The Organellar Genome and Metabolic Potential of the Hydrogen-Producing Mitochondrion of Nyctotherus ovalis

It is generally accepted that hydrogenosomes (hydrogen-producing organelles) evolved from a mitochondrial ancestor. However, until recently, only indirect evidence for this hypothesis was available. Here, we present the almost complete genome of the hydrogen-producing mitochondrion of the anaerobic ciliate Nyctotherus ovalis and show that, except for the notable absence of genes encoding electron transport chain components of Complexes III, IV, and V, it has a gene content similar to the mitochondrial genomes of aerobic ciliates. Analysis of the genome of the hydrogen-producing mitochondrion, in combination with that of more than 9,000 genomic DNA and cDNA sequences, allows a preliminary reconstruction of the organellar metabolism. The sequence data indicate that N. ovalis possesses hydrogen-producing mitochondria that have a truncated, two step (Complex I and II) electron transport chain that uses fumarate as electron acceptor. In addition, components of an extensive protein network for the metabolism of amino acids, defense against oxidative stress, mitochondrial protein synthesis, mitochondrial protein import and processing, and transport of metabolites across the mitochondrial membrane were identified. Genes for MPV17 and ACN9, two hypothetical proteins linked to mitochondrial disease in humans, were also found. The inferred metabolism is remarkably similar to the organellar metabolism of the phylogenetically distant anaerobic Stramenopile Blastocystis. Notably, the Blastocystis organelle and that of the related flagellate Proteromonas lacertae also lack genes encoding components of Complexes III, IV, and V. Thus, our data show that the hydrogenosomes of N. ovalis are highly specialized hydrogen-producing mitochondria

