Wolfgang Nacken

Genetic Control of Flower Development by Homeotic Genes in Antirrhinum majus

Homeotic mutants have been useful for the study of animal development. Such mutants are also known in plants. The isolation and molecular analysis of several homeotic genes in Antirrhinum majus provide insights into the underlying molecular regulatory mechanisms of flower development. A model is presented of how the characteristic sequential pattern of developing organs, comprising the flower, is established in the process of morphogenesis.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

The Ca2+‐ and arachidonic acid‐binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil‐like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9−/− mice. The impaired oxidase activation could also be mimicked in a cell‐free system by pretreatment of neutrophil cytosol with an S100A9‐specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein‐protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Structural analysis of Tam3, a transposable element from Antirrhinum majus, reveals homologies to the Ac element from maize

Transposable elements have been successfully used to tag interesting genes in Antirrhinum majus [ 1, 2, 3]. One of the elements used for tagging is Tam3 which was cloned from the nivea locus of line 98 (niv-98) encoding the chalcone synthase [8, 7]. It is a 3.6 kb long element that has 12 bp inverted repeats [7]. Here we describe the molecular structure of Tam3. The element was characterized by sequence analysis of genomic an cDNA clones prepared from poly(A) + mRNA of line 98 (Fig. 1). The Tam3 element is 3629 bp long and contains an open reading frame of 2.48 kb. A 3.0 kb long cDNA clone that is colinear with the genomic sequence confirms that this open reading frame is transcribed and that the element has no intron. Northern analysis with mRNA of line 98, using Tam3-specific fragments as probes, reveals only one transcript 5.5 kb in length that has 2.7 kb in common with Tam3, as determined by analysis of corresponding cDNA clones. Two lines of evidence suggest that this 5.5 kb message is chimeric, derived from a Tam3 copy that is inserted in a transcribed gene, and not from a larger Tam3 element that possibly could represent an autonomous element. Firstly, a genomic Southern blot with DNA of line 98, restricted with BstNI (an enzyme that cuts in both ends of Tam3) shows when probed with Tam3 no band larger than 3.2 kb, the expected size of the internal BstNI fragment of the Tam3 copy of the nivea locus. Secondly, the Tam3 element is able to transpose in tobacco plants indicating that it is an autonomous element [6]. Tam3, the P and the hobo element from Drosophila [4, 9] and the Ac/Ds transposon system from maize can be grouped into a family by the similarity of their terminal inverted repeats and the generation of a 8 bp target site duplication upon integration. Moreover, patches of strong homology (60 -65~) were found between the deduced Tam3 protein and the amino acid sequence of the Ac gene product (Fig. 1). The overall homology between both amino acid sequences is 30~ over a stretch of 520 amino acids. The homologous part comprises the region between amino acids 220 to 740 of the Ac-ORFA and 190 to 710 of the deduced Tam3 protein. The Ac gene product encoded by ORFA binds to subterminal repeats of the element [ 5 ]. The same is true for the so-called transposase of the P-element [4]. It seems probably that the Tam3 gene product as well binds to subterminal motives of the element. The sequence homology found between Ac and Tam3 strengthens the view that these elements may have a common mechanism of transposition.

The transposable element Tam1 from Antirrhinum majus shows structural homology to the maize transposon En/Spm and has no sequence specificity of insertion

SummaryWe present the genomic structure of Tam1, a transposable element from Antirrhinum majus. The Taml element is 15.2 kb long and includes two genes that are transcribed to produce a 2.4 kb (tnpl) and a 5 kb mRNA (tnp2). These transcripts partially overlap and the exons are scattered over the whole element. Tnp1 encodes a 53 kDa protein as deduced from the cDNA sequence. The 5 kb transcript of tnp2 contains an open reading frame that shares 45% homology with part of the tnpD gene of En/Spm from maize and 48% homology with an open reading frame of the Tgm element from Glycine max. We discuss the possible functions of these genes by analogy with En/Spm. Additionally, a number of flanking sequences of Taml insertions were analysed to investigate the sequence specificity of insertion. From these studies we conclude that Taml transposes predominantly into AT-rich regions that can be unique as well as repetitive. No specific target sequence of insertion could be found.

Analysis of the MRP8-MRP14 Protein-Protein Interaction by the Two-hybrid System Suggests a Prominent Role of the C-terminal Domain of S100 Proteins in Dimer Formation

Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways. They consist of two helix-loop-helix, calcium-binding EF-hand domains. A characteristic feature is their tendency to form homo- and/or heterodimeric complexes. This report presents for the first time a functional “in vivo” approach to the analysis of S100 protein dimerization. Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells. It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse. The ability to homodimerize, however, appears to be restricted to the murine MRPs. Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization. Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.

