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A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells.

Author(s) : Mollereau B, Wernet M, Beaufils P, Killian D, Pichaud F, Kuhnlein R, Desplan C,
Journal : Mech Dev
The Drosophila ommatidia contain two classes of photoreceptor cells (PR's), the outer and the inner PR's. We performed an enhancer trap screen in order to target genes specifically expressed in PR's. Using the UAS/GAL4 method with enhanced green fluorescent protein (eGFP) as a vital marker, we screened 180000 flies. Out of 2730 lines exhibiting new eGFP patterns, we focused on 16 lines expressing eGFP in particular subsets of PR's. In particular, we describe three lines inserted near the spalt major, m-spondin and furrowed genes, whose respective expression patterns resemble those genes. These genes had not been reported to be expressed in the adult eye. These examples clearly show the ability of our screen to target genes expressed in the adult Drosophila eye.

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection.

Author(s) : Elliott D, Bourgeois C, Klink A, Stevenin J, Cooke H,
Journal : Proc Natl Acad Sci U S A
RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBMprotein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor formore ubiquitously expressed pre-mRNA splicing activators.

A mutated PML/RARA found in the retinoid maturation resistant NB4 subclone, NB4-R2, blocks RARA and wild-type PML/RARA transcriptional activities.

Author(s) : Duprez E, Benoit G, Flexor M, Lillehaug J, Lanotte M,
Journal : Leukemia
The fusion protein PML/RARA, associated with acute promyelocytic leukemia behaves as an abnormal retinoic acid (RA) receptor with altered transactivation properties but is still inducible by RA. The chimeric protein is thought to promote leukemogenesis but also paradoxically to mediate the sensitivity to ATRAof APL cells. This has been supported by works reporting that in vitro ATRA resistance is characterized by defects in the RARA/E-domain of PML/RARA. In the present report, we identified a new mutation in the E domain of PML/RARA which is associated with a RA-resistant subline of NB4 cells; NB4-R2. This mutation, identical to the Gln411 mutation found in HL60-R, changes the amino acid Gln903 to an in-phase stop codon, generating a truncated form of PML/RARA which has lost 52 amino acids at its C-terminal end. We have studied the effect of the truncated PML/RARA protein on PML NB formation and RARA and PML/RARA transcriptional activity. We show here that the fusion mutant exerts a dominant negative effect on wild-type PML, PML/RARA and RARA transcription activity. These findings highlight the important role of the RARA E-domain of PML/RARA in mediating RA sensitivity in APL cells.

Expression and cellular localization of the Toucan protein during Drosophila oogenesis.

Author(s) : Grammont M, Berson G, Dastugue B, Couderc J,
Journal : Mech Dev
The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Tocprotein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.

Genomic organization of the JEM-1 (BLZF1) gene on human chromosome 1q24: molecular cloning and analysis of its promoter region.

Author(s) : Tong J, Fant X, Benoit G, Chen S, Chen Z, Lanotte M,
Journal : Genomics
The Jem-1 (JEM-1, HGMW-approved symbol BLZF1) gene mapping to human chromosome 1q24 codes for a ubiquitously expressed 3-kb mRNA, translated in a 45-kDa nuclear protein. Recent studies have shown a deficient expression of this gene in acute promyelocytic leukemia (APL). However, treatment with retinoids was able to upregulate JEM-1 mRNA in maturing NB4 leukemia cells. Here, we report the characterization of the structural organization of JEM-1. By hybridization screening of a human genomic library derived from blood mononuclear cells, five overlapping genomic DNA clones were isolated. These clones extend over 34 kb of the human genome and comprise the complete JEM-1 gene and a 4-kb 5'flanking region. Determination of the exon-intron structure of Jem-1 revealed seven exonswhose junctions with introns exhibited typical splice sequences. A shorter transcript (Jem-1s, 1.3 kb) generated by exon 3 extension and polyadenylation was identified. Its translation generated a 23-kDa protein that exhibited a cytoplasmic localization. 5'RACE-PCR identified a major transcription start site(TSS) located at 403 nt upstream of the ATG. Computer analysis of the 1. 8-kb 5'flanking region showed that it lacks a TATA box, Inr motifs or DPE motifs, butit contains a typical CCAAT box located 95 bp upstream of the TSS. Sequencing also revealed potential cis-acting elements for multiple transcription regulators including Sp1, GATA, C/EBP, AP-1, and Pu1. No retinoic acid receptor elements orretinoic X receptor elements were detected. This 1.8-kb DNA sequence showed a strong constitutive promoter activity determined by a luciferase-reporter gene assay in transiently transfected HeLa cells. Retinoids further increased luciferase expression 2.7-fold. We demonstrated that the 1-kb distal sequence contains yet unidentified elements reducing constitutive transcription. Thus, the maximal constitutive promoter activity was assigned to a -432 + 101 region overlapping the TSS. These data support the idea of a constitutive expression ofJEM-1, but a negative regulation in APL released by retinoids.

Long distance charge transport through DNA: quantification and extension of the hopping model.

Author(s) : Giese B, Spichty M,
Journal : Chemphyschem
Long distance charge transport through DNA occurs by a hopping mechanism. If thepositive charge is injected into a guanine base, all guanines act as charge carriers. Because of the strong influence that the distance has on the charge-transfer step, DNA strands with long adenine:thymine sequences also involve adenine as charge carriers. A prerequsite for this mechanism is that theelectron transfer to an adjacent adenine base is faster than the H2 O trapping reaction of the guanine radical cation. We have developed a model that can explain and qualitatively predict the product yields.

Mechanistic studies in the radical induced DNA strand cleavage—Formation and reactivity of the radical cation intermediate

Author(s) : Glatthar R, Spichty M, Gugger A, Batra R, Damm W, Mohr M, Zipse H, Giese B,
Journal : Tetrahedron
In order to understand the heterolytic cleavage of 4′-DNA radical 1 and the regioselective attack of nucleophiles at the intermediate DNA radical cation 3, the chemistry of model radical 8 was studied. It turned out that the heterolytic cleavage in water is favored over homolysis because of the effective solvation of the ions 9 and 10. The regioselectivity of the nucleophilic attack at radical cation 10 can be explained with the valence bond configuration mixing (VBCM) model.

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge.

Author(s) : Liu X, Grammont M, Irvine K,
Journal : Dev Biol
The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong alleleof scalloped fail to proliferate within the wing pouch, but grow normally in thewing hinge and notum. Cells overexpressing scalloped fail to proliferate in bothnotal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibitionand a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the developmentof surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.

The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site.

Author(s) : Del Gatto-Konczak F, Bourgeois C, Le Guiner C, Kister L, Gesnel M, Stevenin J, Breathnach R,
Journal : Mol Cell Biol
Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site. We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1.Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site. Our results identify TIA-1 as a novel splicing regulator,which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.

Theoretical investigations on the stereoselective selenenylation reaction of alkenes

Author(s) : Spichty M, Fragale G, Wirth T,
Journal : Journal of the American Chemical Society
Ab initio calculations have been performed to study the stereoselective selenenylation reaction of alkenes with chiral selenium electrophiles. The interaction of the heteroatom of the chiral side chain with the selenium atom in the reagent has been investigated by a detailed conformational analysis. Calculations of the seleniranium intermediates with different substituted alkenes have been performed as well. The reversal of selectivity changing from aromatic to aliphatic alkenes as substrates in the selenenylation reaction has already been proven by experiment and is now supported by the calculations described herein.