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Methylierung von Cytosin zu 5-Methylcytosin (5mC) dient der epigenetischen Kontrolle der Gentranskription in Saugetieren und hat direkte Auswirkungen auf die Entwicklung und Pathogenese des Menschen. Insbesondere die Diagnose und Prognostik von Krebspatienten profitierte in den letzten Jahren enorm von der Entwicklung neuer Methoden zur Analyse von DNA-Methylierungsmustern. Wir zeigen hier, dass zwei hitzestabile DNA-Polymerasen – die DNA-Polymerase KlenTaq aus Thermus aquaticus und die KOD-DNA-Polymerase aus Thermococcus kodakaraensis – 3′-fehlgepaarte Oligonukleotid-Primer gegenuber 5mC effektiver verlangern als gegenuber nichtmethyliertem C. Wir konnten auserdem eine DNA-Polymerase-Mutante mit weiter verbesserter 5mC/C-Diskriminierung erzeugen. Diese Mutante wurde erfolgreich in einer neuartigen 5mC-Detektierungsmethode eingesetzt, die es ermoglicht, den Methylierungszustand eines Cytosins direkt durch PCR von unbehandelter genomischer DNA zu bestimmen.

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

Antibacterial proteins like lysozyme are important components of the insect non-specific immune response against bacteria. The complementary deoxyribonucleic acid (cDNA) encoding a new lysozyme from Triatoma infestans, named lysozyme2, has been amplified by polymerase chain reaction and the rapid amplification of cDNA ends technique. The gene is expressed in the small intestine of the insect. The deduced protein sequence shows up to 70% similarity to lysozymes from other species. Furthermore, the protein exhibits significant structural concordance to other insect lysozymes. A striking feature of the lysozyme2 protein is the replacement of the conserved amino acid residues of the active site of classical c-type lysozymes, glutamate and aspartate, by valine and tyrosine.

Background: Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. Methods: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. Results: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. Conclusion: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO2 acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO2-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO2/bicarbonate regulation of Cyr1p. Disruption of the CO2/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO2 sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO2-signalling system., Author Summary Pathogenic microorganisms can produce a variety of secondary metabolites and signalling molecules which can affect the host, or provide them with a selective advantage against competing commensal organisms. We demonstrate that gaseous, metabolically generated CO2 can serve as a signalling molecule to enhance the organism's virulence during infection establishment by using the fungal pathogen Candida albicans as a model. Furthermore, we identified a CO2 receptor site within the catalytic domain of the soluble adenylyl cyclase, Cyr1p, which is critical for CO2 sensing and hence virulence of the organism. CO2 sensing is conserved in a variety of pathogenic species, and increased levels have been shown to suppress the host's immune system. Thus, CO2 sensing may represent a mechanism to enhance C. albicans virulence when the host's immune system is suppressed.