Table Of ContentThe Bronze Age and Early Iron Age Peoples
of Eastern Central Asia
Victor H. Mair
Volume Two
Genetics and Physical Anthropology, Metallurgv, Textiles,
Geography and Cliinatology, Histow,
and Mythology and Ethnology
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Genetics and Physical Anthropology
DNA Analysis on Ancient Desiccated Corpses from
Xinjiang (China): Further Results
Paolo Francalacci
Universita di Sassan
There are two different approaches to the study of the biological
history of human population: physical anthropology and population
genetics. The former studies the remains of past populations: it allows
diachronic studies, but it is often limited by the scarcity of the fossil
record and to the lack of knowledge about the modality of heredity of
the macroscopical characters (such as the skeletal and dental
features) considered. The latter is based on well-known models of
genetic transmission and can be carried out on a large number of
individuals, but it has to interpret a historical process, evolution,
relying only on the present-day situation. An emerging field, called
"molecular archeologv", tries to fill the gap between the two
methodologies, analyzing ancient samples (as physical anthropology
does) at the molecular level (as in genetic analysis).
Early works extracted and characterized ancient proteins, while
more recent studies involve the analysis of ancient DNA. This exciting
new possibility was promoted by recent advancements in molecular
biology, and in particular by the devising of the technique known as
Polvmerase Chain Reaction (PCR) (Saiki et al. 1985). This technology,
which is a kind of in vitro clonation, allows the enzymatic
amplification in billions of copies of an informative DNA sequence
selected by the researcher, starting from a very small amount of DNA.
It is necessary to define two primers (short sequences of nucleotides)
flanking the region of interest, that can promote with extreme
specificity the subsequent amplification. Because of its high
stringency, the presence of very different sequences, like bacterial and
fungine DNA molecules, does not influence the reaction, since the
two primers permit the researcher to pick out selected human
sequences even amidst a huge number of exogenous DNA molecules.
Neither the amount of nucleic acid, nor its physical integrity in
extremely long molecules are an insurmountable problem, although
they do affect the efficiency of the reaction. Since ancient DNA is
usually present in very low amounts, degraded, and mixed with
nucleic acids from microorganisms, the PCR technique, widely
employed in many other fields of Molecular Biology, is the
methodology of choice (virtually the only one) in the ancient DNA
field.
Unfortunately, its application to ancient remains is not as
Thp Bronzp Agp and Early Iron Agr PpopIps of Eastnn Cmtral Asia
538 Paolo Francahcn'
straightfornard as to modern samples. In fact, researchers dealing
with ancient molecules must face several additional problems that are
of' little or no importance when working with fresh tissues. Among
others, the most noticeable are the poor presewation of nucleic acids
in ancient material, often degraded to very short fragments of a few
hundreds of base pairs (this implies that only short sequences can be
successfully retrieved), the presence of inhibitory factors in extracts
from ancient tissues, and possible contamination from modern
human DNA. The last problem is felt particularly when studying
anthropological remains because of the coincidence between subject
and object of the research: in both cases a human being.
In this regard, the very advantage of PCR, i.e., sensitivity and
specificity, is also a disadvantage for ancient DNA studies. This
paradox is due to the fact that there are a lot of possible sources (dead
skin cells from the hands, dandruff, saliva, sweat, blood) that contain
enough DNA to contaminate a precious ancient sample. The more a
specimen has been handled in a museum, the less will it be free from
human modern molecules. These molecules, even if in very low
concentration, are obviously in better condition than the ancient
ones, and they will be preferentially amplified by the enzyme during
PCR. In fact, the kinetics of the reaction are faster with undamaged
molecules, and if intact DNA is present, it is very unlikely that any
ancient molecule will be amplified. Obviously, the laboratory
environment must be kept as clean as possible, but the phases prior to
laboratory analysis (excavation and museum storage), usually not
controlled by the analyst, are crucial. For all these reasons, even
though from a technical point of view the analysis of the ancient DNA
can be done in any Molecular Biology laboratory, since no special
equipment is required and the analytical methods used are directly
derived from those applied in fresh samples, it is necessary to exercise
additional care and caution in extracting and amplifying DNA from
ancient material.
Because of these difficulties, it is appropriate to focus the
attention to a region of the DNA with a suitable ratio between length
and informativeness, the ideal being a short region that is highly
polymorphic. In addition, a DNA molecule present in a large number
of copies per cell has an increased chance that at least one copy could
survive through the years. Fortunately, a region of DNA with these
features does exist, and it is not in the chromosomes, but in the
genome of the cellular organelles called mitochondria.
