The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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The Mitochondrial Free Radical Theory of Aging
mutations are much rarer than deletions, because quantitation of deletions is much more
accurate with existing technology (though still by no means ideal); 5 but there is no evidence
that this is so, other than the study of a region of ATPase 6 6 which SOS anyway indicates that
we should discount (see Section 8.5.4).
The second is methodological. There are potentially about 50,000 possible point
mutations in the mitochondrial genome, since each of the 16,569 base pairs can mutate to
any of three others. PCR, unfortunately, can only search for particular ones,* rather than all
at the same time. It would be something of a shame, therefore, if one designed an experiment
to search for a mutation which was of no consequence to the function of the “mutated”
gene—was phenotypically silent, in other words. In order to avoid this possibility, most
workers have chosen to look for mutations that are known to cause severe diseases
(see Section 6.6.4). Quite a few such diseases are known; they are not very hard to identify,
because they exhibit maternal inheritance (see Section 2.4.2). This certainly protects against
the possibility that the mutation being sought is silent. Unfortunately, however, it also almost
certainly eliminates the possibility that it is null—a complete loss of function of the gene in
question. This is because any mutation which has a really severe effect cannot be identified
as a (maternally) inherited defect, because it won’t be inherited at all—any embryo unlucky
enough to be carrying it will die long before birth, as will be discussed further in Section
10.3.2. Thus, experimenters who focus on disease-causing sequence variants are probably
limiting themselves to extremely mild mutants, which may therefore not tell the whole story.
See Section 6.6.5 for evidence that they do indeed behave qualitatively differently to null
mutations.
In view of this, it would be attractive if we could identify the level of mutations by some
other means. Luckily there is one. The discovery that entire cells are taken over by copies of
a single mutation allows one to examine them not only for their mtDNA composition, but
also for their enzymatic activity. A number of laboratories have analyzed non-dividing or
rarely-dividing tissues—usually muscle—by assaying for either the presence (by antibody
staining) or the activity (by histochemistry) of certain proteins encoded by the mtDNA at
the level of the individual cell. 7,8a This gives a value towards, but not beyond, the high end of
the range that was estimated by sequence analysis: around 0.1% to 1% at most.**
Is this as complete a show-stopper for the mitochondrial free radical theory of aging as
it seems? The fact that this book was written answers the question, of course. In mid-1997 I
formulated a hypothesis that appears able to reconcile the low levels of anaerobic cells with
the idea that those cells are the main promoters of oxidative stress; it was published in
February 1998. 9 I began, as with my work leading to SOS, with consideration of a related
paradox, in the hope that the two problems would provide clues to each other’s solution.
9.2. The Creation of mtDNA-Less Human Cell Lines
Given our utter reliance, as organisms, on a steady supply of oxygen, it might be guessed
that each individual cell is similarly reliant on it. That is, that cells which have—for whatever
* Some variants of PCR, such as that used by Schon and coworkers, 6 can identify a dozen or so potential point
mutations, but there is no way to identify more than that in one experiment.
** Recently there has been a spate of reports of extremely high levels of mtDNA deletions in the elderly, 8b-8d
resulting in much controversy. 8e The main criticism that has been levelled against these studies is that they
used long PCR, whose quantitative accuracy is still in grave doubt. But the argument given here — that clonal
amplification of mtDNA makes serious intracellular heteroplasmy for deletions impossible, since it would
not be stable — is a much more cast-iron argument that these recent findings 8b-8d must be methodologically
flawed.