THYMIDINE FREE MEDIA
Doughert at synapse.bms.com
Mon Feb 5 18:07:01 EST 1996
In article <4f31u4$5do at kadri.ut.ee>, kisand at madli.ut.ee (Veljo Kisand) says:
>Tom Dougherty (Doughert at synapse.bms.com) wrote:
>: In article <4ei11i$ioo at kadri.ut.ee>, kisand at madli.ut.ee (Veljo Kisand) says:
>: >an456680 at anon.penet.fi wrote:
>: >>: Hi all,
>: >: I'm trying to grow some plasmids in E.coli and hopefully radio label
>: >: the DNA. I'm told I need some thymidine (thymine??) free media, so
>: >: that I can add some radio labelled thymidine to it.
An outstanding discussion of the problems and pitfalls of
thymine/thymidine metabolism is in Bacteriol. Revs. 34:278-343. It is still the
best discussion of this topic. The amount of radiolabeled thymidine incorporated
is very sensitive to the internal pool level of deoxyribose sugar, and to whether
the E. coli strain is a thymidine requiring mutant.
>Well, I didn't want to say that internal pools of thymidine/thymine don't
>influence use of external pools. And I'm not specialist of E.colis but at least
>i know most of bacteria prefer salvage pathway to de novo synthesis...
> If specific activity of yourisotope is high enough your label
some of DNA It doesn't really matter if thymidine is degradated or not,
> labelling site is bound to aromatic ring.
>Worser is situation when thymidine is used for energetic purposes, then your
>nice label happens to be who knows where. That means "real" degradation.
>Olny thing with this internal pools is that you should grow bacteria long
>enough on label so some isotope can reach to DNA...
Again, I will say, please read the review. E. coli normally makes dTMP from
dUMP. This is subsequently phosphorylated to dTDP and finally dTTP.
Thymidine is NOT in the normal biosynthetic pathway. It is present only when
added exogenously. Check out the diagram on page 464 in "Escherichia
coli and Salmonella typhimurium, cellular and molecular biology" ASM Press.
The discussion in the same book on page 466 sums up utilization of
thymine and thymidine. The label on the thymidine is on the base, and not the
sugar. This means that radomization of the label does not occur, as the
sugar moiety is the part that is broken down and metabolised extensively.
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