A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL) promoter from S. cerevisiae. By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucosecarbon source) from the GAL) promoter, centromere function can be switched off or on, respectively. With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids. Moreover, it was possible to selectively induce the loss of a single chromosome from a mitoticafly dividing population of cells. We observed that the induction of chromosome HI aneuploidy resulted in a dramatic change in cell morphology. The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.Chromosome segregation in most eucaryotic organisms involves the attachment of a spindle apparatus to a specific chromosomal domain, the kinetochore. While the assembly and disassembly of the mitotic spindle can be visualized by a variety of methods, the mechanism by which chromosomes become attached to this apparatus is not understood. The isolation of the DNA sequences required for chromosome segregation in the budding yeast Saccharomyces cerevisiae has provided the first insights at the molecular level into the function of this unique chromosomal domain (7,12,13,18,24,30,34,38).There are two major components involved in chromosomal segregation: the spindle apparatus and the kinetochore. The spindle apparatus provides the framework for chromosome movement in mitosis through spindle fibers that are connected to the chromosomes. The kinetochore includes the structural components at the site of attachment of the spindle fibers and is located within a unique chromosomal domain, the centromere. During mitosis, the centromere region is observed as the primary constriction along the chromosome fiber in higher eucaryotic cells. The centromeric region in the yeast cannot be seen microscopically due to the absence of mitotic chromosome condensation in S. cerevisiae. Examination of the molecular structure of yeast centromeres by nuclease digestion has revealed that centromeric DNA (CEN) is organized into a 220-to 250-base-pair (bp) protected chromatin structure (4). The centromere core particle, including DNA and associated protein, is therefore structurally distinct from the typical 146-bp DNA-histone core particle that constitutes the bulk of eucaryotic chromatin. Centromere DNA, whether in situ, in heterologous chromosomes or in autonomously replicating plasmids introduced into yeast, is always organized into a 220-to 250-bp core complex (3). Furthermore, point mutations at select sites within this region result in both inactivation of centromere function (28) and complete disruption of the protected chromatin core (M. J. Saunders and K. S. ...

Precise spatial and temporal control of developmental genes is crucial during embryogenesis. Regulatory mutations that cause the misexpression of key developmental genes may underlie a number of developmental abnormalities. The congenital abnormality preaxial polydactyly, extra digits, is an example of this novel class of mutations and is caused by ectopic expression of the signalling molecule Sonic Hedgehog (SHH) in the developing limb bud. Mutations in the long-distant, limb-specific cis-regulator for SHH, called the ZRS, are responsible for the ectopic expression which underlies the abnormality. Here, we show that populations of domestic cats which manifest extra digits, including the celebrated polydactylous Hemingway's cats, also contain mutations within the ZRS. The polydactylous cats add significantly to the number of mutations previously reported in mouse and human and to date, all are single nucleotide substitutions. A mouse transgenic assay shows that these single nucleotide substitutions operate as gain-of-function mutations that activate Shh expression at an ectopic embryonic site; and that the sequence context of the mutation is responsible for a variable regulatory output. The plasticity of the regulatory response correlates with both the phenotypic variability and with species differences. The polydactyly mutations define a new genetic mechanism that results in human congenital abnormalities and identifies a pathogenetic mechanism that may underlie other congenital diseases.

SummarySonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).

The maize VP1 protein is a seed-specific regulator of gene expression that effects the expression of a subset of abscisic acid (ABA)-regulated genes that are expressed during the maturation program of the seed. In addition, VP1 has pleiotropic effects on seed development that are not related to ABA. In transient expression assays, VP1 has been shown to transactivate gene expression through at least two distinct promoter elements: the G boxes from the ABA-inducible wheat Em gene and the SphI box from the maize C1 gene. We have investigated how VP1 can transactivate gene expression through diverse promoter elements by analyzing its association in vitro with EmBP-1, a factor that binds the Em promoter. We demonstrate that VP1 can greatly enhance the DNA binding activity of EmBP-1 in a gel retardation assay. This enhancing activity has also been observed on transcription factors as diverse as Opaque-2, Max, Sp1, and NF-B. Deletion of a small but highly conserved region (BR2) in VP1 eliminates the enhancement in vitro as well as the ability of VP1 to transactivate Em gene expression in a transient expression assay. A 40-amino acid fragment from VP1 sandwiched between the maltose-binding protein and LacZ can confer the enhancement function to this fusion protein in vitro. A weak and relatively nonspecific interaction between BR2 and DNA is demonstrated by UV cross-linking. The in vitro properties we observe for VP1 might explain the regulatory effects of VP1 on a diverse set of genes and why mutations in the vp1 locus have pleiotropic effects.

