The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications.
BACKGROUND
Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme.


RESULTS
Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein.


CONCLUSIONS
This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications.
Herrmann
Lutz
L
Cilian AG, Johann-Krane Weg 42, D-48149 Münster, Germany. hartmann@cilian.de
Bockau
Ulrike
U
Tiedtke
Arno
A
Hartmann
Marcus W W
MW
Weide
Thomas
T
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000201:enzymology*; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase