Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one‐cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one‐cell embryo can be an important model for understanding the regulation of DNA synthesis. J. Cell. Biochem. 107: 214–223, 2009. © 2009 Wiley‐Liss, Inc.     

Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR

We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.     

Genetic Variability of Phytopathogenic Fusarium proliferatum Associated with Crown Rot in Asparagus officinalis

Fusarium proliferatum (teleomorph: Gibberella intermedia ) is a causal agent of crown rot of Asparagus officinalis and is one potential fumonisin-producing species within the genus Fusarium . It colonizes roots and crowns of asparagus plants, but could also be isolated from symptomless asparagus spears. Fusarium proliferatum isolates obtained from perennial asparagus plantings from Austria and Germany were included in a study on detectability and variability of two essential genes of the fumonisin-gene cluster. Genetic fingerprinting of 45 isolates revealed 14 different fingerprint groups, indicating genetic heterogenicity of F. proliferatum . Most isolates differentiated into three main fingerprint clusters, but no association was found between fingerprint group and origin of the isolates. By gene-specific PCR it was shown that, in 25 isolates tested, both initial genes of the fumonisin biosynthetic pathway – FUM1 , encoding a polyketide synthase and FUM8 , a gene for a putative aminoacyl transferase – were detectable. This suggests that these isolates were able to produce fumonisins and could contribute to the detected contamination in originating asparagus spears with this mycotoxin. Thus, early detection of FUM -genes in F. proliferatum -colonized asparagus may be suited to prevent uptake of fumonisin contaminated food with the human diet. Restriction fragment length polymorphism analysis (PCR-RFLP) of the amplified FUM gene fragments revealed little sequence variability, suggesting a conserved structure of these genes within this species. However, sequence analysis confirmed intraspecific nucleotide polymorphisms of these genes.     

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3)

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC₆(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.     

Approaches to defining dual-targeted proteins in Arabidopsis

A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana . These predictions were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of subcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using a variety of prediction programs on a biochemical pathway or process known to contain at least one dual-targeted protein. Several technical parameters need to be taken into account before assigning subcellular localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is required if more than one model exists.     

ADAM-17 regulates endothelial cell morphology, proliferation, and in vitro angiogenesis

Modulation of angiogenesis is a promising approach for treating a wide variety of human diseases including ischemic heart disease and cancer. In this study, we show that ADAM-17 is an important regulator of several key steps during angiogenesis. Knocking down ADAM-17 expression using lentivirus-delivered siRNA in HUVECs inhibited cell proliferation and the ability of cells to form close contact in two-dimensional cultures. Similarly, ADAM-17 depletion inhibited the ability of HUVECs to form capillary-like networks on top of three-dimensional Matrigel as well as in co-culture with fibroblasts within a three-dimensional scaffold. In mechanistic studies, both baseline and VEGF-induced MMP-2 activation and Matrigel invasion were inhibited by ADAM-17 depletion. Based on our findings we propose that ADAM-17 is part of a novel pro-angiogenic pathway leading to MMP-2 activation and vessel formation.     

The amino acid residues of transmembrane helix 5 of multidrug resistance protein CaCdr1p of Candida albicans are involved in substrate specificity and drug transport

In view of the importance of Candida Drug Resistance Protein (Cdr1p) of pathogenic Candida albicans in azole resistance, we have characterized its ability to efflux variety of substrates by subjecting its entire transmembrane segment (TMS) 5 to site directed mutagenesis. All the mutant variants of putative 21 amino acids of TMS 5 and native CaCdr1p were over expressed as a GFP-tagged protein in a heterologous host Saccharomyces cerevisiae. Based on the drug susceptibility pattern, the mutant variants could be grouped into two categories. The variants belonging to first category were susceptible to all the tested drugs, as compared to those belonging to second category which exhibited resistance to selective drugs. The mutant variants of both the categories were analyzed for their ATP catalysis and drug efflux properties. Irrespective of the categories, most of the mutant variants of TMS 5 showed an uncoupling between ATP hydrolysis and drug efflux. The mutant variants such as M667A, F673A, I675A and P678A were an exception since they reflected a sharp reduction in both K_m and V_max values of ATPase activity when compared with WT CaCdr1p-GFP. Based on the competition experiments, we could identify TMS 5 residues which are specific to interact with select drugs. TMS 5 residues of CaCdr1p thus not only impart substrate specificity but also selectively act as a communication link between ATP hydrolysis and drug transport.     

NMR structure of the Wnt modulator protein Sclerostin

Sclerostin has been identified as a negative regulator of bone growth. Initially it was considered that Sclerostin performs its regulatory function via acting as a modulator of bone morphogenetic proteins (BMPs) similar to known examples such as Noggin, Chordin, and members of the DAN family. Recent findings, however, show that Sclerostin interferes with the Wnt signaling pathway due to binding to the Wnt co-receptor LRP5 thereby modulating bone growth. As Sclerostin is exclusively produced by osteocytes located in bones, neutralization of its bone-inhibiting functions makes it a highly interesting target for an osteoanabolic therapeutic approach in diseases characterized by bone loss, such as osteoporosis. Despite the huge interest in Sclerostin inhibitors the molecular basis of its function and its interaction with components of the Wnt signaling cascade has remained unclear. Here, we present the NMR structure of murine Sclerostin providing the first insights how Sclerostin might bind to LRP5.     

Golgi targeting of Drosophila melanogaster β4GalNAcTB requires a DHHC protein family–related protein as a pilot

Drosophila melanogaster β4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcβ1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated β4GalNAcTB together with β4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive β4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of β4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with β4GalNAcTB and, when expressed with an ER retention signal, holds active β4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs β4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of β4GalNAcTB.