No abstract

The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (␤-glucuronidase) gene and showing that ␤-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.

In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided. The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin. Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function. We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ). In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4. A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus. In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes. We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)). Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle.

The A mating-type locus of the mushroom Coprinus cinereus contains three or more paralogous pairs of genes encoding two families of homeodomain proteins (HD1 and HD2). A successful mating brings together different allelic forms of at least one gene, and this is sufficient to trigger initial steps in sexual development. Previous studies have suggested that development is regulated by heterodimerization between HD1 and HD2 proteins. In this report, we describe 5[prime] gene deletions and 5[prime] end exchanges showing that the N-terminal regions of the proteins are essential for choosing a compatible partner but not for regulating gene transcription. Using an in vitro glutathione S-transferase association assay, we demonstrated heterodimerization between HD1 and HD2 proteins and found that heterodimerization only occurs between compatible protein combinations. The N-terminal regions of the proteins were sufficient to mediate dimerization, and N-terminal swaps resulted in a predicted change in dimerization specificity. By analyzing the N-terminal amino acid sequences of HD1 proteins, we identified two potential coiled-coil motifs whose relative positions vary in paralogous proteins but are both required for in vivo function.

SUMMARYThe surface structure of foot-and-mouth disease virus (FMDV) and the interaction of the individual capsid proteins with the virus RNA have been examined using modification reagents. By measuring the extent of modification of the lysine residues of intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter reagent it was found that 54~o of the residues of VP 1, 15 ~o of the residues of VP2 and 37~ of the residues of VP3, equivalent to five, two and four lysine residues respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not modified in intact virus particles, indicating that it has no lysine residues on the surface of the virus. Modification with sodium metabisulphite, which causes a specific transamination reaction between cytidylic acid residues in ssRNA and closely associated basic amino acids, cross-linked all four structural proteins to the virus RNA. Both fragments of VPI, produced by treatment of the virus particle with trypsin, are also cross-linked to the RNA. These observations have been combined with the evidence that the immunogenic activity of VP 1 may be contained in two discontinuous sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the arrangement of this polypeptide in the virus particle.