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- Clendenning, M, et al.
(författare)
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A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome
- 2008
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Ingår i: Journal of Medical Genetics. - : BMJ. - 0022-2593 .- 1468-6244. ; 45:6, s. 340-345
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Tidskriftsartikel (refereegranskat)abstract
- Background: When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (<2% of all identified mutations), yet the immunohistochemical analysis of tumour samples indicates that approximately 5% of Lynch syndrome cases are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. Methods: Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based on immunohistochemical analysis. Results: We have identified a frequently occurring frame-shift mutation (c.736_741del 6ins11) in 12 ostensibly unrelated Lynch syndrome patients (20% of patients we have identified with a deleterious mutation in PMS2, n = 61). These individuals all display the rare allele (population frequency <0.05) at a single nucleotide polymorphism (SNP) in exon 11, and have been shown to possess a short common haplotype, allowing us to calculate that the mutation arose around 1625 years ago (65 generations; 95% confidence interval 22 to 120). Conclusion: Ancestral analysis indicates that this mutation is enriched in individuals with British and Swedish ancestry. We estimate that there are >10 000 carriers of this mutation in the USA alone. The identification of both the mutation and the common haplotype in one Swedish control sample (n = 225), along with evidence that Lynch syndrome associated cancers are rarer than expected in the probands' families, would suggest that this is a prevalent mutation with reduced penetrance.
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- Alinezhad, S., et al.
(författare)
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Expression of keratinase gene in Bacillus megaterium using an expression vector of pHIS1525.SPlipA and utilization of the resulting recombinant strain for chicken feather degradation prior to biogas production
- 2009
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- An increasing quantity of chickens is being utilized annually in the poultry industry, producing a huge volume of chicken feather waste which presents a high quality supply of keratin. Keratinases possessing high level of keratinolytic activity on insoluble keratin play a crucial role in hydrolyzing chicken feathers. Ever since the discovery of proteolytic ability as well as water solubility of keratinase, many industrial processes regarding keratinase application have been developed. A recently invented application to handle poultry waste is to utilize feathers for biogas production. Obviously, large amount of keratinase is required to break down the keratin prior to further conversion to biogas. Previously, several researches have shown that certain bacteria are able to produce keratinase but it is still a challenge to find out which bacteria is the most reliable source for the production with high efficiency. These challenges gave rise to the molecular biologists to bring the focus on gene cloning to develop recombinant strains resulting in overproduction of keratinase. Over the course of various cloning and expression experiments of similar proteins, it was found that Bacillus megaterium could be a susceptible host cell for keratinase production. In our study, the keratinase gene from the chromosomal DNA of Bacillus licheniformis ATCC®53757 was PCR amplified and subsequently cloned into Bacillus megaterium expression vector, pHIS1525.SPlipA. Bacillus megaterium ATCC®14945 strain was transformed with the recombinant plasmid, pKERHIS1525.SPlipA. The KER gene was expressed under xylose inducible promoter, and the product was then purified using Ni-NTA affinity chromatography. After 18 h of incubation an extracellular keratinase activity of 29U ml-1 was achieved (one unit of activity was determined as the amount of enzyme required to an increase of 0.01 in A420 after 30 min of incubation at 37°C). The recombinant strain was further examined for feather degradation using intact chicken feather waste as carbon source. The chopped chicken feathers were partially degraded by the recombinant strain after three days of incubation and the total macroscopic digestion was ultimately observed after seven days resulting in a yellowish peptide rich fermentation broth. The biogas potential of the hydrolysate will be compared with that of untreated feathers by performing anaerobic batch digestion experiments.
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- Kron, J., et al.
(författare)
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Measurements and modelling of air gap formation in aluminium based alloys
- 2005
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Ingår i: International Journal of Cast Metals Research. - : Maney Publishing. - 1364-0461 .- 1743-1336. ; 18:1, s. 29-40
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Tidskriftsartikel (refereegranskat)abstract
- The formation of an air gap has been experimentally studied during solidification of several aluminium based alloys. Air gap widths and temperature distribution have been measured during solidification in a cylindrical mould. The effect of grain refinement on heat transfer and air gap formation has been studied. Mathematical modelling has been performed to increase the understanding of the solidification process and air gap formation. A model was developed for description of air gap formation in alloys solidifying with varying solidification intervals. The model includes the effect of formation and condensation of lattice defects on the solidification process and air gap formation. The calculated shrinkage using this model shows good agreement with the experimental data.
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- Mirabdollah, A., et al.
(författare)
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Optimization of a protoplast transformation method for Bacillus Subtilis, Bacillus megaterium, and Bacillus Cereus by a plasmid pHIS1525.SplipA
- 2009
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- During the past years of gene cloning studies, Escherichia coli has always been a foremost host cell for exogenous genes expressions owing to its high level of protein production and excretion. However, problems relating to low level of extracellular production of some proteins specially the accumulation of cloned proteases within the cells have moved the attentions from E.coli to bacilli bacteria such as B. megaterium, B.subtilis, and B.cereus due to their secretion ability of many different enzymes. Bacillus megaterium is widely used for high-level expression of heterologous proteins with little or no degradation. Bacillus subtilis is a naturally competent host cell for uptake of exogenous DNA, resulting in attractive industrial applications. Bacillus cereus has sporulation capability which makes it suitable for several industrial uses. A conventional approach for transferring DNA into protoplasts or intact cells of bacillus bacteria is chemical transformation, using chemicals through chilling and then shock-heating of the suspension of cells to induce reversible permeabilization of the cell membrane to make it possible for the external DNA to enter into the cells. In most cloning experiments, the transformation with plasmid DNA is performed using Polyethylene glycol (PEG)-induced competence cells. In this study, a PEG-induced protoplast transformation protocol was developed for three different bacillus strains of Bacillus megaterium ATCC®14945, Bacillus Subtilis ATCC®6051, and Bacillus Cereus ATCC®14579. In all cases a plasmid pHIS1525.SPlipA, well working vector in B.megaterium, was applied. Protoplasts were formed in RHAF medium after treating the cells with lysozyme. Two factors, the incubation time and the lysozyme concentration have been found to play the most important role in effective protoplast formation. These two factors were further optimized in this study to elaborate a chemical transformation procedure which can possibly work for other bacillus strains as well. The optical density (A420) and the number of colony-forming units (CFUs) were determined to find the optimal conditions for each strain. The results indicate that PEG-induced protoplast transformation is a sufficient technique when using a plasmid pHIS1525.SPlipA in Bacillus genus.
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- Thuswaldner, Sophie, 1976-, et al.
(författare)
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Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis
- 2007
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Ingår i: J Biol Chem. - 0021-9258 .- 1083-351X. ; 282:12, s. 8848-59
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Tidskriftsartikel (refereegranskat)abstract
- In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.
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