Jean-Antoine Lepesant

Structural features critical to the activity of an ecdysone receptor binding site

Two ecdysone-response elements from the hsp27 (hsp27 EcRE) and the Fbp1 (D EcRE) genes of Drosophila melanogaster were used as probes in a gel shift assay to investigate the interactions of the ecdysone receptor (EcR) with its cognate DNA response element. The source of EcR was a nuclear extract from the late third-larval instar fat body. The hsp27 and D EcREs share a sequence similarity at 12 positions over a 15bp region including an imperfect palindromic structure consisting of two pentamer half-sites separated by a single intervening nucleotide. We have shown that a short oligonucleotide containing this 11bp imperfect palindrome of the hsp27 EcRE and three flanking bp on each side is an efficient EcR binding site. Mutational analysis confirms that the integrity of both these half-sites as well as their 1bp spacing are critical for binding of the ecdysone receptor. The D EcRE behaved as a much weaker EcR binding site than the hsp27 EcRE but a single bp substitution was sufficient to confer upon it a binding capacity equivalent to that of the hsp27 EcRE. These results have led us to propose the sequence PuG(G/T)T(C/G)A(N)TG(C/A)(C/A)(C/t)Py as a revised version of a previously proposed EcRE consensus sequence.

Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

Characterization of two sucrase activites in Bacillus subtilis marburg

Summary Two proteins responsible for sucrase activity in Bacillus subtilis Marburg have been separated by chromatography on hydroxyapatite. These enzymes have been characterized as transfructosidases, namely a levansucrase and a sucrase. Their molecular weights are very similar (40,000). These enzymes differ by their catalytic properties, their sensitivity to trypsin and SH reagents and by their immunological properties. They are both induced by sucrose ; only sucrase synthesis is decreased in the presence of glucose. Sucrase is endocellular while levansucrase is excreted in the culture medium.

Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis : constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168

A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg. This enzyme can not be detected in strain 168. However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.

TRANSCRIPTIONAL AND TRANSLATIONAL CIS-REGULATORY SEQUENCES OF THE SPERMATOCYTE-SPECIFIC DROSOPHILA JANUSB GENE ARE LOCATED IN THE 3' EXONIC REGION OF THE OVERLAPPING JANUSA GENE

SummaryThe janus locus of Drosophila melanogaster displays a very unusual organization. It comprises two partially overlapping genes, janA and janB, which are transcribed in the same orientation; the start of transcription of janB, the downstream gene, is located in the 3′ exonic region of janA. Both genes are expressed during spermatogenesis. Transcription of janB is restricted to this developmental process, whereas janA is ubiquitously transcribed in both the somatic and germinal tissues of males and females. In order to delimit the cis-acting sequences regulating the transcription of janB, the expression of four chimeric janB-lacZ genes was examined in transgenic lines by Northern blot analysis, in situ hybridization and in situ histochemical staining for β-galactosidase activity. Results showed that the testis-specific expression of the janB gene is mediated by a short DNA sequence (positions −174 to + 107) which is located entirely within the last exon of the upstream janA gene. The tissue specificity of the expression of the janB gene is maintained when most of the janA coding and upstream sequences are deleted. Yet the presence in cis of an active janA gene leads to reduced accumulation of the janB-lacZ hybrid mRNA. This supports the hypothesis that janA transcription interferes with the function of the janB cis-regulatory elements. Our results also demonstrate that the 5′ untranslated leader of the janB mRNA contains translational cis-acting elements, which completely block the translation of the janB-lacZ transcripts during the premeiotic stages of sperm development. A janB-lacZ construct was used to examine the sexual phenotype of the germline cells of masculinized XX transformer-2 (tra-2) flies. This has enabled us to confirm at the molecular level previous observations that the germline cells of these flies can enter the spermatogenic pathway of differentiation.

Dual Requirement for the EcR/USP Nuclear Receptor and the dGATAb Factor in an Ecdysone Response in Drosophila melanogaster

ABSTRACT The EcR/USP nuclear receptor controls Drosophilametamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit.

