Archaeological Chemistry - American Chemical Society

Chapter 28

Ancient Nucleic Acids in Prehispanic Mexican Populations 1

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R. Vargas-Sanders , Z. Salazar , and Ma. C. Enriquez

Downloaded by UNIV OF GUELPH LIBRARY on June 29, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0625.ch028

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Laboratorio de Antropologia Molecular, Instituto de Investigaciones Antropológicas, Universidad Nacional Autónoma de México, C.P. 04510, México, D.F., México Departmento de Bioquimica Vegetal, Facultad de Quimica Universidad Nacional Autónoma de México, C.P. 04510, México, D.F., México

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Ancient Mexican populations have been studied by archaeologists and physical anthropologists who have described cultural and some biological characteristics. Now, we report the isolation of high molecular weight DNA from bones of Mexican Tolteca culture. The results of the present study show a polymorphic pattern with the Elongation Factor 1-α (EF1-α) gene. These results open a new perspective on the knowledge of past Mexican populations and their relationships with present ethnic groups. The physical anthropology of Mesoamerican prehispanic populations has been studied amply. Since the end of the last century interest in knowing who the ancestral inhabitants of the land of Mexico were, led to the early studies in osteology. Although these studies were initially descriptive, they were able to infer essential physical characteristics as well as the evaluation of other parameters, such as growth and nutrition, of ancient populations. More recent, anthropometric and paleodemographic investigations of Mesoamerican prehispanic bone remains have revealed data on sex, age, height and population features (1-4). Phenomena such as longevity graphs, mortality per age group and sex have been inferred, as well as some aspects of living conditions. Some examples are certain pathologies, nutritional characteristics and the appraisal of economic and social fluctuations (5-7). Furthermore, some studies have employed immunological techniques to attempt a classification of prehispanic bone remains based on blood groups (8, 9) and histological composition (10-12). Presently, molecular archaeology is interested, in among other things, the search for nucleic acids present in human tissues from hundreds to thousands of years ago to attempt molecular genetics analysis, paleopathology, individual characterization, population studies , etc. (13-39). These investigations have demonstrated that genetic material may be preserved under certain environmental conditions. Therefore,

0097-6156/96/0625-0391$12.00/0 © 1996 American Chemical Society

In Archaeological Chemistry; Orna, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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molecular studies of prehispanic Mexican populations presents the possibility of finding answers to old questions and proposing new ones. In Mexico, this line of investigation started with the identification and characterization of human DNA recovered from Tomb 1, Penon del Marquez, Iztapalapa, Mexico wich is approximately 650-750 years-old (11, 23). The present study employed bone materialfroma prehispanic population of 130 humans found at the archaeological site of Tula, Hidalgo, Mexico aged approximately 750-1050 years old (40). The aim of this study was to isolate and characterize DNA and RNA for future systematic studies at the population level. Materials and Methods Human bone remains were obtained from the archaeological site of Tula, Hidalgo, Mexico. The areas studied were Malinche, Museum (Charnay square)fromhabitions, from civic centers and from palaces, Zapata Π, Viaducto, Plazas and Juego de Pelota. The populations consisted of 130 individuals whose remains are about 750-1050 years old (40), belonging to Tolteca and Mexican cultures. Histological Samples. Bone remains were decalcified in formic acid in neutral formalin for 24 h, submerged in paraffin, and sectioned in 5 μπι thicknesses with a manual microtome (American Optical). The paraffin was removed, and the sections were washed and stained with May-Greenwald-Giemsa stain. DNA Isolation. Bone remains consist of several anatomical pieces in diverse preservation states. The bones were always handled with gloves or forceps to avoid contamination by skin cells or perspiration. Excess soil was removed by scraping with a scalpel blade. Modem chicken bone and prehispanic osseous tissues were processed in parallel for comparative experiments. Bone samples of 1-3 g were crushed and genetic material extracted according to a modified version of Maniatis' method (11, 12). Nucleic Acids Quantification. The amount of nucleic acids was quantified following Spirin's method (41); DNA and RNA were measured by Scheinder's (42) and Burton's (43) methods, respectively. Recovery of High Molecular Weight DNA. DNA extracted from prehispanic bone was analyzed by agarose gel electrophoresis. The high molecular weight DNA was determined by a long wavelength (300-360 nm) from an UV lamp and was extracted following three methods: (1) Electroelution into dialysis bags, as described by Maniatis (44). (2) Separation by DEAE-81 (Whatman) or NA-45 (Schleicher & Schuell) membranes following a modification of the method described by Dretzen and collaborators (45). (3) Electroelution into troughs was prepared by a modification of the Maniatis method (44): the high molecular weight band was located on the 1% agarose gel. In front of the leading edge of the band a pit was made . This trough was filled with 50% glycerol in TBE IX, and electrophoresis resumed. Every 5 or 8 minutes (min) the fluid was recovered. The trough was refilled with fresh solution and


