Refine
Year of publication
Document Type
- Journal article (1370)
- Doctoral Thesis (483)
- Conference Proceeding (24)
- Review (15)
- Book article / Book chapter (14)
- Preprint (13)
- Report (3)
- Book (2)
- Master Thesis (2)
- Working Paper (1)
Language
- English (1927) (remove)
Keywords
- Biochemie (80)
- Physiologische Chemie (46)
- Taufliege (45)
- Drosophila (36)
- Biologie (34)
- evolution (29)
- biodiversity (28)
- cancer (26)
- Biene (23)
- Drosophila melanogaster (23)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (1927) (remove)
Sonstige beteiligte Institutionen
- Institut für Tierökologie und Tropenbiologie (2)
- Mildred-Scheel-Nachwuchszentrum (2)
- Ökologische Station Fabrikschleichach (2)
- Albert-Ludwigs-Universität Freiburg (1)
- Boehringer Ingelheim Pharma GmbH & Co. KG (1)
- Core Unit Systemmedizin (1)
- DNA Analytics Core Facility, Biocenter, University of Wuerzburg, Wuerzburg, Germany (1)
- DNA Analytics Core Facility, Biocenter, University of Würzburg, Würzburg, Germany (1)
- Department of Animal Ecology and Tropical Biology, University of Würzburg, Würzburg, Germany (1)
- Deutsches Krebsforschungszentrum Heidelberg (1)
ResearcherID
- D-1221-2009 (1)
- J-8841-2015 (1)
- N-2030-2015 (1)
In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.
The phylum Tardigrada consists of about 1000 described species to date. The animals live in habitats within marine, freshwater and terrestrial ecosystems allover the world. Tardigrades are polyextremophiles. They are capable to resist extreme temperature, pressure or radiation. In the event of desiccation, tardigrades enter a so-called tun stage. The reason for their great tolerance capabilities against extreme environmental conditions is not discovered yet. Our Funcrypta project aims at finding answers to the question what mechanisms underlie these adaption capabilities particularly with regard to the species Milnesium tardigradum. The first part of this thesis describes the establishment of expressed sequence tags (ESTs) libraries for different stages of M. tardigradum. From proteomics data we bioinformatically identified 144 proteins with a known function and additionally 36 proteins which seemed to be specific for M. tardigradum. The generation of a comprehensive web-based database allows us to merge the proteome and transcriptome data. Therefore we created an annotation pipeline for the functional annotation of the protein and nucleotide sequences. Additionally, we clustered the obtained proteome dataset and identified some tardigrade-specific proteins (TSPs) which did not show homology to known proteins. Moreover, we examined the heat shock proteins of M. tardigradum and their different expression levels depending on the actual state of the animals. In further bioinformatical analyses of the whole data set, we discovered promising proteins and pathways which are described to be correlated with the stress tolerance, e.g. late embryogenesis abundant (LEA) proteins. Besides, we compared the tardigrades with nematodes, rotifers, yeast and man to identify shared and tardigrade specific stress pathways. An analysis of the 50 and 30 untranslated regions (UTRs) demonstrates a strong usage of stabilising motifs like the 15-lipoxygenase differentiation control element (15-LOX-DICE) but also reveals a lack of other common UTR motifs normally used, e.g. AU rich elements. The second part of this thesis focuses on the relatedness between several cryptic species within the tardigrade genus Paramacrobiotus. Therefore for the first time, we used the sequence-structure information of the internal transcribed spacer 2 (ITS2) as a phylogenetic marker in tardigrades. This allowed the description of three new species which were indistinguishable using morphological characters or common molecular markers like the 18S ribosomal ribonucleic acid (rRNA) or the Cytochrome c oxidase subunit I (COI). In a large in silico simulation study we also succeeded to show the benefit for the phylogenetic tree reconstruction by adding structure information to the ITS2 sequence. Next to the genus Paramacrobiotus we used the ITS2 to corroborate a monophyletic DO-group (Sphaeropleales) within the Chlorophyceae. Additionally we redesigned another comprehensive database—the ITS2 database resulting in a doubled number of sequence-structure pairs of the ITS2. In conclusion, this thesis shows the first insights (6 first author publications and 4 coauthor publications) into the reasons for the enormous adaption capabilities of tardigrades and offers a solution to the debate on the phylogenetic relatedness within the tardigrade genus Paramacrobiotus.
The ATP synthase occurs in remarkably conserved form in procaryotic and eucaryotic cells. Thus, our present knowledge of ATP synthase is derived from sturlies of the enzyme from different organisms, each affering specific experimental possibilities. In recent tim es, research on the H\(^+\) -conducting F0 part of the ATP synthase has been greatly stimulated by two developments in the Escherichio coli system. Firstly, the purification and reconstitution of the whole ATP synthase as weil as the proton conductor Fa from E. coli have been achieved. These functionally active preparations are well defined in terms of subunit composition, similar to the thermophilic enzyme from PS-3 studied by Kagawa's group.u Secondly, the genetics and the molecular cloning of the genes of all the F\(_0\) subunits from E. coli yielded information on the function of subunit polypeptides and essential amino acid residues. Furthermore, the amino acid sequence of hydrophobic F\(_0\) subunits, which are difficult to analyze by protein-chemical techniques, could be derived from the nucleotide sequence of the genes. These achievements, which shall be briefly summarized in the next part of this communication, provide the framework to study specific aspects of the structure and function of the F\(_0\) subunits.
The isolated H\(^+\) conductor, F\(_0\) , of the Escherichia co1i ATP-synthase consists of three subunits, a, b, and c. H\(^+\) -permeable liposomes can be reconstit~ted with F\(_0\) and lipids; addition of F\(_1\)-ATPase reconstitutes a functional ATP-synthase. Mutants with altered or misslng F\(_0\) subunits are defective in H\(^+\) conduction. Thus, all three subunits are necessary for the expression of H\(^+\) conduction. The subunits a and b contain binding sites for F\(_1\)• Computer calculations, cross-links, membrane-permeating photo-reactive labels, and proteases were used to develop tentative structural models for the individual F\(_0\) subunits.
The accessibility of the three F\(_0\) subunits a, b and c from the Escherichia coli Kll A TP synthase to various proteases was studied in F\(_1\)-depleted inverted membrane vesicles. Subunit b was very sensitive to all applied proteases. Chymotrypsin produced a defined fragment of mol. wt. 1S 000 which remained tightly bound to the membrane. The cleavage site was located at the C-terminal region of subunit b. Larger amounts of proteases were necessary to attack subunit a (mol. wt. 30 000). There was no detectable deavage of subunit c. It is suggested that the major hydrophilic part of subunit b extends from the membrane into the cytoplasm and is in contact with the F\(_1\) sector. The F\(_1\) sector was found to afford some protection against proteolysis oftheb subunit in vitro andin vivo. Protease digestion bad no influence on the electro-impelled H\(^+\) conduction via F\(_0\) bot ATP-dependent H\(^+\) translocation could not be reconstituted upon binding of F\(_1\)• A possible role for subunit b as a linker between catalytic events on the F\(_1\) component and the proton pathway across the membrane is discussed.
The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the A TP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 8011/o fonnie acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 5011/o homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 genein Aspergillus nidulans.
Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa
(1984)
A cDNA complementary to the mRNA of the ADPIATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cellfree translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony fdter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRl fragment of total Neurospora DNA, and the start of the mRNA was determined by Sl nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP I ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rieb promoter region. The mRNA has a 46-bp 5 1 end and a 219-bp 3 1 end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier.
Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9
(1984)
Subunit 9 (dicyclohexylcarbodümide binding protein, 'proteolipid') of the mitochondrial F 1F0-ATPase is a nuclearly coded protein in Neurospora crassa. lt is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved ofT after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2 + for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two dcavage sites in the precursor molecule were determined. The data indicate that: {a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleueine in position -31. The cleavage sites show similarity ofprimary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NHrterminal part of these polypeptides) into the matrix space of mitochondria.
Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo
(1985)
A method has been developed that allows the isolation of genomic clones from a cosmid library by homologaus recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into A. phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologaus plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 genewas restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.
The c, b and ö subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro andin vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating A TP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit ö. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching > 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage A is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of A TP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons.
The gene for the FeS protein of the Rhodopseudomonas sphaeroides b/c1 complex was identified by means of crosshybridization with a segment of the gene encoding the corresponding FeS protein of Neurospora crassa. Plasmids (pRSF1-14) containing the cross-hybridizing region, covering in total 13.5 kb of chromosomal DNA, were expressed in vitro in a homologous system. One RSF plasmid directed the synthesis of all three main polypeptides of the R. sphaeroides blc1 complex: the FeS protein, cytochrome b and cytochrome c1• The FeS protein and cytochrome c1 were apparently synthesized as precursor fonns. None of the pRSF plasmids directed the synthesis of the 10-kd polypeptide found in b/c1 complex preparations. Partial sequencing of the cloned region was performed. Several sites of strong homology between R. sphaeroides and eukaryotic polypeptides of the b/c1 complex were identified. The genes encode the three b/c1 polypeptides in the order: (5') FeS protein, cytochrome b, cytochrome c1• The three genes are transcribed to give a polycistronic mRNA of 2.9 kb. This transcriptional unit has been designated the jbc operon; its coding capacity corresponds to the size of the polycistronic mRNA assuming that only the genes for the FeS protein (jbcF), cytochrome b (jbcß) and cytochrome c1 (jbcC) are present. This could indicate that these three subunits constitute the minimal catalytic unit of the b/c1 complex from photosynthetic membranes.
The structure of the F0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [125I]TID. In the E. co/i F0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeted amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologaus proteins suggested the existence of tightly packed cx-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling patternwas observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membrancs were pretrcated with DCCD prior to the reaction with [125I]TID an additionally labeled amino acid appeared at the position of Glu·65 which binds DCCD covalently, indicating the Jocation of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit a or b and thus enables proton translocation. Conserved residues in subunit a, probably located in the Iipid bilayer, might participate in the pro· ton translocation mechanism.
Three F0 subunits and the F\(_1\) subunit P of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[\(^{125}\)I]iodophenyl)diazirine ([\(^{125}\)I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confmed to five residues at the NH2-terminus and five residues at the C-terminus ofthe protein. Labeling occurred at similar positions compared with the homologaus protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent oflabeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodümide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F\(_0\) subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F\(_0\) subunits.
A cDNA clone of about 2500 basepairswas prepared from the human osteosarcoma cellline U-2 OS by hybridizing with a v-sis probe. Sequence analysis showed that this cDNA contains the coding region for the PDGF-B chain. Here we report that the mitogen secreted by these osteosarcoma cells contains the PDGF-B chain and is probably a homodimer of two B-chains.
Understanding the emergence of species' ranges is one of the most fundamental challenges in ecology. Early on, geographical barriers were identified as obvious natural constraints to the spread of species. However, many range borders occur along gradually changing landscapes, where no sharp barriers are obvious. Mechanistic explanations for this seeming contradiction incorporate environmental gradients that either affect the spatio-temporal variability of conditions or the increasing fragmentation of habitat. Additionally, biological mechanisms like Allee effects (i.e. decreased growth rates at low population sizes or densities), condition-dependent dispersal, and biological interactions with other species have been shown to severely affect the location of range margins. The role of dispersal has been in the focus of many studies dealing with range border formation. Dispersal is known to be highly plastic and evolvable, even over short ecological time-scales. However, only few studies concentrated on the impact of evolving dispersal on range dynamics. This thesis aims at filling this gap. I study the influence of evolving dispersal rates on the persistence of spatially structured populations in environmental gradients and its consequences for the establishment of range borders. More specially I investigate scenarios of range formation in equilibrium, periods of range expansion, and range shifts under global climate change ...
The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes.
In recent years high-throughput experiments provided a vast amount of data from all areas of molecular biology, including genomics, transcriptomics, proteomics and metabolomics. Its analysis using bioinformatics methods has developed accordingly, towards a systematic approach to understand how genes and their resulting proteins give rise to biological form and function. They interact with each other and with other molecules in highly complex structures, which are explored in network biology. The in-depth knowledge of genes and proteins obtained from high-throughput experiments can be complemented by the architecture of molecular networks to gain a deeper understanding of biological processes. This thesis provides methods and statistical analyses for the integration of molecular data into biological networks and the identification of functional modules, as well as its application to distinct biological data. The integrated network approach is implemented as a software package, termed BioNet, for the statistical language R. The package includes the statistics for the integration of transcriptomic and functional data with biological networks, the scoring of nodes and edges of these networks as well as methods for subnetwork search and visualisation. The exact algorithm is extensively tested in a simulation study and outperforms existing heuristic methods for the calculation of this NP-hard problem in accuracy and robustness. The variability of the resulting solutions is assessed on perturbed data, mimicking random or biased factors that obscure the biological signal, generated for the integrated data and the network. An optimal, robust module can be calculated using a consensus approach, based on a resampling method. It summarizes optimally an ensemble of solutions in a robust consensus module with the estimated variability indicated by confidence values for the nodes and edges. The approach is subsequently applied to two gene expression data sets. The first application analyses gene expression data for acute lymphoblastic leukaemia (ALL) and differences between the subgroups with and without an oncogenic BCR/ABL gene fusion. In a second application gene expression and survival data from diffuse large B-cell lymphomas are examined. The identified modules include and extend already existing gene lists and signatures by further significant genes and their interactions. The most important novelty is that these genes are determined and visualised in the context of their interactions as a functional module and not as a list of independent and unrelated transcripts. In a third application the integrative network approach is used to trace changes in tardigrade metabolism to identify pathways responsible for their extreme resistance to environmental changes and endurance in an inactive tun state. For the first time a metabolic network approach is proposed to detect shifts in metabolic pathways, integrating transcriptome and metabolite data. Concluding, the presented integrated network approach is an adequate technique to unite high-throughput experimental data for single molecules and their intermolecular dependencies. It is flexible to apply on diverse data, ranging from gene expression changes over metabolite abundances to protein modifications in a combination with a suitable molecular network. The exact algorithm is accurate and robust in comparison to heuristic approaches and delivers an optimal, robust solution in form of a consensus module with confidence values. By the integration of diverse sources of information and a simultaneous inspection of a molecular event from different points of view, new and exhaustive insights into biological processes can be acquired.
The responsiveness to IL-4 with and without costimulation with anti-IgM antibodies or phorbolester was studied in 35 cases of low grade non-Hodgkin Iymphoma by analyzing enhancement of CD23 and HLA dass li expression. The predominant phenotype responds directly to IL-4. Separate differentiation states can be distinguished according to coordinate or differential upregulation of CD23 and HLA dass II molecules by IL-4 alone, and differences in responsiveness to anti-IgM antibodies. A particular subgroup of B-lymphoma cells defines a separate stage of B-eeil differentiation. They fail to express high affinity binding sites for IL-4 and accordingly do not respond to IL-4- mediated signals. Cross-linking membrane lgM receptors or direct activation of protein kinase C via phorbolester induces IL-4 receptor expression and subsequent IL-4 reactivity.
