity and approximately normal levels of Adh expression in transformed flies. We used the same 8.6-kb fragment derived from KREITMAN’S Wa-s and Wa-f genomic clones in a previous experiment in which the transformants showed average ADH activity and CRM levels very similar to those of the original Wa-f and Wa-s stocks from which the clones were derived (LAURIE-AHLBERG and STAM 1987).

The 1490 amino acid replacement affects ADH activity but not CRM level: The 1490 site was analyzed first because it seemed plausible that the amino acid replacement might be affecting ADH concentration through an effect on protein stability in vivo. The

8.6-kb Adh fragment from Wa-s was altered by changing the A at 1490 to a C to produce the mutant Wa- s-l490C, which codes for an ADH protein with Fast mobility (;.e., the codon for amino acid residue 192 was changed from AAG, which specifies lysine, to ACG, which specifies threonine). Similarly, the 8.6- kb Adh fragment from Wa-f was altered by changing the C at 1490 to an A to produce the mutant Wa-f- 1490A, which codes for an ADH protein with Slow mobility. A total of 83 single insert transformant stocks were analyzed: 25 Wa-s wild-type, 18 Wa-s- 1490C, 2 1 Wa-f wild-type and 19 Wa-f-l490A. These stocks were analyzed for ADH activity and CRM levels, along with the Wa-s and Wa-f isochromosomal stocks from which the clones were originally derived.

Figure 2 shows a plot of ADH activity us. CRM level for the 83 transformant stocks. Black symbols represent alleles that code for a Fast ADH protein and open symbols represent alleles that code for a Slow ADH protein. A striking feature of this plot is that there is clearly a difference in the slope of the regression of activity on CRM for the two electrophoretic classes. This difference in slope represents the difference in catalytic efficiency between the two forms of the enzyme, which was described earlier on the basis of naturally occurring variants that affect CRM level (see LAURIE and STAM 1988).

The black and open squares in Figure 2 represent alleles that differ by a single nucleotide, the 1490 amino acid replacement, on the Wa-f background. There is clearly a highly significant difference in activity between these two groups, but there is no significant difference in CRM level. Similarly, the black and open circles in Figure 2 represent alleles that differ by a single nucleotide at 1490 on the Wa-s background. Again, there is a marked difference in activity level, but no significant difference in CRM level. Figure 3 provides the same information in the form of average values for the four transformant classes, along with a comparison to the original isochromosomal Wa-s and Wa-f stocks. The latter comparison shows that the two wild-type transformant classes are very similar to the original stocks from which the Wa clones were derived.

The results of this experiment clearly show that the amino acid replacement at 1490 has a marked effect on catalytic efficiency of ADH, but it has no detectable effect on the concentration of ADH protein (CRM level). The catalytic efficiency effect was, of course, expected because only one amino acid distinguishes the Fast and Slow forms of ADH. The informative result is that the threonine/lysine substitution has no detectable affect on protein stability because the two forms of the enzyme have the same steady state level when no other nucleotides in the Adh region differ. At this point in the project, it appeared that the only


Adh

I I Born HI Sac I Hpa I

\ A Distal mRNA / FIGURE ].-The Adh transcriptional unit, which is 1.9 kb in length,

soc I HDa 1 Cla I lies within the 8.6-kb SacllClaI fragment used in the transformation experiments.

2.3 kb I X.6 kb *

I

1443 1490

Slow ACI: AAG Lys Fast A G A S Thr

80

70

60

so

20

IO

0 20 40 60 80 100 120 140 160

CRM

FIGURE 2.-Plot of ADH activity us. CRM for transformant lines lion1 the I490 in vitro mutagenesis experiment. FAch point represents the average of four observations. There is no significant difference in slope for the regression of activity on CRM for the comparison of Wa-f wild-type 0 us. Wa-s-1490C (0) or for the comparison of Wa-s wild-type (0) us. Wa-f-1490A (0). Therefore, the points for each of the two homogenous pairs were pooled to c.;dculate two regression lines, one for Fast proteins (R'= 0.99) and one for Slow proteins (R'= 0.98). The difference in slope between the Fast and Slow groups is highly significant (P < 0.0001). Units o f activity are nanomoles NAD'reduced per minute per milligram total protein multiplied by 0.1 and units of CRM are Hochi-R fly rquivalents per nlg total protein multiplied by 10.

likely candidate remaining to account for the CRM level difference is the 1443 silent substitution, so we proceeded with another site-direct mutagenesis experiment to test the effect of that substitution.

