Maurice Wegnez

Detoxification of cadmium Ultrastructural study and electron-probe microanalysis of the midgut in a cadmium-resistant strain ofDrosophila melanogaster

SummaryThe midgut of a cadmium-resistant strain ofDrosophila melanogaster has been studied at the ultrastructural level and by electronprobe microanalysis (EPMA). Chronic exposure to cadmium leads to a concentration of the metal in a lysosomal system developed in both anterior and posterior segments of the midgut, where it coexists with copper and sulfur. This mechanism apparently ensures a permanent cadmium detoxification and prevents cellular injury. Wild-type flies fed on a cadmium-contaminated medium manifest the same detoxification process. As a result of contamination, copper is stored along the entire length of the midgut, including a part of the middle-midgut previously named ‘copper-accuumulating region’. Our data demonstrate that the midgut, particularly the posterior segment, is an accumulative organ for both cadmium and copper. The involvement of the metallothionein system in the detoxification process is discussed.

Metallothionein Mto gene of Drosophila melanogaster: structure and regulation.

We report the sequence of the Mto gene, one of the two known metallothionein genes of Drosophila melanogaster, and compare its structure with that of the other metallothionein gene, Mtn. The main structural features are the presence of a small intron (61 base-pairs), the presence of four potential MREs (metal regulatory elements) and the absence of a TATA box in the promoter region. Of all metals tested, Hg2+, Cd2+ and Cu2+ are the most efficient ions for inducing an increase in Mto gene transcription. The Mto and Mtn genes are differentially regulated during normal development. Transcription of Mto is detected early in embryogenesis (0 to 3 h) and persists to the third larval instar, while Mtn expression starts later in embryogenesis (12 to 15 h) and is thereafter maintained throughout larval development and adult stages. Sequencing of the Mto protein is in good agreement with the nucleic acid data. Surprisingly, attempts to isolate and characterize the Mtn protein were unsuccessful. Several lines of evidence suggest that this metallothionein is rapidly incorporated after its synthesis into lysosomes, where it would be processed in a way that would not permit its purification. The function of the Mtn protein thus appears to be mainly related to detoxification processes. The pattern of expression of Mto suggests that this gene may be involved in the control of metal homeostasis during development.

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.

Biochemical research on oogenesis: Oocytes and liver cells of the teleost fish Tinca tinca contain different kinds of 5S RNA

Abstract Previtellogenic oocytes of Tinca tinca accumulate very large amounts of 5S RNA. We show here that 5S RNA stored in oocytes differs from liver 5S RNA in 3 out of 120 nucleotides. Liver and oocyte 5S RNAs, therefore, are produced by different genes. Both kinds of 5S genes are active in oocytes. However, only 5S RNA of the oocyte type accumulates in these cells. In Tinca tinca as in Xenopus laevis , oocyte-type and somatic-type 5S RNAs differ by three properties, ie., primary structure, conformation, and metabolic stability. Nucleotide substitutions occur in different positions in oocyte and somatic 5S RNAs of Tinca tinca and Xenopus laevis . We do not understand how different sets of nucleotide substitutions confer to 5S RNAs of both species similar properties in vivo , namely, increased metabolic stability.

Biochemical research on oogenesis. RNA accumulation in the oocytes of teleosts

Abstract RNA accumulation was studied in the oocytes of nine species of teleosts, belonging to nine different families. Immature ovaries of all animals examined contained high amounts of transfer RNA and 5 S RNA. In four species transfer RNA and 5 S RNA made up more than 90% of the RNA content of the ovaries. In three of these species the 28 + 18 S RNA content was so low that it could not be measured. In previtellogenic oocytes of the teleosts, as in those of Xenopus laevis , transfer RNA and 5 S RNA are stored in nucleoprotein particles sedimenting at 42 S. Aminoacyl-tRNA synthetase activity is associated with these particles. The molar ratio of transfer RNA to 5 S RNA is higher in the 42 S particles of the teleosts than in those of Xenopus laevis . Somatic 5 S RNA from the fresh-water fish Tinca vulgaris was eluted from columns of methylated albumin-Kieselguhr ahead of oocyte 5 S RNA. A similar difference was previously reported in Xenopus laevis and shown to be due to nucleotide substitutions in the two types of 5 S RNAs. It was concluded that most of the 5 S genes that are active in the oocytes of Xenopus laevis are repressed in somatic cells. This conclusion might also be valid for the fish Tinca vulgaris .

