Molecular Biology

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not available. I S002 I BASjC TECHNIQUES AND SIMPLIFIED TERMINOLOGY OF MOLECULAR BIOLOGY Ch.C. Zouboulis. Department of Dermatology, University Medical Center Benjamin franklin. The Free University of Berlin, Berlin, Germany The impressive development in molecular genetics and molecular biology led from laboratory use to their clinical application into a short period of time. Therefore, clinicians are nowadays gut advised to become familiar with the basics of these new methods. Molecular biology includes research on three cellular molecule groups, namely DNA, RNA, and proteins. Isolation of chromosomal DNA from cells or human tissue and characterization of certain DNA fragments lead to determination of changed DNA (mutation) or foreign DNA introduced in human cells (infection). Detection of mutated or transfected DNA is not a proof for active disease; for that purpose transcription of DNA to messenger RNA (mRNA) is essential. It is noticeable that only 3-5% of the total DNA is transcribed to RNA. Detection of certain mRNA fragments after isolation of total RNA from human cells or tissue determines expression of its gene at this specific time point, probably followed by protein encoding. Common methods of DNA and RNA detection are fragmentation according to molecular size by gel electrophoresis. Southern (DNA) and Northern (RNA) blotting, and hybridization by radioactive or nonradioactive-labeled complementary DNA (cDNA) probes. DNA and RNA fragments can be analysed immediately after DNA or RNA isolation or after exponential amplification by polymerase chain reaction (PCR) or reverse transcription-PCR (reverse transcription of RNA to DNA and consequent PCR). Sequencing of DNA fragments is capable of resolving single-stranded oligonucleotides and identify consequent deoxynucleotides. RNA can be localized in cells or tissue by in situ hybridization. Proteins are responsible for gene function and are synthesized after translation of mRNA. Common methods of protein identification are oneor two-dimensional gel electrophoretic fragmentation according to protein molecular size and pH value and Western blotting, i.e. hybridization with specific monoclonal antibodies. Protein sequencing leads to identification of consequent aminoacids. I S003 I MOLECULAR GENETICS IN PEDIATRIC DERMATOLOGY G. Zambruno. Laboratory of Molecular and Cellular Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy Substantial progress has been made recently in unravelling the molecular basis of various inheritable skin disorders, such as ichthyoses, albinism, xeroderma pigmentosum, etc. Inherited epidermolysis bullosa (EB), a group of mechanobullous disorders of the skin and mucous membranes, will be examined as a paradigm of the latest advances in molecular genetics applied to pediatric dermatology. EB are divided into three major forms (i. e. simplex, junctional and dystrophic), on the basis of the level of cleavage within the cutaneous basement membrane zone (BMZ). In the simplex forms, tissue separation occurs within the basal keratinocyte as a result of mutations in the genes encoding the basal keratins, K5 and K14. In junctional EB, separation takes place within the lamina lucida of the BMZ, and mutations in the genes encoding different components of the hemidesmosome-anchoring filament complexes (laminin 5, BP180, /34 integrin subunit) have recently been identified. Mutations in the type VII collagen gene, the major component of anchoring fibrils, have been shown to underlie dystrophic EB which is characterized by tissue separation below the lamina densa. Identification of the genes responsible for the different EB forms has had a direct clinical application, enabling DNAbased, first trimester prenatal diagnosis rather than the more risky and delayed fetal skin biopsy. Moreover, characterization of the genetic abnormality has allowed genotype-phenotype relationships to be established and represents the basis for future gene or drug therapy approaches. I S004 I MOLECULAR METHODS IN THE DIAGNOSIS OF MICROBIAL DISEASES E. Tschachler. Department of Dermatology, University of Vienna, Vienna, Austria During the last decade the introduction of molecular-biological methods has initiated a revolution in the diagnosis of infectious diseases. New technologies, e.g. the polymerase chain reaction (PCR), the ligase chaine reaction (LCR) and the in situ hybridization, have made it possible to quickly and reproducibly detect minute amounts of infectious agents in tissue samples and excretions. In contrast to long and tedious culturing procedures for bacteria and viruses and difficult to interpret serological tests, direct and accurate identification of various pathogens can be achieved nowadays within a few hours. Examples for tests which are widely used by now in routine laboratories include the diagnosis of chlamydial infection by PCR and LCR, as well as the identification and typing of human papilloma viruses, herpes viruses, certain mycobacteria and spirochetae by PCR and Southern hybridization. In addition to their importance for S6 Symposia — Dermatopharmacology routine diagnostic, molecular methods have boosted the search for new microbial pathogens linked to dermatological diseases. The detection of /?. henseiae in bacillary angiomatosis and human herpes virus type 8 in Kaposi's sarcoma are the most prominent examples which, without doubt, will be followed by many more in the near future. I S005 I MOLECULAR GENETIC ANALYSIS OF CUTANEOUS