Response of Drosophila metallothionein promoters to metallic, heat shock and oxidative stresses.

The metallothionein system in Drosophila melanogaster is composed of two genes, Mtn and Mto. In order to compare the induction properties of these genes, we transformed D. melanogaster with P-element vectors containing Adh and lacZ reporter genes under the control of Mtn and Mto promoters, respectively. Mtn and Mto transgenes are mainly expressed in digestive tract. However, Mtn expression has been detected also in the fat body. Mtn and Mto transgenes respond differently to metallic, heat-shock and oxidative stresses. These data confirm that both genes are in part functionally different.

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.

The organization of Drosophila genes.

This study was designed to examine the range of size variations in the major functional elements of Drosophila genes and to test whether those size variations occur independently of each other. In a sample of 111 genes the following median values occur: leaders, 123 base pairs (bp); coding regions, 1242 bp; 3' untranslated regions (3'UTR), 246 bp; mRNAs, 1803 bp; 3' terminal exons 843 bp; and exons upstream of the last one 233 bp. Introns show a bimodal distribution with medians of 62 and 595 bp. Unexpected size correlations are evident for several of these elements. The size of the leader, for example, is correlated with the sizes of the coding region and the 3'UTR with very high levels of significance, and the size of the first intron is similarly correlated with the sizes of each of the individual components of the mature mRNA.

Recent evolutionary history of the metallothionein gene Mtn in Drosophila.

A new allele of one of the metallothionein genes of D. melanogaster, Mtn.3, sheds light on the recent evolution of this gene. In comparison to the previously studied Mtn1 allele found in Canton S, this new allele, Mtn.3, produces a transcript that is 49 bases longer and 65-70% less abundant. We detected Mtn.3 in several laboratory strains as well as in isofemale lines derived from natural populations. Sequence comparison showed that Mtn.3 differs from Mtn1 in that it has: (a) base-pair substitution and an extra 49 bp-segment in the 3' untranslated region, (b) a substitution in the coding region that replaces the terminal Glu40 in Mtn1 with Lys40, and (c) two base-pair substitutions in the promoter region. The Mtn.3-type was detected in six species of the melanogaster group by restriction analysis, and this result was confirmed by sequencing the D. simulans Mtn gene. Thus Mtn.3, which produces a less abundant transcript, appears to be the oldest of the two alleles. We also found that the duplications previously isolated from natural populations all derived from Mtn1, the more recent allele. Thus, two evolutionary steps: Mtn.3 to Mtn1 and Mtn1 to Dp(Mtn1), are accompanied by an overall 5- to 6-fold increase of RNA accumulation. The two changes seem to have occurred in non-African populations since Mtn.3 but not Mtn1 was detected in our sample from tropical Africa, while Mtn1 and Dp (Mtn1) are prevalent in European and North American samples.

Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells.

The Drosophila metallothionein promoter (Mtn) was used to obtain efficient, regulated expression of foreign gene products inserted in high copy numbers into Drosophila melanogaster Schneider 2 cells. An expression unit comprised of a reporter gene [Escherichia coli galactokinase (galK)] fused to the Mtn promoter was stably introduced into Schneider 2 cells in up to several hundred copies per cell in a single transfection-selection event. This system contrasts dramatically with other eukaryotic systems that permit only a few copies of a gene to be stably inserted in a single transfection-selection event. The transfected Drosophila S2 cell lines expressed high levels of both galK mRNA and protein in response to metal induction. Most important, and in contrast to mammalian cells, expression remained fully regulated even at high copy number, with low basal expression maintained in the absence of inducer. This regulated system was used to obtain efficient expression in Drosophila cells of an otherwise lethal or growth-inhibitory gene product, the human H-ras oncogene. The ability to obtain regulated high-level expression of potentially lethal foreign proteins is unique to the Drosophila cell system.

Metallothionein gene duplications and metal tolerance in natural populations of Drosophila melanogaster.

A search for duplications of the Drosophila melanogaster metallothionein gene (Mtn) yielded numerous examples of this type of chromosomal rearrangement. These duplications are distributed widely--we found them in samples from four continents, and they are functional--larvae carrying Mtn duplications produce more Mtn RNA and tolerate increased cadmium and copper concentrations. Six different duplication types were characterized by restriction-enzyme analyses using probes from the Mtn region. The restriction maps show that in four cases the sequences, ranging in size between 2.2 and 6.0 kb, are arranged as direct, tandem repeats; in two other cases, this basic pattern is modified by the insertion of a putative transposable element into one of the repeated units. Duplications of the D. melanogaster metallothionein gene such as those that we found in natural populations may represent early stages in the evolution of a gene family.

