Molecular analyses of the “Pharaos

” Feasibility of molecular studies in ancient Egyptian material

Bibliographic Data

ID8315161
AuthorsAlbert Zink (0000-0002-1461-747X, München Klinik Bogenhausen), Andreas G Nerlich (München Klinik Bogenhausen, corresponding author)
Year2003
Volume121
Issue2
Pages109-111
Publication date2003-06-01
Open AccessYes
TypeARTICLE
VenueAmerican Journal of Physical Anthropology (JOURNAL)
Journal identifiersISSN: 0002-9483 • E-ISSN: 1096-8644
PublisherWiley (PUBLISHER • GB)
DOI10.1002/ajpa.10213
PMID12740953
OpenAlexW2022778960
LanguageEN
Citations received6
References cited17

In a recent paper, Marota et al. (2002) described a study on plant chloroplast DNA in modern and ancient papyri specimens in order to evaluate the half-life of ancient DNA (aDNA) in papyri. In parallel, they investigated the racemization of aspartatic acid in those samples, as the extent of racemization is linked to DNA decay (Poinar et al., 1996). The results were compared to the findings in animal and human remains from ancient Egypt in the literature. As a result, a strong and rapid destruction of the aDNA was assumed. On the basis of high temperature conditions in Egypt, an upper limit for DNA preservation of about 700–800 years was calculated. Thereby, the authors suggested that aDNA analyses of the human remains of the “Pharaos” (and other ancient Egyptians) make no sense, and any DNA that would be amplified should be regarded as contamination by more recent DNA. This paper is an interesting contribution to the long-standing discussion on how long authentic DNA can be retrieved from ancient specimens (Höss et al., 1996). This is a very important point, as the work with ancient DNA is a growing branch of paleobiologic research, and this is permanently endangered by the risk of producing false-positive results due to contamination with modern DNA. The avoidance of any contamination and the confirmation of aDNA authenticity are essential for the reliability of ancient DNA analyses. However, it seems that Marota et al. (2002) did not take several important points adequately into consideration, which deserve clarification. The fundamental work by Krings et al. (1997) on mitochondrial (mt) aDNA in human Neanderthal remains describes the successful amplification of authentic aDNA in this approximately 30,000–100,000-year-old material. The presence of authentic aDNA in this setting has been assumed to be the result of low-temperature conservation in Middle European soil. In offering a premise for the calculation of aDNA-stability, Pääbo and Wilson (1991) utilized Lindahl's constants, leading to an estimated half-life of DNA of about 125 years at 15°C and pH 7.0. This fits pretty well with recent data on DNA stability which showed a rapid postmortem decrease of DNA fragments of 700–500 bp length within approximately 30 years after death (Bachmeier et al., 2001), suggesting rapid postmortem DNA fragmentation. However, this study also clearly showed, like others, that smaller fragments can be found for a very long period of time (Lassen et al., 1994). In fact, the microclimate within Egyptian tombs is significantly different from the outside temperature. Thereby, we can assume that the temperature inside Egyptian tombs lies at least 10–20°C on average below the outside temperature. This may depend on local conditions and differences in burial practices, which underwent some modifications during Egyptian history. For instance, during the restoration of the tomb of Nefertari in the “Valley of the Queens,” the temperature and humidity were measured over a long period (Maekawa and Preusser, 1993). Here, the temperature ranged between 15–25°C during the whole year, and thereby was significantly (10–20°C) under the assumed average of 35°C. This means that the half-life of aDNA in Egyptian mummies can be expected to be significantly longer than calculated by Marota et al. (2002). Depending on the burial place, most of the ancient Egyptian biomaterial is kept under constantly dry conditions, which represents a further crucial factor for DNA survival. This holds mainly true at least for the rock tombs at the desert interface, but not for burial places, which were inundated every year by the flooding of the Nile. Therefore, one must take location of burial place into account. Likewise, burials in the Valley of the Kings or the necropoles of Thebes or Aswan (Qubbet-el-Hawa) remained dry, while burials in the delta region (e.g., Minshat Abu Omar; Parsche, 1991) were flooded and therefore show significantly poorer biomaterial conservation. This is of major significance, since a wet environment enhances the hydroxilation of DNA and leads to a rapid degradation of DNA. Kumar et al. (2000) explained their findings of insufficient aDNA retrieval in ancient Indian specimens by the fact that the specimens came from open-air sites which were exposed to heat and moisture. This stimulated the authors to recommend the use of samples from nontropical environments or cave sites, as seen in the pharaonic Egyptian rock tombs. Furthermore, it seems very important also to take the mummification procedure into account. The rapid dehydration of a dead body significantly retards or even inhibits the decomposition process of soft and hard tissues. The mummies were dried by using a salt mixture called natron. Natron, a naturally occurring substance made of four different sodium salts, has two major effects during the mummification procedure. It works as a drying agent, drawing the water out of the body and at the same time increasing the pH. The latter is an important factor for the preservation of DNA, as acidic conditions are poor for DNA preservation. Therefore, the use of natron creates a neutral or mildly alkaline environment that significantly enhances DNA preservation. The ancient Egyptian practice of embalming reached a high level of expertise and led to a remarkable conservation of the entire body, so that inner organs are frequently very well-preserved (although shrunken). Various histological analyses of these tissues (including bone) often revealed a good preservation state of the organic matrix, and even the remains of antigenic structures of various proteins were repeatedly demonstrated by immunohistochemical techniques (Tapp, 1986; Nerlich et al., 1995a; Parsche and Nerlich, 1997). Well in line with a presumed influence of