Molecular characterization of two stamen-specific genes, tap1 and fil1, that are expressed in the wild type, but not in the deficiens mutant of Antirrhinum majus

SummaryDeficiens, a homeotic gene involved in the genetic control of flower development, codes for a putative transcription factor. Upon mutation of the gene, petals are transformed to sepals and stamens to carpels, indicating that deficiens is essential for the activation of genes required for petal and stamen formation. In a search for putative target genes of deficiens, several stamen- and petal-specific genes were cloned that are expressed in wild type but not in the deficiensglobiferamutant. In this report the molecular characterization of two of these genes, tap1 and fil1, is presented. They are transiently expressed during flower development. In situ hybridization data demonstrate that tap1 is expressed in the tapetum of the anthers and fill in the filament of the stamen and at the bases of the petals. Both genes encode small proteins with N-terminal hydrophobic domains suggesting that they are secreted. We discuss possible functions of the gene products and their relationship to the deficiens gene.

Zinc binding reverses the calcium-induced arachidonic acid-binding capacity of the S100A8/A9 protein complex

Analysis of the calcium‐induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF‐hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn2+, Cu2+, and Mg2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn2+ or Cu2+ in the presence of calcium, whereas Mg2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein‐protein interaction assay. Fluorescence measurements gave evidence that both Zn2+ and Cu2+ induce different conformational changes thereby affecting the calcium‐induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.

The calcium binding protein S100A9 is essential for pancreatic leukocyte infiltration and induces disruption of cell–cell contacts†

Leukocyte infiltration is an early and critical event in the development of acute pancreatitis. However, the mechanism of leukocyte transmigration into the pancreas and the function of leukocytes in initiating acute pancreatitis are still poorly understood. Here, we studied the role of S100A9 (MRP14), a calcium binding protein specifically released by polymorph nuclear leukocytes (PMN), in the course of acute experimental pancreatitis. Acute pancreatitis was induced by repeated supramaximal caerulein injections in S100A9 deficient or S100A9 wild‐type mice. We then determined S100A9 expression, trypsinogen activation peptide (TAP) levels, serum amylase and lipase activities, and tissue myeloperoxidase (MPO) activity. Cell–cell contact dissociation was analyzed in vitro with biovolume measurements of isolated acini after incubation with purified S100A8/A9 heterodimers, and in vivo as measurement of Evans Blue extravasation after intravenous application of S100A8/A9. Pancreatitis induced increased levels of S100A9 in the pancreas. However, infiltration of leukocytes and MPO activity in the lungs and pancreas during acute pancreatitis was decreased in S100A9‐deficient mice and associated with significantly lower serum amylase and lipase activities as well as reduced intrapancreatic TAP‐levels. Incubation of isolated pancreatic acini with purified S100A8/A9‐heterodimers resulted in a rapid dissociation of acinar cell–cell contacts which was highly calcium‐dependent. Consistent with these findings, in vivo application of S100A8/A9 in mice was in itself sufficient to induce pancreatic cell–cell contract dissociation as indicated by Evans Blue extravasation. These data show that the degree of intrapancreatic trypsinogen activation is influenced by the extent of leukocyte infiltration into the pancreas which, in turn, depends on the presence of S100A9 that is secreted from PMN. S100A9 directly affects leukocyte tissue invasion and mediates cell contact dissociation via its calcium binding properties. J. Cell. Physiol. 216: 558–567, 2008.

Molecular analysis of tap2, an anther-specific gene from Antirrhinum majus

Deficiens is a floral homeotic gene of Antirrhinum majus, mutation of which results in transformation of petals to sepals and stamens to carpels. In a search for putative target genes, controlled by this regulatory locus, cDNA clones representing genes, that are expressed in wild type but not in the deficiens mutant flowers, were isolated by differential screening. The molecular structure and the expression pattern of one of these genes, tap2, is described. Tap2 is transiently and tissue‐specifically expressed in the tapetum of the anthers. It encodes a 131 amino‐acids‐long protein with a hydrophobic N‐terminus, displaying all characteristic features of a signal peptide. This indicates that the TAP2 protein may be secreted.

Double-Stranded RNA Induces MMP-9 Gene Expression in HaCaT Keratinocytes by Tumor Necrosis Factor-α

Viral double-stranded RNA (dsRNA) and its synthetic analog poly (I:C) are recognized via multiple pathways and induce the expression of genes related to inflammation. In the present study, we demonstrate that poly (I:C) specifically induced the expression of matrix metallo-proteinase-9 (MMP-9) in HaCaT keratinocytes. Studies using specific pharmacological inhibitors revealed the involvement of NF-κB, p38 MAPK, and PI-3K signal transduction pathways in poly (I:C)-induced MMP-9 gene expression. MMP-9 gene induction was sensitive toward treatment with the macrolide antibiotic bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, and with the lysosomotropic agent chloroquine. However, cycloheximide treatment only partially blocked poly (I:C)-induced MMP-9 gene expression. Although HaCaT keratinocytes produce a number of cytokines and chemokines in response to poly (I:C), stimulation experiments revealed that exclusively TNFα strongly promoted MMP-9 gene expression. During the antiviral response MMP-9 expression may be of importance for the tissue injury phase.