The 1443 silent substitution has no detectable effect on activity or CRM level: In this experiment, the 8.6-kb Adh fragment from Wa-f was altered by changing the G at 1443 to a C to produce Wa-f-l443C and the reciprocal change was made in the Wa-s fragment to produce Wa-s-l443G. These changes produce a silent substitution in a threonine codon (ACC to ACG) at amino acid residue 176. A total of 54 transformant stocks were analyzed: 20 Wa-f wild- type, 9 Wa-f-l443C, 19 Wa-s wild-type and 6 Wa-s- 1443G. These stocks were analyzed for ADH activity and CRM levels, along with the Wa-s and Wa-f isochromosomal stocks.

100

I 0

50

n " . . . . . . . Waf wap F W F M S M S W

T 1

c Waf was FW F M S M S W

FIGURE 3.-Activity and CRM level means for the four classes of transformants from the 1490 mutagenesis experiment (FW, FM, SW, SM) and means for the isochromosomal Wa-s and Wa-f stocks. Error bars represent the least significant difference (LSD) at the 5% level. Because of unequal sample sizes, each LSD applies to one paired comparison as follows: Wa-s us. Wa-f, FW us. FM, SW us. SM. FW refers to the Wa-f wild-type transposon, FM to Wa-f- 1490A. SW to Wa-s Wild-type and SM to Wa-s-1490C. See Figure 2 legend for units. Fast protein (r); Slow protein (0).

Figure 4 shows a plot of ADH activity us. CRM level for the 54 transformant stocks and Figure 5 shows the mean values for the four classes of transformants and for the isochromosomal Wa-s and Wa-f stocks. These data show that there is no detectable effect of the silent substitution on either activity or CRM level.

DISCUSSION

The results of the two in vitro mutagenesis experiments reported here show that neither the 1490 amino acid replacement nor the 1443 silent substitu-

486 M. Choudhary and C. C . Laurie

1443 G C

0 20 40 60 80 lW 120 140 160

CRM

FIGURE 4.-Plot of ADH activity vs. CRM for transformant lines from the 1443 in vitro mutagenesis experiment. Each point represents the average of four observations. There is no significant difference in slope for the regression of activity on CRM for the comparison of Wa-f wild-type (.) vs. Wa-f-1443C (0) or for the comparison of Wa-s wild-type (0) vs. Wa-s-1443G (0). Therefore, rhe points for each of the two homogenous pairs were pooled to c;dculate two regression lines, one for Fast proteins (R2= 0.97) and one for Slow proteins ( R S = 0.90). See Figure 2 legend for units.

T

150 .

T r SM Waf was F W P M

ll sw

1

E: U

20

0

Waf was FW FM SM SW

FIGURE 5,"Activity and CRM level means for the four classes of transformants from the 1443 mutagenesis experiment (FW, FM, SW, SM) and means for the isochromosomal Wa-s and Wa-f stocks. Error bars represent the least significant difference (LSD) at the 5 % level. Because of unequal sample sizes, each LSD applies to one paired comparison as follows: Wa-s vs. Wa-f, FW vs. FM, SW vs. SM. FW refers to the Wa-f wild-type transposon, FM to Wa-f- 1443C, SW to Wa-s Wild-type and SM to Wa-s-1443G. See Figure 2 legend for units. Fast protein (W); Slow protein (0).

tion has a detectable effect on the level of ADH protein (CRM). These two sites were identified as the only likely candidates to account for the CRM level difference between the allozymic classes on the basis of a P element-transformation experiment, which mapped the CRM effect to a 2.3-kb HpaI/CZaI fragment, and on the basis of sequence differences within this fragment between the Fast and Slow classes.

1490 A 1 1 1 Wa-s-1443G Wa-f-1490A - -Low High Wa-s-wildtype - Low c Wa-f-wild type - High Wa-s-14% - Low

wa-f-143C - High

FIGURE 6.-ADH CRM levels for the six classes of transformants are designated either high or low. The nucleotides at sites 1443 and 1490 are given for each class.

There are two possible explanations for this. (1) The sequence comparisons might have been misleading somehow, causing us to incorrectly eliminate a poten- tially important site from consideration. (2) No single site substitution causes the difference in CRM level, but rather it comes about through an interaction among two or more different sites.

The first possibility is discussed at length in the accompanying paper by LAURIE, BRIDGHAM and CHOUDHARY (1 99 l) , in which new sequence and Adh expression data are brought to bear on this problem. One sequence difference within the adult intron, V1 (KREITMAN 1983), was previously eliminated from consideration based on a single line, F1-f, which has the Slow-typical form of V1, but has a significantly higher CRM than F1-2s (which was collected at the same geographic location in Florida). A reexamina- tion of the CRM level of F1-f in relation to Kreitman's other Florida Slow allele (Fl- 1 s) and several other Slow and Fast lines shows that F1-f has an unusually low CRM level for a Fast line. In particular, FI-f has a lower CRM level than FI-1s. In addition, new data show that two other second chromosome lines from the collection described by LAURIE-AHLBERG et al. (1980) have unusual CRM levels for their allozymic type and they also have unusual haplotypes with respect to the 1490 amino acid replacement and the V1 sites. The conclusion of the analysis by LAURIE, BRIDGHAM and CHOUDHARY (1991) is that the V1 sequence difference may account for the CRM level difference between the allozymic classes and we are in the process of testing that hypothesis directly through in vitro mutagenesis.

It is also possible that no single site difference causes the CRM level effect, in which case combinations of substitutions will have to be tested. The 1443 plus 1490 double mutants have not been tested, but it is already clear that knowledge of the haplotype at these two sites is not sufficient to predict the CRM level of an allele. For example, both the Wa-s-1443G and the Wa-f-1490A mutants have the same haplotype at these two sites (1443G, 1490A), but when this haplotypes occurs on the Wa-s background, a low CRM level results, and when it occurs on the Wa-f background, a high CRM level results (Figure 6). The same is true of the 14436, 1490C haplotype. There is a cluster of


six substitutions between 1443 and 1557 (which includes the 1490 amino acid replacement site) that all differ between the consensus Slow and the consensus Fast alleles (and between Wa-s and Waf) (KREITMAN 1983). It is possible that either the 1490 or 1443 substitutions must occur within the correct context of the other substitutions within this region in order for CRM level to be affected. This hypothesis can be tested by making all six substitutions at once.

Based on the observation that the allozymic classes differ in level of ADH protein, but not in level of Adh RNA, we previously concluded that the protein level difference comes about either through a difference in translation rates of the two mRNAs or through a difference in protein stability. The results reported here eliminate protein stability as a possible mecha- nism. The 1490 amino acid replacement is the only difference in primary structure of the two allozymic classes and it has no detectable effect on the level of ADH protein. Therefore, through a process of elimi- nation, it appears likely that the ADH protein level difference is caused by a difference in translational efficiency. LAURIE, BRIDCHAM and CHOUDHARY (1 991) discuss some possible mechanisms by which the intronic variant V1 might affect translational efficiency.

We would like to thank C. H. LANGLEY and C. F. AQUADRO for providing helpful comments about the manuscript. We are also grateful for the expert technical assistance of JAMIE BRIDGHAM, ELLEN FLANAGAN, DAVID KEYS and JUSTINA WILLIAMS. This work was supported by National Science Foundation grant BSR 861- 15632.

LITERATURE CITED

ANDERSON, S. M., and J. F. MCDONALD, 1983 Biochemical and molecular analysis of naturally occurring Adh variants in Dro- sophila melanogaster. Proc. Natl. Acad. Sci. USA 8 0 4798- 4802.

AQUADRO, C. F., S. F. DEESE, M. M. BLAND, C. H. LANGLEY and C. C. LAURIE-AHLBERG, 1986 Molecular population genetics of the alcohol dehydrogenase gene region of Drosophila melanogaster. Genetics 114: 1165-1 190.

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CHAMBERS, G. K., 1988 The Drosophila alcohol dehydrogenase gene-enzyme system. Adv. Genet. 25: 39-107.

C:HAMBERS, G. K., T. S. FLETCHER and F. J. AYALA, I984 Purification and partial characterization of alcohol dehydrogenase, fructose l ,6-bisphosphate aldolase and the cyto- plasmic form of malate dehydrogenase from Drosophila melanogaster. Insect Biochem. 14: 359-368.

CORBIN, V., and T. MANIATIS, 1989 The role of specific enhancer- promoter interactions in the Drosophila Adh promoter switch. Genes Dev. 3: 21 91 -2200.

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Communicating editor: A. G. CLARK

mutagenesis to analyze the molecular basis of the adh ... · the bamhi site of the plasmid pbsm 13-...

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