American population studies using DNA techniques ------------------------------------------------ ... Concerning the question of a possible settlement (in Easter Island) from South America, recent surveys of hypervariable region of mitochondrial DNA (mtDNA) of native Americans have shown much greater mtDNA heterogenity in America than in Polynesia, reflecting multiple waves of migration and greater depth of prehistory, whereas the Polynesian type that we observed in Easter Island has not been detected. Although the 9-bp deletion exists throughout the Americas, Asia and the Pacific, often with the T-->C transition in position 16,217, this simply support the view of the shared common origin of the ncestral settlers of Oceania and the New World. The absence of the other two Polynesian substitions (mtDNA sites 16,247 and 16,261) in the New World points to a lack of significant contact between Polynesia and the America... - Hagelberg, E.; Clegg, J.B. (Hagelberg. E. University of Cambridge, Department of biological anthropology, Downing street, Cambridge CB2 3DZ,UK). Genetic Polymorphisms in Prehistoric Pacific Islanders Determined by Analysis of Ancient Bone DNA. Proceedings of the Royal Society of London Series B - Biological Sciences: 1993, 252: 163 - 170 - Nature, 5 may 1994, 369: 25 -26: DNA from ancient Easter Islanders, by Erika Hagelberg et al. (a group of Anthropologists from Cambridge UK, Santiago de Chile, Barcelona Spain and a molecular biologist of Oxford UK) The paper is accompanied by 10 references. - 2 g of powdered bones per each PCR analysis (Hagelberg et al Nature 1989, 342: 485) - Horai, S., Kondo, R., Nakagawahattori, Y., Hayashi, S., Sonoda, S., Tajima, K. (Natl Inst Genet, Dept Human Genet, Mishima, Shizuoka 411, Japan) Peopling of the Americas, Founded by 4 Major Lineages of Mitochondrial DNA. Molecular Biology and Evolution: 10(1993)1,p.23-47 Abstract: Nucleotide sequence analysis of the major noncoding region of human mitochondrial DNA from various races was extended with 72 Native Americans from 16 different local populations (nine populations from Chile, four from Colombia, and one each from Brazil and from Maya and Apache Indians). The sequences were determined directly from the polymerase chain reaction products. On the basis of a comparison of the 482-bp sequences in the 72 Native Americans, 43 different types of mitochondrial DNA sequences were observed. The nucleotide diversity within the Native Americans was estimated to be 1.29%, which is slightly less than the value of 1.44% from the total human population including Africans, Europeans, and Asians. Phylogenetic analysis revealed that most Native American lineages are classified into four major distinct clusters. Individuals belonging to each cluster share at least two specific polymorphic sites that are nearly absent in other human populations, indicating a unique phylogenetic position of Native Americans. A phylogenetic tree of 193 individuals including Africans, Europeans, Asians, and Native Americans indicated that the four Native American clusters are distinct and dispersed in the tree. These clusters almost exclusively consist of Native Americans - with only a few Asians, if any. We postulate that four ancestral populations gave rise to different waves of migration to the New World. From the estimated coalescence time of the Asian and Native American lineages, we infer that the first migration across the Bering landbridge took place approximately 14,000-21,000 years ago. Furthermore, sequence differences in all pairwise comparisons of Native Americans showed a bimodal distribution that is significantly different from Poisson. These results suggest that the ancestral Native American population underwent neither a severe bottleneck nor rapid expansion in population size, during the migration of people into the Americas. - Torroni, A., Schurr, T.G., Cabell, M.F., Brown, M.D., Neel, J.V., Larsen, M., Smith, D.G., Vullo, C.M., Wallace, D.C. (Emory Univ, Dept Genet & Molec Med, Atlanta, GA 30322). Asian Affinities and Continental Radiation of the Four Founding Native American mtDNAs.American Journal of Human Genetics: 53(1993)3,p.563-590 Abstract: The mtDNA variation of 321 individuals from 17 Native American populations was examined by high-resolution restriction endonuclease analysis. All mtDNAs were amplified from a variety of sources by using PCR. The mtDNA of a subset of 38 of these individuals was also analyzed by D-loop sequencing. The resulting data were combined with previous mtDNA data from five other Native American tribes, as well as with data from a variety of Asian populations, and were used to deduce the phylogenetic relationships between mtDNAs and to estimate sequence divergences. This analysis revealed the presence of four haplotype groups (haplogroups A, B, C, and D) in the Amerind, but only one haplogroup (A) in the Na-Dene, and confirmed the independent origins of the Amerinds and the Na-Dene. Further, each haplogroup appeared to have been founded by a single mtDNA haplotype, a result which is consistent with a hypothesized founder effect. Most of the variation within haplogroups was tribal specific, that is, it occurred as tribal private polymorphisms. These observations suggest that the process of tribalization began early in the history of the Amerinds, with relatively little intertribal genetic exchange occurring subsequently. The sequencing of 341 nucleotides in the mtDNA D-loop revealed that the D-loop sequence variation correlated strongly with the four haplogroups defined by restriction analysis, and it indicated that the D-loop variation, like the haplotype variation, arose predominantly after the migration of the ancestral Amerinds across the Bering land bridge. (3) Torroni, A., Sukernik, R.I., Schurr, T.G., Starikovskaya, Y.B., Cabell, M.F., Crawford, M.H., Comuzzie, A.G., Wallace, D.C.