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.

Deletions and amplifications of tandemly arranged ribosomal 5S genes internal to a P element occur at a high rate in a dysgenic context.

We observed unusual kinds of rearrangements within tandemly clustered 5S genes internal to a P element in dysgenic context. Rearranged P transposons, initially containing eight 5S genes, were found to display discrete numbers of 5S genes, from 4 up to 17 units. Precise deletions and amplifications occurred at a high rate (40%), at both original and new insertion sites. These events can be explained by a "cut and paste" transposition model. Possible links between rearrangements due to dysgenic-like processes and concerted evolution are discussed.

Rearrangements involving repeated sequences within a P element preferentially occur between units close to the transposon extremities.

In a previous report we described rearrangements occurring at a high rate (30% of the progeny of dysgenic flies) within a cluster of 5S genes internal to a P element. These events were characterized as precise amplifications and deletions of 5S units. Here we analyze recombination events within P elements containing two repeated arrays of 5S genes flanking a central white gene. Deletions (50%) and duplications (3%) of the white gene together with various amounts of flanking 5S genes were observed. These recombinations occur preferentially between the most external 5S units of P transposons. Such rearrangements could be favored by interactions between the proteins bound to the P terminal sequences.

Bipartite structure of the 5S ribosomal gene family in a Drosophila melanogaster strain, and its evolutionary implications.

Knowledge of multigenic family organization should provide insight into their mode of evolution. Accordingly, we characterized the 5S ribosomal gene family in the Drosophila melanogaster strain ry506. The 5S genes in this strain display a striking HindIII restriction difference compared to the "standard" D. melanogaster 5S genes. The sequence of three ry506 5S genes was determined. We show that the HindIII restriction site heterogeneity within the ry506 5S family most probably results from the same point mutation, suggesting that a single 5S variant was propagated into the 5S cluster of this strain. Furthermore, we demonstrate that the structural organization of the 5S genes in ry506 is a bipartite structure, i.e., that about 40% of the 5S genes constitute a HindIII+/HindIII- mixed cluster, while those remaining constitute an homogeneous HindIII- cluster. The events which might lead to such an heterogeneous pattern are discussed from an evolutionary point of view.

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.

Xenopus elav-like genes are differentially expressed during neurogenesis.

In Xenopus, three neural-specific elav-like genes (ELGs) have been identified, elrB/Xel-1, elrC and elrD. With the aim to highlight possible differences in the regulation of these genes, we compared their expression patterns during development. We had previously shown that elrB is expressed from the early tailbud stage onwards, in both the central and peripheral nervous system. Here we show that both elrC and elrD are expressed earlier than elrB in the developing neural tube and the cranial ganglia, with different temporal specificities. Double in situ hybridizations on brain cross-sections allowed us to define precisely the expression domains of elrB, elrC and elrD in the brain at the tailbud stage. What emerges from this study is a differential distribution of ELGs transcripts in the hindbrain. Also, double labeling with a motor neuron marker shows that in stage 41 tailbud embryos, elrD remains strongly expressed in motor neurons whereas elrC is mostly expressed in non-motor neuron cells.

Misexpression of the RNA-binding protein ELRB in Xenopus presumptive neurectoderm induces proliferation arrest and programmed cell death.

Proteins of the ELAV/Hu family share the presence of three RNA binding domains. In Xenopus, three nervous system-specific elav/Hu related genes, elrB, elrC and elrD, have been identified so far. The temporally regulated expression patterns of elrB, elrC and elrD suggest their involvement at different steps of neural differentiation. In the present study we misexpressed elrB by RNA injection in early Xenopus embryos and analyzed morphologically and molecularly its effects on neural development. We showed that heterochronous expression of elrB in presumptive neurectoderm down-regulates the expression of neural markers, such as N-tubulin, as well as that of other Xenopus elav-like genes, elrC and elrD, whereas ectopic expression of elrB in presumptive mesoderm has no effect on MyoD. Misexpression of elrB also induces severe defects in neural tube development, associated with massive cell loss resulting from early cell cycle arrest and programmed cell death. Our results are discussed in the context of early neural differentiation.

Expression of the Xenopus laevis metallothionein gene during ontogeny.

Expression of the Xenopus laevis metallothionein (MT) gene was studied by in situ hybridization throughout development. MT mRNA was detected from the tailbud stage onwards. MT expression was observed in bucco-pharyngeal epithelium, pronephros and liver anlagen, aswell as in lens and periventricular areas of the encephalon. MT transcripts, in both larvae and adults, were detected in diverse regions of the central nervous system and in differentiating tissues implicated in detoxification processes: liver hepatocytes, small intestine epithelia and kidney tubules. These data are discussed in the context of MT functions and support a physiological role for MT in growth processes.

Thyroid hormone regulation of germ cell-specific EF-1 alpha expression during metamorphosis of Xenopus laevis.

In situ hybridization was used to follow the distribution of the mRNAs encoding the somatic form of elongation factor 1 alpha (EF-1 alpha S) and the germinal counterparts of this factor, thesaurin a and EF-1 alpha O, throughout metamorphosis in the gonads of Xenopus laevis tadpoles. EF-1 alpha S mRNA is detected before metamorphosis in both the somatic and germ cells of the gonads. In contrast, thesaurin a and EF-1 alpha O mRNAs are first detected in spermatogonia and oogonia at stages 60-62, corresponding to the climax of metamorphosis and to the peak of circulating thyroid hormone. To determine whether thyroid hormone, the instigator of metamorphosis, is involved in regulating the expression of the germinal gene EF-1 alpha O, Xenopus XTC cells were transfected with an EF-1 alpha O promoter sequence inserted in front of the luciferase reporter gene. Addition of T3 to the cell culture medium induced a dose-dependent increase in transcription from the EF-1 alpha O promoter. This effect was enhanced when the construct was cotransfected with an expression vector for a Xenopus thyroid hormone receptor. Our data show that germ cells switch from a somatic to a germ-cell specific mode of expression during metamorphosis. Furthermore, this switch appears to be induced by thyroid hormone.

Expression of the Drosophila melanogaster metallothionein genes in yeast.

The metallothionein system in Drosophila melanogaster is composed of two genes, Mto and Mtn, 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.

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.

Relationship between posttranslational modification of transaldolase and catalase deficiency in UV-sensitive repair-deficient xeroderma pigmentosum fibroblasts and SV40-transformed human cells.

Xeroderma Pigmentosum (XP) is a rare recessively inherited human disease associated with a hypersensitivity to ultraviolet radiation. The ultraviolet component of sunlight can initiate and promote the formation of cutaneous tumors as seen in the skin cancer-prone XP patients. Previously, we have found that the low activity of the NADPH-dependent antioxydant enzyme, catalase, which we have observed in XP diploid fibroblasts and SV40-tranformed cells, could be restored by the addition of NADPH. Here we have analyzed transaldolase, which regulates NADPH levels produced by the pentose phosphate pathway in order to examine how it influences the catalase activity regulated in XP and SV40-transformed cells. We find that transaldolase activity is high in XP and SV40-transformed human fibroblasts, whereas transaldolase transcription is unchanged, suggesting that modification of transaldolase activity is due to a posttranslational modification of the protein. Two-dimensional electrophoresis analysis has allowed us to identify a complex set of transaldolase isoforms and to postulate that the phosphorylation of specific isoforms could be correlated with the different enzymatic activities seen. Our results show that high transaldolase activity corresponds to a low catalase activity in SV40-transformed cells and in fibroblasts from XP patients who have a high predisposition to develop skin cancer.

Subcellular distribution of Xenopus XEL-1 protein, a member of the neuron-specific ELAV/Hu family, revealed by epitope tagging.

Drosophila and vertebrate elav/Hu genes are involved in the development and the maintenance of the nervous system. They all encode proteins that contain three RNA recognition motifs (RRM) and are thus expected to play a role in RNA metabolism. Drosophila ELAV and RBP9 proteins were reported to be exclusively distributed in nuclei of neurons, whereas known human Hu proteins display a bipartite nuclear and cytoplasmic distribution. We have previously isolated a member of this family in Xenopus, Xel-1, that is exclusively expressed in neural tissues from the early tailbud stage onward. In the present study, we report on the subcellular distribution of XEL-1 protein using myc epitope tagging, a strategy allowing the study of a single member of the ELAV/Hu family. We show that the subcellular distribution of exogenous XEL-1 protein in neural tissues depends on developmental stages. In the neural tube at the neurula stage, where endogenous Xel-1 is not expressed, exogenous tagged XEL-1 protein is localized in both the nucleus and the cytoplasm. At the tailbud stage, where endogenous Xel-1 is expressed, exogenous tagged XEL-1 protein is localized essentially in the cytoplasm of neural tube cells. In contrast, exogenous Drosophila ELAV protein localizes to the nucleus at all stages in Xenopus embryos. The variability in the subcellular localization of ELAV/Hu proteins in different species may have functional implications.

Molecular cloning and expression of ferritin mRNA in heavy metal-poisoned Xenopus laevis cells.

In a search for genes transcriptionally regulated by metal ions, we have isolated a Xenopus laevis ferritin cDNA clone, XL2-17, from cadmium-poisoned XL2 cells. The large size of the corresponding ferritin mRNA (1.4 kb) is due to the presence of a 629-nucleotide 5'-untranslated region. The Xenopus ferritin sequence is highly isologous with other vertebrate ferritins. In particular, there is a complete sequence identity for the iron-responsive element (IRE) located in the 5'-untranslated region in both XL2-17 and Rana catesbeiana ferritin mRNAs. The position of this IRE is unusual since it is located 489 nucleotides from the 5' end of the ferritin mRNA. Our analysis of phylogenetic relationships among ferritins indicates that all amphibian ferritins thus far sequenced would be more closely related to the mammalian H-type ferritin than to the L-type. The level of ferritin mRNA in XL2 cells rises 10- to 15-fold following exposure of cells to cadmium or copper. This increase is due to both transcriptional and translational regulation. A 10-fold increase was also found at the protein level. These results suggest that ferritin may be a primary detoxification response to heavy metals in Xenopus cells.

Molecular cloning and expression of a metallothionein mRNA in Xenopus laevis.

Metallothioneins (MT) are expressed during early development in species of several groups. To understand MT function in developmental processes, we studied the MT system of Xenopus laevis, a model vertebrate species in experimental embryology. We first purified and sequenced the liver MT from copper-treated animals. This 62-amino-acid protein shares the main structural properties of known vertebrate MT, and is more closely related to avian than to fish or mammalian MT. Using this sequence, we designed oligonucleotide primers to amplify and isolate a MT clone from a XL2 cell line cDNA library. This 752-bp cDNA encodes a putative 62-amino-acid-long protein that is 100% identical with the sequenced MT. Zinc, cadmium, and copper ions are very efficient inducers of MT mRNA accumulation in Xenopus liver and cell lines.