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The establishment of genomic approaches including the sequence determination of complete bacterial genomes started a new era in microbiological research. Since then more than two hundred prokaryotic and eukaryotic genomes have been completely sequenced, and there are additional complete genome projects including different bacterial species and strains in progress (http://www.tigr.org, http://www.sanger.ac.uk). The continously growing amount of bacterial DNA sequence information gives us also the possibility to gain deeper insight into bacterial pathogenesis. With the help of comparative genomics, microbiological research can focus on those DNA sequences that are present in pathogenic bacteria but are absent in non-pathogenic strains. With this knowledge and with the help of molecular biological methods such as PCR,DNA-chip technology, subtractive hybridisation, transcriptomics and proteomics we can analyse in detail what makes a particular bacterial strain pathogenic. This knowledge also gives us the possibility to develop new vaccines, therapeutic approaches or diagnostic tools. The aim of this work was the structural and functional analysis of DNA regions of uropathogenic Escherichia coli strain 536 that belong to the flexible E. coli gene pool. The first part of this thesis focused on the identification and structural characterisation of pathogenicity island V of strain 536 (PAI V536). PAI V536 is integrated at the pheV tRNA gene at 64 minutes of the E. coli K-12 chromosome. In addition to the intact pheV tRNA gene, a truncated copy ('pheV) that represents the last 22 bp of this gene’s 3'-end was identified 49 kb downstream of pheV on PAI V536. The analysis of the DNA sequence flanked by pheV and 'pheV revealed characteristics that are typical of PAIs. This DNA region exhibits homology to IS-elements and prophages and also comprises determinants coding for the Pix fimbriae, a phosphoglycerate transport system, an autotransporter, as well as for hypothetical proteins. Downstream of 'pheV, the K15 capsule determinant (kpsK15) of this strain is located. Structural analysis of the 20-kb kpsK15 locus revealed a so far unknown genetic organisation indicative of recombination events between a group 2 and group 3 capsule gene cluster. Downstream of the capsule determinant, the genes encoding a type II secretion system (general secretion pathway -GSP) are located on PAI V536. The K15 capsule locus was functionally characterized. Specific inactivation of each of the regions 1 to 3 of the kpsK15 gene cluster, and the use of a K15 capsule-specific antiserum demonstrated that this determinant is the functional K15 capsule locus of strain 536. It has been shown in an experimental murine model of ascending urinary tract infection with suckling mice that the K15 capsule contributes to urovirulence. Interestingly, the K15 capsule is not involved in serum resistance of strain 536. Inactivation of the PAI V536-encoded type II secretion system excluded a role of this general secretion pathway for capsule biosynthesis and virulence of strain 536 in the murine ascending urinary tract infection model. In the second part of the thesis, the transferability of PAIs was further investigated. Using PAI II536 as a model, mobilisation of this island from strain 536 into suitable recipient strains was investigated. For this purpose, an antibiotic resistance cassette, the R6K origin of replication as well as plasmid pGP704 carrying the mobilisation region of plasmid RP4 have been inserted into PAI II536. Transformation with the helper plasmid RP4, resulted a derivative of strain 536 that was used as a donor for conjugation experiments, while for recipient the pir + laboratory strain SY327 was used. After deletion the circularised PAI II536 was mobilised with the help of the conjugative helper plasmid (RP4) into the recipient laboratory strain SY327. The frequency of this event was about 10-8. It was also demonstrated that in the transconjugant strains the mobilized PAI II536 could be permanently present as a circular form and also can be integrated into the chromosome at the same chromosomal insertion site (leuX) as in the donor strain 536. Furthermore, after mobilisation and chromosomal integration of PAI II536 it was possible to remobilise this PAI back to a PAI II536-negative derivative of strain 536. The results obtained in this thesis increase our knowledge of the structure and function of a pathogenicity island of uropathogenic E. coli strain 536 and shed some light on the mechanisms contributing to genome plasticity and evolution of pathogenic E. coli variants.
