The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin.

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.

A role for heterodimerization in nuclear localization of a homeodomain protein.

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 (beta-glucuronidase) gene and showing that beta-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.

Heterodimerization between two classes of homeodomain proteins in the mushroom Coprinus cinereus brings together potential DNA-binding and activation domains.

The A mating type-genes of the mushroom, Coprinus cinereus, encode two classes of homeodomain-containing proteins distinguished as HD1 and HD2 on the basis of conserved, but distinctly different motifs. Compatible mating partners bring together versions of the proteins that can heterodimerize, thereby generating an active transcription factor complex that commits mated cells to sexual development. We have previously described a rare mutation in which an HD2::HD1 gene fusion generates a 'fused dimer' lacking much of HD1 including the homeodomain yet capable of constitutively promoting development [Kües et al., EMBO J. 13 (1994b) 4054-4059]. Here, we exploit this mutation to help identify contributions made by each protein class to normal heterodimer function. We show that the HD2 homeodomain is essential; deletion within the HD1 homeodomain can be tolerated in a normal heterodimer, as well as in the mutant fusion protein, but not substitution of a critical amino acid. We define, by deletion analysis, an essential C-terminal region of the HD1 and demonstrate its potential activation function by the ability to activate transcription in yeast when fused to the GAL4 DNA-binding domain. We also identify a potential role in transcriptional repression for the predicted C-terminal helix of HD1 proteins.

Patients' knowledge concerning their medications on discharge from hospital.

Fifty patients were interviewed, on discharge from hospital, about their medications. Nine (18%) patients did not know, and a further four (8%) had inappropriate beliefs about why they were taking at least one of their discharge medications. Very few patients knew of significant side-effects which they might expect, or precautions which they should take, and over half did not know how long they were to continue taking their medicines. A small proportion was unable to read the bottle or open the container. Thus, even patients who, by virtue of an in-patient stay, have had a prolonged opportunity for education regarding their medicines have very little knowledge of their medicines upon discharge from hospital.

Surface structure and RNA-protein interactions of foot-and-mouth disease virus.

The 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% of the residues of VP1, 15% 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 VP1, 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 VP1 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.

Analysis of the secondary structure of the poly(C) tract in foot-and-mouth disease virus RNAs.

Sodium bisulphite modification of foot-and-mouth disease virus (FMDV) RNA in solution indicates that the majority of the poly(C) tract in the RNA is single-stranded in concordance with previous results with encephalomyocarditis virus RNA. The reaction kinetics are biphasic; 60% of the cytidylic acid in the poly(C) tract reacts like synthetic poly(C), and the remainder with the kinetics of the cytidylic acid in the rest of the RNA. The reactivity of the poly(C) tract with poly(I) indicates that it is looped out and exposed in the RNA. The deamination reaction has also been used to investigate the structure of the replicative form (RF) and replicative intermediate (RI) isolated from infected cells. Analysis by gel electrophoresis of the long RNase A- and T1-resistant oligonucleotides of RI suggests that it has five single-stranded poly(C) tracts to every one which is base-paired. Bisulphite reactivity of the poly(C) tract and gel electrophoresis of the ribonuclease-resistant oligonucleotides of RF indicate that the poly(C) is base-paired to a poly(G) tract in this molecule. The presence of a poly(G) tract in RF and RI provides unequivocal evidence that the poly(C) is replicated via poly(G) in the negative strand.