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DNA in Prehispanic Mexican Populations

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the electrophoresis was continued until all the DNA in the band had been removed from the gel and was recovered in the fluid taken from the trough. The high molecular weight band DNA was extracted once with phenol and once with chloroform before being precipitated by ethanol. Southern Blot Analysis. Purified genomic high molecular weight DNA samples (approximately 10 μg/lane) were digested according to conditions recommended by manufacturers with EcoRI (Gibco BRL), at 37 °C overnight and subjected to electrophoresis in a 0.8% agarose gel in 0.089 M Tris-borate, 0.089 M boric acid and 0.002 M EDTA (TBE IX). Phage lambda DNA preparations, digested with Hindm (Gibco BRL), were included as molecular size markers. Gels were run at 25V for 1822 h and then stained with ethidium-bromide and photographed. Gels were denatured with 0.5 M NaOH/1.5 M NaCl for 60 min and neutralized with 1 M Tris-HCl pH 8.0, 1.5 M NaCl, and the DNA fragments were transferred to Nytran membranes (Schleicher & Schuell) using the method first described by Southern (46). After transfer, filters were rinsed in 2 X standard saline citrate (300 mM NaCl/30 mM sodium citrate) and then irradiated with a standard ultraviolet ligth for 5 min. Nucleic Acid Hybridization. Filters were prehybridized for 16 h at 42 °C in 50% formamide, 5 X SSC, 10 X Denhardt's solution, 50 mM Na-phosphate (pH 7.2), sodium dodecyl sulfate (SDS) 0.1% and 100μg/ml denatured sonicated herring sperm DNA. Filters were hybridized overnight at 42 °C in the same solution with the addition of 1-2 χ 10 cpm/ml P probe labeled according to conditions recommended by manufactures with Random Primer Kit (BRL) to specific activity 1 χ 10^ dpm^g. Filters were washed at room temperature once, with 2 X SSC, 0.1% SDS for 30 min at 50 °C, twice with 0.2 X SSC, 0.1% SDS for 30 min, and finally at 55 °C with 0.1 X SSC, 0.1% SDS for 15 min. Filters were then exposed to Cronex film (Dupont) with two intensifying screens at -70°C for 3-6 days. The probe corresponded to the gene HEF-lcc (human elongation factor la) cloned at site PstI of PBr322 in E. coli C-600 supplied by Dr. Mario Castaneda from a donation by Dr.Win Moller. 6

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Results and Discussion Histological examination shows that the prehispanic bone tissue between 750-1050 years old preserves cellular debris, including osteocytes and remains of blood vessel epithelium (Figure 1). Nuclei were also observed, and their distinctness could be increased with a specific stain (May-Greenwald-Giemsa) for nucleic acids instead of hematoxylin-eosine. Similar results have been reported in mummified tissue and in ancient brain tissue, where various cell types and nuclei can be recognized (17-20, 26). Romano and collaborators (10), using eosin-hematoxylin, have observed the presence of blood cells in different stages of differentiation in prehispanic bone remains dated to 500 years of age. Likewise, in a Mexican female skeleton osteocytes and blood vessels have been identified (77). These results indicated that some cells and their components can be preserved in prehispanic human remains under varying ecological conditions.



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Figure 1. Tissue section from prehispanic bone. May-Greenwald-Giemsa staining. Magnification 200x.

Absorbance Spectrum of DNA Samples

1.4 τ

Wavelength(nm)

Figure 2. Absorbance spectrum of DNA from human fibroblast grown in culture (BAZD) and DNA from prehispanic bone remains.


