Biomedical subjects

Christian Dumas

Publications and source records attributed to Christian Dumas.

32 records · Page 2Linked to original sources

PubMed2003

Female control of male gamete delivery during fertilization in Arabidopsis thaliana.

Fertilization in both animals and plants relies on the correct targeting of the male gametes to the female gametes. In flowering plants, the pollen tube carries two male gametes through the maternal reproductive tissues to the embryo sac, which contains two female gametes. The pollen tube then releases its two male gametes into a specialized receptor cell of the embryo sac, the synergid cell. The mechanisms controlling this critical step of gamete delivery are unknown. Here, data based on the new sirène (srn) mutant of Arabidopsis thaliana provide the first evidence for female control over male gamete delivery. Live imaging of fertilization shows that wild-type pollen tubes do not stop their growth and do not deliver their contents in srn embryo sacs.

Arabidopsis↗

PubMed2003

Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing.

A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.

Animals↗

PubMed2003

ZmEBE genes show a novel, continuous expression pattern in the central cell before fertilization and in specific domains of the resulting endosperm after fertilization.

Two novel maize genes expressed specifically in the central cell of the female gametophyte and in two compartments of the endosperm (the basal endosperm transfer layer and the embryo surrounding region) were characterized. The ZmEBE (embryo sac/basal endosperm transfer layer/embryo surrounding region) genes were isolated by a differential display between the upper and the lower half of the kernel at 7 days after pollination (DAP). Sequence analysis revealed ORFs coding for two closely related proteins of 304 amino acids (ZmEBE-1) and 286 amino acids (ZmEBE-2). This size difference was due to differences in the splicing of the two genes. Both protein sequences showed significant similarity to the DUF239 family of Arabidopsis, a group of 22 proteins of unknown function, a small number of which are putative peptidases. ZmEBE genes had a novel cell type-specific expression pattern in the central cell before and the resulting endosperm after fertilization. RT-PCR analysis showed that the expression of both genes started before pollination in the central cell and continued in the kernel up to 20 DAP with a peak at 7 DAP. In situ hybridization revealed that the expression in the kernel was restricted to the basal transfer cell layer and the embryo surrounding region of the endosperm. The expression of ZmEBE-1 was at least 10 times lower than that of ZmEBE-2. Similarly to other genes expressed in the endosperm, ZmEBE-1 expression was subject to a parent-of-origin effect, while no such effect was detected in ZmEBE-2. Sequence analysis of upstream regions revealed a potential cis element of 33 bp repeated 7 times in ZmEBE-1 and ZmEBE-2 between positions -900 and -100. The 1.6 kb ZmEBE-2 upstream sequence containing the seven R7 elements was able to confer expression in the basal endosperm to a Gus reporter gene. These data indicate that ZmEBE is potentially involved in the early development of specialized domains of the endosperm and that this process is possibly already initiated in the central cell, which is at the origin of the endosperm.

Alternative Splicing↗

PubMed2003

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells.

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.

Chromosomes, Plant↗

PubMed2003

Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase.

Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.

Amino Acid Sequence↗

PubMed2003

Sperm cells of Zea mays have a complex complement of mRNAs.

Although double fertilization in angiosperm was discovered in 1898, we still know nothing about the proteins that mediate gamete recognition and fusion in plants. Because sperm are small and embedded within the large vegetative cell of the pollen grain, mRNAs from sperm are poorly represented in EST databases. We optimized fluorescence-activated cell sorting (FACS) in order to isolate Zea mays sperm free of contaminating vegetative cell cytoplasm, and constructed a cDNA library. Sequencing of over 1100 cDNAs from the unamplified library revealed that sperm have a diverse complement of mRNAs. Most transcripts were singletons; the most abundant was sequenced only 17 times. About 8% of the sequences are predicted to encode secreted or plasma membrane-localized proteins and are therefore candidates that might mediate gamete interactions. About 8% of the sequences correspond to retroposons. Plant sperm have condensed chromatin and are thought to be transcriptionally inactive. We used RT-PCR and in situ hybridization to determine when selected sperm mRNAs were transcribed. Sperm transcripts encoding proteins involved in general cell functions were present throughout pollen development and were more abundant in tricellular pollen than in sperm cells, suggesting that these transcripts were also present in the larger vegetative cell. However, several transcripts, which encode proteins that are most similar to hypothetical Arabidopsis proteins, appeared to be present exclusively in the sperm cells inside mature pollen, but were already present in unicellular microspores. This suggests that certain transcripts might be transcribed early during pollen development and later partitioned into the sperm cells.

DNA, Complementary↗

PubMed2002

Crystallization and preliminary X-ray crystallographic analysis of human Geminin coiled-coil domain.

Initially discovered in Xenopus laevis, Geminin is a DNA replication initiation inhibitor found in higher eukaryotes. The coiled-coil domain of Human Geminin (termed GemH-37) has been crystallized by the vapor-diffusion sitting-drop method. A complete 1.74 A data set has been collected on a single orthorhombic crystal with unit cell parameters a = 25.25, b = 44.35, c = 68.58 A. Successful molecular replacement shows that GemH-37 is a dimeric parallel coiled-coil. Structural analysis is now in progress.

Cell Cycle Proteins↗

PubMed2002

Biosynthesis of the major scent components 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose O-methyltransferases.

In Chinese rose species and in many modern varieties, two methylated phenolic derivatives, 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene, are major scent components. We show that cell-free extracts of rose petals catalyse the synthesis of 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by methylation of precursor molecules. An expressed sequence tag approach was used to identify four highly similar O-methyltransferase sequences expressed specifically in petals and anthers. Thin layer chromatography analysis showed that the activities of these enzymes with different substrates and the proportions of reaction products produced closely mimicked those observed using cell-free petal extracts, indicating that orcinol O-methyltransferases are responsible for the biosynthesis of 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene from un-methylated precursors in this organ.

