Basal cell carcinomas (BCC), which are the most common form of skin malignancy, are invariably associated with the deregulation of the Sonic Hedgehog (Shh) signalling pathway. As such, BCC represent a unique model for the study of interactions of the Shh pathway with other genes and pathways. We constructed a tissue microarray (TMA) of 75 paired BCC and normal skin and analysed the expression of b-catenin and RUNX3, nuclear effectors of the wingless-Int (Wnt) and bone morphogenetic protein/ transforming growth factor-b pathways, respectively. In line with previous reports, we observed varying subcellular expression pattern of b-catenin in BCC, with 31 cases (41%) showing nuclear accumulation. In contrast, all the BCC cases tested by the TMA showed RUNX3 protein uniformly overexpressed in the nuclei of the cancer cells. Analysis by Western blotting and DNA sequencing indicates that the overexpressed protein is normal and full-length, containing no mutation in the coding region, implicating RUNX3 as an oncogene in certain human cancers. Our results indicate that although the deregulation of Wnt signalling could contribute to the pathogenesis of a subset of BCC, RUNX3 appears to be a universal downstream mediator of a constitutively active Shh pathway in BCC.

No abstract

The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNA Trp which are scattered on five chromosomes. Other, non-functional tDNA Trp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNA Trp , as well as a 34-nucleotide intervening sequence, are identical. However, the 5 and 3 flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNA Trp to search for functional differences among these genes. Previous studies with one tDNA Trp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNA Trp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNA Trp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5-flanking sequences of the tRNA Trp genes.

A gene from Saccharomyces cerevisiae was sequenced that encodes a protein with homology to a family of putative ATPases. These homologous proteins include the yeast cell division cycle protein Cdc48p and its mammalian homologues VCP and p97; Sec18p and its mammalian homologue NSF, proteins necessary for fusion of transport vesicles to target membranes in the secretory pathway; Pas1p, a protein necessary for peroxisome biosynthesis in yeast; Yme1p, a yeast mitochondrial protein that influences the rate of DNA escape from mitochondria; and TBP-1, MSS1 and Sug1p, proteins that interact with transcription factors. This newly sequenced gene, named AFG2 for ATPase family gene, is located on chromosome XII 5' to the SLP1/VPS33 open reading frame and encodes an essential protein of 780 amino acids that is most homologous to Cdc48p.