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Entwicklung, Implementierung und Evaluierung eines neuen Alignmentalgorithmus f��r paarweises Sequenzalignment von Nukleotid- und Proteinsequenzen.

Early-onset generalized dystonia represents the severest form of dystonia, a hyperkinetic movement disorder defined by involuntary twisting postures. Although frequently transmitted as a single-gene trait, the molecular basis of dystonia remains largely obscure. By whole-exome sequencing a parent-offspring trio in an Austrian kindred affected by non-familial early-onset generalized dystonia, we identified a dominant de novo frameshift mutation, c.6406delC (p.Leu2136Serfs∗17), in KMT2B, encoding a lysine-specific methyltransferase involved in transcriptional regulation via post-translational modification of histones. Whole-exome-sequencing-based exploration of a further 30 German-Austrian individuals with early-onset generalized dystonia uncovered another three deleterious mutations in KMT2B—one de novo nonsense mutation (c.1633C>T [p.Arg545∗]), one de novo essential splice-site mutation (c.7050−2A>G [p.Phe2321Serfs∗93]), and one inherited nonsense mutation (c.2428C>T [p.Gln810∗]) co-segregating with dystonia in a three-generation kindred. Each of the four mutations was predicted to mediate a loss-of-function effect by introducing a premature termination codon. Suggestive of haploinsufficiency, we found significantly decreased total mRNA levels of KMT2B in mutant fibroblasts. The phenotype of individuals with KMT2B loss-of-function mutations was dominated by childhood lower-limb-onset generalized dystonia, and the family harboring c.2428C>T (p.Gln810∗) showed variable expressivity. In most cases, dystonic symptoms were accompanied by heterogeneous non-motor features. Independent support for pathogenicity of the mutations comes from the observation of high rates of dystonic presentations in KMT2B-involving microdeletion syndromes. Our findings thus establish generalized dystonia as the human phenotype associated with haploinsufficiency of KMT2B. Moreover, we provide evidence for a causative role of disordered histone modification, chromatin states, and transcriptional deregulation in dystonia pathogenesis.

Kappa-casein (CSN3) plays an important role in stabilising the Ca-sensitive caseins in the micelle. The European rabbit (Oryctolagus cuniculus) CSN3 has previously been shown to possess two alleles (A and B), which differ deeply in their intronic regions (indels of 100 and 1550 nucleotides in introns 1 and 4, respectively). Furthermore, a correlation between several reproductive performance traits and the different alleles was described. However, all these data were exclusively collected in rabbit domestic breeds, preventing a deeper understanding of the extensive polymorphism observed in the CSN3 gene. Additionally, the techniques available for the typing of both indel polymorphisms were until now not suitable for large-scale studies. In this report, we describe a simple, PCR-based typing method to distinguish rabbit CSN3 alleles. We analyse both ancient wild rabbit populations from the Iberian Peninsula and France, and the more recently derived English wild rabbits and domestic stocks. A new allele (C) showing another major indel (250 bp) in intron 1 was found, but exclusively detected in Iberian wild rabbits. In addition, our survey revealed the occurrence of new haplotypes in wild populations, suggesting that intragenic recombination is important in creating genetic diversity at this locus. This easy and low cost single-step PCR-based method results in an improvement over previous described techniques, can be easily set up in a routine molecular laboratory and would probably be a valuable tool in the management of rabbit domestic breeds.

The genes that are part of the somatotropic axis play a crucial role in the regulation of growth and development of chickens. The identification of genetic polymorphisms in these genes will enable the scientist to evaluate the biological relevance of such polymorphisms and to gain a better understanding of quantitative traits like growth. In the present study, 75 pairs of primers were designed and four chicken breeds, significantly differing in growth and reproduction characteristics, were used to identify single nucleotide polymorphisms (SNP) using the denaturing high performance liquid chromatography (DHPLC) technology. A total of 283 SNP were discovered in 31 897 base pairs (bp) from 12 genes of the growth hormone (GH), growth hormone receptor (GHR), ghrelin, growth hormone secretagogue receptor (GHSR), insulin-like growth factor I and II (IGF-I and -II), insulin-like growth factor binding protein 2 (IGFBP-2), insulin, leptin receptor (LEPR), pituitary-specific transcription factor-1 (PIT-1), somatostatin (SS), thyroid-stimulating hormone beta subunit (TSH-β). The observed average distances in bp between the SNP in the 5'UTR, coding regions (non- and synonymous), introns and 3'UTR were 172, 151 (473 and 222), 89 and 141 respectively. Fifteen non-synonymous SNP altered the translated precursors or mature proteins of GH, GHR, ghrelin, IGFBP-2, PIT-1 and SS. Fifteen indels of no less than 2 bps and 2 poly (A) polymorphisms were also observed in 9 genes. Fifty-nine PCR-RFLP markers were found in 11 genes. The SNP discovered in this study provided suitable markers for association studies of candidate genes for growth related traits in chickens.

The genetic variability of the mitochondrial D-loop DNA sequence in seven horse breeds bred in Italy (Giara, Haflinger, Italian trotter, Lipizzan, Maremmano, Thoroughbred and Sarcidano) was analysed. Five unrelated horses were chosen in each breed and twenty-two haplotypes were identified. The sequences obtained were aligned and compared with a reference sequence and with 27 mtDNA D-loop sequences selected in the GenBank database, representing Spanish, Portuguese, North African, wild horses and an Equus asinus sequence as the outgroup. Kimura two-parameter distances were calculated and a cluster analysis using the Neighbour-joining method was performed to obtain phylogenetic trees among breeds bred in Italy and among Italian and foreign breeds. The cluster analysis indicates that all the breeds but Giara are divided in the two trees, and no clear relationships were revealed between Italian populations and the other breeds. These results could be interpreted as showing the mixed origin of breeds bred in Italy and probably indicate the presence of many ancient maternal lineages with high diversity in mtDNA sequences.

