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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.

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.