O. Cabré

Use of the drosophila wing spot test in the genotoxicity testing of different herbicides.

Four herbicides, namely propanil, maleic hydrazide, glyphosate, and 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The herbicides were administered by chronic feeding to 3‐day‐old larvae. Two different crosses, a standard (ST) and a high‐bioactivation (HB) cross, involving the flare‐3 (flr3) and the multiple wing hairs (mwh) markers, were used. The HB cross uses flies characterized by an increased cytochrome P‐450–dependent bioactivation capacity, which permits a more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, which are inversion‐free marker heterozygotes and balancer heterozygotes, were analyzed. Maleic hydrazide and glyphosate proved to be more genotoxic in the ST cross, whereas propanil appeared to be slightly more genotoxic in the HB cross. On the other hand, the herbicide 2,4,5‐T increased the mutation frequency for only the small single spots in the ST cross. Environ. Mol. Mutagen. 36:40–46, 2000.

Molecular cloning of the Drosophila Fanconi anaemia gene FANCD2 cDNA.

Fanconi anaemia (FA) is a rare disease characterized by chromosome instability and cancer susceptibility. With the exception of FANCD2, none of the Fanconi anaemia genes are conserved in evolution, limiting the study of the Fanconi anaemia pathway in genetically tractable models. Here we report the cloning and sequencing of a Drosophila full length cDNA homologous to human FANCD2 (dmFANCD2) as a first step in using Drosophila in Fanconi anaemia research. dmFANCD2 is composed of 14 exons coding for a protein of 1478 aminoacids. Southern blot and in situ hybridization analysis indicated that dmFANCD2 is present at single copy in the Drosophila genome and maps at the chromosomal band 92-F3. Sequence and structural biocomputational analysis indicated that, although the aminoacidic sequence, and specially the N-terminus region, is not highly conserved between humans and flies (23% identity and 43% similarity), both proteins are of the same size, globular and compact, with several transmembrane helixes and related to nuclear membrane proteins. Interestingly, the human ATM phosphorylation site at S222 and the complex-dependent monoubiquitination site at K561 are highly conserved in Drosophila at positions S267 and K595, respectively. The same is true for other putative ATM sites and their aminoacidic environment and for two out of three aminoacid mutations associated with human pathology. These results suggest that the key FANCD2 features have been conserved during over 500 million years of divergent evolution, highlighting their biological importance.

Analysis of genomic damage in the mutagen-sensitive mus-201 mutant of Drosophila melanogaster by arbitrarily primed PCR (AP-PCR) fingerprinting

DNA repair mechanisms are important to maintain the stability of the genome. In Drosophila melanogaster, the mus-201 gene is required in the excision repair process. To study the contribution of the mus-201 gene in the stability of the Drosophila genome, we have used the arbitrarily primed PCR fingerprinting method (AP-PCR). We have analysed the changes in the genomic DNA fingerprints from the progeny of wild-type males crossed with mus-201 repair-deficient or repair-proficient females. After induction of DNA damage with 2-acetylaminofluorene (2-AAF) in the wild-type parental males, quantitative and qualitative differences in the AP-PCR fingerprints were detected between the two crosses, and the estimate of the genomic damage detected by AP-PCR has clearly shown that the mus-201 repair deficiency is associated with an increase of genomic damage. The predominant type of alterations detected by AP-PCR under the mus-201 repair-deficient conditions agree with the results obtained in microsatellite PCR analysis, suggesting that the role of the mus-201 gene, necessary in excision repair, is not associated to the mismatch repair process. The work reported here demonstrates that the AP-PCR is a suitable technique to analyse genetic alterations in D. melanogaster and, consequently, can be used to compare the susceptibility to genomic damage of different DNA repair mutants.

Effect of cycloheximide on different stages of Drosophila melanogaster.

Cycloheximide, an antibiotic inhibiting protein synthesis, exerted a toxic effect on different developmental stages egg, larva and adult of Drosophila melanogaster. At the egg stage the early embryos were most sensitive. With larvae, a strong decrease in viability was found, with no sex difference. In adults, there was a dose-effect relationship, mortality increasing with concentration. At 10 and 15 mM, males were more sensitive than females. There were consistent differences between the control and cycloheximide-fed females in respect of the average number of eggs deposited and offspring produced.

An FB-NOF mediated duplication of the white gene is responsible for the zeste1 phenotype in some Drosophila melanogaster unstable strains

The males of the Drosophila melanogaster M115 mutant strain and of its spontaneous revertant strain RM115 are phenotypically similar to those carrying the w+UZ and w+UR alleles. The molecular description of these mutant strains could be extended to the unstable-zeste system, which has been used as a genotoxicity test, and could be of use for a better understanding of the assay. An FB element in the 3′ vicinity of the white gene, actually into the second intron of the newly predicted CG32795 gene, was found and precisely located in our M115 and RM115 strains, and also in w+UZ as expected. We demonstrate the presence of NOF sequences in the M115 and w+UZ insertions. However, we found that the z1 phenotype in these males might not be due to the FB-NOF interference on the zeste–white interaction but to a duplication of the white gene unnoticed in previous studies. The reversion of RM115 correlates with the loss of the duplication, probably by a complex recombination event. Furthermore, a FISH experiment suggests that the two copies of white are nearby or tandemly duplicated.

Overcoming false negatives due to the genomic context in polymerase chain reaction amplification.

In some instances defined genomic regions are so poorly amplified that they seem to be unamplifiable. A protocol was developed which allows good PCR amplifications by the use of restriction digestion combined with the elution of a pool of restriction fragments of defined size range from agarose gel after electrophoresis. We describe the application of the method in the PCR amplification of a region of the white locus of Drosophila melanogaster that otherwise may be considered as a negative result.

FB-NOF is a non-autonomous transposable element, expressed in Drosophila melanogaster and present only in the melanogaster group

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this. In this work we test the hypothesis that FB-NOF is an active transposon nowadays. We search for its expression product, obtaining its cDNA, and propose the ORF and the sequence of its potential protein. We found that the NOF protein is not a transposase as it lacks any of the motifs of known transposases and also shows structural homology with hydrolases, therefore FB-NOF cannot belong to the superfamily MuDR/foldback, as up to now it has been classified, and can be considered as a non-autonomous transposable element. The alignment with the published genomes of 12 Drosophila species shows that NOF presence is restricted only to the 6 Drosophila species belonging to the melanogaster group.

Restriction mapping of phage λ vectors using non-radioactive methods

Abstract In order to take advantage of non-radioactive methods, we have developed two plasmids (pλLE and pλRE) for mapping restriction sites of long inserts cloned in phage λ vectors. These plasmids are constructed by cloning the left 402-bp and right 560-bp phage λ genome ends, respectively. To map restriction sites, the cloned sequences in pλLE and pλRE are labeled with digoxygenin and hybridized to partially digested λ DNA. The ladder of bands detected with these probes can be used to construct restriction maps in the same way as those obtained using radioactively labeled cos complementary oligodeoxyribonucleotides [Rackwitz et al., Gene 30 (1984) 195–200].