Thymine bacterial synthesis

DNA synthesis in irradiated sensitive bacterial cells is permanently inhibited by doses which produce about 5 thymine dimers per single strand of DNA (1000 p.). In the cells of resistant bacteria, DNA synthesis is inhibited only by doses of radiation which produce about 500 dimers per cell. The difference between the two types of bacteria is caused by the presence in the resistant cells of efficient enzymatic mechanisms for repairing the damage done by thymine dimerization. 

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

Chloro-9-cyclopentyl-8-azapurine inhibited synthesis of DNA, RNA, and protein in E. coli. Blockage of thymine-nucleotide formation was the first effect seen. Alkylation of enzymes by the 6-chloro substituent was suggested as a mechanism. This azapurine inhibited the RNA polymerase from E. coli, but not that from M. lysodeikticus. Formyltetrahydrofolate synthetases, of both mammalian and bacterial origins, were strongly inhibited. The same azapurine, at 0.3 mM, markedly inhibited the steroid-induced synthesis of A -3-ketosteroid isomerase in Pseudomonas testoster-oni ... 

Because there is only one pathway for synthesis of TMP, it can be used to synthesize radioactively labeled DNA or to inhibit DNA synthesis selectively. In bacterial thymidylate synthase mutants (thy ), DNA synthesis is not possible without added thymine or thymidine, both of which can be utilized by salvage pathways. Neither thymine nor thymidine engages in synthetic reactions other than production of TMP, so radioactive thymine or thymidine can serve as unique precursors for synthesis of radioactive DNA. The radioactivity usually is in the methyl group since this group is not metabolized, the appearance of radioactivity in any other compound is avoided. 

In bacterial and mammalian cell systems in exponential growth new DNA synthesis occurs in synchrony when the blockage of DNA synthesis by selective thymine starvation is released after a period. The same is true in Tetrahymena cultures. This is seen from Figure 7, in which 2 mM thymidine was added after an exponentially multiplying population had been treated with methotrexate + uridine for 3 hours (3 hours is nearly equal to one generation time). Synthesis of DNA began immediately after thymidine was added, and cell division occurred 110 minutes later. 

Recently the direct use of bacterial thymine for the synthesis of phage DNA has been demonstrated by infection with Tfir" of bacteria labeled with C -thymi-dine and with N in the other nitrogenous components (GoUub-Banks, personal communication). 

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