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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a powerful and widespread analytical tool in all fields of life science. The wide mass range (1-300 kDa), high accuracy, and sensitivity make it a superior method for analysis of all kinds of biomolecules including proteins, nucleic acids, and carbohydrates. In combination with 2D-gelelectrophoresis, MALDI-TOF-MS is particularly suitable for the identification of protein spots via mass fingerprint or microsequencing. Furthermore, the method allows a detailed analysis of posttranslational protein modifications. Recently, the method was also successfully applied to DNA sequencing as well as screening for mutations. Thus, high-throughput genotyping of single nucleotide polymorphisms has the potential to become a routine method for both laboratory and clinical applications.

Hyperekplexia or startle disease (stiff baby syndrome, STHE) is a hereditary neurological disorder characterised by an exaggerated startle response and infantile muscle hypertonia. Several autosomal dominant and recessive forms of the disorder have been associated with point mutations in GLRA1, the human gene encoding the a1 subunit of the inhibitory glycine receptor. Here, we describe a recessive point mutation (C1073G) in exon 7 of GLRA1 leading to an amino acid exchange of serine 231 to arginine in transmembrane region TM1. The mutation was detectable by restriction digest analysis of genomic PCR amplimers by matrixassisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Genotyping of family members was performed using an allele specific primer extension assay in combination with MALDI-TOF-MS and confirmed by conventional DNA sequencing. These studies demonstrate the broad applicability of MALDI-TOF-MS as a comparative screening tool applicable to the analysis of allelic gene variants. In comparison to the wild type a1 subunit, biochemical, electrophysiological, and confocal microscopy data indicate a reduced integration of functional a1 S231R glycine receptors into the cell surface membrane upon recombinant expression. Apparently, the amino acid exchange S231R influences glycine receptor biogenesis and cellular trafficking by introducing a positive charge into transmembrane region TM1.

Motivation: Analyses and algorithmic predictions based on high-throughput data are essential for the success of systems biology in academic and industrial settings. Organizations, such as companies and academic consortia, conduct large multi-year scientific studies that entail the collection and analysis of thousands of individual experiments, often over many physical sites and with internal and outsourced components. To extract maximum value, the interested parties need to verify the accuracy and reproducibility of data and methods before the initiation of such large multi-year studies. However, systematic and well-established verification procedures do not exist for automated collection and analysis workflows in systems biology which could lead to inaccurate conclusions.Results: We present here, a review of the current state of systems biology verification and a detailed methodology to address its shortcomings. This methodology named ‘Industrial Methodology for Process Verification in Research’ or IMPROVER, consists on evaluating a research program by dividing a workflow into smaller building blocks that are individually verified. The verification of each building block can be done internally by members of the research program or externally by ‘crowd-sourcing’ to an interested community. www.sbvimprover.comImplementation: This methodology could become the preferred choice to verify systems biology research workflows that are becoming increasingly complex and sophisticated in industrial and academic settings.Contact: gustavo@us.ibm.com

Thymosin L L 4 possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin L L 4 serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin L L 4 may serve as a useful tool for further investigations in cell biology. Thymosin L L 4 could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin L L 4 concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin L L 4 may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.z 1999 Federation of European Biochemical Societies.

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Background: Inherited defects in the DNA mismatch repair system lead to increased loss or gain of repeat units in microsatellites, commonly referred to as microsatellite instability (MSI). MSIs in coding regions of critical genes contribute to the pathogenesis of DNAmismatch repair-deficient cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). MSI typing is therefore increasingly used to guide the molecular diagnosis of HNPCC. Methods: We used matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify MSIs in mononucleotide repeats within the coding sequences of genes relevant to the pathogenesis of MSI؉ neoplastic lesions. After a primer extension reaction of PCR products encompassing the microsatellites, the molecular masses of the extension products were determined by MALDI-TOF-MS. Results: MSIs were detected by MALDI-TOF-MS in the GART, AC1, TGFBR2, MSH3, and MSH6 genes in neoplastic tissues and MSI؉ colorectal cancer cell lines but not in MSI؊ control tissues. The analysis of peakintegral ratios in a single spectrum of the peaks representing insertions or deletions compared with the full-

The evolution, inheritance and recombination rate of genes located in the pseudoautosomal region 1 (PAR1) is exceptional within the human genome. Pseudoautosomal genes are identical on X and Y chromosomes and are not inherited in a sex linked manner. Due to an obligatory recombination event in male meiosis, pseudoautosomal genes are exchanged frequently between X and Y chromosomes. During the isolation, characterization and sequencing of a novel gene PPP2R3L, which was classified by sequence homology as a novel member of the protein phosphatase regulatory subunit families, it became apparent that cosmids of different origin harboring this gene are highly polymorphic between individuals, both at the nucleotide level and in the number and sequence of tandem repeats.