Anisoles↗

PubMed2002

Analysis of gene expression in rose petals using expressed sequence tags.

Single-pass sequences were obtained from the 5'-ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent.

Contig Mapping↗

PubMed2002

Comparison of the expression patterns of two small gene families of S gene family receptor kinase genes during the defence response in Brassica oleracea and Arabidopsis thaliana.

SFR2, a member of the S gene family of receptor kinases, has been shown to be rapidly induced by wounding and bacterial infection suggesting that this gene may play a role in the defence response in Brassica. In this study we have compared the response of SFR2 to that of two other members of the SFR gene family in Brassica (SFR1 and SFR3) and to the closely-related ARK genes of Arabidopsis. Different patterns of mRNA accumulation were observed for different members of these families. SFR1 transcripts only accumulated in response to bacterial infection and their abundance was not significantly affected by wounding. Neither treatment induced accumulation of SFR3 transcripts. ARK1 and ARK3 resembled SFR2 in that their mRNAs accumulated in response to both wounding and bacterial infection. Both SFR1 and SFR2 mRNAs accumulated in response to exogenously applied salicylic acid (SA) and SA was shown to be required for induction of expression from the SFR2 promoter in Arabidopsis. However, the timing of the increase in endogenous SA levels following bacterial infiltration in Brassica indicates that the accumulation of SFR mRNA in the first few hours after infiltration does not occur in response to an increase in SA levels. We discuss the possibility that induction of SFR gene expression by SA may contribute to potentialization of the defence response. Taken together with previous studies that indicate a possible role during development, the data presented here suggest that the SFR and ARK gene families may have overlapping roles in both defence and during development.

Arabidopsis↗

PubMed2002

Between myth and reality: genetically modified maize, an example of a sizeable scientific controversy.

Maize is a major crop plant with essential agronomical interests and a model plant for genetic studies. With the development of plant genetic engineering technology, many transgenic strains of this monocotyledonous plant have been produced over the past decade. In particular, field-cultivated insect-resistant Bt-maize hybrids are at the centre of an intense debate between scientists and organizations recalcitrant to genetically modified organisms (GMOs). This debate, which addresses both safety and ethical aspects, has raised questions about the impact of genetically modified (GM) crops on the biodiversity of traditional landraces and on the environment. Here, we review some of the key points of maize genetic history as well as the methods used to stably transform this cereal. We describe the genetically engineered Bt-maizes available for field cultivation and we investigate the controversial reports on their impacts on non-target insects such as the monarch butterfly and on the flow of transgenes into Mexican maize landraces.

Animals↗

PubMed2002

Structure of the cathelicidin motif of protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein.

Cathelicidins are a family of antimicrobial proteins isolated from leucocytes and epithelia cells that contribute to the innate host defense mechanisms in mammalians. Located in the C-terminal part of the holoprotein, the cathelicidin-derived antimicrobial peptide is liberated by a specific protease cleavage. Here, we report the X-ray structure of the cathelicidin motif of protegrin-3 solved by MAD phasing using the selenocysteine-labeled protein. Its overall structure represents a fold homologous to the cystatin family and adopts two native states, a monomer, and a domain-swapped dimer. This crystal structure is the first example of a structural characterization of the highly conserved cathelicidin motif and thus provides insights into the possible mechanism of activation of the antimicrobial protegrin peptide.

Amino Acid Motifs↗

PubMed2002

Fertilization in Arabidopsis thaliana wild type: developmental stages and time course.

We describe some previously uncharacterised stages of fertilization in Arabidopsis thaliana and provide for the first time a precise time course of the fertilization process. We hand-pollinated wild type pistils with wild type pollen (Columbia ecotype), fixed them at various times after pollination, and analysed 600 embryo sacs using Confocal Laser Scanning Microscopy. Degeneration of one of the synergid cells starts at 5 Hours After Pollination (HAP). Polarity of the egg changes rapidly after this synergid degeneration. Karyogamy is then detected by the presence of two nucleoli of different diameters in both the egg and central cell nuclei, 7-8 HAP. Within the next hour, first nuclear division takes place in the fertilized central cell and two nucleoli can then be seen transiently in each nucleus produced. In a second set of experiments, we hand-pollinated wild type pistils with pollen from a transgenic promLAT52::EGFP line that expresses EGFP in its pollen vegetative cell. Release of the pollen tube contents into the synergid cell could be detected in living material. We show that the timing of synergid degeneration and pollen tube release correlate well, suggesting that either the synergid cell degenerates at the time of pollen tube discharge or very shortly before it. These observations and protocols constitute an important basis for the further phenotypic analysis of mutants affected in fertilization.

Arabidopsis↗

PubMed2002

Esr proteins are secreted by the cells of the embryo surrounding region.

Three highly homologous Esr genes are expressed specifically in the embryo surrounding region at the micropylar end of the maize endosperm. The proteins belong to a family of small hydrophilic proteins that share a conserved motive with Clv3, the ligand of the receptor-like kinase Clv1. In this study, co-localization of Esr proteins with their mRNAs in the embryo surrounding region was shown with polyclonal antibodies recognizing all three Esr proteins. On a subcellular level the secretion of Esr proteins and their association with the cell wall was shown independently by cell fractionation, immunohistochemistry and transient expression of Gfp fusion proteins. Furthermore, a possible interaction of Esr proteins with a 35 kDa protein present in the lower half of maize kernels was suggested by in vitro affinity chromatography. Therefore Esr proteins share two characteristics with ligands of receptor-like kinases: they are released in the extracellular space and they have the capacity to form protein-protein interactions.

Amino Acid Sequence↗