Molecular and genetic analysis of complex genomes

Titelblatt Inhaltsverzeichnis Abkürzungsverzeichnis 1\. Einleitung 1.1. Modell Organismen 1.1.1. Der Zebrafisch 1.1.2. Der Medakafisch 1.1.3. Der Fugufisch 1.1.4. Lanzettfisch 1.2. Isolierung und Analyse von Genen 1.2.1. Mutagenese-Screen 1.2.2. Genetisches Kartieren 1.2.3. Physikalisches Kartieren 1.2.4. Sequenzanalyse 2\. Material und Methoden 2.1. Materialien und Chemikalien 2.1.1. Chemikalien 2.1.2. Enzyme 2.1.3. Molekulargewichtsstandards 2.1.4. Oligonukleotide 2.1.5. Klonierungsvektoren 2.1.6. E. coli-Stämme, Zellinien und Fische 2.2. Häufig verwendete Lösungen 2.3. Medien und Zellkultur 2.3.1. Bakterienmedien und Zellkultur 2.3.2. Zebrafisch Zellkultur 2.4. Experimentelle Methoden 2.4.1. Nukleinsäure-Extraktionsprotokolle 2.4.2. DNA Manipulationen 2.4.3. Subtraktive Klonierung 2.4.4. Klonierungsverfahren 2.4.5. Elektrophorese 2.4.6. Hybridisierungstechniken 2.4.7. PCR Techniken 2.4.8. Haploide Zebrafisch Embryos 2.5. Daten-Analyse 2.5.1. Sequenz-Analyse 2.5.2. Kopplungs-Analyse 3. Ergebnisse 3.1. Genetisches Kartieren im Zebrafisch 3.1.1. Identifikation polymorpher Zebrafischstämme 3.1.2. Testen der verschiedenen repetitiven Elemente auf ihre Tauglichkeit für IRS-PCR 3.1.3. Verteilung der ´mermaid´ Elemente im Zebrafisch-Genom 3.1.4. Abschätzung der Häufigkeit der ´mermaid´-Elemente im Genom 3.1.5. Konstruktion der ´inter-mermaid´-Marker-Banken 3.1.6. Charkterisierung der ´inter-mermaid´-Marker-Banken 3.1.7. Identifikation polymorpher Klone in den Marker-Banken 3.1.8. Hybridisierung der polymorphen Klone auf Kartierungsfilter 3.1.9. Integration von genetischer, cytogenetischer und physikalischer Karte 3.1.10. Testen der Repräsentativen Differenz Analyse (RDA) als alternative Strategie 3.2. Genetisches Kartieren im Medakafisch 3.2.1. IRS-PCR-Strategie 3.2.2. Analyse mittels AFLP-Hybridisierung 3.3. Analyse eines Amphioxus Cosmids 3.3.1. Cosmidbank 3.3.2. Sequenzanlyse 3.3.3. Gen-Inhalt des Amphioxuscosmids MPMGc117B0533 3.3.4. Repetitive Elemente innerhalb des Amphioxuscosmids MPMGc117B0533 4\. Diskussion 4.1. Wahl der Organismen 4.2. Genetische Kartierung im Zebrafisch 4.2.1. Wahl der ´Stämme´ für das Kartierungs-Panel 4.2.2. IRS-PCR-Strategie 4.2.3. Identifikation polymorpher Klone in den Markerbanken 4.2.4. Kopplungsanalyse 4.2.5. Integration von genetischer und physikalischer Karte 4.2.6. Alternative Hybridisierungsstrategie - RDA 4.2.7. Abschließende Bewertung und Ausblick 4.3. Genetische Kartierung im Medaka 4.3.1. IRS-PCR im Medaka 4.3.2. Anwendung einer modifizierten AFLP-Strategie im Medaka 4.3.3. Abschließende Beurteilung und Ausblick 4.4. Analyse eines Amphioxus Cosmids 4.4.1. Bedeutung des Amphioxusgenoms für die Analyse des Vertebratengenoms 4.4.2. Basenzusammensetzung des Cosmids 4.4.3. Gengehalt des Amphioxuscosmids 4.4.4. Repetitive Elemente innerhalb des Amphioxuscosmids MPMGc117B0533 4.4.5. Kombination von EST-Projekt und genomischer Sequenzanalyse 5a Zusammenfassung 5b Summary 6\. Danksagung 7\. Literaturverzeichnis 8\. Anhang 8.1 Sequenzvergleich von mermaid-Elementen aus dem Zebrafisch 8.2. Sequenzvergleich repetitiver Elemente des Medaka-Fisches 8.3. Verteilungsschlüssel für die Kartierungsfilter 8.4. Sequenzdaten einiger ´inter-mermaid´-Marker 8.5. Publikationsliste 2 4 4 6 9 10 11 13 13 16 23 24 26 26 26 26 26 26 28 29 30 32 32 33 33 33 37 40 43 45 46 49 50 50 50 51 52 52 52 53 54 54 55 56 58 59 62 63 64 64 69 71 71 71 72 77 79 79 80 80 80 81 82 83 84 85 86 86 87 88 89 89 90 91 92 92 94 95 96 97 104 104 106 107 108 125Innerhalb dieser Doktorarbeit wurden verschiedene Aspekte der Analyse komplexer Genome untersucht. Zunächst wurde die Anwendbarkeit verschiedener, auf Hybridisierung beruhender Techniken für das genetische Kartieren in den Genomen von Zebra- und Medakafisch getestet. Diese sollten ein effizientes Kartieren mit einer hohen genetischen Auflösung erlauben. Die einfachste Möglichkeit Marker zu generieren, die durch Hybridisierung kartiert werden können, ist die IRS-PCR. Diese PCR-Technik nutzt die im Genom eingestreuten repetitiven Elemente aus, um die nicht repetitive Frakton des Genoms zu amplifizieren, die zwischen zwei dieser Elemente liegt. Dieses komplexe PCR- Produkt wurde kloniert und etwa 1000 individuelle Klone einer rund 16.000 Klone umfassenden Marker-Bibliothek wurden auf kleine ´Southern´-blots hybridisiert, die IRS-PCR-Produkte verschiedener Zebrafischstämme enthielten. Die Klone, die auf den Southernblots einen +/- -Polymorphismus zeigten, wurden anschließend auf "Kartierungsfilter" hybridisiert, die IRS-PCR-Produkte von vier verschiedenen Kreuzungen enthielten. Bei dieser Vorgehensweise traten zwei Schwierigkeiten auf: Zum einen wurden aufgrund einer gewissen Redundanz der Marker-Bank wiederholt die gleichen Klone analysiert. Dieses Problem konnte teilweise gelöst werden, indem die gesamten Marker-Bank durch ´Oligo- fingerprinting´ analysiert wurde, und Klone mit ähnlichen ´fingerprints´ in Clustern zusammengefaßt, und dann repräsentative Klone verschiedener Cluster untersucht wurden. Das größere Problem war jedoch der hohe Grad an Variabilität innerhalb der Zebrafisch-Stämme, so daß schließlich nur 30% der Marker, die zuvor als polymorph identifiziert wurden, auf der Karte plaziert werden konnten. Insgesamt konnten aber dennoch rund 80 verschiedene Marker auf einer der Referenz-Kreuzungen ausgewertet werden, und 50 davon zeigten genetische Kopplung zu