André Mazabraud

Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli

Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR). In Plasmodium falciparum, antifolate resistance has been associated with point mutations in the gene encoding DHFR. Recently, mutations at homologous positions have been observed in the P. vivax gene. Since P. vivax cannot be propagated in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escherichia coli. Induced expression yielded a protein product that precipitated as an inclusion body in E. coli. The soluble, active DHFR recovered after denaturation and renaturation was purified to homogeneity by affinity chromatography. Kinetic properties of the recombinant P. vivax DHFR showed that the wild-type DHFR (Ser-58 and Ser-117) and double mutant DHFR (Arg-58 and Asn-117) have similar K(m) values for dihydrofolate and NADPH. Antifolate drugs (pyrimethamine, cycloguanil, trimethoprim, and methotrexate), but not proguanil (parent compound of cycloguanil) inhibit DHFR activity, as expected. The kinetics of enzyme inhibition indicated that point mutations (Ser58Arg and Ser117Asn) are associated with lower affinity between the mutant enzyme and pyrimethamine and cycloguanil, which may be the origin of antifolate resistance.

Early aspects of gonadal sex differentiation in Xenopus tropicalis with reference to an antero-posterior gradient.

In an effort to contribute to the development of Xenopus tropicalis as an amphibian model system, we carried out a detailed histological analysis of the process of gonadal sex differentiation and were able to find evidence that gonadal differentiation in X. tropicalis follows an antero-posterior gradient. Although the main reason for the presence of a gradient of sex differentiation is still unknown, this gradient enabled us to define the early events that signal ovarian and testicular differentiation and to identify the undifferentiated gonad structure. Given the various advantages of this emerging model, our work paves the way for experiments that should contribute to our understanding of the dynamics and mechanisms of gonadal sex differentiation in amphibians.

Regulation of XSnail2 Expression by Rho GTPases

We analyzed the effects of Rho GTPases on XSnail2 expression during neural crest (NC) ontogeny in Xenopus laevis embryos. The ectopic expression of both dominant‐negative (N−) and constitutively active (V−) Rho GTPase mutants after RNA or DNA microinjection disrupted the endogenous expression of XSnail2, XFoxD3, and XSnail1. V14RhoA and N17Rac1 were inhibitory, whereas N19RhoA and V12Rac1 increased NC marker gene expression. In reporter assays using a XSnail2 promoter–green fluorescent protein (GFP) construct (α700BA‐GFP), the ectopic expression of V14RhoA, N17Rac1, or the Rac1 inhibitor NSC 23766 decreased reporter expression in NC‐neural plate, whereas N19RhoA or the RhoA inhibitor Y27632 and V12Rac1 enhanced it. Similarly, transgenic embryos expressing Rho GTPase mutants and GFP under control of the α700BA promoter displayed variations similar to those observed for ectopic RNA and DNA expression. These results show that Rho GTPases can regulate the expression of XSnail2 during NC ontogeny. Developmental Dynamics 236:2555–2566, 2007.

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 .

Thesaurin a, the major protein of Xenopus laevis previtellogenic oocytes, present in the 42 S particles, is homologous to elongation factor EF-1α

We have purified in SDS X. laevis thesaurin a (M r 50 000) which is part of the 42 S storage particles. Its N‐terminal amino acid is blocked and several peptides obtained by V8 protease treatment were purified and sequenced. As expected from one of the functional roles of the 42 S particles (tRNA binding, protection against deacylation and exchange with the ribosome), the amino acid sequence of thesaurin a was found to be closely related to that of the elongation factor EF‐1α. We suggest that all three proteins involved in 5 S RNA and tRNA storage in previtellogenic oocytes, TFIIIA, thesaurin a and thesaurin b, have a dual function: storage and a role in transcription or in protein synthesis.

Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis

BackgroundThe western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in X. tropicalis.ResultsA gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers.ConclusionThese cDNA sequences permit in silico cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study X. tropicalis biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.