Somites are repeated epithelial segments that are generated in a rhythmic manner from the presomitic mesoderm (PSM) in the embryonic tailbud. Later, they differentiate into skeletal muscle, cartilage and dermis. Somitogenesis is regulated by a complex interplay of different pathways. Notch/Delta signaling is one of the pathways well characterized in zebrafish through mutants affected in its different components. Previous work in mouse, chicken and zebrafish has shown that also additional components are required during somitogenesis, most importantly through an FGF and Retinoic acid (RA) gradient, as well as Wnt signaling. However, no zebrafish mutants with defects in these pathways showing specific somite malformations are described. This was explained by functional redundancies among related genes that have resulted from a whole genome duplication which occurred in a teleost fish ancestor 350 million years ago. As distinct duplicates exist in different teleost species, a large scale mutagenesis screen in the medaka (Oryzias latipes) has been performed successfully in Kyoto, Japan. I analyzed nine of the isolated medaka mutants that show variable aspects of somitic phenotypes. This includes a complete or partial loss of somite boundaries (e.g. bms and sne), somites with irregular sizes and shapes (e.g. krz and fsl) or partially fused and enlarged somites (e.g. dpk). Although some of these medaka mutants share characteristics with previously described zebrafish somite mutants, most of the mutants represent unique phenotypes, not obtained in the zebrafish screens. In-situ hybridization analyses with marker genes implicated in the segmentation clock (e.g. her7), establishment of anterior-posterior (A-P) polarity (e.g. mesp) and differentiation of somites (e.g. myf5, lfng) revealed that the medaka mutants can be separated into two classes. Class I shows defects in tailbud formation and PSM prepatterning, and lateron somite boundary formation was impaired in these mutants. A unique member of this class with a novel phenotype is the doppelkorn (dpk) mutant that has single fused or enlarged somites. This phenotype has not been reported till now in zebrafish somite mutants. In-situ analyses on dpk showed that stabilization of the cyclically expressed somitogenesis clock genes must be affected in this mutant. This is accompanied by a disrupted regulation of A-P polarity genes like mesp. This suggests that dpk is a mutant deficient in the wave front, which is necessary for the down-regulation of oscillating genes in the anterior PSM. Furthermore, as the initiation of oscillation of all three cyclic her genes was unaffected in dpk embryos, I could exclude that this mutant in affected in the Notch/Delta pathway. Another mutant that belongs to this class is the samidare (sam) mutant. Morphologically, sam mutants are similar to zebrafish after eight (aei). In both cases, the first 7-9 somites are formed properly, but after this somite formation ceases. Different to the situation in aei, sam mutant embryos presented an additional defect in the mid-hindbrain boundary (MHB) region. Similar MHB defects were described in the zebrafish fgf8 mutant acerebellar (ace). In ace zebrafish mutant, somites were only slightly defective, although FGF signaling has been shown to be important for somite formation in chicken, mouse and zebrafish. This was explained by functional redundancy between fgf8 and fgf24 ligands in the tailbud of zebrafish. Thus, it is interesting to suggest that the sam mutant, based on the parallel defects in somites and MHB, is a potential member of the FGF signaling pathway muatnts. It was shown that FGF plays a crucial role during MHB formation in medaka. In addition, I showed that fgf8 acts non-redundantly during tailbud formation and somitogenesis in medaka. Furthermore, I showed that FGF signaling regulates somite size also in medaka and that fgfr1 is the only FGF receptor expressed in the tailbud and somites. In class II medaka somite mutants, PSM prepatterning appears normal, whereas A-P polarity, boundary formation, epithelialization or the later differentiation of somites appears to be affected. Such mutants have not been isolated so far in zebrafish, mice or chicken. Therefore, medaka class II somite mutants seem to be a novel group of mutants that opens new perspectives to analyze A-P polarity regulation, determination and boundary formation in the presence of a normally functioning clock in the PSM. Identifying the encoding genes for all analyzed medaka somite mutants will contribute to the understanding of the molecular interactions of different signaling pathways involved during somitogenesis, and is expected to result in the identification of new components.