The mitochondria are formerly independent organisms, similar
to bacteria, that entered in symbiosis with the first nucleate cells at the
very beginning of the evolution of the life on Earth. The early cell
provided them nutrition and protection, while the mitochondria gave
in exchange the management of energy. All superior organisms
possess many mitochondria per cell which still maintain a sort of
Victol- H. Alair, editor
DNA Analysis on Ancient Corpses from Xinjiang: Furthm h u h 539
autonomy with a separate duplication cycle and their own particular
DNA. During the course of evolution, the mitochondrial genome
(mtDNA) has been simplified by the migration of many of its genes in
the nuclear chromosomes, and presently only a few genes, carried by a
very small number of base pairs (bp), are present (human mtl)NA has
37 genes in 16,569 bp compared to hundreds of thousands of genes in
3 billion base pairs for the nuclear genome). These genes play an
important role in cellular metabolism and they are suictly in
the genome, with no gaps but existing in a region of about 1,000 bases
with no special genetic expression, called a "control region" (it has a
regulatory function for mtDNA duplication), that contains two
domains of 400 bp each which can vary almost freely in their sequence
without consequences for the functionalin. of the organelle. To be of
evolutionary interest, a mutation (in the simplest case just a change in
the DNA sequence of the 4 bases constituting DNA; for example a
Guanine instead of an Adenine, or a Timine instead of a Citosinc)
should be neutral, or, in other words, not influenced bv the
environment, since, otherwise, two unrelated populations living in
similar environment could present similar genetic features simply
because of convergence (such as the dark color of the skin in tropical
Africa and in Oceania). This is the case of two short portions of the
control region, called hypervariable segment I and 11, that presumably
show the highest mutation rate (the first being twice as high as the
second) of the whole genome. Even within a homogeneous
population it is rather difficult to find identical individuals for the
entirety of the hypervariable regions (excluding relatives from the
maternal side, see below) and there is almost no sharing among
different populations (obviously some overlapping can be observed if
we consider short portions of the control region). Other interesting
characteristics, besides its simplicity, abundance, and variability,
account for the attention paid to this molecule by evolutionary
geneticists. The mtDNA has no recombination and it is transmitted
only by the maternal lineage since the mitochondria present in the
spermatozoa do not enter into the egg but only those of the latter are
present in the offspring: both features that drastically simplify the
study of its evolution.
For all these reasons, the hypervariable control regions of
mtDNA (especially region I) are by far the most often investigated in
ancient DNA studies, and the data which they yield can be compared
with relevant data derived from modern populations from all around
the world. The high mutation rate of this region is at the same
moment both an advantage, because it allows for fast differentiation
among individuals and populations, but also a difficult)., because of
the possible occurrence of the same mutation in independent
lineages, thus confusing the reconstruction of the evolution of the
mitochondria1 types in a given population. In addition, the huge
The Bronrp Age mixi Early Iron Age P~opleso f Eartmn Cmiral dsin
540 Paolo Francalacci
variability induces a background noise of many rare and often
conflicting mutations that sometimes hampers adequate statistical
treatment of the data. Apparently, not all the mutations are equally
important, and my current work, in collaboration with Antonio
Torroni, a geneticist of the University of Rome, is aimed to
individuate, among the changes in the control region, those of
phylogenetic interest (Torroni et al., 1996, in press). The coding
portions of the mtDNA are more stable, and a change there is likely to
occur only once in the evolutionary history of a population. Our
preliminary results show that the parallel analysis of both the
sequences of the control region and the various point mutations in
the coding areas allows us to define groups of lineages with changes in
the control region that are population-specific, at least at a
continental level.
It is apparent that no evolutionary interpretation of the DNA
extracts from ancient samples can leave out of consideration both the
nature and the extent of the variability of mtDNA in modern
populations. More precise phylogenetical affiliation of the desiccated
corpses from Xinjiang, the object of this study, could be established
only when the variability patterns of the mtDNA will be fully
understood and the polymorphic sequences from modern individuals
from Xinjiang (presently studied by Du Ruofu of the Institute of
Genetics of the Chinese Academy of Sciences in Beijing) and from
other regions of Eurasia will be known.
Drawings of desiccated tissues were carried out on several
individuals naturally mummified, dated 3,200 BP, from the graveyard
at Qizilchoqa near Qumul (Hami),i n eastern Xinjiang (far northwest
China) and from the Museum of Archeology in ~riimchi.A ll
together, 25 specimens from 11 individuals were collected, but up to
now only 5 samples belonging to 2 individuals are available for
analysis. Every effort has been made to keep these samples free from
modern human DNA contamination. The samplings were made
wearing disposable rubber gloves to avoid skin contact and a mask to
prevent contamination from saliva when speaking or breathing. The
drawings were made by disposable sterile scalpels. The gloves and the
tools were changed when sampling a new individual to prevent cross
contamination among ancient corpses. The specimens, about 1-2
grams of desiccated tissue, of different types-muscle, skin, bone,
etc.-and from various parts of the body, were stored in sterile plastic
tubes, immediately labelled and sealed, to avoid the growth of micro-
organisms. The least exposed parts of the body, such as the inner
thighs or underarms, were selected, with the aim of analyzing tissue
with limited handling. In some cases, especially at the necropolis
where many mummies where unearthed and reburied shortly after, it
was possible to draw bone and soft tissue from below the woollen
clothes, ensuring protection from handling in those places.
Victor H. ilkair, editor
