Genetic control of cadmium tolerance in Drosophila melanogaster.

Files from a transgenic line of Drosophila melanogaster with two copies of the metallothionein allele Mtn3 were more tolerant to cadmium than strains with only one copy of the gene. However, flies with the Mtn3 allele were as tolerant as flies with the Mtn3 allele, despite the level of expression of Mtn3 being three times higher than of Mtn3. We propose that the substitution of Lys-40 (in Mtn3) for Glu-40 (in Mtn1) accounts for a reduction in binding affinity of Mtn1, which offsets the increased expression levels.

fers from Mtn3 in three respects: 1) it has two base substitutions in the promoter region, 2) it lacks a 49-base pair segment in the 3' untranslated region, and 3) it has a base substitution in the C-terminal codon. Studies in sibling species suggest that Mtn3 is the ancestral allele. RNA measurements indicate that Mtn] strains accumulate mRNA at a level approximately three times greater than do Mtn.3 strains, probably the consequence of one of the first two differences listed above (2).
Metallothionein seems to be a very monomorphic protein in most species. To our knowledge, Drosophila presents the only case in which genetic polymorphism of metallothionein has been studied; we have observed polymorphism both in the coding sequence and the presence of duplications (2,4). The occurrence of Lys-40 in Mtn3 in place of the Glu-40 found in Mtn' may have a profound effect. This C-terminal residue is next to one of the Cys-X-Cys groups responsible for metal binding. Coordination of Cd2+ ions by the thiolate groups of four cysteine residues creates an excess negative charge that is thought to be neutralized by basic amino acids in the vicinity (5). Thus, the presence of Glu in that position may affect the binding capacity of the metallothionein produced by Mtn'. Despite numerous attempts, it has not been possible to purify Drosophila metallothionein to test this hypothesis, so we resorted to the indirect approach of measuring tolerance to metal toxicity in flies of various genotypes.
Increased transcriptional activity in any of several duplications of the Mtn1 allele is always accompanied by increased tolerance to cadmium and copper ions (4). The lower level of expression of Mtn 3, therefore, should lead to reduced metal tolerance, unless its potentially more efficient protein counters this effect. This argument assumes that the two alleles have comparable translational efficiency. To discover whether altered levels of Mtn 3 product lead to correspondingly modified tolerance to toxic metals, we generated a transgenic line of Drosophila melanogaster with two copies of the Mtn3 allele. As in the case of Mtn' natural duplications, flies with this synthetic duplication were more tolerant to cadmium than strains with only one copy of the gene. Comparison of the cadmium tolerance of Mtn' and Mtn3 strains, however, showed that the two strains are quite similar; this suggests that the increased transcript level in Mtn' exactly compensates for a reduction in either its metal-binding efficiency or its stability, caused by the amino acid substitution.

Materials and Methods
Flies of a Canton S strain with allele Mtn] were crossed to a Samarkand strain (S500) with allele Mtn 3, and females of the progeny were back-crossed to Samarkand males. Females of this second generation were then individually mated to Samarkand males. After allowing these females to lay eggs for a few days, DNA was extracted from them and tested for presence of the Mtn1 allele by Southern blotting. Vials with progeny from females that were heterozygous were kept, and vials in which the female proved to be homozygous for Mtn3 were discarded. This procedure was repeated for 11 generations, at which time Mtn' homozygous stocks were established.
Thus, stocks were created in which the majority of the genetic background was the same as that in the Samarkand Mtn 3 stock but which were homozygous for the Mtnl allele. Two of these lines, SCSI and SCS2, were used for tests of metal tolerance.
Stocks with a duplication for the Mtn3 allele were produced by P-element-mediated transformation with a vector carrying a copy of the Mtn3 allele including 370 base pairs (bp) of the promoter region. We have shown that this segment of the promoter is sufficient for normal expression of the Mtn gene (6). Transformed flies were crossed to a strain carrying Mtn3, and the third-chromosome marker, "red" (C301). The strain derived from this cross, C3 13, was homozygous for the Mtn3 allele at two different sites. Level of expression was determined by northern analysis as previously done (4); we determined that the amount of RNA loaded in all lanes was comparable by visual inspection of ethidium-bromide-stained rRNA after electrophoresis.
Males, eclosed over a 24-hr period, were sorted in groups of 20 and kept on normal food for a 6-hr period. At this point they were transferred to treatment vials containing Instant Drosophila Medium (Carolina Biologicals, Burlington, North Carolina) supplemented with 0, 0.1, 0.5, or 1.0 mM CdCI2. Surviving flies were counted every 24 hr and transferred to fresh vials with the corresponding medium every 3 days. We determined the half-life of different genotypes in the various treatments from survival curves. All genotypes and concentrations tested were tested simultaneously, with 4 vials of 20 flies for each genotype and each treatment. This unit experiment was repeated three times for each comparison.

Results and Discussion
Using P-element-mediated transformation, we produced flies carrying an extra copy of the Mtn 3 allele. Estimates of metallothionein RNA, by northern analysis, confirmed that these transformed flies (C313) Articles * Cadmium tolerance in Drosophila Is Z P.
with a single copy, as is shown in Figure 2 and Table 1. Tolerance here is measured as the half-life of adult males in various media; Figure 2A shows a set of survival curves of the type used to obtain the halflife values reported in Figure 2B. These results indicate that the allele Mtm3 is capable of participating in metal detoxification, and we propose that the failure to find duplications for this allele in natural populations is probably due to its infrequent occurrence in Europe. It is in Europe where duplications of Mtn' seem to have originated, in response to the agricultural use of copper salts for antimicrobial purposes (2).
In addition to studying flies with one   and two copies of Mtn3, we also determined the tolerance of a new allele, MtnK45 (CK45), which has the same sequence and RNA level as Mtn3 (data not shown) but which has an insertion of 14 bp (GTTA-CAATCGTTAC) in the 3' untranslated region (1 1 1 bp downstream of the termination codon). With respect to cadmium tolerance, MtnK45 is indistinguishable from Mt3. Table 2 and Figure 3 show that there is no significant difference between Mtn' (SCS1 and SCS2) and Mtn 3 (S500) flies with respect to their tolerance to various concentrations of cadmium. At the lowest cadmium concentration (0.1 mM), Mtnl appears to have a slight advantage, but analysis of variance indicates that overall the two alleles are not significantly different. Table 2 also shows that both Mtn' and Mtn3 are more sensitive to cadmium than a duplication for Mtn' (statistically significant difference), as was expected based on our previous work (4). In Table 2, the halflife of flies was standardized as a percent of their half-life in control medium. Despite a threefold difference in expression level of Mtnl and Mtn3, the half-life in cadmium of flies carrying these alleles is not significantly different. Since the only known difference between the two alleles, other than in the level of expression, is in the substitution of Lys-40 (in Mtn3) with Glu-40 (in Mtn), this result supports the hypothesis that the protein of Mtn' has reduced binding affinity or stability, which is compensated by its increased expression levels. It should be noted that metallothionein is probably quickly removed from the cytosol, either by aggregation or degra- Borates and boric acid, which through gross medical misuse years ago gained a reputation for acute poisonings and fatalities, have in recent years received growing attention from two separate, major groups of investigators. One group has been pursuing evidence that boron is an essential element to humans, with a regulatory role in calcium metabolism and energy substrate use. The other group has been studying the reproductive and developmental toxicity of boron and the borates and has bound boric acid at high doses to be a model cormpound for the study of mechanisms of reproductive and developmental toxicity.