A large variety of sex determination systems have been described in fish. However, almost no information is available about sex determination in the classical fish models, the zebrafish Danio rerio and the pufferfish Takifugu rubripes. A DNA-binding protein gene called dmrt1bY (or DMY) has been recently described as an outstanding candidate for the primary sex-determining gene in the medaka fish Oryzias latipes. But this gene is not the universal master sex-determining gene in teleost fish, since dmrt1bY is not found in most other fishes. Hence, other fish models need to be examined including the platyfish Xiphophorus maculatus. Xiphophorus maculatus has three types of sex chromosomes (X, Y and W; females are XX, WX or WY; males are XY or YY). Its gonosomes are at an early stage of differentiation. The sex-determining locus on the sex chromosomes is flanked by two receptor tyrosine kinase genes, the Xmrk oncogene and its protooncogenic progenitor gene egfrb, which both delimit a region of about 0.6 centiMorgans. This situation should allow the positional cloning of the sex-determining gene (SD) of the platyfish. For this purpose, Bacterial Artificial Chromosome (BAC) contigs were assembled from a BAC library of XY males constructed in our laboratory, using the oncogene Xmrk, egfrb, as well as a Y-specific pseudogene called ps-criptY as starting points. The ps-criptY sequence was found to be closely linked to the SD gene, since no recombination was observed between SD and ps-criptY in more than 400 individuals tested. Two major BAC contigs for the X chromosome (about 2.5 Mb) and three major BAC contigs for the Y chromosome (about 3.5 Mb) were built up and analyzed by strategic sequencing. These are some of the largest contigs ever assembled for the sex chromosomes of a non-mammalian vertebrate species. The molecular analysis of the ps-criptY contig was the major objective of this work. The Y-specific ps-criptY contig has been extended over 1 Mb in this work with 58 identified molecular markers. Approximatively 700 kb of non-redundant sequences has been obtained from this contig by strategic sequencing. Numerous Y-linked markers from the contig including ps-criptY were also detected on the X chromosome. Nevertheless, major structural differences were observed between the X and Y chromosomes. Particularly, a large region, which is present at one copy on the X chromosome and contains several candidate genes, was found to be duplicated on the Y chromosome. Evidence for an inversion in the sex-determining region and for the Y-specific accumulation of a repeated sequence called XIR was also obtained. Such events might correspond to an initiation of differentiation between both types of gonosomes. Accumulation of transposable elements was also observed in the ps-criptY contig. A DNA transposable element, helitron, was isolated from the sex-determining region of X. maculatus. Three copies of helitron are located on the ps-criptY contig and one copy on the X-linked contig (helitron has roughly 15 copies per haploid genome). No in-frame stop codon, truncation or intron was found in these four copies, which present high nucleotide identities to each other. This suggests that helitron elements might be active or have been recently active in X. maculatus. A consensus open reading frame of helitron was also assembled from medaka (Oryzias latipes) genomic sequences. Two candidate genes from the ps-criptY contig are also located on the W chromosome in the X. maculatus Usumacinta strain (heterogamety). These markers show the relationship between the different types of gonosomes and allow to compare the male and female heterogameties in the platyfish. Several gene candidates were identified in the ps-criptY contig. However, some of them such as msh2, cript, igd and acr probably correspond to pseudogenes. Interestingly, a novel gene, called swimy, is exclusively expressed in spermatogonia of the adult testis. Swimy is a gene encoding a DNA-binding protein with several putative DNA-binding domains. The data suggest that swimy is a very promising candidate for the master SD gene. Another novel gene, which is called fredi and encodes a novel helix-turn-helix protein, is predominately expressed in the adult testis and currently under scrutiny. There is no doubt that the master SD gene of X. maculatus will be identified by positional cloning. Further molecular analysis of the contigs built in this work will shed new light on the molecular mechanism of sex determination and the evolution of sex chromosomes in fish.
Cloning and functional characterization of novel genes expressed preferentially in the human retina
(2005)
The human retina is a multi-layered neuronal tissue specialized for the reception and processing of visual information. The retina is composed of a great diversity of neuronal cell types including rod and cone photoreceptors, bipolar cells, ganglion cells, amacrine cells, horizontal cells and Müller glia. In response to light, a coordinated series of molecular events, the so-called phototransduction cascade, is triggered in photoreceptor cells and the signals from the photoreceptors are further processed by the bipolar and ganglion cells to the higher centers of the brain. The retina as highly complex system may be greatly susceptible to genetic defects which can lead to a wide range of disease phenotypes. Therefore, isolation and characterisation of the genes active in the human retina will facilitate our deeper understanding of retinal physiology and mechanisms underlying retinal degeneration and provide novel candidates for the retinal disease genes. To identify novel genes that are