To investigate the possible hemodynamic efl'ects of interleukin-6 (IL-6), a single dose of 15 mcg/kg of recombinant IL-6 isolated from Escherichia coli was injected intravenously in six pentobarbital-anesthetized dogs. After 30 min, saline infusion was performed to maintain the - pulmonary artery balloon-occluded pressure at baseline Ievel. The animals were observed for up to 5 hours. No other hemodynamic alteration was observed than a gradual decline in cardiac output attributed to anesthesia. Hematologic variables, blood glucose, and total serum proteins were also constant. IL-6 levels were markedly elevated in the blood, bot no tumor necrosis factor activity was detected. Thus a primary role for IL-6 in the early cardiovascular alterations associated with septic shock seems unlikely.
Background: In populations of most social insects, gene flow is maintained through mating between reproductive individuals from different colonies in periodic nuptial flights followed by dispersal of the fertilized foundresses. Some ant species, however, form large polygynous supercolonies, in which mating takes place within the maternal nest (intranidal mating) and fertilized queens disperse within or along the boundary of the supercolony, leading to supercolony growth (colony budding). As a consequence, gene flow is largely confined within supercolonies. Over time, such supercolonies may diverge genetically and, thus, also in recognition cues (cuticular hydrocarbons, CHC’s) by a combination of genetic drift and accumulation of colony-specific, neutral mutations. Methodology/Principal Findings: We tested this hypothesis for six supercolonies of the invasive ant Anoplolepis gracilipes in north-east Borneo. Within supercolonies, workers from different nests tolerated each other, were closely related and showed highly similar CHC profiles. Between supercolonies, aggression ranged from tolerance to mortal encounters and was negatively correlated with relatedness and CHC profile similarity. Supercolonies were genetically and chemically distinct, with mutually aggressive supercolony pairs sharing only 33.1%617.5% (mean 6 SD) of their alleles across six microsatellite loci and 73.8%611.6% of the compounds in their CHC profile. Moreover, the proportion of alleles that differed between supercolony pairs was positively correlated to the proportion of qualitatively different CHC compounds. These qualitatively differing CHC compounds were found across various substance classes including alkanes, alkenes and mono-, di- and trimethyl-branched alkanes. Conclusions: We conclude that positive feedback between genetic, chemical and behavioural traits may further enhance supercolony differentiation through genetic drift and neutral evolution, and may drive colonies towards different evolutionary pathways, possibly including speciation.
Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.
Mutant proteins (muteins) of human lnterleukin-4 (llA) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. co/1, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted. in a nearly co•mplete loss · of biological actiyity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of · the single tryptophane had smaller etTects.
Cell kinetic studies of T cells stimulated with the interleukin 2 (11-2), D-4, or both lymphokines were performed with conventional [3H] thymidine incorporation and with the bivariate BrdU/Hoechst technique. 11-2 and 11-4 are able to drive phytohemagglutininactivated T cells through more than one cell cycle. Neither synergistic nor inhibitory efl'ect on T -cell proliferationwas seen for the stimulation with both 11-2 and 11-4 as compared with the effect ofll-2 alone. The quantitative data ofthe cell cycle distribution ofphytohemagglutininactivated T cells suggestthat the population ofll-4-responsive cells is at least an overlapping population, if not a real subset of the ·population of the 11-2-responsive cells.
Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement
(1992)
lnterleukin-4 (IL-4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B-cells and the proliferation of T- and B-cell, and other cell types of the lymphoid system. An antagonist of human IL-4 was discovered during the studies presented here after Tyr124 of the recombinant proteinbad been substituted by an aspartic acid residue. This IL-4 variant, Y124D, bound with high affinity to the IL-4 receptor (K\(_D\) = 310 pM), but retained no detectable proliferative activity for T -<:ells and inhibited IL-4-dependent T -cell proliferation competitively (K\(_i\) = 620 pM). The loss of efficacy in variant Y124D was estimated to be > 100-fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B-cells. The subsitution of Tyr124 by either phenylalanine, histidine, asparagine, Iysine or glycine resulted in partial agonist variants with unaltered receptor binding atTmity and relatively small deficiencies in efficacy. These results demoostrate that high affinity binding and signal generation can be uncoupled efticiently in a Iigand of a receptor betonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL-4 receptor system can be derived from the IL-4 protein.
Interleukin 4 (IL-4) exerts a decisive role in the coord.ination of proteelive immune responses against parasites, particularly helminths. A disregulation of ll.r4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human ll.r4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4-helix-bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL-4 variants deficient in binding to the extracellular domain of the ll.r4 receptor (IL-4ReJ. In parallel, up to 1000-fold increased concentrations of this type of variant were required to induce T -cell proliferation and B-eeil CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild-type).ß.A variants affected at site 2 exhibited partial agonist activity during T -cell proliferation; however, they still bound with high affinity to IL-4Rex. [The generation of an IL-4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO }., 11, 3237-3244)]. These findings indicate that IL-4 functions by bind.ing IL-4Rex via site 1 which is constituted by residues on helices A and C. They further suggest that the association of a second, still undetined receptor protein with site 2 in helix D activates the receptor system and generates a transmembrane signal.
Cytokines are hormones that carry information from ceJI to ceH. This information is read from their surface upon binding to transmembrane receptors and by the subsequent initiation of receptor oligomerization. An inftuence on this process through mutagenesis on the hormone surface is highly desirab)e for medical reasons. However, an understanding of hormone-receptor interactions requires insight into the structural changes introduced by the mutations. In this line structural studies on human TL-4 and the medically important IL-4 antagonists YI24D and Y124G are presented. The site a.round YI24 is an important epitope responsible for the a.bility of 11-4 t.o ca.use a signal in the target cells. It is shown that the local main-chain structure around residue 124 in the variants remains unchanged. A strategy is presented here which allows the study of these types of proteins and their variants by NMR which does not require carbon Iabeiied sa.mples.
The effects of the interlenkins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. 8oth IL· 7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 wasmorepotent on cell cycle progression than IL-9. Neither synergistic nor inhibitory effects were seen between various combinations of the lymphokines IL-7, IL-9 and IL-4 compared to each lymphokine alone. When T cells are activated with phytohemagglutinin for 3 days, all or most IL-4 responsive cells respond to IL-7 as weil, whereas only a part of IL-7 responders are IL-4 responders. In contrast, when T cells are activated with phytohemagglutinin for 7 days, the quantitative data of the cell cycle distribution soggest that the population of IL-7 responders is at least an overlapping, if not a real subset of the population of the IL-4 responders.
Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis
(1994)
Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-a-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormonereceptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 Iack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors.
Neutralizing monoclonal antibodies define two different functional sites in human interleukin-4
(1994)
Human interleukin-4 (IL-4) is a small four-helix-bundle protein which is essential for organizing defense reactions against macroparasites, in particular helminths. Human IL-4 also appears to exert a pathophysiological role during various IgE-mediated allergic diseases. Seven different monoclonal antibodies neutralizing the activity of human IL-4 were studied in order to identify functionally important epitopes. A collection of 41 purified IL-4 variants was used to analyse how defined amino acid replacements affect binding affinity for each individual mAb. Specific amino acid positions could be assigned to four different epitopes. mAbs recognizing epitopes on helix A and/or C interfered with IL-4 receptor binding and thus inhibited IL-4 function. However, other mAbs also inhibiting IL-4 function recognized an epitope on helix D of IL-4 and did not inhibit IL-4 binding to the receptor protein. One mAb, recognizing N-terminal and C-terminal residues, partially competed for binding to the receptor. The results of these mAb epitope analyses confirm and extend previous data on the functional consequences of the amino acid replacements which showed that amino acid residues in helices A and C of IL-4 provide a binding site for the cloned IL-4 receptor and that a signalling site in helix D interacts with a further receptor protein.
Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.
