Hereditary pancreatitis (HP) is a rare, early-onset genetic disorder characterized by epigastric pain and often more serious complications. We now report that an Arg-His substitution at residue 117 of the cationic trypsinogen gene is associated with the HP phenotype. This mutation was observed in all HP affected individuals and obligate carriers from five kindreds, but not in individuals who married into the families nor in 140 unrelated individuals. X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is probably part of a fail-safe mechanism by which trypsin, which is activated within the pancreas, may be inactivated; loss of this cleavage site would permit autodigestion resulting in pancreatitis.

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As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification.Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo.Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vphl-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pepl2::LEU2 mutant appeared defective by fluorescence-ratio microscopy and qulnacrineuptake assays, but the vacuolar pH in thepepl2::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.Vacuoles in Saccharomyces cerevisiae are analogous to lysosomes of other organisms. Both types of organelles contain similar sets of proteases and other hydrolytic enzymes (1, 2), and both are energized and acidified by similar membrane-bound, proton-translocating ATPases (H+-ATPase) (3-6). Vacuolar proteases are required for intracellular protein turnover induced by nitrogen starvation (7). Apart from that typically "lysosomal" activity, a variety of additional functions have been ascribed to the vacuole, including maintenance of cytosolic amino acid homeostasis (8, 9), regulation of cellular calcium metabolism (8, 10), and others (11). Genetic analysis of protease-deficient mutants has been instrumental in delimiting the physiological significance of the vacuolar proteases (7 METHODSStrains and Culture Conditions. The wild-type strain was the diploid formed by mating strains X2180-1A and X2180-1B. Mutant strains and genotypes were BJ4895, a/a vphl-J/ vphl-1 trpl/+ leu2/+ +/ura3-52 and BJ4984, a/a pepl2:: LEU2/pepl2::LEU2 ura3-52/ura3-52 leu2-1/leu2-1 hisl1+ ade6/+. Growth media YPD and SC have been described (12). Cells in the logarithmic-growth phase were prepared by overnight growth in YPD or SC (10 ml) at 30'C in roller tube cultures. Culture densities were maintained below 2 x 107 cell per ml by dilution with YPD or SC as required.Labeling with Fluorescent Dyes. Medium for labeling with 6-CF diacetate (6-CFDA; C1362; Molecular Probes) was prepared by adding 50 mM citric acid to YPD, adjusting the pH to 3.0 with 12 M HCl, autoclaving, and then filtering (0.2 gm membrane filter); 6-CFDA was added to the medium immediately before use by dilution from 5 mM stock solutions in dimethyl sulfoxide; unless otherwise noted, the final 6-CFDA concentration was 5 1LM. Logarithmic-phase cells were harvested by membrane filtration, resuspended at 2 X 107 cell per ml in the labeling medium, and incubated with shakin...

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.Dupuytren's contracture (DC) (OMIM 126900) is the most common connective tissue disease of mankind and has both heritable and sporadic forms. The inherited form is most frequently observed among the xanthochroi peoples of Northern Europe where its most common manifestations are thickening of the palmar fascia and contracture of the fingers. We ascertained a five-generation Swedish family in which DC is inherited in an autosomal dominant manner with high, but incomplete, penetrance by the end of the fifth decade. Blood was collected from all affected and informative unaffected family members for the performance of a genome-wide scan at a resolution of approximately 8 cM for all autosomes. Linkage was established to a single 6 cM region between markers D16S419 and D16S3032 on chromosome 16. A maximal two-point logarithm of odds (LOD) score of 3.18 was achieved at microsatellite marker D16S415 with four other markers in the region producing LODs of >1.5.

The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This

These data suggest that a gene for severe pediatric GER maps to chromosome 13q14. JAMA. 2000;284:325-334

Genetic epidemiological studies have shown that genetic factors are important in the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn disease (CD), and ulcerative colitis (UC). A genome screen in the United Kingdom found linkage of IBD to a 41-cM region of chromosome 12, surrounding D12S83. We aimed to replicate this linkage and to narrow the region of interest. Nonparametric linkage analyses at microsatellites surrounding D12S83 were performed in 122 North American Caucasian families containing 208 genotyped IBD-affected relative pairs. Transmission/disequilibrium tests (TDTs) were also performed. We confirmed that IBD is linked to chromosome 12 (peak GENEHUNTER-PLUS LOD* score 2.76 [P = .00016] between D12S1724 and D12S90). The evidence for linkage is contributed by both the group of CD-affected relative pairs (peak GENEHUNTER-PLUS LOD* score 1.79 [P = .0021] between D12S1724 and D12S90) and the group of UC-affected relative pairs (peak GENEHUNTER-PLUS LOD* score 1.82 [P = .0019] at D12S335). The TDT is positive at the D12S83 locus (global chi2 = 16.41, 6 df, P = .012). In conclusion, we have independently confirmed linkage of IBD to the chromosome 12 region that we investigated. A positive TDT at D12S83 suggests that we have greatly narrowed the chromosome 12 region that contains an IBD locus.

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus Ela zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. CeUls bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely smal vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37C, and were incapable of sporulating (as homozygotes). Fractionation of ceUs expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cels and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.The vacuole in Saccharomyces cerevisiae is a polymorphic organelle that has similarities both to vacuoles in plants and to lysosomes in animal cells. Like lysosomes, yeast vacuoles are relatively acidic organelles that contain a number of proteases and other hydrolases that have relatively broad substrate specificities. Mutants that lack particular vacuolar proteases have been useful for determining the functions of specific enzymes (23,25). One search for protease-deficient mutants unexpectedly identified 16 genes, mutations in any of which gave rise to deficiencies of two or three different vacuolar hydrolase activities (PEP genes) (21). Because pep mutations are pleiotropic, the phenotypes of the mutants provide very limited information about the primary functions of the PEP gene products. At the time the genes were identified, several general hypotheses were advanced to account for the pleiotropy of the mutations: that (i) these are regulatory mutations, (ii) the mutations cause changes in the structure of the compartments containing the enzymes, or (iii) the mutations alter components of the system that places the enzymes in the compartments (21). Thus, the PEP genes appeared to provide access to cellular "capital equipment" common to the expression of more than one vacuolar protease, but it was not clear in biochemical terms what type of equipment that might be. All three of the mechanisms mentioned above (or others not thought of)...