Distribution and Evolution of Introns in Drosophila Amylase Genes

While the two amylase genes ofDrosophila melanogaster are intronless, the three genes ofD. pseudoobscura harbor a short intron. This raises the question of the common structure of theAmy gene in Drosophila species. We have investigated the presence or absence of an intron in the amylase genes of 150 species of Drosophilids. Using polymerase chain reaction (PCR), we have amplified a region that surrounds the intron site reported inD. pseudoobscura and a few other species. The results revealed that most species contain an intron, with a variable size ranging from 50 to 750 bp, although the very majoritary size was around 60–80 bp. Several species belonging to different lineages were found to lack an intron. This loss of intervening sequence was likely due to evolutionarily independent and rather frequent events. Some other species had both types of genes: In theobscura group, and to a lesser extent in theananassae subgroup, intronless copies had much diverged from intron-containing genes. Base composition of short introns was found to be variable and correlated with that of the surrounding exons, whereas long introns were all A-T rich. We have extended our study to non-Drosophilid insects. In species from other orders of Holometaboles, Lepidoptera and Hymenoptera, an intron was found at an identical position in theAmy gene, suggesting that the intron was ancestral.

XSEB4R, a novel RNA-binding protein involved in retinal cell differentiation downstream of bHLH proneural genes.

RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression but so far they have not been studied extensively in the context of developmental processes. We report on the molecular cloning and spatio-temporal expression of a novel RNA-binding protein, XSEB4R, which is strongly expressed in the nervous system. This study is focused on the analysis of Xseb4R in the context of primary neurogenesis and retinogenesis. To study Xseb4R function during eye development, we set up a new protocol allowing in vivo lipofection of antisense morpholino oligonucleotides into the retina. The resulting XSEB4R knockdown causes an impairment of neuronal differentiation, with an increase in the number of glial cells. By contrast, our gain-of-function analysis demonstrates that Xseb4R strongly promotes neural differentiation. We also showed a similar function during primary neurogenesis. Consistent with this proneural effect, we found that in the open neural plate Xseb4R expression is upregulated by the proneural gene XNgnr1, as well as by the differentiation gene XNeuroD, but is inhibited by the Notch/Delta pathway. Altogether, our results suggest for the first time a proneural effect for a RNA-binding protein involved in the genetic network of retinogenesis.

Isolation and embryonic expression of Xel-1, a nervous system-specific Xenopus gene related to the elav gene family.

We have identified a new member of the elav gene family in Xenopus laevis. This gene, Xel-1, like the other elav-related genes, encodes a putative RNA-binding protein that contains three RNA Recognition Motifs and is solely expressed in the nervous system. Xel-1 is most likely the Xenopus homologue of Hel-N1, one of the three known human genes related to elav. Xel-1 is not expressed in early neural precursors but rather in differentiating neurons of the central nervous system, as well as in the cranial and the spinal ganglion cells. Xel-1 thus appears to be an early differentiation marker for both the central and the peripheral nervous system of Xenopus laevis.

Structural evolution of the Drosophila 5S ribosomal genes.

We compare the 5S gene structure from nine Drosophila species. New sequence data (5S genes of D. melanogaster, D. mauritiana, D. sechellia, D. yakuba, D. erecta, D. orena, and D. takahashii) and already-published data (5S genes of D. melanogaster, D. simulans, and D. teissieri) are used in these comparisons. We show that four regions within the Drosophila 5S genes display distinct rates of evolution: the coding region (120 bp), the 5′-flanking region (54–55 bp), the 3′-flanking region (21–22 bp), and the internal spacer (149–206 bp). Intra- and interspecific heterogeneity is due mainly to insertions and deletions of 6–17-bp oligomers. These small rearrangements could be generated by fork slippages during replication and could produce rapid sequence divergence in a limited number of steps.

Expression of the Drosophila melanogaster metallothionein genes in yeast

The metallothionein system in Drosophila melanogaster is composed of two genes, Mto and Mn, that code for distinctly different proteins. In order to compare the properties of Mto and Mtn, we transformed yeast with several fusion plasmids. The Mto and Mtn cDNAs, when placed under the control of CUP1 or PGK promoters, can confer a copper‐resistance phenotype to copper‐hypersensitive cells. Both Mto and Mtn proteins can be characterized in extracts from transformed yeast cells.