Scheffler Immo E. Mitochondria

was performed with mtDNA from a baby tooth extracted 20 years earlier, and

the sequence was matched to mtDNA from the now adult, his sister, and his

mother. Baby teeth may be one of the best ways to preserve DNA samples for

posterity and future cloning, should that become socially acceptable.

The technology was subsequently applied to a problem illustrating in a

marvelous and touching way the application of science to seek justice and

uphold human rights. An Interamerican Commission on Human Rights, upon

a request by the National Commission on the Disappearance of Persons

formed by the newly elected democratic government of Argentina in 1983,

assisted the Grandmothers of the Plaza de Mayo and other surviving relatives

to be reunited with their kidnapped children. These children had been born

to couples and women who subsequently “ disappeared ” — murder victims of

the military junta that ruled Argentina from 1976 to 1983. The children were

often given away or adopted by the military or police, but Argentinian courts

were willing to promote a reuniﬁ cation of the families, if objective scientiﬁ c

proof could establish the identity of these children. Since mtDNA is

maternally inherited, haplotyping of mtDNAs from the children and relevant

surviving relatives and speciﬁ cally maternal grandmothers became a legally

accepted method to test kinship in the absence of surviving parents.

While the science was objective and frequently decisive in relating children

to grandmothers, a host of legal, ethical, sociological, and psychological prob-

lems arose, because by the time the tests became possible, the children were

no longer small, they had developed relationships with their foster parents,

and it was not always very clear who had the right to know, nor was it clear

what to do with the new knowledge. Should a child be informed that the foster

parents were among the killers of their parents? Should they be coerced back

to their original families? Even the political climate has changed in Argentina

and the judicial system is no longer so supportive of the Grandmothers. As

King pointed out, once the “ children ” reach the age of 21 years, they will obtain

the legal right to ﬁ nd out their identities if they wish.

Another headline - producing story involving mtDNA sequencing was the

recent authentication of the remains of the Tsar Nicholas II of Russia, his wife

and children, and some of his closest servants (57, 58) . The Russian imperial

family had been executed during the revolution and buried in a mass grave in

the vicinity of Yekaterinburg. Nine sets of skeletal remains were recovered in

1991, but it remained to prove that these indeed included the remains of the

Tsar, his wife, and three of their children. Tissue from the bones yielded sufﬁ -

cient mtDNA to allow ampliﬁ cation and sequencing of D - loops, to be com-

pared with those of living and dead relatives (Figure 8.2 ). The Tsar ’ s brother

was exhumed from his grave in St. Peter and Paul Cathedral in St. Petersburg,

where he had rested since 1899. Prince Philip, a known maternal relative, had

mtDNA that matched that of the presumed Tsarina. Sequences of the Tsar

matched those of two living maternal relatives, but in an additional twist to

the story it was found that the Tsar was heteroplasmic at position 16169

(72%C/28%T). There were suspicions of contamination after the ﬁ rst

FORENSIC APPLICATIONS 431

432 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

publication which could be removed by independent repeats of the sequencing

as well as by the ﬁ nding that the Tsar ’ s brother was also heteroplasmic at the

same position (38%C/62%T). Countess Xenia Cheremeteff - Sﬁ ri and the Duke

of Fife were homoplasmic, but had the same sequence at the other six posi-

tions where substitutions relative to the standard reference sequence had been

found (Figure 8.3 ). The existence of heteroplasmy in two siblings (but with

different ratio), along with the progression to homoplasmy within four genera-

tional events, is another interesting example illustrating rapid mtDNA

segregation. It adds evidence in support of the bottleneck mechanism (see

Chapter 7 ).

The use of mtDNA polymorphisms in forensics has attracted the attention

of the FBI and the U.S. Militiary (the Armed Forces DNA Identiﬁ cation Labo-

ratory in Rockville, Maryland). Thus, a considerable number mtDNA control

region sequences from hundreds of individuals representing more than 120

Figure 8.2 Pedigrees of European aristocracy showing maternal inheritance of

mtDNA polymorphisms.

Prince Philip

Duke of Edinburgh

Tsarina

Alexandra

Tsar Nicholas II

Princess Alice

daughter of Queen

Victoria

Alexei Maria Tatiana Olga Anastasia

families have been analyzed recently, and the results have caused surprise and

consternation. Contrary to expectations and previous estimates, the mutation

rate was much larger than expected. Instead of a rate of one mutation every

12,000 years ( ∼ 600 generations), deduced from the differences between the

great apes and humans and their divergence ∼ 5 million years ago, the observed

differences among contemporary humans yielded an estimate of one mutation

every 800 years. The controversy received fuel from additional supporting

studies, as well as from studies failing to conﬁ rm the high mutation rate (see

reference 59 for further discussion). It is clear that the acceptance of a much

higher mutation rate would also upset various estimates of landmarks in

human evolution (emergence from Africa, expansion of modern Europeans,

immigration into North America).

