Practice question

Survival in HAT environment demands functional purine and pyrimidine salvage enzymes inherited from normal B parent. B lymphocytes express HPRT1 locus on X chromosome encoding 24 kDa hypoxanthine-guanine phosphoribosyltransferase that catalyzes phosphoribosyl transfer from PRPP to hypoxanthine yielding IMP plus pyrophosphate and to guanine yielding GMP, bypassing de novo steps requiring folate. They also express cytosolic thymidine kinase 1 cell-cycle regulated peaking in S phase and mitochondrial kinase 2 constitutive, phosphorylating thymidine supplied in medium to TMP using ATP. Hybridoma cells retain active alleles, enabling uptake of exogenous bases via equilibrative nucleoside transporters ENT1 and ENT2 and concentrative transporters CNT, generating nucleotides sufficient for DNA synthesis even when de novo synthesis ablated by aminopterin. Energy charge maintained allowing progression through G1/S checkpoint via cyclin E CDK2. Myeloma parents selected with 8-azaguanine lack HGPRT activity, cannot recycle hypoxanthine, undergo purine starvation, decrease in ATP and GTP pools activates p53 and causes mitochondrial depolarization. Unfused B cells possess salvage enzymes but inherently short lived due to withdrawal of BAFF and CD40L, undergoing apoptosis within seven days, leaving only hybrids as long-term proliferating population capable of indefinite expansion in selective medium.