59. Aminopterin in HAT medium inhibits:
1. Thymidine kinase
2. Hypoxanthine – guanine phosphoribosyltransferase
3. Ribonucleotidereductase
4. Dihydrofolatereductase
Aminopterin in HAT Medium: Inhibiting DNA Synthesis for Hybridoma Selection
In the widely used HAT medium (Hypoxanthine-Aminopterin-Thymidine), aminopterin plays a crucial role in selecting hybrid cells, particularly in monoclonal antibody production using hybridoma technology.
What Does Aminopterin Do?
Aminopterin is a folic acid analog and acts as a potent inhibitor of:
Dihydrofolate Reductase (DHFR)
This enzyme is essential for the de novo synthesis of purines and thymidine, which are required for DNA synthesis. By blocking DHFR, aminopterin effectively shuts down the de novo nucleotide synthesis pathway.
Why Is This Important in HAT Medium?
HAT medium selects for hybridomas by exploiting the metabolic deficiencies of the fusion partners:
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Myeloma cells used in fusion lack HGPRT (hypoxanthine-guanine phosphoribosyltransferase) and cannot use the salvage pathway.
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B lymphocytes (which do have HGPRT) die naturally after a few days.
So, the only surviving cells are hybrids that:
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Get HGPRT from B cells (to survive aminopterin’s effects via salvage pathway)
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Divide indefinitely like myeloma cells
Let’s Review the Options:
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Thymidine kinase – Important in salvage pathway, but not directly inhibited by aminopterin.
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HGPRT – Also part of the salvage pathway, but not aminopterin’s target.
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Ribonucleotide reductase – Not the primary target here.
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Dihydrofolate reductase – Correct. This is the direct target of aminopterin, shutting down de novo purine and thymidine synthesis.
Summary:
Correct Answer: 4. Dihydrofolate reductase
Aminopterin in HAT medium inhibits DHFR, forcing cells to rely on the salvage pathway for nucleotide synthesis, which is the basis for selecting hybridoma cells.
This biochemical strategy is essential in monoclonal antibody production and a cornerstone of cell fusion selection techniques.