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Nucleic acids were estimated as described in the Materials and Methods section from two anatomical pieces of the same individual. DNA and RNA values obtained from rib, vertebra, phalanx, femur or tibia were similar and fairly reproducible. The recovery of nucleic acids obtained from 130 prehispanic bone remains collected in various sites of excavation have been summarized in Table I. Table I. Nucleic Acids from Prehispanic Bones Yield Recovery Diphenylamin Orcinol e Sample (pg/g) (Percent) (pg/g) (pg/g) dry tissue fresh tissue dry tissue dry tissue rib/vertebra 159 ±24 7-10


a

rib/vertebra

1.0

rib/vertebra

10.0

13±4 145±22

rib/vertebra 9.0 2.5 Spirin's method quantification; ^Treatment with Ribonuclease b

a

The total amount of nucleic acids from different samples is 136-182 μg/g of dry tissue as determined by Spirin's method (41). However, only 7-10 percent is DNA and the rest is RNA. The amount of DNA in the presence of Ribonuclease during DNA extraction is 9 μ/g per dry tissue which corresponds to 6 percent. These results show, that on the order of 9-17 μg DNA are recovered typically from lg from osseous tissue. Therefore the DNA yield obtained from prehispanic skeletons is similar to values reported for other ancient tissues (18-20, 34, 37). The recovery of DNA and RNA obtained from lg from modem chicken bone is 1,400 μg/g and 1,200 μg/g respectively. Values obtained from prehispanic skeletons yield about 1 percent of DNA, and between 9-12 percent of RNA with respect to that recovered from fresh bone (Table I). The presence of RNA in human bone remains had not been reported previously. The RNA recovery is 123-167 μg/g of dry tissue, which represents 93 percent of total nucleic acids from ancient bone. Other authors have reported the presence ofribosomalRNA (rRNA) in com seeds (47), plants (48), marsupial wolf, 12S mithocondrial rDNA (49) and 18S and 28S rRNA in mummies (26). In order to prove that the isolated material corresponds to nucleic acids, the following criteria were used: (1) UV absorbance spectrum. The absorbance spectrum of the DNA is shown in Figure 2, where the spectrum of contemporary DNA, extracted from human fibroblast grown in culture (BAZD) was compared to that of DNA purified from ancient bone. In bone tissue, a color shade ranging from yellow to a dark brown was observed that modified the typical absorbance spectrum of nucleic acids (Figure 2). This pattern is similar to the fulvic acids ultraviolet absorbance spectrum (50). To eliminate the pigment, samples were subjected to gel filtration chromatography (79), dialysis and the different treatments to obtain high molecular weight DNA as described in Materials and Methods.



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The quality of the absorbance spectrum after gel column chromatography and different treatments improves and results are typical of the nucleic acids absorbance spectrum (Figure 2). Through gel filtration chromatography, approximately 25 percent of the genetic material is lost in the process. Dialysis and electroelution also eliminated the pigmentfromthe sample, but the loss of genetic material is greater: up to 60 percent (results not shown). However, recovery with DE-81 filters, NA-45 membranes or by electroelution into troughs is between 65-85 percent (Figure 2). Nevertheless, all DNA recovered by any one of these methods shows a typical absorbance spectrum of nucleic acids (Figure 2). This pattern is different from the DNAfromEgyptian mummies (76). These pigments are not only present in bone remains but have also been reported in other ancient tissues (77, 72, 16, 21, 27, 34, 51). Pàabo (27) suggested that the pigments consist of Maillard products; however these could not be identified in the present study (results not shown). Furthermore, during 1% agarose electrophoresis, the pigments migrated faster than bromophenol blue. Other authors have suggested that this pigment could be derived from humic acids (57). The origin of the variations among the pigments is unknown: They may be related to the kind of soil where the remains were buried, to length of time and the type of pit, as well as other physical and chemical factors. Although the pigments have not been chemically characterized, their presence is independent of the mechanism by which the remains were preserved (77, 16, 21, 27, 34, 51) and also interferes with the polymerase chain reaction (PCR) amplification (22, 34, 52, 53). (2) Agarose gel electrophoresis. An alternative procedure to identify genetic material was agarose gel electrophoresis. The pattern of DNA obtained from prehispanic bone shows that the molecular weight of this genetic material ranges between 0.125-23.1 Kilobases (Kb) (results not shown). These values contrast with those obtained for genetic material from other human remains which have exhibited DNA molecular weights ranging from 0.120-12 Kb, in remains preserved under conditions of extreme anaerobiosis (19) or in other environments (79, 27-29, 33, 34, 37). Low molecular weight DNA seems to be common in human remains. The cause of the degradation of the DNA may be attributed to processes such as the effect of environmental factors like radioactivity, post-mortem autolytic processes, hydrolysis and oxidation of sugar and nitrogenous bases (18, 21, 27, 54) or instability of DNA glycosyl bonds under different solvent conditions as a function of temperature, pH, ionic strength and nucleic acid secondary structure and also non-enzymatic DNA methylation (54). The transformation of nitrogenous bases is also important, as exemplified in the alterations found in pyrimidines in mummified tissue (18). However results from the present study show that high molecular weight DNA from ancient human remains is not exclusively related to the burial conditions found at the moment of unearthing. The burial undergoes many environmental modifications with time. In this sense importance must be given to such aspects as the archaeological context, location of the burial, type of dwelling, primary and secondary nature of burial, presence of offerings, type of soil, as well as the following factors: land displacement, temperature, filtration, humidity, uses given to the land, pH, oxide reduction potential, presence of organic and inorganic salts and the presence of




