Ziel
The aim of our project is the construction of artificial chromosomes to be used for directed engineering of higher eukaryotic cells. This will be accomplished by assembling biologically relevant DNA sequences (replication origins, telomeric and centromeric sequences) from eukaryotic chromosomes and naturally occurring linear plasmids.
The general objective of the proposed project is the construction of artificial chromosomes for mammalian cells by assembling biologically relevant deoxyribonucleic acid (DNA) sequences from chromosomes and naturally occurring linear plasmids. The reason for creating such constructs is that so far no satisfactory autonomously replicating vectors exist for higher eucaryotic cells and organisms and it may well be that the ideal eucaryotic vector may have to mimic a chromosome. There are 3 functional elements necessary to turn a segment of DNA into an artificial chromosome: telomeres, replicators and centromeres or other stabilizing sequences. The major effort therefore was made to identify and isolate such sequences from different sources as well as to assemble the different elements and test them in several organisms.
If mammalian cells are transfected with yeast artificial chromosomes (YAC), the YAC integrates into the genome. Therefore, mammalian telomeres were ligated to a YAC carrying the neomycin resistance gene under the control of the TK promoter. Cells transfected with this construct became G418 resistant, ie the neomycin resistance gene is expressed. Experiments to study the requirements to keep a linear DNA molecular physically stable were performed by injecting constructs with different telomeric structures in Xenopus oocytes. It could clearly be shown that the native DNA structure is sufficient to prevent a linear DNA molecule from exonucleolytic digestion.
In contrast to the autonomous replicating sequences (ARS) in yeast nothing is known about mammalian origins of replication. However, cytological data indicate that in S phase initiation of replication occurs about every 100 kb apart. Since a YAC easily can span this length, a mammalian YAC library was constructed and is now tested for replication capacity. Using a different approach, mammalian DNA was restricted to about 20 kb and ligated to the pMAMneo vector. After introduction into mammalian c ells, they were selected for G418 resistance. None of the cells survived for longer than one generation. These data are consistent with the notion that the information for replication initiation is present on long DNA fragments but that is not sufficient for maintenance of replication. A simple and efficient method for the mapping of eucaryotic origins of replication was established. In an attempt to isolate sequences involved in copy number control, the mouseamplification promoting sequences (APS) were inserted in a BPV-1 containing vector. Insertion of these sequences results in a drastic reduction of plasmid copy number. As working hypothesis it was assumed, that the amplification promoting sequences act with the aid of BPV coded transacting factors as an origin of replication. Such constructs then would carry 2 origins of replication (from BPV and APS) and replication would be badly disturbed. This hypothesis is now experimentally tested.
Further experiments concentrated on the study of the structural organization of pericentromeric satellite DNA sequences and the construction of an extrachromosomally replicating expression vector for Xenopus. The structural organization of mouse and Drosophila satellite DNA was studied and analyzed. From mouse nuclear extracts proteins were isolated that bind specifically to these sequences. In this context the structural analysis of a d(GA.CT)22 sequences was completed. Based on the observation that a BPV-1 containing vector replication extrachromosomally in Xenopus embryos an expression vector containing actin sequences in antisense orientation was constructed and injected into Xenopus embryos. It could be shown that transcripts produced by this vector specifically inhibit actin gene expression. This kind of vector may prove useful for the identification of as yet uncharacterized sequences.
The aim of our proposal is the construction of artificial
chromosomes for higher eucaryotic cells by assembling biologically relevant DNA sequences from chromosomes and naturally occurring linear plasmids. The reason for creating such constructs is that so far no satisfactorily autonomously replicating vectors exist for higher eucaryotic cells and organisms and it may well be that the ideal eucaryotic vector may have to mimic a chromosome. This kind of construct will allow directed genetic engineering of higher eucaryotes, will help to isolate other biologically important sequences, will be useful in the production of medically important molecules, will help to understand differentiation processes and may prove to be a safe and reproducible method for the somatic therapy of some genetic diseases. The minimal requirements for an artificial chromosome are sequences to allow and regulate its autonomous replication, centromeric sequences to guarantee the mitotic and meiotic stability of the chromosome, and telomeric sequences needed for its replication as a linear DNA molecule. Appropriate expression cassettes can be included in the construct. All these sequences will be isolated from different sources, characterized structurally, tested in homologous and heterologous systems, and assembled into artificial chromosomes functioning in a variety of higher eucaryotic cells.
Wissenschaftliches Gebiet (EuroSciVoc)
CORDIS klassifiziert Projekte mit EuroSciVoc, einer mehrsprachigen Taxonomie der Wissenschaftsbereiche, durch einen halbautomatischen Prozess, der auf Verfahren der Verarbeitung natürlicher Sprache beruht. Siehe: Das European Science Vocabulary.
CORDIS klassifiziert Projekte mit EuroSciVoc, einer mehrsprachigen Taxonomie der Wissenschaftsbereiche, durch einen halbautomatischen Prozess, der auf Verfahren der Verarbeitung natürlicher Sprache beruht. Siehe: Das European Science Vocabulary.
- Naturwissenschaften Biowissenschaften Genetik DNS
- Medizin- und Gesundheitswissenschaften Medizinische Biotechnologie Gentechnologie
- Naturwissenschaften Biowissenschaften Biochemie Biomoleküle Proteine
- Naturwissenschaften Biowissenschaften Genetik Chromosom
- Naturwissenschaften Biowissenschaften Genetik Genom eukaryotische Genome
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Mehrjährige Finanzierungsprogramme, in denen die Prioritäten der EU für Forschung und Innovation festgelegt sind.
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Aufforderungen zur Einreichung von Vorschlägen sind nach Themen gegliedert. Ein Thema definiert einen bestimmten Bereich oder ein Gebiet, zu dem Vorschläge eingereicht werden können. Die Beschreibung eines Themas umfasst seinen spezifischen Umfang und die erwarteten Auswirkungen des finanzierten Projekts.
Aufforderung zur Vorschlagseinreichung
Verfahren zur Aufforderung zur Einreichung von Projektvorschlägen mit dem Ziel, eine EU-Finanzierung zu erhalten.
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Verfahren zur Aufforderung zur Einreichung von Projektvorschlägen mit dem Ziel, eine EU-Finanzierung zu erhalten.
Finanzierungsplan
Finanzierungsregelung (oder „Art der Maßnahme“) innerhalb eines Programms mit gemeinsamen Merkmalen. Sieht folgendes vor: den Umfang der finanzierten Maßnahmen, den Erstattungssatz, spezifische Bewertungskriterien für die Finanzierung und die Verwendung vereinfachter Kostenformen wie Pauschalbeträge.
Finanzierungsregelung (oder „Art der Maßnahme“) innerhalb eines Programms mit gemeinsamen Merkmalen. Sieht folgendes vor: den Umfang der finanzierten Maßnahmen, den Erstattungssatz, spezifische Bewertungskriterien für die Finanzierung und die Verwendung vereinfachter Kostenformen wie Pauschalbeträge.
Koordinator
72074 Tübingen
Deutschland
Die Gesamtkosten, die dieser Organisation durch die Beteiligung am Projekt entstanden sind, einschließlich der direkten und indirekten Kosten. Dieser Betrag ist Teil des Gesamtbudgets des Projekts.