More data will resolve whether the discrepancy is due to “ statistical ﬂ uctua-

tions ” (low sample size). There is also debate about the existence and meaning

of “ hot spots. ” If hot spots exist, mutations will be observed over the short

term, but over the long term such mutations can revert back to the original

sequence, in effect giving an average long - term mutation rate that is signiﬁ -

Figure 8.3 Pedigrees of European aristocracy showing maternal inheritance of

mtDNA polymorphisms.

Duke of Fife

Xenia Cheremeteff-Sfiri

Tsar

Nicholas II

Georgij

Romanov

FORENSIC APPLICATIONS 433

434 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

cantly slower. Another issue to be resolved is whether the DNA in the control

region is not subject to selection pressure. Since it is noncoding, it has been

assumed that mutations in this region are neutral. On the other hand, such

mutations may in subtle ways affect the initiation of transcription and replica-

tion of mtDNA, even if none of the identiﬁ ed sequence elements for protein

binding are directly involved.

8.6 FUTURE CHALLENGES

It is possible that the harvesting of information from the human mtDNA tree

is almost complete, except for the outermost branches that will yield informa-

tion about very regional migrations such as those on the Indian subcontinent,

in the Canadian Arctic (60) , or in the Paciﬁ c Islands (61) . The challenge will

be to account for discrepancies in time estimates derived from the mtDNA

tree and from Y chromosome sequences, and ultimately from sequences on

autosomes and the X chromosome.

Another challenge will be to provide solid evidence in support of specula-

tions that certain mitochondrial haplotypes may confer a selective advantage

under certain environmental conditions and to correlate such haplotypes with

speciﬁ c nuclear polymorphisms. Furthermore, correlations may not be sufﬁ -

cient or convincing unless biochemical and physiological properties of the

mitochondria in question can be shown to be responsible for the selection (in

the same way in which the sickle cell allele confers resistance to malaria in

heterozygotes, or the CFTR Phe508 mutation may allow heterozygotes to

survive cholera epidemics).

Clearly, the molecular clock will require continuous re - calibration — for

example, for different primate species and perhaps even for different segments

of the mt genome (41) .

Forensic applications have been illustrated with some spectacular and

widely publicized examples. The challenge in the future will be to ﬁ nd “ cases ”

of sufﬁ ciently broad interest to warrant publication of the results in journals

like Nature , since the technology appears to be on a ﬁ rm footing. Hetero-

plasmy and rapid ﬂ uctuations in the proportion of genomes may add some

complications in speciﬁ c situations.

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437

9

Mitochondria, Second Edition, by Immo E. Schefﬂ er

MITOCHONDRIA AND

PHARMACOLOGY

9.1 INTRODUCTION

Studies on mitochondria have for a long time beneﬁ ted from the judicious use

of speciﬁ c drugs, antibiotics, inhibitors, and/or analogues that have helped in

deﬁ ning pathways, or in focusing on a particular complex, or even in creating

phenocopies of speciﬁ c mutations. A number of these have been introduced

in previous chapters in the context of the reactions or complexes being dis-

cussed. The following is intended to be more of a summary and compilation

for easy reference and identiﬁ cation of the targets of both the common and

less common drugs being employed in this ﬁ eld. Drugs that inhibit speciﬁ c

enzymes in biochemical pathways and metabolic cycles (for example, ﬂ uoroci-

trate) will not be covered exhaustively, unless there is a special relevance to

mitochondrial function.

One can subdivide the collection of drugs/inhibitors into several categories.

A summary of information with some relevant, recent references is presented

in following Table 9.1 .

438 MITOCHONDRIA AND PHARMACOLOGY

TABLE 9.1 Common Inhibitors Used in Mitochondrial Studies

Name Target Reference

Discussion

in This

Book

(page)

1. inhibitors of electron transport

Rotenone Complex I

Amytal Complex I

Piericidin A Complex I

N - Methylquinolinium derivatives

Complex I 1

N - Methyl - pyridinium derivatives (MPP

+

)

Complex I

Others Complex I 2

Malonate Complex II

Antimycin A Complex III 3, 4

Quinoline N - oxides

Complex III 3, 4

Myxothiazol Complex III 3, 4

Azide, carbon monoxide, cyanide Complex IV

2. uncouplers/ionophores

Dinitrophenol

Valinomycin

Carbonylcyanide - p -

triﬂ uoromethoxyphenylhydrazone (FCCP)

3. inhibitors of ATP synthase

Oligomycin B Complex V 5, 6

Dicyclohexylcarbodiimide (DCCD) Complex V

Venturicidin Complex V

4. inhibitors of transport systems

Atractyloside ANT 7, 8

Bongkrekic acid ANT 7, 8

5. sensors of membrane potential

Rhodamine123 9

3,3 ′ - Dihexylooxacarbocyanine iodide

DIOC

6

(3)

9

5,5 ′ ,6,6 ′ - Tetrachloro - 1,1 ′ ,3,3 ′ -

tetraethylbenzimidazolcarbocyanine

(JC - 1)

9

Chloromethyl - X - rosamine (CMXRos) 9

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Scheffler Immo E. Mitochondria

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