Identification of the structural gene of levansucrase in Bacillus subtilis Marburg

SummaryTwo mutations affecting either the temperature sensitivity or the catalytic properties of the levansucrase of Bacillus subtilis Marburg were characterized. The relevant altered levansucrases were purified and their properties compared with those of the unmodified levansucrase. The results of this analysis and the genetic mapping of these mutations indicates that the structural gene of levansucrase is located in the sacB locus on the Bacillus subtilis chromosome between the cysB3 and hisA1 markers.

Drosophila fat body protein P6 and alcohol dehydrogenase are derived from a common ancestral protein

SummaryDrosophila melanogaster alcohol dehydrogenase is an example of convergent evolution: it is not related to the ADHs of other organisms, but to short-chain dehydrogenases, which until now have been found only in bacteria and in mammalian steroid hormone metabolism. We present evidence that theDrosophila ADH is phylogenetically more closely related to P6, another highly expressed protein from the fat body ofDrosophila, than it is to the short-chain dehydrogenases. The polypeptide sequence of P6 was inferred from DNA sequence analysis. Both ADH and P6 polypeptides have retained a high structural similarity with respect to the Chou-Fasman prediction of secondary structure and hydropathy. P6 is also homologous to the 25-kd protein from the fat body ofSarcophaga peregrina, whose sequence we have reexamined. The evolution of the P6-ADH family of proteins is characterized by a dramatic increase in the methionine content of P6. Methionine accounts for 20% of P6 amino acids. This is in contrast with the absence of this amino acid in mature ADH. There is evidence that P6 and the 25-kd protein have undergone a parallel and independent enrichment in methionine. When corrected for this, the rate of amino acid replacement shows that the P6-25-kd lineage diverged from insect ADH shortly before the divergence of the ADH gene (Adh) from its 3′-duplication (Adhdup).

Isolation and characterization of novel mutations of the Broad-Complex, a key regulatory gene of ecdysone induction in Drosophila melanogaster

Seven new alleles of the Broad-Complex gene of Drosophila melanogaster, which encodes a family of four zinc finger protein isoforms BR-C Z1, Z2, Z3 and Z4, were generated by transposase-induced mobilization of a P[Zw] element inserted in either the first intron downstream from the P165 promoter or the exon encoding the Z2-specific zinc finger domain. They were characterized by genetic complementation tests, molecular mapping and cytogenetic analysis of their effect on ecdysone-induced puffing and BR-C proteins binding to polytene chromosomes. Four mutations that correspond to three overlapping deletions and one tandem insertion of the P[Zw] element are located in the intron. They provide evidence that regulatory elements essential for a correct expression of the BR-C Z2 and BR-C Z3 transcripts are located within the intron downstream from the P165 promoter. Three mutations correspond to internal deletions of the locus and exhibit a complete loss of all BR-C(+) genetic functions in the complementation and cytogenetic tests. They thus provide well characterized new amorphic reference alleles of the BR-C gene. The precise cytogenetic location of more than 300 binding sites of BR-C proteins on larval salivary gland polytene chromosomes was determined by immunostaining using specific antibodies. Sites were found in big ecdysone inducible puffs, constitutively active small puffs as well as interbands. A complete list of the major sites on all four salivary gland polytene chromosomes of BR-C(+) larvae is presented.

Spatial distribution of the Sm antigen in Drosophila early embryos

Summary— Anti‐Sm antibodies recognize the major small nuclear RNA‐protein particles (snRNPs) involved in pre‐mRNA processing. The spatial distribution of the snRNPs has been investigated in Drosophila embryos up to the cellularization stage (cycle 14), using the Y12 anti‐Sm antibody. Our results show that: 1) all or most of the Sm antigen is localized in the cytoplasm of the syncitial blastoderm until the 12th cycle of division, in both the nuclear and cytoplasmic compartments at cycle 13, and then in the nuclei at cycle 14 and later. This relocalization takes place when zygotic transcriptional activation occurs; 2) at the subcellular level, the Sm antigen localizes in a speckled pattern and in foci‐like structures within the nucleus of Drosophila blastoderm embryos; 3) strikingly, some nuclei of embryos at the 14th cycle appear to contain more snRNPs than others. The position of these nuclei differs from one embryo to another, and their distribution does not resemble any known developmental pattern of Drosophila embryogenesis. We propose that random differences in snRNP concentration may serve as an epigenetic signal for stochastic events occurring during development.