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

SUMMARYA late phase of HoxD activation is crucial for the patterning and growth of distal structures across the anterior-posterior (A-P) limb axis of mammals. Polycomb complexes and chromatin compaction have been shown to regulate Hox loci along the main body axis in embryonic development, but the extent to which they have a role in limb-specific HoxD expression, an evolutionary adaptation defined by the activity of distal enhancer elements that drive expression of 5Ј Hoxd genes, has yet to be fully elucidated. We reveal two levels of chromatin topology that differentiate distal limb A-P HoxD activity. Using both immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limb buds, and analysis in E10.5 dissected limb buds themselves, we show that there is a loss of polycomb-catalysed H3K27me3 histone modification and a chromatin decompaction over HoxD in the distal posterior limb compared with anterior. Moreover, we show that the global control region (GCR) long-range enhancer spatially colocalises with the 5Ј HoxD genomic region specifically in the distal posterior limb. This is consistent with the formation of a chromatin loop between 5Ј HoxD and the GCR regulatory module at the time and place of distal limb bud development when the GCR participates in initiating Hoxd gene quantitative collinearity and Hoxd13 expression. This is the first example of A-P differences in chromatin compaction and chromatin looping in the development of the mammalian secondary body axis (limb).

Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in keratins K5 (keratin 5) and K14 (keratin 14), with fragility of basal keratinocytes leading to epidermal cytolysis and blistering. Patients present with widely varying severity and are classified in three main subtypes: EBS Weber-Cockayne (EBS-WC), EBS Köbner (EBS-K), and EBS Dowling-Meara (EBS-DM), based on distribution and pattern of blisters. We could identify K5/K14 mutations in 20 out of the 43 families registered as affected by dominant EBS in Scotland; with previous studies this covers 70% of all Scottish EBS patients, making this the most comprehensively analyzed EBS population. Nine mutations are novel. All mutations lie within five previously identified rod domain hotspots and the severest blistering was associated with mutations in the helix boundary motifs. In some cases, the same mutation caused symptoms of EBS-WC and/or EBS-K, both within and between families, suggesting a contribution of additional factors to the phenotype. In some patients, no mutations were found in K5, K14, or K15, suggesting involvement of other genes. The results confirm that EBS is best considered as a single disorder with a spectrum of phenotypic variations, from severe EBS-DM at one extreme to mild EBS-WC at the other.

The introduction of a conditional centromere into chromosome III of Saccharomyces cerevisiae provided an opportunity to evaluate phenotypic and karyotypic consequences in cells harboring dicentric chromosomes upon entry into mitosis. A mitotic pause ensued, and monocentric derivatives of chromosome III were generated at a high frequency.The transmission of chromosomes in Saccharomyces cerevisiae can be manipulated effectively with a conditional centromere. The conditional centromere consists of a centromere that is regulated by the inducible GAL] promoter in yeast cells (6). Utilization of growth conditions that favor transcriptional induction (galactose as the carbon source) or repression (glucose as the carbon source) from the GAL] promoter results in centromere function that is switched off or on, respectively. In this paper, we describe the construction of a conditional dicentric chromosome. Observations made in a number of different organisms suggest diverse behaviors for dicentric chromosomes. These range from the extreme instability of chromosomes that undergo a breakage-fusion-bridge cycle through subsequent mitotic cell divisions (9,10) to the stable transmission of a chromosome with eight centromeres (13). With an increased understanding of the molecular structure of the centromere, this diversity of behavior may be explained.Acquisition of a dicentric chromosome results in mitotic lag. A conditional centromere was introduced into the left arm of chromosome III at the HIS4 locus, approximately 45 kilobases (kb) from CEN3 ( Fig. 1). Logarithmically growing cultures of strains containing this construction (J178-1D#7 MATa adel metl4 ura3 leu2 his3 HIS4::pBR322; URA3; GALCEN3) were switched from galactose-containing to glucose-containing medium. Cells appeared to be synchronous for mitosis after approximately 1.5 to 2 generations in the glucose-containing medium. The parent strain, J178-1D (MATa adel metl4 ura3 leu2 his3), did not exhibit a similar mitotic pause. A majority of the cells had buds that were equivalent in size to those of the mother cells. Staining with the fluorescent dye revealed that cells in the nuclear division stage were approximately three times more numerous in the experimental cultures than in control cultures raised in the galactose-containing medium.Monocentric derivatives. Figure 2 shows the electrophoretic karyotype of chromosomes after the carbon source switch. After two generations in the glucose-containing medium, a new chromosome band smaller than chromosome III was evident (Fig. 2, lane 1). This chromosome appeared when the mitotic lag was first observed. The intensity of this chromosome increased over time, while that of its wild-type counterpart decreased. It was apparent after eight generations (Fig. 2, lane 4) to the GALJ-GALJO probe, while the 370-kb chromosome III band in all lanes in Fig. 2 (WT to 6), as well as the 325-kb chromosome in lanes 1 to 6, contained sequences homologous to CEN3 (data not shown). The major rearrangement involving the dicentric chromosome i...