SummarySister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin’s Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin’s association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase-independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation.

Mutation screening of the major autosomal dominant polycystic kidney disease (ADPKD) locus, PKD1, has proved difficult because of the large transcript and complex reiterated gene region. We have developed methods, employing long polymerase chain reaction (PCR) and specific reverse transcription-PCR, to amplify all of the PKD1 coding area. The gene was screened for mutations in 131 unrelated patients with ADPKD, using the protein-truncation test and direct sequencing. Mutations were identified in 57 families, and, including 24 previously characterized changes from this cohort, a detection rate of 52.3% was achieved in 155 families. Mutations were found in all areas of the gene, from exons 1 to 46, with no clear hotspot identified. There was no significant difference in mutation frequency between the single-copy and duplicated areas, but mutations were more than twice as frequent in the 3' half of the gene, compared with the 5' half. The majority of changes were predicted to truncate the protein through nonsense mutations (32%), insertions or deletions (29.6%), or splicing changes (6.2%), although the figures were biased by the methods employed, and, in sequenced areas, approximately 50% of all mutations were missense or in-frame. Studies elsewhere have suggested that gene conversion may be a significant cause of mutation at PKD1, but only 3 of 69 different mutations matched PKD1-like HG sequence. A relatively high rate of new PKD1 mutation was calculated, 1.8x10-5 mutations per generation, consistent with the many different mutations identified (69 in 81 pedigrees) and suggesting significant selection against mutant alleles. The mutation detection rate, in this study, of>50% is comparable to that achieved for other large multiexon genes and shows the feasibility of genetic diagnosis in this disorder.

Abstract. The severity of renal cystic disease in the major form of autosomal dominant polycystic kidney disease (PKD1) is highly variable. Clinical data was analyzed from 324 mutation-characterized PKD1 patients (80 families) to document factors associated with the renal outcome. The mean age to end-stage renal disease (ESRD) was 54 yr, with no significant difference between men and women and no association with the angiotensin-converting enzyme polymorphism. Considerable intrafamilial variability was observed, reflecting the influences of genetic modifiers and environmental factors. However, significant differences in outcome were also found among families, with rare examples of unusually late-onset PKD1. Possible phenotype/genotype correlations were evaluated by estimating the effects of covariants on the time to ESRD using proportional hazards models. In the total population, the location of the mutation (in relation to the median position; nucleotide 7812), but not the type, was associated with the age at onset of ESRD. Patients with mutations in the 5' region had significantly more severe disease than the 3' group; median time to ESRD was 53 and 56 yr, respectively (P ϭ 0.025), with less than half the chance of adequate renal function at 60 yr (18.9% and 39.7%, respectively). This study has shown that the position of the PKD1 mutation is significantly associated with earlier ESRD and questions whether PKD1 mutations simply inactivate all products of the gene.

A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesionDNA has been suggested to enter ring-shaped cohesin complexes via a structural maintenance of chromosome (SMC) protein dimerization interface. Structure-based analyses now show that a positively charged channel within this hinge region, while being dispensable for SMC dimerization and cohesin loading, is essential for cohesin acetylation and establishment of sister chromatid cohesin during S phase.

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.

Dictyostelium discoideum is an excellent system in which to study developmental decisions. Synchronous development is triggered by starvation and rapidly generates a limited number of cell types. Genetic and image analyses have revealed the elegant intricacies associated with this simple development system. Key signaling pathways identified as regulating cell fate decisions are likely to be conserved with metazoa and are providing insight into differentiation decisions under circumstances where considerable cell movement takes place during development.

GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.GSK-3 is a multifunctional serine/threonine protein kinase that regulates a large number of key eukaryotic cellular processes, including intermediate metabolism, cytoskeleton maintenance, and development (12). A significant question is how GSK-3 can be active in a broad range of signal pathways yet retain pathway specificity. For example, in the same cell, insulin can control glycogen synthesis and Wnt can control ␤-catenin protein levels, both in a GSK-3-dependent manner but without crossover between the two pathways (10

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis.