Identification of nodal signaling targets by array analysis of induced complex probes.

Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals.

A crucial component of the endoderm formation pathway, CASANOVA, is encoded by a novel sox-related gene.

casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene, 10J3, which is expressed in the endoderm. The cas phenotype is rescued by overexpression of 10J3 and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role for cas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of cas activity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals.

Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.

Posttranscriptional gene silencing (PTGS) in plants resuits from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, definitively proving that PTGS is an antiviral defense mechanism that can also target transgene RNA for degradation.

Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing.

Homology-dependent gene silencing is a regulatory mechanism that limits RNA accumulation from affected loci either by suppression of transcription (transcriptional gene silencing, TGS) or by activation of a sequence-specific RNA degradation process (post-transcriptional gene silencing, PTGS). The P1/HC-Pro sequence of plant potyviruses and the 2b gene of the cucumber mosaic virus have been shown to interfere with PTGS. The ability of these viral suppressors of PTGS to interfere with TGS was tested using the 271 locus which imposes TGS on transgenes under 35S or 19S promoters and PTGS on the endogenous nitrite reductase gene (Nii). Both P1/HC-Pro and 2b reversed PTGS of Nii genes in 271-containing tobacco plants, but failed to reverse TGS of 35S-GUS transgenes in the same plant. P1/HC-Pro expression from a transgene also failed to suppress either the initiation or maintenance of TGS imposed by the NOSpro-silencing locus, H2. These results indicate that PTGS and TGS operate through unlinked pathways or that P1/HC-Pro and 2b interfere at step(s) in PTGS that are downstream of any common components in the two pathways. The data suggest a simple assay to identify post-transcriptionally silenced transgenic lines with the potential to be stably converted to high expressing lines.

DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis.

In plants, transgenes can be silenced at both the transcriptional [1] and post-transcriptional levels [2]. Methylation of the transgene promoter correlates with transcriptional gene silencing (TGS) [3] whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS) [4]. In animals, TGS requires methylation and changes in chromatin conformation [5]. The involvement of methylation during PTGS in plants is unclear and organisms with non-methylated genomes such as Caenorhabditis elegans or Drosophila can display RNA interference (RNAi), a silencing process mechanistically related to PTGS [6]. Here, we crossed Arabidopsis mutants impaired in a SWI2/SNF2 chromatin component (ddm1 [7]) or in the major DNA methyltransferase (met1 [8] and E. Richards, personal communication) with transgenic lines in which a reporter consisting of the cauliflower mosaic virus 35S promoter fused to the beta-glucuronidase (GUS) gene (35S-GUS) was silenced by TGS or PTGS. We observed an efficient release of 35S-GUS TGS by both the ddm1 and met1 mutations and stochastic release of 35S-GUS PTGS by these two mutations during development. These results show that DNA methylation and chromatin structure are common regulators of TGS and PTGS.

Arabidopsis mutants impaired in cosuppression.

Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S-uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and beta-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S-uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S-hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S-Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing.

ActA is a dimer.

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Expression of contact, a new zebrafish DVR member, marks mesenchymal cell lineages in the developing pectoral fins and head and is regulated by retinoic acid.

Contact, a new zebrafish transforming growth factor-beta (TGF-beta) member is most closely related to mouse GDF5 and to human CDMP-1 responsible, when mutated, for limb brachypodism phenotype and Hunter-Thompson syndrome, respectively. Contact exhibits a dynamic spatial expression pattern in the pharyngeal arches and the pectoral fin buds that much prefigures cartilage formation. Within the fin buds, contact expression is detected in the proximal mesenchyme from which the endoskeleton will develop. Exogeneously applied retinoic acid (RA) induces duplication of the pectoral fin rudiment in zebrafish embryos as well as contact expression along the proximal margin of the fin mesenchyme showing that both endoskeleton and exoskeleton can be duplicated.

Nitrite reductase silencing as a tool for selecting spontaneous haploid plants.

Using tobacco as a model species, we have developed a simple procedure for the selection of spontaneous haploid plants under horticultural conditions, which does not require the use of any selective agent. One transgenic tobacco plant, homozygous for an antisense transgene able to silence the expression of nitrite reductase host genes, and encoding the second enzyme of the nitrate assimilation pathway, was used to pollinate two different cultivars of wild type tobacco plants. Seeds were sown at high density in the greenhouse and watered with a nutrient solution containing nitrate. Green plants able to develop normally emerged at a frequency of 5.10(-4) in a mass of chlorotic retarded plants. Phenotypic and genetic analysis, chloroplast counting in stomatal guard cells and molecular hybridizations revealed that 22% of these plants were gynogenetic haploid plants exhibiting the maternal phenotype whereas the remaining 78% were true diploid plants that have lost the antisense transgene. These results demonstrate that a transgene able to silence the expression of a housekeeping gene can be utilized as a counter-selectionable marker for the rapid and easy selection of spontaneous haploid plants in transformable species.