moisture, this holds true only for rock tomb material (such as from Thebes), but not for delta material (e.g., from Minshat Abu Omar; Parsche and Nerlich, 1997). Taking these observations together, we strongly suggest that aDNA investigation in pharaonic mummy tissue samples is indeed not senseless. This assumption is further corroborated by our own extensive studies of the molecular identification of bacterial DNA in ancient Egyptian mummies, which should have undergone similar decay as human aDNA. In several studies, we were able to amplify mycobacterial (and other bacterial) aDNA in several tissue specimens from Egyptian mummified and skeletonized material (Nerlich et al., 1997; Zink et al., 2000, 2001, 2002). Most of our samples were from the necropolis of Thebes West (Middle/New Kingdom), and the oldest derived from the Predynastic and Early Dynastic burial site of Abydos (3500–2800 BC). The authenticity of bacterial DNA was confirmed by several observations, such as the identification of the correct gene sequence of the mycobacterial DNA (Nerlich et al., 1997; Zink et al., 2001, 2002). Furthermore, recent experiments using the spoligotyping technique clearly provided evidence for differences in the strains of mycobacteria among different individuals, time periods, and locations, so that both recent or historic contaminations are ruled out (Zink et al., 2002). In parallel, in these studies we had amplified human DNA in order to test for the presence of DNA by amplifying a 202-bp fragment of the β-actin multicopy gene. Furthermore, the extracted DNA was used to determine the individual gender by amplifying a 93-bp fragment of the SRY gene and a 106-bp and/or 112-bp fragment of the amelogenin gene (located either on the X- and Y-chromosome). The resulting observations (when compared to the morphological sex determination using the criteria by Ferembach et al., 1979) showed a concordant sex determination (Zink et al., 2001). Besides our own studies on ancient Egyptian material, several additional studies support these findings. Likewise, Crubézy et al. (1998) identified M. tuberculosis complex DNA in a 5,400-year-old Egyptian case of Pott's disease. In the ongoing research at the Dakhleh oasis located in Egypt's western desert, Graver et al. (2001) analyzed the mitochondrial DNA (mtDNA) variation within a Roman period cemetery sample (Kellis 2) associated with the town of Kellis (100–450 AD), and compared the results with mtDNA of contemporary inhabitants of the oasis. In their study, they successfully recovered ancient DNA from 34 of 50 skeletal samples from Kellis 2. Subsequently, they performed mtDNA restriction site analysis and direct sequencing of these samples. The preliminary data of the restriction site analysis and the control region sequence variability provided evidence that there exist significant genetic differences between the ancient and modern population of the Dahkleh oasis. Although the authors presented a preliminary report, they provided clear evidence that they had recovered sufficient authentic ancient mtDNA to perform successful analyses. A similarly good state of preservation of human biomaterial can also be deduced from previous studies on ancient tissue samples from other areas. Thus, the investigation by Filon et al. (1995) clearly identified the molecular defect of thalassemia in an old Near East bone sample. This indicates that human genomic aDNA must have been adequately preserved to provide the mutation during molecular analysis. We therefore clearly support the notion that aDNA may be sufficiently preserved to allow authentic molecular studies on ancient Egyptian material. This is not in conflict with the statement of Poinar et al. (1996), who indicated that the survival of DNA is limited to a few thousand years in warm regions such as Egypt, but clearly rejects the assumption of Marota et al. (2002). Interestingly, Marota et al. (2002) based their assumption on the study of a 2,300-year-old monkey bone, and thereby on the study of only one single individual. Keeping in mind that the success rate for amplifying DNA in ancient samples ranges from about 50–60% (Zink et al., 2001), it is not convincing to rely on the negative results from one single bone. Furthermore, Marota et al. (2002) cited the study of Krings et al. (1999), who investigated 132 mummies and skeleton samples from Late Predynastic and Early Dynastic Egypt. This material came from Minshat Abu Omar in the Nile Delta Valley. The individuals were buried in sand, without evidence of artificial mummification. The archaeological site is located in a region which was annually flooded by the Nile (Parsche, 1991). The preservation state of these bones was therefore very poor, as already shown by chemical and histological analyses of the bone matrix (Nerlich et al., 1995b). The burial practice in that necropolis (close to the surface, no artificial mummification) and the temporarily wet environment due to the flooding explain the high degradation rate and nearly complete loss of DNA in these samples. Nevertheless, this material is not comparable to mummies from later periods, which were artificially dried and embalmed and buried in ramified tomb complexes deep under the surface. This is the case for the Tombs of the Nobles in Thebes West. The five human femurs investigated by Poinar et al. (1996) were probably from the same site. Taking these arguments together, we oppose the assumption by Marota et al. (2002) that the molecular analysis of ancient Egyptian biomaterial is unsuitable for aDNA analysis. In contrast, we have good evidence that the careful and critical application of various molecular approaches may significantly further our knowledge of ancient Egyptian populations and certain aspects of their living conditions and diseases. Nevertheless, we concur with the authors of all studies that intend to raise critical aspects of aDNA research. This is absolutely necessary, as the danger of false-positive results is very high, and several studies raise serious questions concerning the authenticity of the aDNA retrieved

Ancient DNA · Ancient egypt · Ancient history · Archaeology · Biology · DNA · Evolutionary biology · Forensic and Genetic Research · Forensic Anthropology and Bioarchaeology Studies · Genetics · History · Medicine · Paleopathology and ancient diseases

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