Gustavo Maroni

Uptake and binding of cadmium, copper and zinc by Drosophila melanogaster larvae

Abstract Drosophila melanogaster larvae reared on food containing radioactive cadmium retained over 80% of ingested radioactivity; more than 95% was located in the intestinal epithelium. The majority of this radioactivity associated with a macromolecule (probably a protein) of less than 10,000 molecular weight. Synthesis of this cadmium-binding protein (CBP) was induced by the metal as demonstrated by incorporation of radioactive cysteine and the synthesis proceeded at such a rate that within a few hours there was enough CBP to bind 90–95% of the ingested cadmium. Most of the copper ingested by larvae was also found to associate with a low molecular-weight, inducible protein, but a significant fraction of this metal was found in an insoluble fraction. Zinc was unable to bind or induce the synthesis of a similar protein or did so very inefficiently. The cadmium- and copper-binding protein(s) are probably metallothioneins.

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.

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 the 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 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.

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 sum 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. -/3 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.

X-Linked gene activity in metamales (XY; 3A) of Drosophila

In order to determine the level of X-linked gene activity in metamales (XY; 3A) of Drosophila melanogaster, RNA synthesis was monitored as a function of 3H-uridine incorporation in nuclei of isolated larval salivary glands by standard autoradiographic means. These data show that the rate of RNA synthesis by the single X chromosome in metamales is approximately 90% of the rate achieved by the three X chromosomes of triploid females (3X; 3A). As an independent measure of chromosome activity, two X-linked enzymes and two autosomal enzymes were monitored in crude extracts by spectrophotometric assay procedures. The results reveal that X-linked enzyme activity levels in metamales constitute 80% of the levels exhibited by control triploid females or intersexes (XXY; 3A). We conclude that metamales exhibit substantial compensation beyond that which occurs in regular, diploid males. This finding establishes a new level of transcriptional activity possible for the X chromosome of Drosophila melanogaster.

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. ImagesFIGURE 2.FIGURE 6.FIGURE 9.

RNA synthesis in Drosophila melanogaster polytene chromosomes indications of simultaneous dosage compensation and dosage effect in X chromosomes

AbstractIt is shown that the apparent incompleteness of dosage compensation when RNA synthesis is measured autoradiographically is not due to the existence of contiguous dosage compensated and non-dosage compensated genes. Rather this seems to be the result of peculiarities in the coordination of RNA synthesis between the X chromosomes and autosomes. The slope of the line defined by

Metal-regulated synthesis of a bacterial galactokinase in Drosophila larvae

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