specifically or predominantly expressed in the human retina, a cDNA library enriched for retina specific transcripts was generated using suppression subtractive hybridization (SSH) technique. In total, 1113 clones were randomly isolated from the retina SSH cDNA library and partially sequenced. On the basis of BLASTN algorithm analysis these clones were classified into four categories including those with I) significant homology to known human genes (766/1113), II) significant homology to partial transcripts and hypothetical gene predictions (162/1113), III) no homology to known mRNAs (149/1113), and IV) vector sequences and clones derived from mitochondrial genes (36/1113). After correcting for redundancy, category I represented 234 known human genes and category II a total of 92unknown transcripts. Clones from category I, were selected for expression analysis by RT-PCR in a great number of human tissues. This resulted in the identification of 16 genes which were expressed exclusively in the retina, 13 which were highly expressed in the retina compared to other tissues, 12 genes which were specifically expressed in neuronal tissues and 48 ubiquitously expressed genes. Thus, our expression analysis resulted in the identification of 29 genes exclusively or abundantly transcribed in the human retina. Of those, retina specific genes L25,L33, L35, L37, L38 and L40 were selected for further analysis. To characterize the complete mRNA sequences of these transcripts a full-length human retina cDNA library was constructed. The analysis of the L25 gene revealed three splicing variants of the ABCC5 gene, consequently named ABCC5_SV1 (SV1), ABCC5_SV2 (SV2) and ABCC5_SV3 (SV3).These isoforms comprise the first five exons of ABCC5 and additional novel exons named 5a, 5b and 5c, generated by differential exon usage. The determined lengths of the three transcripts are 2039 bp, 1962 bp, and 1887 bp in size, respectively. RT-PCR, real-time PCR and Northern blot analysis of ABCC5 as well as the isoforms SV1, SV2 and SV3demonstrated high levels of expression for all transcripts in the retina compared to other tissues. Analysis of their nucleotide sequences revealed that inclusion of exon 5a in splicing variant SV1 produced a frame shift and premature termination codon (PTC). Our data show that this splice variant is the target of nonsense mediated mRNA decay (NMD). This was shown by inhibition of protein synthesis with antibiotics puromycin and anisomycin in human cell lines A-RPE 19 and Y79. Our analysis resulted in an increase of the PTC containing transcript and a decrease of the ABCC5 transcript. Conversely, the amount of both transcripts (SV1 and ABCC5) returned to pre-treatment levels after removal of the inhibitors. Together, our results suggest that alternative splicing of the ubiquitously expressed ABCC5 gene in addition to NMD is involved in retina-specific transcriptional regulation of the mRNA level of ABCC5. In contrast, additional experiments demonstrated that the levels of expression ofSV2 and SV3 isoforms do not appear to influence ABCC5 transcription. Several of the cloned genes were selected for additional genotyping of single nucleotide polymorphisms (SNPs) in order to construct their SNP maps which are going to be used for future association studies of complex disease AMD. Thus, identification of novel retinal genes and their functional characterization will further our elucidation of retinal physiology in general and in the diseased state in particular, by providing candidate retinal disease genes.
In the present thesis, two projects on the use of microarray technology for molecular epidemiology of Neisseria meningitidis have been followed. The first one evaluated microarrays based on polymorphism-directed oligonucleotide design for typing of N. meningitidis adopting the multilocus sequence typing (MLST) concept. The number of oligonucleotides needed to cover all known polymorphisms was much lower compared to the number needed if a tiling strategy would have been chosen. Initial experiments using oligonucleotides 28-32 nucleotides in length, revealed that the applied hybridisation protocols were highly specific. However, despite of several optimisation steps, the rate of misidentification of oligonucleotides remained >1.8% in consecutive validation experiments using arrays representing the genetic diversity at three MLST loci. This finding led to the assumption that the high density of polymorphic sites and extensive GC-content variations at N. meningitidis MLST loci hindered the successful implementation of MLST microarrays based on polymorphism-directed oligonucleotide design. In the 1980s, the ET-15 clone emerged within the ST-11 complex of N. meningitidis. This new clone was associated with severe meningococcal disease and outbreaks world-wide. Therefore, the goal of the second project was to identify genetic differences between ET-15 strains and other ST-11 strains using whole genome microarray technology. Three genes encoding hypothetical proteins were identified to be present in all ET-15 strains but absent in other ST-11 strains. This finding together with unpublished observation from our group suggested that several genome alterations occurred before the clonal expansion of the ET-15 clone started. The role that these three genes play in the pathogenicity of the ET-15 clone is unclear. The genome comparisons revealed furthermore that studies of the ET-15 clone displayed approximately two-fold less gene content variation than ST-11 strains not belonging to the ET-15 clone. This finding is in accordance with the recent emergence and clonal expansion of the ET-15 variant.
