Tags

Type your tag names separated by a space and hit enter

UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein.
Somat Cell Mol Genet. 1995 May; 21(3):161-75.SC

Abstract

Segments of the human UMP synthase cDNA coding for the orotate phosphoribosyl transferase (OPRT) and orotidylate decarboxylase (ODC) domains of the bifunctional protein UMP synthase were produced by polymerase chain reaction techniques and cloned into a eukaryotic expression vector. The separate OPRT and ODC vectors, along with a selectable marker, were cotransfected into UMP synthase-deficient hamster cells (Urd-C) that require exogeneous uridine for growth. Transfected Urd-C cells surviving selection in media without added uridine were isolated and designated transferase decarboxylase Urd-C (TDU). All of the selected colonies contained DNA corresponding to the OPRT and ODC expression vectors. Two cell lines (TDU3 and TDU5) integrated many more copies of the OPRT and ODC vectors into their genomes compared to the other TDU lines. A 28.6-kDa ODC protein band and a 24.4-kDa OPRT band were detected on western blots with UMP synthase-specific polyclonal antiserum. The OPRT activity of the TDU lines was up to 8.7 times the OPRT activity of control CHL cells, and the ODC activity was up to 12.5 times control levels. Both OPRT and ODC activities in the monofunctional proteins were less heat stable than in the bifunctional UMP synthase protein. The monofunctional OPRT protein was less stable than the ODC protein at 45 degrees C. Growth of transfected cells in 6-azauridine resulted in striking increases in activity and temperature stability for the monofunctional ODC protein. A UMP synthase bifunctional protein was constructed with a deletion of the suspected linker region joining the two catalytic domains. The linker-deleted UMP synthase showed no significant change in either OPRT or ODC activity or temperature stability. The increased stability of the bifunctional protein may be a factor in its evolutionary selection in mammalian cells.

Authors+Show Affiliations

Lin T
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Suttle DP
No affiliation info available

MeSH

AnimalsBase SequenceCatalysisCricetinaeDNA PrimersEnzyme StabilityHot TemperatureHumansMolecular Sequence DataMultienzyme ComplexesOrotate PhosphoribosyltransferaseOrotidine-5'-Phosphate DecarboxylasePlasmidsSequence DeletionTransfection

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7482031

Citation

Lin, T, and D P. Suttle. "UMP Synthase Activity Expressed in Deficient Hamster Cells By Separate Transferase and Decarboxylase Proteins or By Linker-deleted Bifunctional Protein." Somatic Cell and Molecular Genetics, vol. 21, no. 3, 1995, pp. 161-75.
Lin T, Suttle DP. UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein. Somat Cell Mol Genet. 1995;21(3):161-75.
Lin, T., & Suttle, D. P. (1995). UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein. Somatic Cell and Molecular Genetics, 21(3), 161-75.
Lin T, Suttle DP. UMP Synthase Activity Expressed in Deficient Hamster Cells By Separate Transferase and Decarboxylase Proteins or By Linker-deleted Bifunctional Protein. Somat Cell Mol Genet. 1995;21(3):161-75. PubMed PMID: 7482031.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein. AU - Lin,T, AU - Suttle,D P, PY - 1995/5/1/pubmed PY - 1995/5/1/medline PY - 1995/5/1/entrez SP - 161 EP - 75 JF - Somatic cell and molecular genetics JO - Somat Cell Mol Genet VL - 21 IS - 3 N2 - Segments of the human UMP synthase cDNA coding for the orotate phosphoribosyl transferase (OPRT) and orotidylate decarboxylase (ODC) domains of the bifunctional protein UMP synthase were produced by polymerase chain reaction techniques and cloned into a eukaryotic expression vector. The separate OPRT and ODC vectors, along with a selectable marker, were cotransfected into UMP synthase-deficient hamster cells (Urd-C) that require exogeneous uridine for growth. Transfected Urd-C cells surviving selection in media without added uridine were isolated and designated transferase decarboxylase Urd-C (TDU). All of the selected colonies contained DNA corresponding to the OPRT and ODC expression vectors. Two cell lines (TDU3 and TDU5) integrated many more copies of the OPRT and ODC vectors into their genomes compared to the other TDU lines. A 28.6-kDa ODC protein band and a 24.4-kDa OPRT band were detected on western blots with UMP synthase-specific polyclonal antiserum. The OPRT activity of the TDU lines was up to 8.7 times the OPRT activity of control CHL cells, and the ODC activity was up to 12.5 times control levels. Both OPRT and ODC activities in the monofunctional proteins were less heat stable than in the bifunctional UMP synthase protein. The monofunctional OPRT protein was less stable than the ODC protein at 45 degrees C. Growth of transfected cells in 6-azauridine resulted in striking increases in activity and temperature stability for the monofunctional ODC protein. A UMP synthase bifunctional protein was constructed with a deletion of the suspected linker region joining the two catalytic domains. The linker-deleted UMP synthase showed no significant change in either OPRT or ODC activity or temperature stability. The increased stability of the bifunctional protein may be a factor in its evolutionary selection in mammalian cells. SN - 0740-7750 UR - https://www.unboundmedicine.com/medline/citation/7482031/UMP_synthase_activity_expressed_in_deficient_hamster_cells_by_separate_transferase_and_decarboxylase_proteins_or_by_linker_deleted_bifunctional_protein_ L2 - https://antibodies.cancer.gov/detail/CPTC-ODC1-1 DB - PRIME DP - Unbound Medicine ER -

Related Citations

More