DNA in Prehispanic Mexican Populations 397

microorganisms, fungi, plants and animals. It is therefore difficult to explain differences between nucleic acid recovery that belong to human remains which have been exposed to similar, if not equal, "environmental conditions" as exemplified in several prehispanic DNA samples (results not shown). Even so, high molecular weight DNA was isolated from human bone remains between 750-1050 years-old (11,12). The next step was to demonstrate that the high molecular weight DNA samples were of human origin. This was tested by Southern blot analysis with the human gene of elongation factor-la (HEF-la). Figure 3 depicts Southern hybridization of high molecular DNA from 14 randomly chosen individuals from the archaeological sites (750-1050 years-old) of Malinche, Zapata Π and Charnay (Museum Area) which were compared to present day human DNA from human cell-line grown in culture (BAZD). The DNA was digested with EcoRI and hybridized as explained in Materials and Methods. Results

Figure 3. Southern blot analysis of elongation factor 1-oc (HEF-la) DNA. Contemporany DNA (Lane 1), prehispanic DNA (Lanes 2-15), λ phage DNA digested with Hindm (Lane 16). Molecular size markers are indicated in numbers of base pairs. Kilobases (Kb).



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show that ten fragments invariably appear both prehispanic DNA and in contemporary DNA.. However, 19 Kb fragments are only present in prehispanic samples, 3.7 Kb fragments in only 6 of the prehispanic samples and 2 Kb in both. Polymorphism of the human gene HEF-la has been detected in Caucasian populations (55). The present study revealed EcoRI RFLPs of 3.7 Kb in 6 out of 14 studied individuals from the archaeological site of Tula, Hidalgo, which suggests a possible polymorphism of the gene HEF-la in this prehispanic Mexican population. Thus, the possibility arises of studying polymorphisms and genetic variability in ancient Mexican populations, until now only described in living population (56, 57). These studies may be applied to determine kinship, migration patterns, relationships to present day ethnic groups, sex and genetic diseases of ancient Mexican populations. The systematic application of these studies will enrich the knowledge derived from the classical methodologies of physical anthropology. Acknowledgments Authors wish to thank Dr. Mario Castaneda for supplied HEF-la probe, Ernesto Guerrero for help with preparation of histological sections and Blanca Paredes have generously provided bone samples. Literature Cited 1. Genovés, S. Introducción al diagnóstico de la edad y del sexo en restos óseos prehispάnicos. Instituto de Historia: Universidad Nacional Autónoma de México. México, México, D.F., 1962. 2. Genovés, S. La proporcionalidad de los huesos largos y su relación con la estatura en restos mesoamericanos; Cuademos; Serie Antropológica, Núm. 19; Instituto de Investigaciones Históricas: Universidad Nacional Autónoma de México, México, D.F., 1962. 3. Jaen, M . T. ; López-Alonso, S. En Antropologίa Fίsica. Época Prehispánica; Romero J., Ed.; Panorama Historico Cultural, III; Instituto Nacional de Antropología e Historia / Secretaria de Educación Pública, México, D.F., 1974; pp 113-118. 4. Comas, J. Manual de Antropologίa Fίsica. Instituto de Investigaciones Antropológicas: Universidad Nacional Autónoma de México, México, D. F., 1976. 5. Dávalos-Hurtado, Ε. In Handbook of Middle American Indians: Physical Anthropology; Stewart, T. D., Ed.;University of Texas Press: Austin, 1970;p 9. 6. Jaen, M. T. ; Serrano, C. En Antropologίa Fίsica: Epoca Prehispánica; Romero, J., Ed.; Panorama Histórico Cultural, III; Instituto Nacional de Antropología e Historia / Secretaria de Educación Publica, México, D.F., 1974; pp 153-168. 7. Serrano, C . ; Ramos, R. Ma. Perfil bioantropológicode la población prehispάnica de San Luis Potosί. Serie Antropológica, Núm 40; Instituto de Investigaciones Antropológicas: Universidad Nacional Autónoma de México, México, D.F., 1984.





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