Human interleukin-4 possesses two distinct sites for receptor activation. A signaHing site, comprising residues near the C-terminus on helix D, determines the efficacy of interleukin-4 signal transduction without affecting the binding to the interleukin-4 receptor a subunit. A complete antagonist and a series of low-efficacy agonist variants of human interleukin-4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin-4, with replacements of böth Arg121 and Tyr124 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y124D]interleukin-4 is estimated to be larger than 2000-fold. Variant [R121D,Y124D]interleukin-4 was also a perfect antagonist for inhibition of interleukin-13-dependent responses in B-cells and the TF-1 cellline with a K\(_i\) value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleuk.in-13- induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Rm the extracellular domain of the interleuk.in-4 receptor a subunit. These results indicate that efficient interleukin-4 antagonists can be designed on the basis of a sequential two-step activation model. In addition, the experiments indicate the functional participation of the interleukin-4 receptor a subunit in the interleukin-13 receptor system.
Monoclonal hBMP/NCP (human bone morphogenetic protein anrl associaterl noncollagenous proteins) antiborlies of the lgG class were prorlucerl. In vitro, 12 of 19 hBMP/NCP antiborlies showerl functional inhibition of hBMP/ NCP-induced chondroneogenesis in a neonatal muscle tissue assay. Inducing factors were characterized by their inhibiting antibodies with immunoblotting. Several peptide factors seem to be involved in the cascade of inducerl chondro- and osteogenesis.
Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection / immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation, which in turn is a critical parameter for male fertility.
Yersinia enterocolitica subsp. palearctica serobiotype O:3/4 comprises about 80-90 % of all human patient isolates in Germany and Europe and is responsible for sporadic cases worldwide. Even though this serobiotype is low pathogenic, Y. enterocolitica subsp. palearctica serobiotype O:3/4 is involved in gastroenteritis, lymphadenitis and various extraintestinal sequelae as reactive arthritis. The main animal reservoir of this serobiotype are pigs, causing a high rate of O:3/4 contaminations of raw pork in butcher shops in Germany (e.g. Bavaria 25 %) and countries in north-east Europe. As Y. enterocolitica O:3/4 is geographically and phylogenetically distinct from the so far sequenced mouse-virulent O:8/1B strain, complete genome sequencing has been performed for the European serobiotype O:3/4 DSMZ reference strain Y11, which has been isolated from a patient stool. To gain greater insight into the Y. enterocolitica subspecies palearctica group, also draft genome sequences of two other human O:3/4 isolates (strains Y8265, patient isolate, and Y5307, patient isolate associated with reactive arthritis), a closely related Y. enterocolitica palearctica serobiotype O:5,27/3 (strain Y527P), and two biotype 1A strains (a nosocomial strain of serogroup O:5 and an environmental serogroup O:36 isolate) have been performed. Those strains were compared to the high-pathogenic Y. enterocolitica subsp. enterocolitica serobiotype O:8/1B strain 8081 to address the peculiarities of the strain Y11 and the Y. enterocolitica subspecies palearctica group. The main focus was to unravel the pathogenic potential of strain Y11 and thus to identify novel putative virulence genes and fitness factors, especially those that may constitute host specificity of serobiotype O:3/4. Y. enterocolitica subspecies palearctica serobiotype O:3/4 strains lack most of the mouse-virulence-associated determinants of Y. enterocolitica subsp. enterocolitica serotype O:8, for example the HPI, Yts1 type 2 and Ysa type three secretion systems. In comparison, serobiotype O:3/4 strains obviously acquired a different set of genes and genomic islands for virulence and fitness such as the Ysp type three secretion system, an RtxA-like putative toxin, insecticidal toxins and a functional PTS system for N-acetyl-galactosamine uptake, named aga-operon. The aga-operon is able to support the growth of the Y. enterocolitica subsp. enterocolitica O:8/1B on N-acetyl-galactosamine after transformation with the aga operon. Besides these genes, also two prophages, PhiYep-2 and PhiYep-3, and a asn tRNA-associated GIYep-01 genomic island might influence the Y. enterocolitica subsp. palearctica serobiotype O:3/4 pathoadaptation. The PhiYep-3 prophage and the GIYep-01 island show recombination activity and PhiYep-3 was not found in all O:3/4 strains of a small strain collection tested. Y. enterocolitica subsp. palearctica serobiotype O:5,27/3 strain Y527P was found to be closely related to all serobiotype O:3/4 strains, whereas the biotype 1A isolates have more mosaic-segmented genomes and share putative virulence genes both with serobiotypes O:8/1B and O:3/4, which implies their common descent. Besides the pYV virulence plasmid, biotype 1A strains lack classical virulence markers as the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. Interestingly, there are no notable differences between the known virulence factors present in nosocomial and environmental strains, except the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital serogroup O:5 isolate.
As one of the disciplines of systems biology, proteomics is central to enabling the elucidation of protein function within the cell; furthermore, the question of how to deduce protein structure and function from the genetic readout has gained new significance. This problem is of particular relevance for proteins engaged in cell signalling. In dealing with this question, I shall critically comment on the reliability and predictability of transmission and translation of the genetic blue print into the phenotype, the protein. Based on this information, I will then evaluate the intentions and goals of today’s proteomics and gene-networking and appraise their chances of success. Some of the themes commented on in this publication are explored in greater detail with particular emphasis on the historical roots of concepts and techniques in my forthcoming book, published in German: Von Molekülen zu Zellen. 100 Jahre experimentelle Biologie. Betrachtungen eines Biochemikers
Background: The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C.floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals. Results: We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of Blochmannia. In adult animals the number of bacteriocytes generally decreased. Conclusions: During the life cycle of the animals the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of Blochmannia for its host in particular during metamorphosis.
Understanding of complex interactions and events in a nervous system, leading from the molecular level up to certain behavioural patterns calls for interdisciplinary interactions of various research areas. The goal of the presented work is to achieve such an interdisciplinary approach to study and manipulate animal behaviour and its underlying mechanisms. Optical in vivo imaging is a new constantly evolving method, allowing one to study not only the local but also wide reaching activity in the nervous system. Due to ease of its genetic accessibility Drosophila melanogaster represents an extraordinary experimental organism to utilize not only imaging but also various optogenetic techniques to study the neuronal underpinnings of behaviour. In this study four genetically encoded sensors were used to investigate the temporal dynamics of cAMP concentration changes in the horizontal lobes of the mushroom body, a brain area important for learning and memory, in response to various physiological and pharmacological stimuli. Several transgenic lines with various genomic insertion sites for the sensor constructs Epac1, Epac2, Epac2K390E and HCN2 were screened for the best signal quality, one line was selected for further experiments. The in vivo functionality of the sensor was assessed via pharmacological application of 8-bromo-cAMP as well as Forskolin, a substance stimulating cAMP producing adenylyl cyclases. This was followed by recording of the cAMP dynamics in response to the application of dopamine and octopamine, as well as to the presentation of electric shock, odorants or a simulated olfactory signal, induced by acetylcholine application to the observed brain area. In addition the interaction between the shock and the simulated olfactory signal by simultaneous presentation of both stimuli was studied. Preliminary results are supporting a coincidence detection mechanism at the level of the adenylyl cyclase as postulated by the present model for classical olfactory conditioning. In a second series of experiments an effort was made to selecticvely activate a subset of neurons via the optogenetic tool Channelrhodopsin (ChR2). This was achieved by recording the behaviour of the fly in a walking ball paradigm. A new method was developed to analyse the walking behaviour of the animal whose brain was made optically accessible via a dissection technique, as used for imaging, thus allowing one to target selected brain areas. Using the Gal4-UAS system the protocerebral bridge, a substructure of the central complex, was highlighted by expressing the ChR2 tagged by fluorescent protein EYFP. First behavioural recordings of such specially prepared animals were made. Lastly a new experimental paradigm for single animal conditioning was developed (Shock Box). Its design is based on the established Heat Box paradigm, however in addition to spatial and operant conditioning available in the Heat Box, the design of the new paradigm allows one to set up experiments to study classical and semioperant olfactory conditioning, as well as semioperant place learning and operant no idleness experiments. First experiments involving place learning were successfully performed in the new apparatus.
