Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripeningrelated genes isolated by differential display.

Differential display was used to isolate early ethyleneregulated genes from late immature green tomato fruit in order to obtain a broader understanding of the molecular basis by which ethylene coordinates the ripening process. Nineteen novel ethylene-responsive (ER) cDNA clones were isolated that fell into three classes: (i) ethylene up-regulated (ii) ethylene downregulated, and (iii) transiently induced. Expression analysis revealed that ethylene-dependent changes in mRNA accumulation occurred rapidly (15 min) for most of the ER clones. The predicted proteins encoded by the ER genes are putatively involved in processes as diverse as primary metabolism, hormone signalling and stress responses. Although a number of the isolated ER clones correspond to genes already documented in other species, their responsiveness to ethylene is described here for the ®rst time. Among the ER clones sharing high homology with regulatory genes, ER43, a putative GTP-binding protein, and ER50, a CTR1-like clone, are potentially involved in signal transduction. ER24 is homologous to the multiprotein bridging factor MBF1 involved in transcriptional activation, and ®nally, two clones are homologous to genes involved in post-transcriptional regulation: ER49, a putative translational elongation factor, and ER68, a mRNA helicase-like gene. Six ER clones correspond to as yet unidenti®ed genes. The expression studies indicated that all the ER genes are ripening-regulated, and, depending on the clone, show changes in transcript accumulation either at the breaker, turning, or red stage. Analysis of transcript accumulation in different organs indicated a strong bias towards expression in the fruit for many of the clones. The potential roles for some of the ER clone

Replicating parvoviruses that target colon cancer cells.

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and beta-catenin genes. We have modified the minute virus of mice (MVM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric beta-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of Delta N-beta-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy

A simple p53 functional assay for screening cell lines, blood, and tumors

Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors. We have developed a simple functional assay for p53 mutation in which human p53 expressed in Saccharomyces cerevisiae activates transcription of the ADE2 gene. Consequently, yeast colonies containing wild-type p53 are white and colonies containing mutant p53 are red. Since this assay tests the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations. By combining this approach with gap repair techniques in which unpurified p53 reverse transcription-PCR products are cloned by homologous recombination in vivo it is possible to screen large numbers of samples and multiple clones per sample for biologically important mutations. This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue. In addition, the assay detects temperature-sensitive mutants, which give pink colonies. We show here that this form of p53 functional assay can be used rapidly to detect germline mutations in blood samples, somatic mutations in tumors, and mutations in cell lines