The mammalian sex determining gene, SRY, is the founding member of the new growing family of Sox (SRY-like HMG-box gene) genes. Sox genes encode transcription factors with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the existence of Sox genes in the rice field eel, Monopterus albus, and DNA sequence information of the HMG box region of five Sox genes. The Sox1, Sox4 and Sox14 genes do not have introns in the HMG box region. The Sox9 gene and Sox17 gene, which each have an intron in the conserved region, show strong identity at the amino acid level with the corresponding genes of mammals and chickens. Similar structure and identity of the Sox9 and Sox17 genes among mammals, chickens and fish suggest that these genes have evolutionarily conserved roles, potentially including sex determination and differentiation.

In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 3' regions.

The multifaceted repertoire of non-protein-coding RNAs (ncRNAs) in organisms of all three domains of life emphasizes their fundamental role in biology. Research in my lab focuses on revealing the regulatory and catalytic function of small and large ncRNAs in different model organisms. In particular we are interested in understanding ncRNA/protein complexes such as the vault complex or the ribosome. The ribosome, the central enzyme of protein biosynthesis, is a multifunctional ribonucleoprotein particle composed of two unequal subunits that translates the genome's message into all proteins needed for life. The crucial role the translation machinery plays in gene expression is also mirrored by the fact that the ribosome represents the main target for antibiotics. Decades of genetic, biochemical and recent crystallographic studies revealed the ribosome as an RNA-enzyme with roots in the 'RNA world'. Despite these experimental insights, the catalytic and regulatory mechanisms of the ribosomal RNA are still not fully understood at the molecular level. To unravel the detailed contributions of rRNA nucleotides for protein synthesis we have developed and applied an 'atomic mutagenesis' approach. This tool allows the role of specific 23S rRNA functional groups and even individual atoms to be studied during various stages of the ribosomal elongation cycle with thus far unequalled precision. This experimental approach bridges the disciplines of biochemistry and organic chemistry and has recently revealed specific functional 23S rRNA groups involved in peptide bond synthesis, peptidyl-tRNA hydrolysis, GTPase activation, and tRNA translocation.

Single-molecule spectroscopy allows the direct observation of conformational dynamics in individual biomolecules. Here, we describe how single-molecule Förster resonance energy transfer (smFRET) reveals heterogeneous kinetics in the EBS1*/IBS1* interaction, two RNA sequences that play an important role in group II intron mediated self-cleavage. Further examples of dynamic heterogeneity in functional nucleic acids are provided and the possible origins of this phenomenon are discussed.

Modified nucleoside triphosphates (dNTPs) are a versatile platform for the generation of high-density functionalized nucleic acids. The enzymatic polymerization of dNTPs allows the introduction of sensible functionalities that might not be compatible with the standard automated synthesis of oligonucleotides. Their application to in vitro selections, an elegant chemical approach to Darwinian evolution, delivers modified aptamers and catalytic nucleic acids with potentially enhanced properties. This review article highlights some recent synthetic examples of dNTPs bearing functionalities that are either found in the active site of protein enzymes or have been employed in organocatalysis and further underscores their usefulness in the development of some modified catalytic nucleic acids with special emphasis on M2+-independent RNA-cleaving DNAzymes.

Defects in the FANCJ/BRIP1 helicase gene are associated with genome instability disorders such as familial breast cancer or Fanconi anemia (FA). Although FANCJ has an in vitro activity to resolve G-quadruplex (G4) structures, and FANCJ ortholog in C. elegans prevents G4-associated deletions during replication, how FANCJ loss affects genome integrity in higher organisms remains unclear. Here, we report that FANCJ, but not other FA genes FANCD2 or FANCC, protected against large-scale genomic deletion that occurred frequently at the rearranged immunoglobulin heavy chain (IgH) locus in chicken DT40 cell line, suggesting that FancJ protects the genome independently of the FA ubiquitination pathway. In a more unbiased approach using array-comparative genomic hybridization, we identified de novo deletions as well as amplifications in fancj cells kept in culture for 2 months. A cluster of G4 sequence motifs was found near the breakpoint of one amplified region, but G4 sequence motifs were not detected at the breakpoints of two deleted regions. These results collectively suggest that, unlike in C. elegans, actions of vertebrate FANCJ to promote genome stability may not be limited to protection against the G4-mediated gene deletions.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

For several anatomical localisations of extranodal marginal zone B-cell lymphoma of MALT type (eMZBCL), an association with chronic inflammation caused by microbiological agents (e.g. Helicobacter pylori in the stomach) has been described. In the lung, a link between lymphomagenesis and a defined causative organism is still missing. A comprehensive diversity survey using 16S-rDNA library construction followed by restriction fragment length polymorphism (RFLP) analysis, sequencing, and phylogenetic tree construction was employed for nine cases each of BALT lymphoma and control lung tissues (normal foetal lung, pneumonitis, carcinoid). This highly sensitive method, hereafter termed SHARP screening allowed for identification of the entire bacterial population in the tissue in a cultivation-independent manner. It was noteworthy that in eight of the nine cases of BALT lymphoma, bacteria of the Alcaligenaceae family (Alcaligenes, Achromobacter, AKIW733), were detected, whereas none of the control cases showed the presence of these clades. 16S-rDNA library construction in combination with RFLP screening and phylogenetic analyses, hereafter described as SHARP screening, is a cultivation-independent tool for analysing the microbial environment in chronic inflammation processes giving rise to extranodal marginal zone B-cell lymphomas of MALT-type. Betaproteobacteria of the Alcaligenaceae family may be affiliated with and possibly involved in the lymphomagenesis of BALT lymphomas.

Enzootic bovine leukaemia (EBL) which is caused by the bovine leukaemia virus (BLV) still plays a remarkable role despite a significant success in sanitation programmes. In the Federal Republic of Germany it was not possible to eradicate the disease until today. Sporadically during slaughter or necropsy of cattle neoplastic lesions of the lymphatic tissues are observed that need to be clarified with regard to BLV as etiological agent. Due to the fact that in most instances no serological data are available from the respective animals and blood drawings from the original holdings are not easy to obtain the polymerase chain reaction (PCR) opens new avenues as supplementary diagnostic tool to test unfixed lymphatic tissues for the presence of BLV proviral DNA. Lymph node tissues from 10 naturally or experimentally BLV-infected cattle, which have been monitored virologically and serologically, and tissues from 4 negative animals were processed, DNA was extracted and subjected to PCR to amplify BLV env gene specific sequences. The results show that in cattle with BLV-induced leukosis as well as in cattle, which were clinically healthy and unsuspicious at slaughter or at post-mortem, either with persistent lymphocytosis (PL) or without, BLV proviral DNA could be detected easily in samples of lymphatic tissues and in high concordance with serological data. In this article data from the National and OIE reference laboratory for EBL at the Friedrich-Loeffler-Institut (FLI, Germany) are presented. Elaborated laboratory protocols for processing of tissue samples and performing of BLV-PCR are recommended.

During regular health status monitoring of the colony of amphibian, Mycobacterium (M.) gordonae were isolated from granulomatous lesions of the tiptoes from the South African clawed frog (Xenopus laevis) maintained at the Tierforschungszentrum of the University of Ulm. During a period of three years a total of 21 animals of the colony, consisting of 350-400 frogs, showed granuloma of the tip of the toes and a loss of the claws. The general condition and the behavior of the frogs appeared to be unchanged. Using a selective medium one isolate was cultured and identified by sequencing of the 16S rRNA gene. To apply a rapid diagnostic method for detecting mycobacteria, in particular M. gordonae in the health monitoring programme of the Xenopus laevis colony, we established the rpoB gene PCR followed by HaeIII restriction analysis of the PCR product. We identified M. gordonae from granuloma of the tiptoes and from unaltered tissue samples of the lungs and skin by PCR restriction analysis. Since mycobacterial species apparently are widespread in granulomatous lesions of the tiptoes of Xenopus laevis, we hypothesize a pathogenic potential. This view is supported by an increasing number of reports in the literature on infections with nontuberculous, "non-pathogenic" mycobacteria in Xenopus laevis.

GIPC is highly expressed in human pancreatic adenocarcinoma and is a central protein for the stability of IGF-1R in pancreatic adenocarcinoma cell lines (15). The goal of this study was to prove the importance of GIPC in vivo and to evaluate possible therapeutic strategies that target this protein and its PDZ domain. In vivo effects of GIPC knockout were studied after lentiviral transduction of luciferase-expressing MiaPaCa2 pancreatic cancer cells with shRNA against GIPC; growth characteristics were monitored with bioluminiscence. Knockdown of GIPC led to a significant inhibition of pancreatic tumor cell growth in vivo in different mouse models. To test a possible therapeutic approach, the PDZ domain of GIPC was targeted by a short peptide composed of the amino acid sequence PSQSSSEA. This octapeptide was designed based on the C-terminal binding motif of GAIP. Targeting GIPC with this peptide inhibited the association between IGF-1R and GIPC. The subsequent downregulation of IGF-1R decreased proliferation in vitro and in vivo. In conclusion, our findings suggest that targeting GIPC and its PDZ domain-mediated interaction with the tyrosine kinase receptor IGF-1R could be a promising new treatment option for pancreatic cancer.

Lobular carcinoma in situ (LCIS) of the breast typically is an incidental finding in breast biopsies performed for a variety of reasons. As patients with LCIS have a bilaterally increased risk of developing invasive ductal or lobular breast cancer, the lesion is presently considered an indicator rather than a direct precursor of breast cancer. However, this view is challenged by the finding that LCIS often accompanies invasive lobular carcinomas (ILC) and that the frequency of ILC following LCIS is far higher compared to unselected patients. To further examine the role of LCIS in the development of invasive breast cancer, we analysed a series of patients with pure LCIS, who later developed invasive breast cancer. Mitochondrial D-loop sequencing revealed a clonal relationship of LCIS and a subset of ILC occurring in the same breast between 2 and 10 years later. Apparently, LCIS has a chimeric role in breast cancer development and is not only a risk factor but in some cases a precursor of ILC.

Although TP53 is one of the most studied genes/proteins in ovarian carcinomas, the predictive value of TP53 alterations is still ambiguous.We performed analyses of the TP53 mutational status and its protein expression by immunohistochemistry. Moreover, the single nucleotide polymorphism SNP309 in the P2-promotor of the HDM2 gene was investigated. We correlated the results with the age of onset and the outcome of 107 ovarian carcinoma patients.In our study, we identified a large group of patients with TP53 overexpression despite having a wild-type gene (49% of all patients with wild-type TP53). This was associated with a significantly shortened overall survival time (p = 0.019). Patients with TP53 alterations (especially those with overexpression of wild-type TP53) were also more refractory to chemotherapy than patients with normal TP53 (p = 0.027). The Gallele of the SNP309 is associated with an earlier age of onset in estrogen receptor expressing FIGO stage III patients (p = 0.048). In contrast, in FIGO III patients, a weakened TP53 pathway (either G-allele of SNP309 or a TP53 mutation) is correlated with an increased overall survival compared with patients whose tumors are wild-type for TP53 and SNP309 (p = 0.0035).Our study provides evidence that both germ line and somatic alterations of the TP53 pathway influence incidence and survival of ovarian carcinoma, and it underscores the importance of assessing the functionality of TP53 in order to predict sensitivity of platin-based chemotherapies and patient outcome.

The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P (BLOOD, 105, 2535–42, 2005). For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (p

Molecular alterations in the tyrosine kinase (TK) domain of the human epidermal growth factor receptor (EGFR) have been correlated with tumour remission upon treatment with the TK inhibitor Gefitinib in non-small cell lung cancer (NSCLC). We have retrospectively investigated the correlation of point mutations with clinical response and, based on our retrospective results, used predictive molecular assessment as the basis for treatment in one patient.Mutational analysis was performed in 11 NSCLC-patients (10 responders, 1 non-responder) among 62 patients treated with Gefitinib within an expanded access program.Activating molecular alterations were found in 8/11 investigated samples (point mutations in exons 18 and 21, deletions in exon 19). All molecular changes were found in adenocarcinoma or bronchioloalveolar carcinoma. The tumours of two male responders with squamous cell carcinoma showed either a wild-type sequence or carried a nonsense mutation. In one patient treatment with Gefitinib after prospective assessment of mutations resulted in tumour remission and thus proved to be predictive.Mutations in the EGFR TK domain correlate with the clinical response to Gefitinib. The predictive assessment of molecular alterations may thus be helpful for treatment decisions in selected cases. A clinical response to Gefitinib is nevertheless also found in patients with wild-type EGFR.

The goal of the present study was to investigate whether new PCR-methods would improve diagnostic of R. equi. In a first step, sensitivity and specificity of the PCR-methods in respect to the"gold standard" microbiological culture were determined. Secondly, sensitivity and specificity of both microbiological methods were evaluated in respect to the clinical diagnosis. The tracheobronchial secretions of 48 foals with pulmonary abscesses and of 37 healthy foals were evaluated by bacteriological culture as well as by four PCR-methods: aceA-, ideR-, vapA- and VP-PCR. In respect to the"gold standard" microbiological culture, the sensitivity of most PCR methods lay between 63.9 and 69.4 % except the vapA-PCR (27.8 %). The specificity of all PCR methods in this comparison was between 98 to 100 %. In this analysis, clinical diagnosis had a low sensitivity (66.7 %) and a low specificity (51.0 %). In respect to the clinical diagnosis, microbiological culture sensitivity was 50.0 % and specificity 67.7 9%. In this analysis, sensitivity rates of aceA-, ideR and VP-PCR methods lay between 33.3 and 37.5 %, sensitivity of the vapA-PCR was lower (10.4 %). The specificity of all PCR methods ranged from 78.4 to 86.5 %. In conclusion, these results show that the diagnostic potential of the microbiological methods"Culture" and "PCR" is different and that for the diagnosis of R. equi-pneumonia in foals the combination of microbiological culture with PCR should be used for examination of samples of the airways of foals.

The importance of latent zoonoses has increased in recent years in view of foodborne diseases: (i) the "healthy" animal repesents a reservoir for specific pathogens; () no pathological-anatomical changes in the carcass and its organs show the presence of these pathogens; and (iii) these pathogens may enter the food chain via hygienic weak points in the slaughtering process. To estimate the risks involved and to take appropriate measures, analysis of the slaughtering process should be complemented by collecting data relating to the carriage of the animals of latent zoonotic pathogens. From October 2004 to June 2005, fecal samples from 630 slaughtered sheep were enriched and then examied by IMS technique and by PCR to assess the prevalence of E. coli O157 (OE). Seven samples (1.1%), distributed throughout the whole examination period, were found to be positive. To assess the potential pathogenicity for humans, E. coli O157 strains were isolated by colony hybridization and further characterized. The isolated strains fermented Sorbitol, showed four different H tys (H7, H12, H38, H48), and were all negative for stx. One O157:H7 strain harbored the gene for intimin (eae) in combination with ehxA, and paa. In consequence, the potential health hazard from sheep meat related to O157 STEC seems current not to be of particular importance in Switzerland. Results emphasize the fact that E. coli O157 are not always STEC but may belong to other pathotypes as nontraditional EPEC.

Altered glutamatergic neurotransmission is considered a potential etiological factor of schizophrenia (SCZ) and affective disorders. The gene ASCT1 (SLC1A4) coding for a Na+-dependent neutral aminoacid transporter is a member of the glutamate transporter superfamily and is located on 2p13-14, a region showing linkage to both SCZ and bipolar disorder (BD). ASCT1 can thus be considered a candidate gene for both disorders. In a German sample, we tested for association between ASCT1 and both SCZ and BD. Allele and haplotype frequencies, however, did not differ between cases and controls. Recent findings on the associations between brainderived neurotrophic factor (BDNF) and SCZ and between G72/G30 and BD suggest that SCZ patients with a history of major depressive episodes (MDE) outside psychotic episodes and BD cases with a history of persecutory delusions constitute genetically distinct subgroups of these disorders. Thus, we hypothesized that restricting case definition to those 95 SCZ individuals with MDE and to those 107 BD patients with a history of persecutory delusions might clarify the relationship between BD, SCZ and ASCT1. However, these stratification approaches did not yield any significant association either. Allele and haplotype frequencies did not differ between cases and controls. Our results do not support an association of the ASCT1 gene with BD or SCZ in the German population.

Expanding research in the field of modified oligonucleotides demands suitable analytical tools for size and purity verification of known compounds and accurate structure elucidation of unknowns. There is a need for characterization of the types and sites of modifications in oligonucleotides and to identify and sequence selected candidates originating from synthesis. The potential of electrospray tandem mass spectrometry (ESI-MS/MS) for structural characterization and sequencing of oligonucleotides is demonstrated. The fundamental behavior of DNA, RNA, and selected modified oligonucleotides in gas-phase is shown. Since gas-phase dissociation does not demand specific structural prerequisites, the method bears a great potential for rapid and most accurate characterization of modified oligonucleotides, e.g. from combinatorial libraries.

Hyperekplexia (OMIM 138491) is primarily an autosomal dominant disease characterized by exaggerated startle reflex and neonatal hypertonia. If untreated it can be associated with sudden infant death from apnea or aspiration pneumonia and serious injuries. Different mutations of the alpha1-subunit of inhbitory glyzine receptor (GLRA1) could be found. Clonazepame, a gammaaminobutyric acid (GABA) receptor agonist is the therapy of choice. An early diagnose will lead to appropriate treatment and genetic counseling.

Resistance can be overcome by modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. We constructed an adenoviral vector with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter (designated Ad/TRAIL-F/RGD), and evaluated its antitumor activity in vitro and in vivo.The induction of apoptosis by the new vector Ad/TRAIL-F/RGD was evaluated in human carcinoma cells derived from hepatocellular carcinoma (Hep G2, Hep 3b), pancreatic carcinoma (Panc-1, Capan-1), and colon carcinoma (LOVO, SW 620) . Cell viability was measured by the XTT assay and GFP expression and apoptosis induction by fluorescence-activated cell sorting (FACS) and Western blot. In vivo experiments were performed in an orthotopic pancreas tumor model in nu/nu nude mice.Treatment with Ad/TRAIL-F/RGD and Ad/gTRAIL resulted in significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in all examined human carcinoma cell lines. In addition, mice treated with Ad/TRAIL-F/RGD showed a significantly decreased tumor growth than both control groups.Our results suggest that Ad/TRAIL-F/RGD may become a potent therapeutic agent for the treatment of different human solid carcinomas.

Heredity of MEN2 syndromes is caused by a heterozygous germline mutation in the RET proto-oncogene. This study describes families with rare noncysteine codon 790/791 mutations and discusses the genotype-phenotype correlation plus the therapeutic options.Forty-five patients with a putative sporadic MTC were screened for RET germline mutations by direct DNA sequencing. Family members of identified index cases underwent genetic analysis. Gene carriers were examined clinically and biochemicaly and underwent prophylactic thyroidectomy.Five index patients were identified. In the kindreds three L790F and one Y791F carriers were detected. The thyroid gland histology of L790F carriers revealed MTC in 2 patients and C-cell hyperplasia in 2 additional patients. The Y791F carrier had a normal histology.Codon 790/791 mutations had diverse penetrance: prophylactic thyreoidectomy in children is a justifiable approach for codon 790 mutation carriers, but should depend on the clinical course of codon 791 carriers.

The Cromer blood group system consists of eight high incidence and three low incidence antigens carried on decay-accelerating factor (DAF). This report describes the identification and characterization of a new Cromer high incidence antigen, named SERF. Sequence analyses of DNA from a Thai female whose serum contained the alloantibody to a high incidence antigen in the Cromer blood group system (anti-SERF) and from her two children were performed. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis on cDNA from the proband and PCR-restriction fragment length polymorphism analysis on DNA from Thais were also performed. To map the epitope, DAF deletion mutants were tested by immunoblotting with anti-SERF. Sequence analysis revealed a substitution of 647C>T in exon 5 DAF in the proband. The proband's two children and two of 100 Thais were heterozygotes 647C/T. Analysis using DAF deletion mutants revealed the antigenic determinant to be within short consensus repeat 3 (SCR3), which is encoded by exon 5. This study describes a novel high incidence antigen (SERF) in the Cromer blood group system characterized by the amino acid proline at position 182 in SCR3 of DAF. The SERF-negative proband has a substitution mutation that predicts for leucine at this position. SERF has been provisionally assigned the International Society of Blood Transfusion number 021��012 (CROM 12).

We gathered dead bees of 56 Hessian bee colonies following a sudden collapse during winter 2002/03. Viral RNA was purified from ten dead bees per sample. Kashmir bee virus (KBV) was detected by use of a RT-PCR protocol. 13 samples were positive for KBV. The PCR amplicon was sequenced. A BLAST GenBank search clearly identified the Hessian amplicon as a KBV fragment. Similarities of more than 85% were found. Phylogenetic analysis revealed a close genetic relationship of the Hessian isolate to an isolate from New Zealand. The Northamerican, the Russian and Australian notations listed in GenBank did not cluster round with the Hessian isolate. This is the first documented detection of KBV in Middle Europe.

A multiplex-PCR (MPCR) assay was optimised in the framework of an enterococci monitoring project in order to accelerate the previous time-consuming biochemical testing of enterococcal strains isolated from animal materials and food. The MPCR was optimised by variation of annealing temperature, MgCl2-concentration but more over by primer combinations and primer concentrations. Reference strains were used during the optimization. Afterwards 522 enterococcal field strains were examined by the MPCR. Ambiguous results occurred for 20 strains (17 with false-positive and 3 with false-negative results) which could be explained by the minor specificity ("EM"-primers) and sensitivity (ddlE.faecium-primers) respectively. Differences also existed between the genus-specific primers "E" and "Ent". The first mentioned primers showed weaknesses for the identification of enterococci whereas the use of the "Ent" primers inhibited other primers in the MPCR. However, the majority of the examined strains could be detected unambiguously, so that a differentiation of enterococcal isolates can be recommended by MPCR in contrast to the high time-, material- and work-consumption of conventional biochemical testings.

Shigatoxin-producing E. coli (STEC) and their subgroup, the enterohaemorrhagic E. coli (EHEC) are known since about 25 years. Only EHEC can cause diseases in humans. A molecularbiological method cascade is described in this article. It contains the following steps: preenrichment, enrichment, preparation of samples (DNA extraction), screening PCR, specific STEC isolation by using DNA DNA hybridization, verification of isolates as STEC by using PCR, characterization of STEC isolates. Pulsed field gel electrophoresis and DNA sequencing are additional methods for the detection of clonal correlations. They should be performed in special labs, only. Furthermore, some information about an interlaboratory ring trial within Germany and some conclusions are given.

LEOPARD syndrome (MIM #151100) is a rare autosomal dominant condition with characteristic skin anomalies, facial dysmorphism, hypertelorism, cardiac anomalies, and occasional conductive hearing loss. Mutations in the PTPN11 gene are described as the causal gene defect for the clinical features of Noonan syndrome (MIM #163950), but also for LEOPARD syndrome. For confirmation of the clinical diagnosis of multiple lentigines syndrome, the molecular genetic mutation analysis in the PTPN11 gene could be helpful.We report on a family with LEOPARD syndrome in which the mutation analysis in the father and his daughter in the PTPN11 gene was carried out us:ng PCR, DHPLC, and automated sequencing.We could identify both father and daughter as carriers of the mutation Y279C in the PTPN11 gene, which is known as a disease-related mutation.The allelic affinity to Noonan syndrome could thus be further supported.