Deficiency of the peroxy-Y base in oocyte phenylalanine tRNA

The activity of genes coding for 5 S ribosomal RNA has been shown to differ between oocytes and somatic cells of amphibians and fishes. This has been done by comparing in Xerzopus laevis [ 1,2] and in Tinca tinca [3] the primary sequences of somatic 5 S RNA with its major counterpart in oocytes. Somatic 5 S RNA differs by 8 bases from the major oocyte species. Whereas probably all the 5 S genes are transcribed in the oocyte in Xenopus and Tinca, only -10% is active in somatic cells. Since transfer RNA accumulates synchronously with 5 S RNA in the oocytes of these species [4,5], a difference in the activity of somatic and oocyte tRNA genes could also be looked at by comparing the sequences of the corresponding RNA transcripts. Other differences between somatic and oocyte tRNAs could also exist at the level of modified bases, which are formed after transcription. We have compared the chromatographic properties of tRNA from previtellogenic oocytes and somatic cells [6]. Except for tRNAmetl, all tRNA species we tested on RPCs or BD cellulose columns had a chromatographic behaviour characteristic of their somatic or oocyte origin. Such differences also occur between tRNA of normal and tumoral cells [7-121. So far, the only well documented difference is the deficiency of the Y base in tumoral tRNAPhe [13-l 51. This residue, a tricyclic imidazo derivative of guanine to which is attached a 4 carbon side chain [ 171, is a highly fluorescent and hydrophobic base. It has been found in all normal eukaryotic cells, next to the 3’-end of the anticodon [ 161. Some variations occur between species in terms of the sidechain [ 171.

Origin of several abundant proteins of amphibian oocytes

SummaryPrevious studies indicate that the genes controlling cell-specific functions in extant metazoans derive from housekeeping genes of their unicellular ancestors. Traces of such relationships can be found in the gene families controlling signal reception at cell surfaces and light condensation in eye lens. We present other examples of gene remodeling taken in the field of germ cell-specific proteins. In amphibian oocytes several proteins contribute to edification of an efficient translation machinery for the future embryo. Some RNA components of this machinery have to be protected against degradation during growth of the oocytes in the ovary. The protective function is served by a small group of RNA-binding proteins deriving from universal transcription or translation factors. Several of those proteins are bifunctional.

Functional role of nucleoside diphosphate kinase during development and oncogenesis

We have shown the presence of at least two genes specifying the nucleoside diphosphate kinase (NDPK or NM23, metastasis suppressor) in the toad Xenopus laevis, as in mammals. Gel retardation assays showed that enzyme Xl, but not X3, binds to pyrimidine-rich regions of nucleic acids, such as “CT” repeats. It also recognizes the “CCCACCC” motif of the human c-myc promoter, just like the human NDPK B, characterized as the PUF19 transcription activator factor (Postel E.H., et n1.(1996), Proc. Natl. Acad. Sci.,93,6892-6897). This motif is also found in the first exon of c-myc and ki-ras Xenopus genes, as well as in the 3’ noncoding sequence of Xenopus NDPK mRNA, suggesting a self post-transcriptional regulation of the different isozymes. During embryonic development, NDPK is heterogeneously distributed, being abundant in rapidly dividing cells and in the nuclei of migrating embryonic cells. In toto hybridization with anti-mRNA revealed isozyme mRNA specific patterns during somite formation. Immunodetection of NDPK in Xenopus 693N immortalised cells and T30-derived invasive cells detected a single isoform migrating as the NDPK Xl hexamer in T30. These results indicate that there may be functional differences between the two NDPK. We have also tested the capacity of Xenopus NDPK to rescue mutant Drosophila aw& larvae lacking NDPK. Xenopus NDPK Xl-X3 rescues these larvae, but the fertility of the resulting females was not restored. Presently, we are investigating the functions of the CCCACCC motif in stabilisation, polyadenylation or translation.of NDPK Xenopus mRNA and in the expression of c-myc and ki-ras Xenopus genes, both in vitro and in vivo. Our preliminary results suggest that NDPK Xl is able to bind to its own mRNA and stabilise it in a S 100 Xenopus oocyte extract.

The nucleotide sequence of phenylalanine tRNA of Xenopus laevis.

The nucleotide sequence of Xenopus laevis phenylalanine tRNA extracted from oocytes was determined to be: pGCCGAAAUAm2GCUCm1AG DDGGGAGAGCm22 G psi psi AGACmUGmAAYA psi C UAAAGm7GDCm5CCUGGT psi CGm1AUCCCGG GUUUCGGCACCAoH. This result was achieved by analysing, with classical procedures [6], the oligonucleotides obtained after digestion by T1 or pancreatic ribonuclease. This sequence is identical to the mammalian sequence. It has been entirely conserved during 10(8) years, the time lapse between the divergence of amphibians and mammals in evolution. In contrast to 5S RNA, no important heterogeneity has been found in the oocyte sequence, suggesting that there is only a single sequence for tRNAphe in X. laevis. Small differences are seen in the elution pattern from RPC-5 columns for immature oocyte and somatic tRNAphe. They are probably due to a submodification of methyl-5-cytidine residues, which appear to be about half methylated in tRNAphe as well as in total tRNA from immature oocytes.