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Mutations affecting the structure of hypoxanthine: guanine phosphoribosyltransferase in cultured Chinese hamster
Abstract:
14 Clones resistant to 8-azaguanine, isolated from mutagenically treated cultured cells from Chinese hamsters, were tested for loss of enzymic and immunological activities of hypoxanthine: guanine phosphoribosyltransferase. Three of the clones had no enzymic activity but reacted strongly with antiserum prepared against the enzyme (CRM(+)), indicating the presence of a defective enzyme protein, probably caused by a mutation in its structural gene. The other 11 mutants had little or no enzymic or immunologic activity. Revertant clones with positive enzymic and crossreacting immunologic activities were derived from both classes of mutants. The antiserum has been used for rapid purification of the phosphoribosyltransferase from Chinese hamster cells by immunoadsorbant affinity chromatography.
Insights
Researchers studied Chinese hamster cells resistant to 8-azaguanine to understand mutations affecting hypoxanthine: guanine phosphoribosyltransferase (HGPRT). Some mutants retained a defective enzyme, while others lost all activity, aiding in enzyme purification.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Hypoxanthine: guanine phosphoribosyltransferase (HGPRT) is crucial for purine metabolism.
- Mutations in HGPRT can lead to Lesch-Nyhan syndrome and resistance to certain drugs.
- Understanding HGPRT mutations is key to cellular and genetic research.
Purpose of the Study:
- To investigate the enzymatic and immunological properties of HGPRT in 8-azaguanine-resistant Chinese hamster cell clones.
- To characterize mutations affecting HGPRT activity and structure.
- To explore the utility of anti-HGPRT antiserum in enzyme purification.
Main Methods:
- Isolation of 8-azaguanine-resistant clones from mutagenically treated Chinese hamster cells.
- Assays for hypoxanthine: guanine phosphoribosyltransferase (HGPRT) enzymic activity.
- Immunological testing using antiserum against HGPRT.
- Derivation and analysis of revertant clones.
- Purification of HGPRT using immunoadsorbent affinity chromatography.
Main Results:
- 14 resistant clones were analyzed for HGPRT activity and immunological properties.
- Three clones showed no enzymic activity but retained cross-reacting immunologic material (CRM(+)), indicating a defective enzyme protein.
- Eleven clones exhibited minimal or no enzymic or immunologic activity.
- Revertant clones with restored HGPRT function were obtained from both mutant classes.
- Anti-HGPRT antiserum facilitated rapid purification of the enzyme.
Conclusions:
- Mutations affecting HGPRT in Chinese hamster cells can result in either a defective enzyme protein or a complete loss of the enzyme.
- The study provides insights into the genetic basis of drug resistance and HGPRT deficiency.
- Anti-HGPRT antiserum is a valuable tool for the purification and study of HGPRT.