The frequency and importance of APC gene mutations in patients with adenocarcinoma in Barrett's esophagus were evaluated. Tissue samples were obtained by endoscopic biopsy or after surgery in 43 patients. DNA analysis was performed with PCR SSCP and DNA sequencing of the mutation cluster region (Exon 15) of the APC gene. Our analysis demonstrated an infrequent occurrence of APC gene mutations in Barrett's cancer (n = 3) and dysplastic Barrett's mucosa (n = 1). Therefore, the functional significance of the frequently observed APC allelic losses (LOH) must be questioned, as a single allelic loss is not sufficient for a complete gene inactivation. It might however be, that a target gene responsible for the molecular pathogenesis of Barrett's cancer is located outside the APC region on chromosome 5q21. 5q allelic losses could however, serve as a marker for the malignant potential of Barrett's epithelium, as they occur with a high frequency in an early stage of carcinogenesis.

Approximately 1 out of 1000 children is affected by severe or profound hearing impairment at birth. In the last years it has been shown that more than 50 % of inherited prelingual, sensorineural hearing impairment may be attributed to genetic defects. Most commonly, the GJB2 gene (chromosome 13q11) that encodes connexin 26 (Cx26) is affected. Cx26 is crucial for the formation of gap junctions which play an important role in the intercellular exchange of electrolytes. A variety of autosomal recessive GJB2 mutations associated with inherited hearing impairment has meanwhile been identified. The most common GJB2 mutation in Caucasian populations, 35delG accounts for the majority of cases and has a carrier frequency of more than 2.5 %. Other distinct mutations account for hearing impairment in other parts of the world.We examined in 59 Caucasian and Ghanaian individuals whether DNA recovered from buccal smears was appropriate for genetic testing by polymerase-chain reaction (PCR) based DNA-sequencing.Buccal smears could be taken conveniently in all cases, even from small babies. In 53 out of 59 samples the material recovered from buccal smears could be subjected to PCR of the second exon of the GJB2 gene and subsequent DNA-sequencing. GJB2 mutations were identified in 34 patients. 13 Caucasian individuals exhibited the most common mutation 35delG. In addition, four cases of the rare W24X and each one heterozygous case of the V153I- and the L90P mutation were found. In two African individuals the 35insG mutation was detected. All other African patients had mutations exclusively identified in Ghana so far with the exception of R143W. R143W accounts for most cases of profound deafness in Ghana and has been identified in low frequencies in other ethnic groups as well.Screening for GJB2 mutations in DNA recovered from buccal smears of individuals with inherited hearing impairment offers an easy, non-invasive method for early diagnosis and a basis of genetic counselling.

Hepatitis C virus (HCV) isolates from 12 patients with chronic hepatitis C underwent the sequence analysis. Among the 12 patients, 9 obtained a complete response (CR), 2 partial response (PR) and 1 non--response (NR) after the treatment with IFN-alpha. The results showed that only single amino acid (aa) substitution in iIFN sensitivity determining region (ISDR) (aa 2213 and aa 2218) in 6 cases with CR was observed, while 3 cases of CR and all PR and NR cases had no aa mutation of ISDR. It is suggested that the ISDR in HCV NS5A was yet not identified by our patients.

Several candidate genes were selected, based on their critical roles in the host's response to intracellular bacteria, to study the genetic control of the chicken response to Salmonella enteritidis (SE). The candidate genes were: inducible nitric oxide synthase (INOS), tumor necrosis factor related apoptosis inducing ligand (TRAIL), transforming growth factor β2 (TGF-β2), transforming growth factor β3 (TGF-β3), and immunoglobulin G light chain (IgL). Responses to pathogenic SE colonization or to SE vaccination were measured in the Iowa Salmonella response resource population (ISRRP). Outbred broiler sires and three diverse, highly inbred dam lines produced 508 F1 progeny, which were evaluated as young chicks for either bacterial load isolated from spleen or cecum contents after pathogenic SE inoculation, or the circulating antibody level after SE vaccination. Fragments of each gene were sequenced from the founder lines of the resource population to identify genomic sequence variation. Single nucleotide polymorphisms (SNP) were identified, then PCR-RFLP techniques were developed to genotype the F1 resource population. Linear mixed models were used for statistical analyses. Because the inbred dam lines always contributed one copy of the same allele, the heterozygous sire allele effects could be assessed in the F1 generation. Association analyses revealed significant effects of the sire allele of TRAIL-StyI on the spleen (P < 0.07) and cecum (P < 0.0002) SE bacterial load. Significant effects (P < 0.04) were found on the cecum bacterial load for TGF-β3-BsrI. Varied and moderate association was found for SE vaccine antibody response for all genes. This is the first reported study on the association of SNP in INOS, TRAIL, TGF-β2, TGF-β3, and IgL with the chicken response to SE. Identification of candidate genes to improve the immune response may be useful for marker-assisted selection to enhance disease resistance.

The Black Queen Cell Virus (BQCV) can affect brood of the honey bee (Apis mellifera). In general queen cells are endangered showing dark coloured cell walls as typical symptoms. Worker- and dronebrood can be infected by BQCV but normally without clinical symptoms. This paper describes for the first time a symptomatic BQCV-infection of diseased drone brood found on two bee yards in Hessen/Germany in 2001. The drone larvae were seriously damaged and some of them were dead. Samples of the affected brood were tested for BQCV by the PCR detection method. A BQCV specific nucleic acid fragment was found. The PCR product were sequenced and aligned with the relevant GenBank entry. At the nucleic acid level as well as at the deduced protein level the isolate showed a high similarity with the south african isolate noted in GenBank.