A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn.

Cloned fragments of DNA including the Drosophila melanogaster metallothionein gene Mtn and different amounts of 5' flanking sequences were introduced into flies by P-element-mediated germ line transformation. Comparison of RNA levels in different transformants revealed that metal-regulated and tissue-specific expression of Mtn requires no more than 373 base pairs upstream of the initiation site of transcription. Transformants having an additional, transcribed copy of Mtn could tolerate increased concentrations of cadmium, indicating that Mtn expression is directly related to this phenotype. In separate experiments, these D. melanogaster promoter sequences were fused to the coding sequences of the herpes simplex virus thymidine kinase (TK) gene. After transfection of this fusion into baby hamster kidney cells, increases in TK activity and accumulation of TK RNA were inducible by metals. A series of 5' and 3' deletions showed that D. melanogaster sequences from -130 to -6 were sufficient to confer metal-regulated expression to the TK gene. The function of the D. melanogaster metallothionein promoter in mammalian cells indicates that the mechanism controlling metal regulation is evolutionarily conserved.

The ecdysoneless (ecd1ts) mutation disrupts ecdysteroid synthesis autonomously in the ring gland of Drosophila melanogaster.

Ring glands dissected from homozygous l(3)ecd1ts wandering larvae and upshifted in vitro to the restrictive temperature, 29 degrees C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29 degrees C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29 degrees C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29 degrees C autonomously and may also interfere with PTTH release.

The metallothionein gene of Drosophila.

A chromosomal DNA segment containing the metallothionein gene was isolated from a genomic library of Drosophila melanogaster. A segment of 1543 bp was sequenced and found to include the structural sequence interrupted by one small intron. Within 300 bases upstream of the apparent site of transcription initiation, there are several short intervals very similar to the 12-base-pair segments considered to be responsible for metal regulation in mammalian systems. Several lines of evidence indicate that there is a single copy of the metallothionein gene (Mtn) in Drosophila. Mtn is located in the right arm of the third chromosome in region 85E10-15.

Structure and expression of a tandem duplication of the Drosophila metallothionein gene.

A strain of cadmium-resistant Drosophila containing a chromosomal duplication of the metallothionein gene was isolated. This duplication is stably inherited in the absence of selective pressure, and larvae homozygous for it can produce approximately twice as much metallothionein RNA as wild-type larvae. The entire duplication was cloned within a 5.7-kilobase fragment; this fragment contained a direct, tandem repeat of 2.2 kilobases of DNA: 228 bases of 5' flanking DNA, the entire transcription unit, and 1.4 kilobases of 3' flanking sequences. The 3' region of the first repeated unit is joined to the 5' region of the second unit by a 6-base-pair segment we define as the novel joint. This joint forms part of a 10-base-pair inverted repeat of a segment within the 3' region of the first unit. Comparison of the sequences of the 5' and 3' boundaries revealed no extensive regions of similarity at a position corresponding to the novel joint, thus suggesting that a mechanism other than homologous recombination was involved in the origin of this duplication.

Molecular and cytogenetic characterization of a metallothionein gene of Drosophila.

A chromosomal DNA segment containing the metallothionein gene was isolated from a genomic library of Drosophila melanogaster using a previously characterized cDNA of this species as a probe. A segment of 1543 base pair (bp) was sequenced and found to include the cDNA sequence interrupted by one small intron. Several lines of evidence indicate that there is a single copy of the metallothionein gene (Mtn) in Drosophila; any other related genes, if they occur, must be sufficiently different that they are not detectable by our probe, even under hybridization conditions of reduced stringency. According to in situ hybridization and deletion mapping, Mtn is located in the right arm of the third chromosome in region 85E10-15. Within 300 bases upstream of the apparent site of transcription initiation, there are several short intervals very similar to the 12-bp segments considered to be responsible for metal regulation in mammalian systems.

Effects of heavy metals on Drosophila larvae and a metallothionein cDNA.

Drosophila melanogaster larvae reared on food containing radioactive cadmium retained over 80% of it, mostly in the intestinal epithelium. The majority of this radioactivity was associated with a soluble protein of less than 10,000 molecular weight. Synthesis of this cadmium-binding protein was induced by the metal as demonstrated by incorporation of radioactive cysteine. Most copper ingested by larvae was also found to associate with a low molecular weight, inducible protein, but some of it was found in an insoluble fraction. Zinc was unable to, or very inefficient at, binding or inducing the synthesis of a similar protein. A D. melanogaster cDNA clone was isolated based on its more intense hybridization to copies of RNA sequences from copper-fed larvae than from control larvae. This clone showed strong hybridization to mouse metallothionein-I cDNA at reduced stringency. Its nucleotide sequence includes an open-reading segment which codes for a 40-amino acid protein; this protein was identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The ten cysteine residues present occur in five pairs of near-vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels. Mercury, silver, and zinc induced metallothionein to a lesser extent. Whether (any of) the protein(s) discussed above correspond(s) to that coded by this RNA sequence has not yet been determined.

Nucleotide sequence and expression of a Drosophila metallothionein.

A Drosophila melanogaster cDNA clone was isolated based on its more intense hybridization to RNA sequences from copper-fed larvae than from control larval RNA. This clone showed strong hybridization to mouse metallothionein I cDNA at reduced stringency. Its nucleotide sequence includes an open reading segment which codes for a 40-amino acid protein; this protein is identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The 10 cysteine residues present occur in five pairs of near vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels. Mercury, silver, and zinc induced metallothionein to a lesser extent. The level of metallothionein RNA increased very soon after the initiation of metal treatment and reached a maximum after approximately 36 h.

Genetic control of Adh expression in Drosophila melanogaster.

Natural variants displaying different levels of expression of the gene for alcohol dehydrogenase (Adh) were subjected to genetic mapping experiments. The strains studied carry one of the two common electrophoretic forms of the enzyme. The difference among Adh-fast strains appears to be due to multiple loci with trans-acting effects. Differences among Adh-slow strains are due to modifiers or quantitative sites located very close to the structural gene (less than 0.05 map unit) or part of it. The modifiers detected in the Adhs strains seem to operate only on the structural allele in the cis-position.--A modifier that affects the ratio of ADH levels in larvae and adults was also detected in the Adhs strains. This modifier is also closely linked to Adh and is cis-acting.

Genetic variation in the expression of ADH in Drosophila melanogaster.

Several chromosomes derived from natural populations have been identified that affect the expression of alcohol dehydrogenase (ADH). Second chromosomes, which also carry the structural gene Adh, show a great deal of polymorphism of genetic elements that determine how much enzyme protein accumulates. The level of enzyme was measured in third instar larvae, 6-to-8-day-old males and in larval fat bodies and alimentary canals. In general, activities in the different organs and stages are highly correlated with one another. One line was found, however, in which the ADH level in the fat body is more than twice the level one would expect on the basis of the activity in alimentary canal. We have also found evidence of third-chromosome elements that affect the level of ADH.

Quantitative genetic variation of enzyme activities in natural populations of Drosophila melanogaster.

The genetic component of variation of enzyme activity in natural populations of Drosophila melanogaster was investigated by using two sets of chromosome substitution lines. The constitution of a line of each type is: i(1)/i(1);+(2)/ +(2);i(3)/i(3) and i(1)/i(1);i(2)/ i(2);+(3)/+(3), where i refers to a chromosome from a highly inbred line and + refers to a chromosome from a natural population. The + but not the i chromosomes vary within a set of lines. By use of a randomized block design to test and estimate components of variance, 50 of the second- and 50 of the third- chromosome substitution lines have been screened for variation in the activity levels of seven enzymes. Six of the seven enzymes show a significant genetic component in at least one set of lines, and five of the seven enzymes show activity variations attributable to factors that are not linked to the structural gene. These unlinked activity modifiers identify possible regulatory elements. Analyses of covariance show that most of the genetic variation of enzyme activities cannot be accounted for by genetic variation of live weight or protein content. These results and the lack of strong correlations between the genetic effects on the activities of different enzymes indicate that the effects are mainly specific for individual enzymes.

X-chromosome transcription in Drosophila.

Duplications involving the X chromosome of Drosophila were used to produce individuals with 1.25, 1.50, 1.62 and 1.85 X chromosomes. Rates of transcription in polytene chromosomes were measured autoradiographically after pulse-labeling salivary glands with 3H-uridine. We conclude that: (1) the sume of all transcription occurring on the X elements is constant (relative to autosomal transcription) regardless of how much X-chromosome material is present; (2) this constancy is apparently achieved through a uniform reduction of the rate of synthesis over all X-chromosomal segments as the size of the duplication increases.--Thus, the transcription of a given segment of the X is dependent not just on the number of copies of that segment but also on the number of copies of other regions of the chromosome.

Genetic control of alcohol dehydrogenase levels in Drosophila.

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55--65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.