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The Runx3 transcription factor regulates cell fate decisions during embryonic development and in adults. It was previously reported that Runx3 is strongly expressed in embryonic and adult gastrointestinal tract (GIT) epithelium (Ep) and that its loss causes gastric cancer. More than 280 publications have based their research on these findings and concluded that Runx3 is indeed a tumour suppressor (TS). In stark contrast, using various measures, we found that Runx3 expression is undetectable in GIT Ep. Employing a variety of biochemical and genetic techniques, including analysis of Runx3-GFP and R26LacZ/Runx3Cre or R26tdTomato/Runx3Cre reporter strains, we readily detected Runx3 in GIT-embedded leukocytes, dorsal root ganglia, skeletal elements and hair follicles. However, none of these approaches revealed detectable Runx3 levels in GIT Ep. Moreover, our analysis of the original Runx3LacZ/LacZ mice used in the previously reported study failed to reproduce the GIT expression of Runx3. The lack of evidence for Runx3 expression in normal GIT Ep creates a serious challenge to the published data and undermines the notion that Runx3 is a TS involved in cancer pathogenesis.

Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.

The estrogenic mycotoxin zearalenone (ZON) produced by some Fusarium spp. causes reproductive problems and hyperestrogenic syndromes in mammals. In an effort to elucidate the molecular pathways of ZON production, we present a comparative real-time quantitative polymerase chain reaction expression study of seven contiguous genes in the ZON biosynthetic cluster on sterile rice and during wheat and oat infection. Under ZON production on rice, the polyketide synthase (PKS) genes PKS4 and PKS13, alcohol oxidase FG12056 gene, and transcriptional regulator FG02398 gene showed similarly upregulated patterns, whereas the nonribosomal peptide synthetase (NPS) FG02394, the K(+) channel beta subunit FG12015, and the protein kinase FG02399 displayed a variant pattern. During the same time period under wheat infection when no ZON was produced, the PKS genes and the NPS were downregulated relative to rice whereas the K(+) channel beta subunit gene FG12015 was markedly upregulated, suggesting that it may play a role in the infection process. This is the first expression study of ZON biosynthetic genes in planta. The results give insight into the regulation and activities of the ZON gene cluster under different experimental systems and suggest a connection between ZON and a K(+) channel that could reveal a novel function for ZON in Fusarium spp.

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.The retroviral Gag polyprotein is sufficient to direct virus assembly, as demonstrated by its ability to form virus-like particles in the absence of all other viral gene products (reviewed in reference 39). Previously, it was thought that Gag proteins of type C retroviruses were synthesized on cytosolic ribosomes and then targeted directly to the plasma membrane, where budding occurs. However, we discovered that the Rous sarcoma virus (RSV) Gag polyprotein traffics through the nucleus (32). Nuclear import is mediated by a nuclear localization signal (NLS) within the N-terminal matrix (MA) domain. A CRM1-dependent nuclear export signal (NES) within the p10 domain of Gag mediates nuclear egress, after which Gag travels to the plasma membrane for particle release. Treatment of cells with leptomycin B (LMB), a specific inhibitor of CRM1, results in the accumulation of Gag proteins in the nucleus (32).Nuclear import of macromolecules, including all proteins greater than 50 kDa, occurs by signal-mediated active transport across the nuclear membrane through the nuclear pore complex. The majority of proteins bearing specific NLS sequences are imported through an interaction with soluble transport factors belonging to the importin superfamily (reviewed in reference 9). The "classical" NLSs in...