The species Campylobacter (C.) fetus is divided into the subspecies venerealis and fetus, which differ in epidemiology and clinical importance. The differences between these subspecies make an accurate distinction essential. Differentiation of C. fetus by traditional microbiological methods is only based on two reactions (tolerance to glycin, Na selenite reduction), in which C. fetus ssp. venerealis reacts negatively. However, the value of both reactions is limited. We used a specific PCR-based assay for identifying and differentiating the two C. fetus subspecies, which was recently developed by HUM et al. (1997). In this assay, a 764 bp amplicon is produced using primers MG3F and MG4R for both subspecies of C. fetus. In contrast to HUM et al. (1997), this amplicon was approximately 200 bp smaller. This discrepancy can't be explained. Afterwards, the primers VenSF and VenSR are used for differentiation. The identification of the sub-species venerealis is based on the presence of a 142 bp amplicon, which is not formed with subspecies fetus. The type strains of both C. fetus subspecies were used as positive controls. Non-specific reactions were not observed. In this PCR assay, 73 field strains were investigated (among them 24 C. fetus ssp. veneralis, 26 C. fetus ssp. fetus). In these investigations, the method has proved its diagnostic suitability. The results of the traditional microbiological differentiation of the C. fetus field strains could be confirmed by the PCR assay. In future, the traditional phenotypic characterization of C. fetus subspecies remains indispensable, but this PCR assay constitutes a valuable method for the confirmation of these results.

Since Saikku's investigations in 1988 a direct relation between atherosclerosis and infection with Chlamydia pneumoniae has been suspected. The aim of the present study was to examine histologically normal and atherosclerotic femoral arteries of autopsy cases and to correlate histological findings with the presence of Chlamydia pneumoniae which was tested with nested polymerase chain reaction. The study included all of the femoral arteries (of which 16 male, 15 female, aged between 50 and 88 years) obtained by autopsy from the cases occurring between July 1, 1999 and August 31, 1999 in the Medical Clinic Darmstadt. Each paraffin block supplied us with six consecutive sections of which the first two sectional areas were histologically stained, while the other four were used in PCR examination for DNA extraction. The polymerase chain reaction did not reveal presence of Chlamydia pneumoniae in any of the cases, neither in atherosclerotic lesions, nor in normal arteries. Because of the negative results, an additional PCR test was performed with primers that amplify another region of the Chlamydia genome, but the result did not change. The negative result of our prospective study does not contradict the assumption that infection with Chlamydia pneumoniae may enhance the progress of atherosclerosis in single cases. However, the results do not favour the concept of atherosclerosis as an infectious disorder caused by Chlamydia pneumoniae.

In Switzerland clinical bovine paratuberculosis is registered sporadically with on average seven outbreaks per year. Our present studies are aimed to investigate the prevalence of Mycobacterium avium ssp. paratuberculosis (MAP)-infections in the Swiss cattle population and, therefore methods to culture MAP from bovine feces as well as a commercially available ELISA to detect MAP-specific antibodies are evaluated by using fecal samples and blood sera from herds with cases of clinical paratuberculosis. A series of molecular methods i.e. PCR-coupled RFLP analysis of the IS1311-insertion element of M. avium, PCR-coupled RFLP-analysis of the mycobacterial rpoB-gene, and DNA analysis of the mycobacterial 16S rRNA gene are used to identify mycobacterial isolates grown from bovine feces. Up to now, MAP was detected by culture in 12 of 155 (7.7%) animals from herds with paratuberculosis. A rather striking result is the finding of atypical mycobacteria in feces of 75 cattle (48.3%). Among these isolates, M. avium ssp. avium, M. thermoresistibile, and M. hassiacum/M. buckleii have been identified so far.

Coat color played an important role during domestication and formation of breeds. Livestock breeders often had special preferences for particular color phenotypes because they believed them to be associated with performance or fitness traits. Socio-cultural reasons might have had an influence on color selection as well. Recently genetic tests on DNA level got available to genotype in any individual horse for basic horse coat colors (chestnut, bay, black). In particular, hidden carriers of the recessive chestnut and black allele are recognizable with these tests. A sample of 162 Franches-Montagnes horses from Switzerland was genotyped for the alleles for chestnut and black. The analysis of allele frequencies revealed a high prevalence of the chestnut allele and a low frequency of the black allele in this population. Rare colors are in demand on the market. The statistical analysis of 1369 offspring from five stallions indicate, that darker shades of basic color phenotypes (dark chestnut, dark bay) follow a recessive mode of inheritance in the Franches-Montagnes horse breed.

Bacteria and viruses may be transmitted through contaminated biological materials such as transplantable tumors, cell lines, sera or other biological material. Currently, the screening of biological material is being done using the mouse or rat antibody production (MAP/RAP) test (serological testing). We decided to test and validate an alternative assay using polymerase chain reaction (PCR / RT-PCR) technology to detect viral contamination directly in biological material. The aim of this study therefore is the validation of our new PCR assays and the comparison of PCR and MAP test. For 6/14 viruses the conventional PCR, seems to be more sensitive and more specific in detecting murine viruses. In 12/14 viruses the RT-PCR is more sensitive than MAP-test. In 2/14 cases all detection methods have the same sensitivity. Furthermore, PCR screening clearly contributes to the principles of 3R as a replacement technique as it eliminates the need for using animals to screen for murine viruses in biological material.

