Exjobbsförslag från företag

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Förslaget inkom 2004-06-29

EVOLUTION OF AN ENDONUCLEASE.

OBS! ANSÖKNINGSTIDEN FÖR DETTA EXJOBB HAR LÖPT UT.
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 repair endonucleases (UvrC) and homing endonucleases (recombinases causing horizontal spread of their encoding genes) in sequence and catalytic properties. We have sequenced the homologous gene in a large number of T4-related phage; most encode an identical enzyme. Preliminary experiments have shown that one distantly related enzyme lacked the ds endonucleolytic activity of EndoII; other activities have not been tested yet. Our screen has picked up another 6 distantly related T4-like coliphage that either lack EndoII or encode enzymes with only limited sequence identity. In addition, several recently sequenced T4-like phage infecting other bacteria than E. coli carry reading frames homologous to that encoding EndoII.
(i) One project involves production of these variant enzymes from T4-like phage in vitro, and assaying their catalytic function - ds endonuclease, ss endonuclease, recombinase? Methods to be employed include in vitro transcription-translation, enzyme assays, PCR, DNA-labelling, cleavage assays, electrophoresis.
(ii) A second project involves testing whether EndoII can catalyze recombination. Some substrates are available, others need to be constructed through PCR and cloning. Recombinants arising will be sequenced to determine the crossover points.


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