The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Results: We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Conclusions: Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.
Background: The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system. Results: We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution. Conclusions: Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr ligands. This fish-specific occurrence of Egf receptor specialization offers unique opportunities to study the functions of different Egf receptor-ligand combinations and their biological outputs in vertebrates. Furthermore, our results strongly support the use of homologous ligands in future studies, as sufficient cross-specificity is very unlikely for this ligand/receptor system.
Background: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable overexpression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.
Background: Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods: Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results: Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion: Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.
The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays
(2010)
Background: Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results: The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions: ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see “Additional Files” section) and at: http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/
Background: Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set. Results: Here we present a platform-independent framework based on Java and Java 3D for accelerated rendering of biological images. Our framework is seamlessly integrated into ImageJ, a free image processing package with a vast collection of community-developed biological image analysis tools. Our framework enriches the ImageJ software libraries with methods that greatly reduce the complexity of developing image analysis tools in an interactive 3D visualization environment. In particular, we provide high-level access to volume rendering, volume editing, surface extraction, and image annotation. The ability to rely on a library that removes the low-level details enables concentrating software development efforts on the algorithm implementation parts. Conclusions: Our framework enables biomedical image software development to be built with 3D visualization capabilities with very little effort. We offer the source code and convenient binary packages along with extensive documentation at http://3dviewer.neurofly.de.
For a large fraction of the proteins expressed in the human brain only the primary structure is known from the genome project. Proteins conserved in evolution can be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and characterized with the aim to identify the target antigen. The mAb ab52 was found to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15 (epidermal growth factor receptor pathway substrate clone 15) to be a strong candidate. Another mAb from the library, aa2, was already found to recognize EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and 2D electrophoretic separations revealed similar patterns, hence indicating that both antigens could represent the same protein. Finally absence of the wild-type signal in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52 antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for applications like immunoprecipitation (IP). It has already been submitted to the Developmental Studies Hybridoma Bank (DSHB) to be easily available for the entire research community. The mAb na21 was also found to be an IgM. It recognizes a membrane associated antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to biochemically purify the endogenously expressed protein from the tissue, gave promising results but could not be completed due to lack of time. Thus biochemical purification of the protein seems possible in order to facilitate its identification by mass spectrometry. Several other mAbs were studied for their staining pattern on cryosections and whole mounts of Drosophila brains. However, many of these mAbs stained very few structures in the brain, which indicated that only a very limited amount of protein would be available as starting material. Because these antibodies did not produce signals on Western blots, which made it impossible to enrich the antigens by electrophoretic methods, we did not attempt their purification. However, the specific localization of these proteins makes them highly interesting and calls for their further characterization, as they may play a highly specialized role in the development and/or function of the neural circuits they are present in. The purification and identification of such low expression proteins would need novel methods of enrichment of the stained structures.
Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration.
An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade.
Precise control of mitotic progression is vital for the maintenance of genomic integrity. Since the loss of genomic integrity is known to promote tumorigenesis, the identification of knew G2/M regulatory genes attracts great attention. LINC, a human multiprotein complex, is a transcriptional activator of a set of G2/M specific genes. By depleting LIN9 in MEFs, a core subunit of LINC, Gas2l3 was identified as a novel LINC target gene. The so far uncharacterized Gas2l3 gene encodes for a member of the family of growth arrest specific 2 (GAS2) proteins, which share a highly conserved putative actin binding CH and a putative microtubule binding GAS2 domain. In the present study GAS2L3 was identified as a LINC target gene also in human cells. Gene expression analysis revealed that GAS2L3 transcription, in contrast to all other GAS2 family members, is highly regulated during the cell cycle with highest expression in G2/M. The GAS2L3 protein showed a specific localization pattern during the M phase: In metaphase, GAS2L3 localized to the mitotic spindle, relocated to the spindle midzone microtubules in late anaphase and concentrated at the midbody in telophase where it persisted until the end of cytokinesis. Overexpression of a set of different GAS2L3 deletion mutants demonstrated that the localization to the mitotic microtubule network is dependent on the C-terminus, whereas the midbody localization is dependent on full length GAS2L3 protein. Additionally, exclusive overexpression of the CH domain induced the formation of actin stress fibers, suggesting that the CH domain is an actin binding domain. In contrast, the GAS2 domain was neither needed nor sufficient for microtubule binding, indicating that there must be an additional so far unknown microtubule binding domain in the C-terminus. Interestingly, immunoblot analysis also identified the C-terminus as the domain responsible for GAS2L3 protein instability, partially dependent on proteasomal degradation. Consistent with its specific localization pattern, GAS2L3 depletion by RNAi demonstrated its responsibility for proper mitosis and cytokinesis. GAS2L3 depletion in HeLa cells resulted in the accumulation of multinucleated cells, an indicator for chromosome mis-segregation during mitosis. Also the amount of cells in cytokinesis was enriched, indicating failures in completing the last step of cytokinesis, the abscission. Strikingly, treatment with microtubule poisons that lead to the activation of the spindle assembly checkpoint (SAC) indicated that the SAC was weakened in GAS2L3 depleted cells. Although the exact molecular mechanism is still unknown, fist experiments support the hypothesis that GAS2L3 might be a regulator of the SAC master kinase BUBR1. In conclusion, this study provides first evidence for GAS2L3 as a novel regulator of mitosis and cytokinesis and it might therefore be an important guardian against tumorigenesis.
Monoclonal antlbodies (MAbs) directed against Xiphophorus melanoma cells were deve(oped and tested by lndirect immunofluorescence and Immunoperoxidase staining for reactivity with a panel of I 5 allogeneic tissues and 12 allogeneic cell llnes. The reactivity of such MAbs was restricted to melanoma cells from tumor biopsies and melanoma-derived cell lines. ln addition, all embryonie cells of all histiotypes from developmental stages later than mld·organogenesis and from corresponding short term in vitro cultures reacted with these MAbs. ln contrast, normal tissues and organs from adult fish dlsplayed no reactivity, thus implying that the melanoma-associated antigens detected by the MAbs described are oncofetal antigens.
ln order to construct fish specific expression vectors for studies on gene regulation in vitro and in vivo a variety of heterologous enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial chloramphenicol acetyl transferase reporter gene in three teleost fish cell lines. Several viral enhancers were found to be constitutively active at high Ieveis. The human metallothionein promoter showed inducible expression in the presence of heavy metal Ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient splicing and Iack of translation were observed, pointing to limitations in the use of heterologous genes in gene transfer experiments. On the contrary, some heterologous promoters and enhancers functioned in fish c as weil as in their cell type of origin, indicating t at corresponding transcription factors are sufficient conserved between fish and human over a period of 900 million years of Independent evolution.
Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Same of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern Iod from the platyfish Xiphophorus maculatus the melanoma-inducing gene has been doned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanomapredisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a geneduplication event, thereby acquiring the oncogenic potential. The homology of the melanomainducing Iod points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.
Species of small fish are becoming useful tools for studies on vertebrate development. Wehave investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporaland spatial expression patterns ofbacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as weil as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.
In dooal unisexual vertebrales, the genes specifying the males become dispensable. To study tbe rate of such geoes the gynogeoetic all-female fisb Poecilillfonnolll was treated with androgens. Phenotypic males were obtained that exbibited the complete set of male cbaracteristics of dosely related gooocboristic species, induding body proportions, pigmentation, the extremely complex insemination apparatus of poecilüd fish, sexual bebavior, and spermatogeoesls. Tbe apparent stabllity of such genic structures, induding those involved in androgen regulation, is contrasted by high instability of noncoding sequeaces. Frequent mutations, thelr donal transmission, and at least two truly hypervariable Iod leading to individual difl'ereaces between these othenrise donal organisms were detected by DNA fingerprinting. These observations substantiate the concept that also in "ameiotic" vertebrates certain compartments of the genome are more prooe to mutatiooal alterations than others.
Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecu/ar identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confinn that ES 1 is a distant marker for R ( approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity Ievels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markersthat bridge the large distance between ESl and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding Iandmarks on a physical map of the R-containing chromosomal region.
Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically dernonstrahle sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)\(_n\)/(gaca)\(_m\) are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)\(_4\) probe. The (GATA)\(_4\) oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In cantrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)\(_4\). Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)\(_4\) probe.
Mammalian nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three-dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fi.sh Xiphophorus, with the mammalian homlogues. Genomic clones encoding fish NGF and BDNF were isolated by cross-hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half-maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purifi.ed recombinant BDNF. However, the activity ofrecombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng,lml, 75-fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA-deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF bad reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates ftom a common ancestral gene about 600 million years ago. By RNA gel blot anaJysis NGF mRNA was detected during late embryonie development; BDNF was present in adult brain.
Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential Iigand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properlies and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related Iigand-binding domain sequences of the human epidennal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk proteinwas examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transfonning protein equivalent to other tyrosine kinase oncoproteins.
Pseudosexual behaviour is a rare phenomenon associated with unisexuality in vertebrates. In the gynogenetic, all-female teleost Poecilia formosa, rare individuals occur that resemble males of closely related gonochoristic species both in behaviour and external morphology. These masculinized gynogens and normal gynogens are members of the same clone, as demonstrated by DNA-fingerprinting. The behaviour of these masculinized gynogens is described and compared to the behaviour of the gonochoristic species Poecilia mexicana, P. latipinna and their hybrid as weil as androgen-treated individuals of P. formosa. No statistically significant difTerences were found between masculinized gynogens and hormonetreated individuals nor between the gonochoristic P. mexicana and P. latipinna males. Differences exist between gonochoristic and unisexual species. Passihle causes and effects of masculinized gynogens are discussed.
To investigate the regulation of metallothionein-encoding genes in fish, we have isolated and sequenced the rainbow trout metallothionein-A-encoding gene (tMT-A) by polymerase chain reaction. This gene spans about 1.1 kb, consists of three exons and two introns, and has an A+ T-rieb 5' -region which contains a TATAAA signal, and two metal responsive elements (MREs). The transcription start point is centered around an A residue 81 nt upstream of the ATG codon.
The promoter of the rainbow trout metallothionein B gene ( tMTb) was isolated from genomic DNA by the polymerase chain reaction (PCR ), fused to the bacterial chloramphenicol acetyltransferase (CAT) genein an expression vector, and functionally analyzed in one human cellline and four fish celllines. This promoter exhibited an extremely low basal expression in all celllines and was zincand cadmium-inducible except in the fish melanoma cell line where the promoter was completely inactive. The metal-induced expression patterns were cellline-specific. In general the fish promoter was more species- and cell type-specific than its human counterpart. In a transient assay it was functional in developing embryos of the medaka ( Oryzias /atipes). These properties make this promoter suitable for inducible, tissue-specific expression of transgenes and for in vivo studies of gene function and regulation.
The demonstration ofthe chromosomal mode ofsex determinationvia genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the mostprimitive form of sex chromosornes. In many different taxa the evolutionary process involved in the differentiation ofadvanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hydridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparaus fishes ofthe family Poeciliidae. The hybridization pattern together with specific staining ofthe constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as weil as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.
Hierarchical structures among male indlviduals in a population are frequently reflected ln differences in aggressive and reproductive behavior and access to the females. In general, sodal dominance requires the Investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesls has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Borke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poecUiid fish Limia perugitu, in small matlng groups the dominant male has 8 mating success of 100%, whereas ln larger groups lts contribution to the offspring unexpectedly drops to zero.
Melanoma formation in the teleost Xiphophorus is caused by a dominant genetic locus, Tu. This locus includes the Xmrk oncogene, which encodes a receptor tyrosine kinase. Tumor induction is. suppressed in wild-type fish by a tumor suppressor locus, R. Molecular genetic analyses revealed that the Tu locus emerged by nonhomologaus recombination of the Xmrk proto-oncogene with a previously uncharacterized sequence, D. This event generated an additional copy of Xmrk with a new promoter. Suppression of the new Xmrk promoter by R in parental fish and its deregulation in hybrids explain the genetics of melanoma formation in Xiphophorus.
The silver carp (Hypophthalmichthys molitrix) growth hormone (GH) genewas isolated and sequenced following amplification from genomic DNA by the polymerase chain reaction. The gene spans a region of approx. 2.5 kb nucleotides (nt) and consists of five exons. The sequence predicts a polypeptide of 210 amino acids (aa) including a putative signal peptide of 22 hydrophobic aa residues. The arrangement of exons and introns is identical to the GH genes of common carp, grass carp, and very similar to mammals and birds, but quite different from that for the GH genes of tilapia and salmonids. The silver carp GH gene shares a high homology at the nt and aa Ievels with those of grass carp (95.3% nt, 99.5% aa) and of common carp (81% nt, 95.7% aa).
In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following cunstructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAßgsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter ß-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic ß-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was Iigated to GH cDNA of S. aurata to form the pcAß-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found tobe induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.
Which home for coelacanth?
(1993)
Hereditary melanoma in Xiphophorus hybrids canying the melanoma·induclng Tu-Sd locus is caused by transcriptional activation of the Xmrk gene that resides at the Tu·Sd locus and encodes a novel member of receptor tyrosine kinases (RTK). ln this study, a total of 17 hereditary melanomas from various hybrid genotypes harbouring 7 different Tu alleles were also found to aver-express the correspondlng Xmrlc alleles. The Ievei of over-expression correlated with the degree of malignancy of the melanoma. ln addition, Xsrc expression was high ln many malignant melanomas. Expression pattems and Ieveis of the Xiphophorus EGF-receptor gene (Xerb B), the c-myc (Xmyc), and the PDGF (Xsls) gene(s) were not intriguing. Transcription of the ras gene(s) may be correlated to secondary events of melanoma progression. Expression pattems of Xfms, the Xiphophorus CSF-1 receptor homologue, can be explained by different contents of infiltrating macrophages in the tumors. ln carcinogen-induced tumors includlng one melanoma no significant expression of the Xmrk oncogene could be detected. Xsrc expression, however, was strikingly high. This indicates that activation of oncogenes other than Xmrk ls instrumental in tumorigenesls of neoplasia of non-hereditary origin.
In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA ofmales and females ofthe rainbow trout (XX/XY) and of Leporinus elongatus (ZW /ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinusfriderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.
The male-polymorphic poeciliid fish, Limia perugiae, a small teleostean endemic to the southeast of the Caribbean island Hispafiola, consists of three male size morphs with uniform females. Large males differentiate at a size va:rying between 25 and 38 mm; intermediate males, between 21 and 25 mm. Under competition, !arge males exhibit an elaborate courtship display, whereas small males show only a sneak-chase behavior. Intermediate males adapt their tactics to the respective competitors. However, all malemorphs can switch from courtship display to sneak-chase behavior. In large mating groups with four males of different size and five or six virgin females, large dominant a-males as weil as small subordinate \(\delta\)-males did not produce any offspring. Unexpectedly, all progeny were sired exclusively by the intemediate subordinate ß- and \(\gamma\)-males. Breeding experiments with the three male morphs can best be explained by a model of Y -linked genes for small and !arge size which are both suspended by the activity of an autosomal recessive repressor responsible for the development of intermediate males. The dominant allele of the recessive repressor, in either its homoorits heterozygous state, activates the Y-chromosomal genes for !arge or small size, respectively. Accordingly, intermediate males may produce male offspring of all size classes, depending on the presence of either the Y-linked gene or the autosomal repressor.