Progressive multifocal leukoencephalopathy: investigation of three cases using in situ hybridization with JC virus biotinylated DNA probe.

Using the technique of in situ DNA-to-DNA hybridization, a JC virus biotinylated DNA probe was developed and applied to formalin-fixed, paraffin-embedded, or fixed, frozen sections of brain tissue from three subjects with progressive multifocal leukoencephalopathy (PML). Light microscopy was carried out to correlate the presence of JC virus DNA with the selective infection of oligodendrocytes and astrocytes in PML. Oligodendrocytes (lytically infected) showed the greatest evidence of viral DNA. More astrocytes showing bizarre morphological changes had evidence of viral DNA than did astrocytes that were simply reactive. Viral DNA was not evident in vascular endothelial cells using this technique. Viral DNA replication may be an important initial step which produces the bizarre "transformed" astrocytes of PML. Findings in this study do not support the hypothesis that vascular endothelial replication is important in the pathogenesis of JC virus-induced PML. In situ hybridization with biotinylated JC virus probe may be useful in the diagnosis of PML on brain biopsy specimens.

Establishment of a line of human fetal glial cells that supports JC virus multiplication.

Primary cultures of human fetal brain cells were transfected with plasmid DNA pMK16, containing an origin-defective mutant of simian virus 40 (SV40). Several weeks after DNA treatment, proliferation of glial cells was evident in the culture, allowing passage of the cells at low split ratios. Initially, only 10% of the cells demonstrated nuclear fluorescence staining using a hamster tumor antibody to the SV40 T protein. By the sixth passage, however, 100% of the cells reacted positively to the same antibody. During these early passages, the cells designated SVG began growing very rapidly and acquired a homogeneous morphology. Cell division required only low serum concentrations, was not contact-inhibited, and remained anchorage dependent. These characteristics of the SVG cells have been stable through 25 passages or approximately equal to 80 cell generations. The SV40 T protein is continuously produced in the cells and can direct the replication of DNA inserts in the pSV2 vector, determined by in situ hybridization using biotin-labeled DNA probes, which contains the SV40 replication origin. More importantly, SVG cells support the multiplication of the human papovavirus JCV at levels comparable to primary cultures of human fetal glial cells, producing infectious virus as early as 1 week after viral adsorption. Their brain-cell derivation has been established as astroglial, based on their reactivity with a monoclonal antibody to glial fibrillary acid protein and lack of activity with an anti-galactocerebroside antibody, which identifies oligodendroglial cells. The SVG cells represent a unique line of continuous rapidly growing human fetal astroglial cells that synthesizes a replication-proficient SV40 T protein. Their susceptibility to JC virus (JCV) infection obviates a host restriction barrier that limited JCV studies to primary cultures of human fetal brain and thus should allow for more detailed molecular studies of human brain cells and JCV that infects them.

JC virus-induced owl monkey glioblastoma cells in culture: biological properties associated with the viral early gene product.

JCV induces glioblastomas in owl monkeys 18-24 months or longer following intracranial inoculation (W. London, S. Houff, D. Madden, D. Fuccillo, M. Gravell, W. Wallen, A. Palmer, J. Sever, B. Padgett, D. Walker, G. Zu Rhein, and T. Ohashi, 1978, Science 201, 1246-1248). Cells from one brain tumor, owl monkey 26, were successfully established in culture and analyzed for phenotypic characteristics generally associated with persistence and expression of the papovavirus early region of its genome. Owl monkey 26 cells demonstrated nuclear JCV T protein detected by either SV40 hamster tumor sera or PAb 108, a monoclonal antibody to SV40 T protein. However, the JCV T protein was not detected in a complex with the host cell p53 protein as judged by immunoprecipitation using PAb 122, a monoclonal antibody directed to the mammalian p53 cellular protein. These cells also demonstrated increased plasminogen activator secretion and actin cable disorganization properties not before reported for JCV-induced tumor or transformed cells. Of the primate papovaviruses, JCV is unique in its ability to induce brain tumors in these primates. JCV early gene expression can be shown to persist in brain tumor cells once established in culture and correlates with cell phenotypes typical of papovavirus malignant transformation even though the time between virus inoculation and tumor development is usually several years.