Botulism is one of the most important bird diseases world-wide and is caused by the intoxication with Botulinum-Neurotoxin-C1 (BoNt-C1), which is produced by toxigenic clostridia under appropriate conditions. Avian botulism leads regularly to large losses among the migrating bird populations breeding and resting at the saltwater pools of the Austrian national park Neusiedler See-Seewinkel. Despite of its ethical dubiousness and its high technical expense the mouse-bioassay is still used as the routine standard method for the detection of BoNt-C1. According to the 3R-concept, in vitro alternative methods for the qualitative detection of BoNt-C1 (immunostick-ELISA) and a corresponding BoNt-C1 gene fragment (nested-PCR) were established. In order to estimate the BoNt-C1 production potential the methods were tested with sediment samples from different saltwater pools subjected to cultivation conditions appropriate for in vitro BoNt-C1-production. With the mouse-bioassay, 52 out of 77 samples were found to have a positive toxin production potential. The immunostick-ELISA showed a similar sensitivity as the mouse-bioassay and exhibited a highly significant positive correlation (r=0.94; p0.001) with the mouse-bioassay in detecting BoNt-C1. The nested-PCR approach revealed higher numbers of positive BoNt-C1 gene fragment detections as compared to the direct toxin analysis approaches. A weak correlation (r=0.21; p=0.07) with the mouse-bioassay was discernible, no correlation was found with the immunostick-ELISA (r=0.09; p=0.46). Obviously, the PCR approach detected the BoNt-C1 gene fragment in some of the samples where no toxin expression has occurred. Thus it is suggested that the qualitative immunostick-ELISA represents a potential in-vitro alternative to the mouse-bioassay for assessing the BoNt-C1 production potential in environmental samples. In contrast, qualitative BoNt-C1 gene fragment detection via PCR led to an overestimation of the actual toxin production potential.

Among the genus Cunninghamella, so far C. bertholletiae is known to be the only clinically relevant species. Correct identification of C. bertholletiae is not possible with classical methods. PCR and sequencing of the internal transcribed spacer (ITS) region was used to identify seven of nine clinical isolates as C. bertholletiae and two as C. echinulata. Also an isolate of the surrounding area of one patient infected with C. echinulata could be identified as C. echinulata. High homology in the ITS region was found within the isolates of C. bertholletiae. Within the species C. echinulata and C. elegans a differentiation on subspecies level was achieved by an analysis of restriction fragment length polymorphism of the ITS amplicons after incubation with TaqI and HinfI. Similar results were obtained by PCR fingerprinting of the complete DNA with the single microsatellite DNA primers (GTG)5 and (GAC)5. For the first time C. echinulata could be identified as agent of zygomycosis in humans.

Despite apparently curative resection adenocarcinomas of the pancreas early recur. Thus, the pathological examination should be enriched by sensitive methods to detect minimal residual disease (MRD). Mutant K-ras is the most promising genetic alteration in ductal adenocarcinoma and may serve to detect malignant cells by polymerase chain reaction (PCR) based techniques. Therefore, we set out to detect K-ras mutations by PCR for evaluation of MRD in patients after curative resection of pancreatic adenocarcinoma.Tumor tissue and corresponding paraaortic lymph nodes were obtained from 51 patients, who underwent surgery for pancreatic head tumors. The paraaortic lymph nodes were staged as tumor-free by routine histopathology in all cases diagnosed for ductal adenocarcinoma (study group, n = 40) or other tumors (control group, n = 11). Therefore, DNA of both primary tumors and lymph nodes was extracted and analysed by a PCR-based assay with respect to mutated K-ras. As a positive control the human pancreatic cancer cell line PaTu-8902 was used.K-ras mutations were detected in 73 % (29/40) of primary tumors of ductal adenocarcinomas and in 17 % (5/29) in the corresponding paraaortic lymph nodes, which were diagnosed as tumor-free by routine pathology. The identical type of point mutation was found in primary tumors and corresponding lymph nodes by use of sequence specific primers. In the control group no K-ras mutation was detected.Tumor cell DNA can be detected sensitively in tumor- and lymph node specimen with the described method. Routinely assessed, this method is able to detect MRD and could enrich the pathological examination, in order to determine prognostic relevant subgroups of patients.

Retinoic acid and its natural and synthetic analogs (retinoids) affect a wide array of biological processes. Retinoids are used in the treatment of many skin diseases and are promising drugs for several cancers. Most of their actions are thought to result from changes in gene expression which is done through nuclear retinoic acid receptors and retinoid X receptors. We conducted a study to determine whether the expression of these receptors is different in malignant tumors and tumor cell lines versus normal tissue.We performed reverse transcription PCR from 29 tissue specimens of squamous cell carcinomas and one melanoma and of the head and neck as well as from 13 cell lines established from head and neck cancer. We were looking for the expression pattern of RARalpha, beta, gamma and RXRalpha.Only RARgamma was expressed 100 % in cell lines and tissue specimens. RARbeta showed a 100 % expression only in tissue specimens whereas a 54 % expression in cell lines was seen. All other receptors were diminished in their expression. In the positive controls all receptors were expressed 100 %.The expression of RARalpha and RARbeta was partially lost in cell lines established from squamous cell carcinoma of the head and neck. The 100 % expression of RARbeta in tissue samples versus 54 % in cell lines can be explained by clonal growth of malignant cells in cell lines and also possible "contamination" by normal cells in the tissue specimen. In concordance with the literature it seems that RARalpha and beta play a pivotal role in mediating the response to retinoids.

The insulin receptor (IR) is an essential protein localized on the surface of almost all cell types. IR belongs to the tyrosine-kinase growth factor receptor family. Insulin mediates proliferative responses in a variety of tumor cells, however, the role of the IR molecule in carcinogenesis has not yet exactly been established.Messenger RNA from mucosal keratinocytes and laryngeal carcinoma cells were transcribed in cDNA using reverse transcriptase and amplified by PCR by means of a number of oligonucleotides. PCR products were analyzed electrophoretically.Comparing the electrophoretic pattern of both cell types the overexpression of a 127 bp fragment could be detected in laryngeal carcinoma cells in contrast to benign keratinocytes. Cloning and sequencing this fragment exact homologous match was found with exon-2 of the IR gene. The overexpression of the IR gene in laryngeal carcinoma cells was confirmed by Northern hybridization.The results show up-regulation of IR-mRNA in laryngeal carcinoma cells suggesting that the number of IR is enhanced in these cells. Hence it follows, that the overexpression of IR plays a possible role in laryngeal cancer initiation and/or progression.