According to a changing environment it is crucial for animals to make experience and learn about it. Sensing, integrating and learning to associate different kinds of modalities enables animals to expect future events and to adjust behavior in the way, expected as the most profitable. Complex processes as memory formation and storage make it necessary to investigate learning and memory on different levels. In this context Drosophila melanogaster represents a powerful model organism. As the adult brain of the fly is still quite complex, I chose the third instar larva as model - the more simple the system, the easier to isolate single, fundamental principles of learning. In this thesis I addressed several kinds of questions on different mechanism of olfactory associative and synaptic plasiticity in Drosophila larvae. I focused on short-term memory throughout my thesis. First, investigating larval learning on behavioral level, I developed a one-odor paradigm for olfactory associative conditioning. This enables to estimate the learnability of single odors, reduces the complexity of the task and simplify analyses of "learning mutants". It further allows to balance learnability of odors for generalization-type experiments to describe the olfactory "coding space". Furthermore I could show that innate attractiveness and learnability can be dissociated and found finally that paired presentation of a given odor with reward increase performance, whereas unpaired presentations of these two stimuli decrease performance, indicating that larva are able to learn about the presence as well as about the absence of a reward. Second, on behavioral level, together with Thomas Niewalda and colleagues we focussed on salt processing in the context of choice, feeding and learning. Salt is required in several physiological processes, but can neither be synthesized nor stored. Various salt concentrations shift the valence from attraction to repulsion in reflexive behaviour. Interestingly, the reinforcing effect of salt in learning is shifted by more than one order of magnitude toward higher concentrations. Thus, the input pathways for gustatory behavior appear to be more sensitive than the ones supporting gustatory reinforcement, which is may be due to the dissociation of the reflexive and the reinforcing signalling pathways of salt. Third, in cooperation with Michael Schleyer we performed a series of behavioral gustatory, olfactory preference tests and larval learning experiments. Based on the available neuroanatomical and behavioral data we propose a model regarding chemosensory processing, odor-tastant memory trace formation and the 'decision' like process. It incorporates putative sites of interaction between olfactory and gustatory pathways during the establishment as well as behavioral expression of odor-tastant memory. We claim that innate olfactory behavior is responsive in nature and suggest that associative conditioned behavior is not a simple substitution like process, but driven more likely by the expectation of its outcome. Fourth, together with Birgit Michels and colleagues we investigated the cellular site and molecular mode of Synapsin, an evolutionarily conserved, presynaptic vesicular phosphoprotein and its action in larval learning. We confirmed a previously described learning impairment upon loss of Synapsin. We localized this Synapsin dependent memory trace in the mushroom bodies, a third-order "cortical" brain region, and could further show on molecular level, that Synapsin is as a downstream element of the AC-cAMP-PKA signalling cascade. This study provides a comprehensive chain of explanation from the molecular level to an associative behavioral change. Fifth, in the main part of my thesis I focused on molecular level on another synaptic protein, the Synapse associated protein of 47kDa (Sap47) and its role in larval behavior. As a member of a phylogenetically conserved gene family of hitherto unknown function. It is localized throughout the whole neuropil of larval brains and associated with presynaptic vesicles. Upon loss of Sap47 larvae exhibit normal sensory detection of the to-be-associated stimuli as well as normal motor performance and basic synaptic transmission. Interestingly, short-term plasticity is distorted and odorant–tastant associative learning ability is reduced. This defect in associative function could be rescued by restoring Sap47 expression. Therefore, this report is the first to suggest a function for Sap47 and specifically argues that Sap47 is required for synaptic as well as for behavioral plasticity in Drosophila larva. This prompts the question whether its homologs are required for synaptic and behavioral plasticity also in other species. Further in the last part of my thesis I contributed to the study of Ayse Yarali. Her central topic was the role of the White protein in punishment and relief learning in adult flies. Whereas stimuli that precede shock during training are subsequently avoided as predictors for punishment, stimuli that follow shock during training are later on approached, as they predict relief. Concerning the loss of White we report that pain-relief learning as well as punishment learning is changed. My contribution was a comparison between wild type and the white1118 mutant larvae in odor-reward learning. It turned out that a loss of White has no effect on larval odorant-tastant learning. This study, regarding painrelief learning provides the very first hints concerning the genetic determinants of this form of learning.
DARWIN\(^1\) believed that sexual selection accounts for the evolution of exaggerated male ornaments, such as the sword-like caudal fin extensions of male fishes of the genus Xiphophorus, that appear detrimental to survival. Swordtails continue to feature prominently in empirical work and theories of sexual selection; the pre-existing bias hypothesis has been offered as an explanation for the evolution of swords in these fishes\(^{2,3}\). Based upon a largely morphological phylogeny, this hypothesis suggests that female preference to mate with sworded males arose in ancestrally swordless species, thus pre-dating the origin of the sword itself and directly driving its evolution. Here we present a molecular phylogeny (based on mitochondrial and nuclear DNA sequences) of Xiphophorus which differs from the traditional one: it indicates that the sword originated and was lost repeatedly. Our phylogeny suggests that the ancestor of the genus is more likely to have possessed a sword than not, thus questioning the applicability of the pre-existing bias hypothesis as an explanation for the cvolution of this sexually selected trait.
The melanoma·inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. U sing a highly specific antiserum p~oduced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cellline revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cellsupernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.
DURING vertebrale development, many neurons depend for survival and differentiation on their target cells\(^{1-3}\). The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF)\(^1\). NGF and the other known members of tbe neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT -3) and neurotrophin-4/5 (NT -4/5) are conserved as distinct genes over large evolutionary distances\(^{4 -6}\). Here we report the cloning of neurotrophin-6 (NT -6), a new member of this family from the teleost fish Xiphophorus. NT -6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT -6 from cell surface and extracellular matrix molecules. Recombinant purified NT -6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT -6 is expressed in tbe embryonie valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecuJes may modulate its action in the nervous system .
Honeybees (Apis mellifera) forage on a great variety of plant species, navigate over large distances to crucial resources, and return to communicate the locations of food sources and potential new nest sites to nest mates using a symbolic dance language. In order to achieve this, honeybees have evolved a rich repertoire of adaptive behaviours, some of which were earlier believed to be restricted to vertebrates. In this thesis, I explore the mechanisms involved in honeybee learning, memory, numerical competence and navigation. The findings acquired in this thesis show that honeybees are not the simple reflex automats they were once believed to be. The level of sophistication I found in the bees’ memory, their learning ability, their time sense, their numerical competence and their navigational abilities are surprisingly similar to the results obtained in comparable experiments with vertebrates. Thus, we should reconsider the notion that a bigger brain automatically indicates higher intelligence.
All animals learn in order to cope with challenges imposed on them by their environment. This is true also for both larval and adult fruit flies as exemplified in pavlovian conditioning. The focus of this Thesis is on various aspects of the fruit flies learning ability. My main project deals with two types of learning which we call punishment-learning and pain-relief learning. Punishment learning happens when fruit flies are exposed to an odour which is followed by electric shock. After such training, flies have learned that that odour signals pain and consequently will avoid it in the future. If the sequence of the two stimuli is reversed such that odour follows shock, flies learn the odour as a signal for relief and will later on approach it. I first report a series of experiments investigating qualitative and parametric features of relief-learning; I find that (i) relief learning does result from true associative conditioning, (ii) it requires a relatively high number of training trials, (iii) context-shock training is ineffective for subsequent shock-odour learning. A further question is whether punishment-learning and pain-relief learning share genetic determinants. In terms of genetics, I test a synapsin mutant strain, which lacks all Synapsin protein, in punishment and relief-learning. Punishment learning is significantly reduced, and relief-learning is abolished. Pan-neuronal RNAi-mediated knock-down of Synapsin results in mutant-like phenotypes, confirming the attribution of the phenotype to lack of Synapsin. Also, a rescue of Synapsin in the mushroom body of syn97 mutants restores both punishment- and relief-learning fully, suggesting the sufficiency of Synapsin in the mushroom body for both these kinds of learning. I also elucidate the relationship between perception and physiology in adult fruit flies. I use odour-shock conditioning experiments to identify degrees of similarity between odours; I find that those similarity measures are consistent across generalization and discrimination tasks of diverse difficulty. Then, as collaborator of T. Völler and A. Fiala, I investigate how such behavioural similarity/dissimilarity is reflected at the physiological level. I combine the behaviour data with calcium imaging data obtained by measuring the activity patterns of those odours in either the sensory neurons or the projection neurons at the antennal lobe. Our interpretation of the results is that the odours perceptual similarity is organized by antennal lobe interneurons. In another project I investigate the effect of gustatory stimuli on reflexive behaviour as well as their role as reinforcer in larval learning. Drosophila larvae greatly alter their behaviour in presence of sodium chloride. Increasing salt concentration modulates choice behaviour from weakly appetitive to strongly aversive. A similar concentration-behaviour function is also found for feeding: larval feeding is slightly enhanced in presence of low salt concentrations, and strongly decreased in the presence of high salt concentrations. Regarding learning, relatively weak salt concentrations function as appetitive reinforcer, whereas high salt concentrations function as aversive reinforcer. Interestingly, the behaviour-concentration curves are shifted towards higher concentrations from reflexive behaviour (choice behaviour, feeding) as compared to associative learning. This dissociation may reflect a different sensitivity in the respective sensory-motor circuitry.
Bees are subject to permanent threat from predators such as ants. Their nests with large quantities of brood, pollen and honey represent lucrative targets for attacks whereas foragers have to face rivalry at food sources. This thesis focused on the role of stingless bees as third party interactor on ant-aphid-associations as well as on the predatory potential represented by ants and defense mechanisms against this threat. Regular observations of an aphid infested Podocarpus for approaching stingless bees yielded no results. Another aim of this thesis was the observation of foraging habits of four native and one introduced ant species for assessment of their predatory potential to stingless bees. All species turned out to be dietary balanced generalists with one mostly carnivorous species and four species predominantly collecting nectar roughly according to optimal foraging theory. Two of the species monitored, Rhytidoponera metallica and Iridomyrmex rufoniger were considered potential nest robbers. As the name implies, stingless bees lack the powerful weapon of their distant relatives; hence they specialized on other defense strategies. Resin is an important, multipurpose resource for stingless bees that is used as material for nest construction, antibiotic and for defensive means. For the latter purpose highly viscous resin is either directly used to stick down aggressors or its terpenic compounds are included in the bees cuticular surface. In a feeding choice experiment, three ant species were confronted with the choice between two native bee species - Tetragonula carbonaria and Austroplebeia australis - with different cuticular profiles and resin collection habits. Two of the ant species, especially the introduced Tetramorium bicarinatum did not show any preferences. The carnivorous R. metallica predominantly took the less resinous A. australis as prey. The reluctance towards T. carbonaria disappeared when the resinous compounds on its cuticle had been washed off with hexane. To test whether the repulsive reactions were related to the stickiness of the resinous surface or to chemical substances, hexane extracts of bees’ cuticles, propolis and three natural tree resins were prepared. In the following assay responses of ants towards extract treated surfaces were observed. Except for one of the resin extracts, all tested substances had repellent effects to the ants. Efficacy varied with the type of extract and species. Especially to the introduced T. bicarinatum the cuticular extract had no effect. GCMS-analyses showed that some of the resinous compounds were also found in the cuticular profile of T. carbonaria which featured reasonable analogies to the resin of Corymbia torelliana that is highly attractive for stingless bees. The results showed that repellent effects were only partially related to the sticky quality of resin but were rather caused by chemical substances, presumably sesqui- and diterpenes. Despite its efficacy this defense strategy only provides short time repellent effects sufficient for escape and warning of nest mates to initiate further preventive measures.
Epimutations in Germ-Cell and Embryo Development: Possible Consequences for Assisted Reproduction
(2011)
Assisted reproductive technologies (ART) emerged in the late 1970’s as a therapy for human infertility. Up till now more than 3 million babies have been conceived through ART, demonstrating the safety and efficiency of the technique. Published reports showed an increase in the rate of imprinting disorders (Beckwith Wiedemann Syndrome, Angelman Syndrome, etc.) in babies born after ART. What are the effects imposed through ART and should researchers reassess its safety and implications on the future offspring? Throughout this thesis, I analyzed the methylation patterns of germ cells and embryos to determine whether in vitro maturation and in vitro fertilization have a negative impact on the epigenetic patterns. Furthermore, DNA methylation was compared between sperm of infertile and presumably fertile controls in order to understand whether epigenetic disturbances lead to infertility at the first place. The occurrence of methylation aberrations in germ cells of infertile patients could be transmitted to new-borns and then cause epigenetic disorders. In order to elucidate the imprinting status within single cells, I developed a new technique based on limiting dilution where bisulfite treated DNA is distributed across several wells before amplification. This allowed methylation measurement at the single allele level as well parent of origin detection. In a total of 141 sperm samples from couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) including 106 with male factor or combined infertility and 28 with female infertility, I detected a significant correlation between lower quality of semen parameters (sperm count, percentage of abnormal sperm, and percentage of motile sperm) and the rate of imprinting errors. ALU repeats displayed a higher methylation in sperm DNA of patients leading to a pregnancy and live birth, compared to patients in which pregnancy was not achieved or a spontaneous abortion occurred. A discriminant analysis based on ALU methylation allowed correct classification of >70% of cases. Preliminary data from illumina methylation arrays where more than 27,000 CpGs were analyzed determined that only a single CpG site from the open reading frame C14orf93 was significantly different between the infertile and presumably fertile control group. However, further improvements on data normalization might permit detection of other differentially methylated regions. Comparison of embryos after natural conception, in vitro fertilized embryos from superovulated oocytes, and embryos achieved through fertilization of in vitro cultured oocytes revealed no dramatic effect on the imprinting patterns of Igf2r, H19, and Snrpn. Oocyte cryotop vitrification did not result in a dramatic increase of imprinting mutations in oocytes even though the rate of sporadic methylation errors in single Snrpn CpGs were higher within the in-vitrified group. Collectively, the results I will present within this thesis suggest an increase in the rate of imprinting errors within the germ cells of infertile patients, in addition to a decrease in genome wide methylation of ALU repetitive elements. I did not observe a detrimental effect on the methylation patterns of oocytes and the resulting embryos using in vitro maturation of oocytes and/or standard IVF with in vivo grown superovulated oocytes.
Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by σ<sup>55</sup> of B. subtilis RNA polymerase.
From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertionwas recloned as aPstl fragment into pJKK3-1, a shuttle vector which repücates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no extemal or intemal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at Ievels comparable to those ofthe B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin 0-producing strain of Streptococcus pyogenes or from üsteriolysin-producing strains of Usteria monoeytogenes, no positive hybridization signals were obtained. These data soggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.
A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular Ievel. Nucleotide sequence determination revealed the presence of two open reading frames. 8oth open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.
During an investigation into the substrate specificity and processing of subtilisin Carlsberg from Bacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97-101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, M<sub>r</sub>=42 kDa) was not processed to the mature form (M<sub>r</sub> = 30 kDa) and was not released into the medium by a proteasedeficient B. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants from B. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0-3 h after onset of stationary phase) of a processing intermediate (P38c, M<sub>r</sub> = 38 kDa) oftbis protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.