wenigstens einem weiteren Marker. Im Medakafisch wurde diese Strategie ebenfalls, jedoch mit einer Kombination von Primern, die für drei verschiedene repetitive Elemente komplementär sind, getestet. Aus der 3800 Klone umfassenden Marker-Bank wurden hundert potentielle Marker auf Southern blots getestet. Die Rate an polymorphen Markern lag mit 30 % mehr als doppelt so hoch wie im Zebrafisch. Darüber hinaus waren 50 % der als polymorph identifizierten Marker auch auf einer F2-Kreuzung zweier verschiedener Populationen polymorph. Dies zeigt, daß im Medakafisch die Stämme genetisch gut getrennt und innerhalb eines Stammes relativ homozygot sind. Um weitere genetische Marker zu finden, die außerdem eine gleichzeitige Verknüpfung von genetischen Kartierungsdaten und genomischen Klonen erlaubten, wurde für den Medakafisch eine modifizierte AFLP-Methode entwickelt. Nach Hybridisierung von Amplikons, die aus zwei verschiedenen Medakafischstämmen generiert wurden, auf eine Cosmid-Bank, wurden 80 Klone isoliert, die ein differentielles Hybridisierungsmuster aufwiesen. Aus 20 % der so identifizierten Cosmide ließ sich eine Probe isolieren, die sich auf einer F2-Geschwister-Kreuzung kartieren ließ. Ein weiterer Aspekt bei der Untersuchung komplexer Genome ist die vergleichende Sequenzanalyse. Dadurch können Einblicke in Genomevolution und -organisation gewonnen werden. Innerhalb dieser Arbeit wurde ein 40 kb großer Klon aus einer Amphioxus-Cosmid-Bank sequenziert, mit verschiedenen Exon-Vorhersage-Programmen (Grail, GenScan, Mzef) untersucht und mit zwei öffentlichen (Genbank, Swissprot) und einer internen (Amphioxus-EST) Sequenzdatenbanken verglichen. Dabei konnte unter anderem ein Gen der Aldo- Keto-Reduktase-Familie identifiziert werden. Es konnte ferner gezeigt werden, daß sich genomische Sequenzanalyse und EST-Projekte komplementieren.During the work for this thesis several aspects of the analysis of complex genomes were explored. Firstly, the applicability of different hybridisation based approaches for genetic mapping was tested in the genomes of zebrafish and medakafish. Such approaches should allow efficient mapping of new markers to a high resolution. IRS-PCR is the simplest possibility to generate markers that can be mapped by hybridisation. It takes advantage of interspersed repetitive sequences in the genome to amplify a set of unique DNAs between two of such elements. The PCR-products were cloned and more than 1000 individual clones of a 16 thousand clone containing marker library were tested for polymorphism on small southern blots of complex IRS-PCR product of different Zebrafisch strains. The markers identified polymorphic were subsequently hybridised on mapping filters onto which IRS-PCR-products of individuals of four different mapping crosses were spotted. Two major difficulties were found with this approach: Because of the redundancy of the library clones were tested repeatedly. This problem could be overcome by analysing the whole marker library by oligo-fingerprinting and grouping the clones with similar fingerprints in clusters. Representatives of different clusters were then used as hybridisation probes and sequenced. The major problem, however, was the high degree of variability within the zebrafish strains. Because of this less than 10 % of the markers that were identified polymorphic on Southern blots were informative on a reference cross and could be placed on a map. Despite of the encountered problems around 80 markers could be scored on one of the reference crosses, and 50 of those showed linkage to at least one further clone. The size of the genetic map of the Zebrafish estimated from these experiments is around 2700 cM and thus very close to the size determined from other laboratories (2635 cM). The same strategy was also tested for the Medakafish, however with a combination of primers, specific for three different repetitive elements. From the 3800 clone medaka library hundred clones were tested on Southern blots. The rate of polymorphic markers was almost 30% and compared to the zebra fish more than twice as high. Moreover 15 % of the makers previously identified polymorphic were informative on an F2-intercross of different strains. This indicates a much higher genetic distance between the different strains than in the Zebrafish. In order to generate further markers that could simultaneously be linked to genomic clones a modified AFLP-technique was developed. After differential hybridisation of two amplicons generated from different strains on a cosmid library 80 clones with a differential hybridisation pattern could be identified. It was possible to isolate a probe from 20 % of the cosmids, and more than 50% could be mapped on a F2-intercross. A further aspect of the analysis of complex genomes is the comparative sequence analysis. By comparison of genomic sequences of different species it is possible to get insights into gemone evolution and -organisation. Within this work a 40 kb clone of an amphioxus cosmid-library was sequenced, analysed with different exon prediction programs and the deduced protein sequence was compared with two public and one in house sequence database. This has led to the identification of a gene of the aldo- keto reductase family as well as further exons which gave a significant database match to known genes. Furthermore it could be demonstrated that genomic sequence analysis and EST projects complement each other well

