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Förslaget inkom 2004-06-29

COLIPHAGE T4 ENDONUCLEASE II ¿ A RECOMBINASE DISGUISED AS RESTRICTION ENDONUCLEASE?

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Endonuclease II (EndoII) is a viral-encoded (coliphage T4) enzyme that behaves like a bacterial restriction endonuclease in causing degradation of foreign DNA, but resembles homing endonucleases (recombinases causing horizontal spread of their encoding genes) in sequence and catalytic properties. The overall goal of this project is to understand precisely how EndoII interacts with its target, understand its short-term and long-term biological roles and how what appears to have been a recombinational enzyme has evolved towards restriction functions. We have recently partially purified several mutant enzymes. Although the expression levels are low, very interesting results concerning both DNA binding and catalytic activity are emerging. Projects along the following lines are available:
(i) Sequences corresponding to the DNA-binding parts of related enzymes are lacking in EndoII, indicating that its sequence-specific binding must depend on novel interactions. Different kinds of mutagenesis will be used to isolate mutant enzymes that do not bind to DNA. Results will identify residues involved in the sequence-specific binding of the protein.
(ii) All mutants we have tested appear to form differently-shaped DNA-complexes. What do these complexes look like, what is the stoichiometry of enzyme-DNA-binding, what is the binding affinity and how is it affected by changes in conditions?
(iii) Studies with impure enzymes have suggested that the two strands are cleaved in different reactions, with context and substrate length influencing the two reactions differently. With the pure enzyme preparation now available it will be possible to test whether this is really the case, and work out the biochemistry of this unusual reaction
Methods employed include PCR, cloning, sequencing, electrophoretic mobility shift assays, cleavage assays, various kinds of electrophoresis, PhosphorImager-quantification.
Endonuclease II (EndoII) is a viral-encoded (coliphage T4) enzyme that behaves like a bacterial restriction endonuclease in causing degradation of foreign DNA, but resembles homing endonucleases (recombinases causing horizontal spread of their encoding genes) in sequence and catalytic properties. The overall goal of this project is to understand precisely how EndoII interacts with its target, understand its short-term and long-term biological roles and how what appears to have been a recombinational enzyme has evolved towards restriction functions. We have recently partially purified several mutant enzymes. Although the expression levels are low, very interesting results concerning both DNA binding and catalytic activity are emerging. Projects along the following lines are available:
(i) Sequences corresponding to the DNA-binding parts of related enzymes are lacking in EndoII, indicating that its sequence-specific binding must depend on novel interactions. Different kinds of mutagenesis will be used to isolate mutant enzymes that do not bind to DNA. Results will identify residues involved in the sequence-specific binding of the protein.
(ii) All mutants we have tested appear to form differently-shaped DNA-complexes. What do these complexes look like, what is the stoichiometry of enzyme-DNA-binding, what is the binding affinity and how is it affected by changes in conditions?
(iii) Studies with impure enzymes have suggested that the two strands are cleaved in different reactions, with context and substrate length influencing the two reactions differently. With the pure enzyme preparation now available it will be possible to test whether this is really the case, and work out the biochemistry of this unusual reaction
Methods employed include PCR, cloning, sequencing, electrophoretic mobility shift assays, cleavage assays, various kinds of electrophoresis, PhosphorImager-quantification.


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