We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (⌬per9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced ⌬per9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.Peroxisomes are cell organelles that are present in virtually all eukaryotic cells. They perform specific metabolic functions that are often related to the developmental stage and/or the organism in which they occur (1). The metabolic importance of peroxisomes in humans is demonstrated by the fact that the absence of the organelles leads to severe abnormalities, followed by an early death (e.g. Zellweger syndrome (2)). Consequently, many studies are now devoted to unravel the molecular mechanisms of peroxisome biogenesis and function. Yeasts are excellent model systems for such studies having the advantages that (i) the induction and protein composition of peroxisomes can readily be manipulated by varying growth conditions and (ii) in the absence of peroxisomes, yeasts are viable (3, 4). Hence, peroxisome-deficient mutants have been isolated from different yeast species (4), and the corresponding genes are being cloned and characterized.In yeast, peroxisomes normally develop by growth and fission from pre-existing ones. Peroxisomal matrix proteins are nuclear-encoded, synthesized in the cytoplasm, and directed to the organelle by topogenic signals (PTSs).1 Two PTSs have been identified and are located either at the extreme C terminus (PTS1) or the N terminus of the protein (PTS2) (4). Our knowledge on the sorting of peroxisomal membrane proteins is still limited, and consensus topogenic sequences have yet to be identified (5).In our laboratory, we use the methylotrophic yeast Hansenula po...
An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-l-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50°C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.Both epichlorohydrin (3-chloro-1,2-epoxypropane) and its precursor in chemical synthesis, 1,3-dichloro-2-propanol, are industrial chemicals that may enter the environment because of their volatile character or improper disposal. These compounds are considered important environmental pollutants (12), in part because epichlorohydrin has been shown to be mutagenic and carcinogenic in rats (18).Recently, three bacterial cultures able to grow on epichlorohydrin, 3-chloro-1,2-propanediol, or 1,3-dichloro-2-propanol were isolated (20). Two of these cultures, Pseudomonas sp. strain AD1 and Arthrobacter sp. strain AD2, were examined in more detail. Degradation of epichlorohydrin proceeded via 3-chloro-1,2-propanediol and glycidol (3-hydroxy-1,2-epoxypropane). Arthrobacter sp. strain AD2 was not capable of enzymatic opening of the epoxide ring in epichlorohydrin, but Pseudomonas sp. strain AD1 possessed an epoxide hydrolase, producing 3-chloro-1,2-propanediol., It was shown that both strains dehalogenated haloalcohols by a highly ihducible enzyme which we call haloalcohol dehalogenase (20). The dehalogenase catalyzed the conversion of vicinal haloalcohols to the corresponding epoxides with liberation of inorganic chloride or bromide. The same mechanism of dehalogenation of haloalcohols was found earlier in a Flavobacterium sp. isolated on 2,3-dibromo-1-propanol (1, 3).The products of dehalogenation and the substrate range of the enzyme suggest that haloalcohol dehalogenases do not belong to other established classes of dehalogenases, such as haloacid dehalogenases (4, 15), haloalkane dehalogenases (6-8, 10, 16, 22), and dichloromethane dehalogenases (11,17). Since no information on the biochemical characteristics of haloalcohol dehalogenases is available, we purified and studied the enzyme from Arthrobacter sp. strain AD2 in more detail. We also present evidence that the dehalogenase of Pseudomonas sp. strain AD1 differs from the...
A progressive accumulation of genetic alterations underlies the adenoma-carcinoma sequence of colorectal cancer. This accumulation of mutations is driven by genetic instability, of which there are different types. Microsatellite instability (MSI) is the predominant type present in the tumours of Lynch syndrome patients and in a subset of sporadic tumours. It is generally accepted that MSI can be found in the early stages of tumour progression, such as adenomas; however, the frequencies reported vary widely among studies. Moreover, data on the qualitative differences between adenomas and carcinomas, or between tumours of hereditary and sporadic origin, are scarce. We compared MSI in samples of colorectal adenoma and colorectal carcinoma in order to identify possible differences along the adenoma-carcinoma sequence. We compared germline mismatch repair (MMR) gene mutation carriers and non-carriers, to address possible differences of instability patterns between Lynch syndrome patients and patients with sporadic tumours. We found a comparable relative frequency of mono- and dinucleotide instability in sporadic colorectal adenomas and carcinomas, dinucleotide instability being observed most frequently in these sporadic tumours. In MMR gene truncating mutation carriers, the profile was different: colorectal adenomas showed predominantly mononucleotide instability and in colorectal carcinomas, also more mononucleotide than dinucleotide instability was detected. We conclude that MSI profiles differ between sporadic and Lynch syndrome tumours, and that mononucleotide marker instability precedes dinucleotide marker instability during colorectal tumour development in Lynch syndrome patients. As mononucleotide MSI proves to be highly sensitive for detecting mutation carriers, we propose the use of mononucleotide markers for the identification of possible Lynch syndrome patients.
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