A scientific note on the preparation of high molecular weight DNA from honeybee Apis mellifera L. pupae for PFGE analysis

International audienc

Clone-Based Systematic Haplotyping (CSH): A Procedure for Physical Haplotyping of Whole Genomes

We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing ∼10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual

Isolation and characterization of cold-shock domain protein genes, Oryzias latipes Y-box protein 2 (OlaYP2) and Fugu rubripes Y-box protein 1 (FruYP1), in medakafish and pufferfish

Copyright © 2002 Elsevier Science B.V. All rights reserved.Enchshargal Zend-Ajusch, Ute Hornung, Carola Burgtorf, Götz Lütjens, Zhihong Shan, Manfred Schartl and Thomas Haafhttp://www.elsevier.com/wps/find/journaldescription.cws_home/506033/description#descriptio

Comparative analysis of globin loci in pufferfish and man suggests a common origin of vertebrate globin loci and reveals a novel mammalian globin locus.

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing - and -globin genes from the pufferfish Fugu rubripes. By DNA FISH analysis we show that Fugu contains two distinct hemoglobin loci situated on separate chromosomes. One locus contains only -globin genes ( -locus) while the other also contains a -globin gene ( -locus). This is the first poikilothermic species analysed where the physical linkage of the - and -globin genes has been uncoupled, supporting a model in which the separation of the - and -globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu -locus suggest that a strong distal Locus Control Region regulating the activity of the globin genes, as found in mammalian -globin clusters, may not be present in the Fugu -locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are in close proximity to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the five human globin loci known to date, each encoding proteins with specific functions in specific cell types

Functional and comparative analysis of globin loci in pufferfish and humans

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing - and -globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only -globin genes (-locus), whereas the other also contains a -globin gene (-locus). This is the first poikilothermic species analyzed in which the physical linkage of the - and -globin genes has been uncoupled, supporting a model in which the separation of the - and -globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu -locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian -globin clusters, may not be present in the Fugu -locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types

Characterization and developmental expression of the amphioxus homolog of Notch (AmphiNotch): Evolutionary conservation of multiple expression domains in amphioxus and vertebrates

See http://dx.doi.org/10.1006/dbio.2001.0271 for erratum correcting omission of Robert Kortschak from original author identificationNotch encodes a transmembrane protein that functions in intercellular signaling. Although there is one Notch gene in Drosophila, vertebrates have three or more with overlapping patterns of embryonic expression. We cloned the entire 7575-bp coding region of an amphioxus Notch gene (AmphiNotch), encoding 2524 amino acids, and obtained the exon/intron organization from a genomic cosmid clone. Southern blot and PCR data indicate that AmphiNotch is the only Notch gene in amphioxus. AmphiNotch, like Drosophila Notch and vertebrate Notch1 and Notch2, has 36 EGF repeats, 3 Notch/lin-12 repeats, a transmembrane region, and 6 ankyrin repeats. Phylogenetic analysis places it at the base of all the vertebrate genes, suggesting it is similar to the ancestral gene from which the vertebrate Notch family genes evolved. AmphiNotch is expressed in all three embryonic germ layers in spatiotemporal patterns strikingly similar to those of all the vertebrate homologs combined. In the developing nerve cord, AmphiNotch is first expressed in the posteriormost part of the neural plate, then it becomes more broadly expressed and later is localized dorsally in the anteriormost part of the nerve cord corresponding to the diencephalon. In late embryos and larvae, AmphiNotch is also expressed in parts of the pharyngeal endoderm, in the anterior gut diverticulum, and, like AmphiPax2/5/8, in the rudiment of Hatschek’s kidney. A comparison with Notch1 and Pax5 and Pax8 expression in the embryonic mouse kidney helps support homology of the amphioxus and vertebrate kidneys. AmphiNotch is also an early marker for presumptive mesoderm, transcripts first being detectable at the gastrula stage in a ring of mesendoderm just inside the blastopore and subsequently in the posterior mesoderm, notochord, and somites. As in sea urchins and vertebrates, these domains of AmphiNotch expression overlap with those of several Wnt genes and brachyury. These relationships suggest that amphioxus shares with other deuterostomes a common mechanism for patterning along the anterior/posterior axis involving a posterior signaling center in which the Notch and Wnt pathways and brachyury interact.Linda Z. Holland, Laurent Abi Rached, Richard Tamme, Nicholas D. Holland, Hidetoshi Inoko, Takashi Shiina, Carola Burgtorf, and Michael Lardellihttp://www.sciencedirect.com/science/journal/0012160