The olig family: phylogenetic analysis and early gene expression in Xenopus tropicalis.

The olig genes form a small subfamily of basic helix-loop-helix transcription factors. They were discovered in 2000 as genes required for oligodendrocyte lineage specification. Since then, olig genes have been identified in various vertebrate species and corresponding sequences accumulated within genomic databases. Until now, three groups of olig genes have been characterized. Our phylogenetic analysis demonstrates the existence of a fourth group, which we named olig4. Genes of the four olig groups are present in actinopterygians and amphibians, whereas mammals only possess olig1, 2, and 3. We also found one olig gene in hemichordates, urochordates, and cephalochordates. Our expression study during Xenopus tropicalis embryogenesis shows that the four olig genes have very distinct expression patterns. Olig1 is very faintly expressed before the tadpole stage, whereas olig2, 3, and 4 are expressed from the gastrula stage onward. The olig3 expression during neurulation suggests a role in early anteroposterior patterning of the brain. All these results indicate that olig genes are involved in several developmental processes during early development.

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.

Advantages of a P-element construct containing MtnA sequences for the identification of patterning and cell determination genes in Drosophila melanogaster.

The P[MTW] transposon carries a functional MtnA (metallothionein) gene and a miniwhite reporter gene. When P[MTW] was transformed into Drosophila, many lines were found to show position-dependent expression patterns of the miniwhite or the MtnA transgene. Identification of several of the target genes indicated that this construct behaves as an enhancer or silencer trap. For instance, expression of at least one reporter transgene was shown to correlate with that of the endogenous gene in the case of insertions in Ultrabithorax, four-jointed, and the iroquois complex. The frequency of patterns recovered with P[MTW] is higher than that reported for P[LacW], suggesting that P[MTW] has unusual properties. The possibility of biased insertion of P[MTW] was assayed by screening a sample of 66 MTW lines for modifiers of the extra sex comb phenotype caused by a hypomorphic allele of polyhomeotic. Seven modifiers were recovered, which could be ranked in two classes: genes involved in leg morphogenesis (including four-jointed and spitz), and genes of the Polycomb- or trithorax-Group, including trithorax and batman, a new gene which encodes a product with a BTB/POZ domain. Taken together, these results indicate that P[MTW] allows the tagging of patterning and cell determination genes, and thus provides a useful tool for identifying new developmental functions.

Characterization of two promoters of the Xenopus laevis elrD gene.

The Xenopus laevis elrD gene belongs to the multigenic elav/Hu family. elrD is exclusively expressed in neural cells, where it could be involved in the posttranscriptional control of mRNAs. Here we report the isolation and characterization of the genomic elrD 5'-flanking region. We localized the transcription initiation sites and thus identified two distinct transcripts, elrD1 and elrD2 by 5' RACE PCR. The two transcripts derive from the use of alternative promoters located 915 bp apart. We show that sequences upstream of the elrD1 and elrD2 transcription units can direct expression of the reporter luciferase gene in Xenopus embryos. We also observed length variation of the ELRD first RNA recognition motif (RRM1).

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.

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.

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.

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.

Distribution and evolution of introns in Drosophila amylase genes.

While the two amylase genes of Drosophila melanogaster are intronless, the three genes of D. pseudoobscura harbor a short intron. This raises the question of the common structure of the Amy 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 in D. 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 the obscura group, and to a lesser extent in the ananassae 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 the Amy gene, suggesting that the intron was ancestral.

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.

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.

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.

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 metallothionein genes during the post-embryonic development of Drosophila melanogaster.

Expression of the two Drosophila melanogaster metallothionein genes, Mtn and Mto, has been analyzed by in situ hybridization during post-embryonic development. Mtn and Mto transcripts were detected exclusively in the digestive tract of larvae, pupae and adults reared on standard medium. Mtn and Mto expression domains overlap, but each gene is also expressed at unique sites. Mtn mRNA levels are approximately 10 and 20 times higher than those of Mto in larvae and adults, respectively. Copper and cadmium ions strongly induce Mtn and Mto mRNA accumulation in the midgut. Zinc is a weaker inducer, acting only at high concentrations. Mtn gene expression is induced by these three metals in Malpighian tubules, while Mto gene expression in this organ is induced only by zinc. Iron is a poor inducer of metallothionein mRNA accumulation. Functions of MTN and MTO proteins in metal homeostasis and detoxification are considered.

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.

Developmental variability of metallothionein Mtn gene expression in the species of the Drosophila melanogaster subgroup.

Developmental expression of the Drosophila melanogaster metallothionein Mtn gene has been analysed. Transcripts of this gene accumulate during the vitellogenic phase of oogenesis in a ring of follicular cells at the oocyte-nurse cell margin and in the follicular cells surrounding the oocyte. There is also strong expression of the Mtn gene during the second half of embryogenesis in hemocytes, the endoderm midgut, and Malpighian tubules. A banded expression pattern is observed transiently in the midgut at stage 13. The two Mtn alleles, Mtn and Mtn, show quantitative differences in their expression patterns. Copper intoxication of flies does not induce ectopic expression of the Mtn gene, but rather leads to over-expression of the gene in the structures where it is normally transcribed. Mtn transcription is not altered in homozygous mutants of four genes (lab, wg, dpp, bap) known to be involved in midgut morphogenesis. Expression of Mtn has been also studied in six other species of the melanogaster subgroup. This analysis demonstrates that regulation of Mtn gene transcription has changed during evolution of the Drosophila lineage. For example, Mtn is expressed specifically in the Malpighian tubules of D. melanogaster, while in D. mauritiana and D. sechellia the amnioserosa is a specific location of expression. Nonetheless, expression of Mtn in the midgut is common to the seven species, suggesting a basic role for the MTN protein during embryogenesis in this organ, possibly in the release of metallic ions from vitellogenins. In contrast, two genes also expressed in the embryonic midgut, lab and dFRA, display identical patterns in all species of the melanogaster subgroup. The diversity of Mtn patterns in closely related Drosophila species exemplifies the rapid evolution of a gene regulatory system.

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.

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.

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.

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.