Marine Schwämme (Porifera) sind sessile Invertebraten, deren Biomasse bis zu 60% von assoziierten Mikroorganismen gebildet werden kann. Dieses mikrobielle Konsortium ist phylogenetisch komplex, die monophyletischen Abstammungslinien sind hochgradig wirtsspezifisch und bisher konnte kein Vertreter dieser Mikroflora kultiviert werden. In seiner Zusammensetzung unterscheidet sich dieses Konsortium sowohl von der Mikroflora mariner Sedimente, als auch vom marinen Bakterioplankton. Durch 16S rRNA Sequenzanalysen und Fluoreszenz in situ Hybridisierung (FISH) konnte während dieser Arbeit das neue Candidatus Phylum Poribacteria kultivierungsunabhängig identifiziert werden. Poribacteria bilden definitionsgemäß ein unabhängiges Candidatus Phylum, da sie weniger als 75% Sequenzhomologie innerhalb der 16S rRNA zu anderen prokaryontischen Phyla zeigen. Sie sind verwandt mit Planctomycetes. Der Name „Poribacteria“ wurde gewählt, da diese Organismen spezifisch mit marinen Porifera assoziiert zu sein scheinen. Bisher konnten Poribacteria in Porifera der Ordnungen Verongida, Haplosclerida und Lithistida nachgewiesen werden, während sie in den Ordnungen Poecilosclerida, Agelasida, Halichondrida und Hadromerida nicht nachweisbar waren. Im marinen Sediment und im Bakterioplankton wurden Poribacteria ebenfalls nicht detektiert. Durch FISH Analysen wurde deutlich, dass Poribacteria in A. aerophoba (Verongida) eine abundante Fraktion der assoziierten Mikroflora bilden. Da Vertreter des mikrobiellen Konsortiums mariner Schwämme bisher nicht kultiviert werden konnten, wurde das „Metagenom“ dieser Mikroorganismen durch die ex situ Isolierung hoch molekularer DNA direkt kloniert. Eine Charakterisierung von Metagenomen erlaubt unabhängig von der Kultivierbarkeit der entsprechenden Organismen direkte Einblicke in deren Genotyp und liefert so eine erste Verbindung zwischen phylogenetischer Diversität und physiologischen Eigenschaften. Für die Erstellung der Metagenombank wurde mikrobielle Biomasse aus A. aerophoba vom Mesohyl getrennt und lysiert und die gereinigte DNA in Fosmid Vektoren in E. coli kloniert. Die resultierende Metagenombank APAE02 umfasst ca. 1,1 Gb hoch molekularer prokaryontischer genomischer DNA. Eine Bestimmung der in dieser Metagenombank archivierten mikrobiellen Diversität lieferte zusätzlich zu bekannten 16S rRNA kodierenden Loci aus Cyanobacteria, Chloroflexi, Acidobacteria und Gammaproteobacteria einen 16S rRNA kodierenden poribakteriellen Fosmidklon. Die Annotation der flankierenden genomischen Regionen des 16S rRNA Gens führte zur Detektion eines unterbrochenen rrn Operons, eines wahrscheinlich neuen Transporters, einer neuen Molybdän enthaltenen Oxidoreduktase und orthologer „open reading frames“ (ORFs) aus Rhodopirellula baltica (Planctomycetes) in Poribacteria. Die Charakterisierung dieses 38,7 kb DNA Fragmentes stellt die Basis für weitere genomische Untersuchungen an Poribacteria dar. Metagenombanken repräsentieren eine reichhaltige Quelle zum Nachweis neuer Enzyme oder Biosyntheseoperons. Somit konnten in der Metagenombank APAE02 neuartige Typ I Polyketidsynthasen (PKS) nachgewiesen werden. Phylogenetische Analysen der Ketosynthasedomäne zeigten, dass diese Systeme nicht herkömmlichen Typ I cis-AT bzw. trans-AT (Acyltransferase) PKS Systemen zugeordnet werden können. Die kodierenden Bereiche der PKS Systeme sind mit nur ca. 10 kb relativ klein. Im Gegensatz zu der Organisation sich wiederholender multipler Module herkömmlicher PKS Typ I Systeme bestehen sie nur aus einem einzigen Modul und könnten vermutlich bei der Synthese von Fettsäuren beteiligt sein. Die Struktur und Funktion der Produkte ist bisher unbekannt. Generell ist durch in silico Analysen eine Abbildung des „funktionellen Repertoires“ unkultivierter Mikroorganismen möglich. Es wäre denkbar, dass durch weitere Studien fundierte Einblicke in den Genpool der Poribacteria und anderer Organismen des mikrobiellen Konsortiums aus Poriferen eröffnet werden, um metabolische Eigenschaften zu rekonstruieren und die Mechanismen zur Interaktion mit dem Wirt verstehen zu können.