(Emory Univ, Dept Genet & Molec Biol, Atlanta, GA 30322). mtDNA Variation of Aboriginal Siberians Reveals Distinct Genetic Affinities with Native Americans. American Journal of Human Genetics: 53(1993)3,p.591-608 Abstract: The mtDNA variation of 411 individuals from 10 aboriginal Siberian populations was analyzed in an effort to delineate the relationships between Siberian and Native American populations. All mtDNAs were characterized by PCR amplification and restriction analysis, and a subset of them was characterized by control region sequencing. The resulting data were then compiled with previous mtDNA data from Native Americans and Asians and were used for phylogenetic analyses and sequence divergence estimations. Aboriginal Siberian populations exhibited mtDNAs from three (A, C, and D) of the four haplogroups observed in Native Americans. However, none of the Siberian populations showed mtDNAs from the fourth haplogroup, group B. The presence of group B deletion haplotypes in East Asian and Native American populations but their absence in Siberians raises the possibility that haplogroup B could represent a migratory event distinct from the one(s) which brought group A, C, and D mtDNAs to the Americas. Our findings support the hypothesis that the first humans to move from Siberia to the Americas carried with them a limited number of founding mtDNAs and that the initial migration occurred between 17,000-34,000 years before present. This paper presented below tries to determine if any correlation exists between gene variants (allele in the genetics' jargon) and language distributions among the actual ameridians. Callegari-Jacques, S.M.; Salzano, F.M.; Constans, J.; Maurieres, P. (Univ Fed Rio Grande Sul; Inst Matemat; Dept Estatist; Campus Vale; BR-91540 Porto Alegre; RS, Brazil). GM Haplotype Distribution in Amerindians - Relationship with Geography and Language. American Journal of Physical Anthropology: 90(1993)4,p.427-444 Abstract (the original): review is made of the Gm haplotype distribution in 60 groups of Eskimos, North, Central and South American Indians, totaling 22,808 individuals. Differences were observed in the shapes of the distribution of Gm*ag and the other markers. Nearly identical values for F(ST) and average heterozygosities were obtained in the North + Central/South comparisons. North-South and Southwest/Northeast clinal differences were observed in the Americas using correspondence factorial analysis. The two haplotypes mainly responsible for these differences are Gm*axg and Gm*abOst. When the populations are classified by language groups, besides the recognized differences between Eskimos and Athabaskan (Na-Dene) speakers compared with Amerinds, others are found. For instance, Uto-Aztecan speakers of the United States and Mexico differ in Gm frequencies from the Nuclear Chibchan, Macro-Arawak, and Carib speakers of Central and South America. The notion of a homogeneous Amerind genetic pool does not conform with these and other results. Gm Haplotype Distribution of 27 Amerindian Tribes: description of Mobile Site Method: Hazout, S.; Guasp, G.; Loirat, F.; Maurieres, P.; Larrouy, G.; Dugoujon, J.M. (S Hazout; Univ Paris 07; Inserm; U263; Unite Rech Biomath & Biostat; 2 Pl Jussieu; F-75251 Paris 05, France). A New Approach for Interpreting the Genetic Diversity in Space - Mobile Site Method - Application to Gm Haplotype Distribution of 27 Amerindian Tribes from North and Central America. Annals of Human Genetics: 57(1993) Part 3,p.221-237 Abstract: We present a new approach, called 'Mobile Site Method' (MSM), to the construction of genetic similarity maps' more efficient than that described in a preceding paper (Hazout et al. 1991). After building a triangular mesh between the geographical sites, the method consists of moving these locations at each iteration to reduce the overall differences between the geographic and genetic distances. The genetic similarity map, i.e. the final distorted map, allows the interpretation of the genetic diversity of a population set. We have applied this method to the study of Gm immunoglobulin allotypes of twenty-seven Amerindian groups from North and Central America. By a local weighted linear regression, we have reconstituted the distorted contour of America. This representation completes the observations of the sites during the map distortion. In this study, we have defined a large geographical factor in the genetic data (84% of the variability explained), related to a linguistic factor. - Benerecetti, A.S.S.; Semino, O.; Passarino, G.; Torroni, A.; Brdicka, R.; Fellous, M.; Modiano, G. (ASS Benerecetti; Univ Pavia; Dipartimento Genet &; Microbiola Buzzati Traverso; I-27100 Pavia, Italy) The Common, Near-Eastern Origin of Ashkenazi and Sephardi Jews Supported by Y-Chromosome Similarity. Annals of Human Genetics: 1993, 57: 55 - 64 - Torroni, A.; Chen, Y.S.; Semino, O.; Santachiarabeneceretti, A.S.; Scott, C.R.; Lott, M.T.; Winter, M.; Wallace, D.C. (Wallace, D.C.; EMORY UNIV SCH MED,DEPT GENET & MOLEC MED, ATLANTA, GA 30322 USA) mt DNA and Y-Chromosome polymorphism in 4 Native-American populations from Southern Mexico. American Journal of Human Genetics, (1994)2, 54: 303 - 318 Abstract: mtDNA sequence variation was examined in 60 Native Americans (Mixtecs from the Alta, Mixtecs from the Baja, Valley Zapotecs, and Highland Mire) from southern Mexico by PCR amplification and high-resolution restriction endonuclease analysis. Four groups of mtDNA haplotypes (haplogroups A, B, C, and D) characterize Amerind populations, but only three (haplogroups A, B, and C) were observed in these Mexican populations. The comparison of their mtDNA variation with that observed in other populations from Mexico and Central America permits a clear distinction among the different Middle American tribes and raises questions about some of their linguistic affiliations. The males of these population samples were also an analyzed for Y-chromosome RFLPs with the probes 49a, 49f, and 12f2. This analysis suggests that certain Y-chromosome haplotypes were brought from Asia during the colonization of the Americas, and a differential gene flow was introduced into Native American populations from European males and females. - AUTHOR: Serrano, Carlos TITLE: Grupos sanguineos (sistema ABO) y mestizaje en los Tojolabales de Chiapas / by Carlos Serrano PUBLISHED IN: Los Legitimos hombres. Mario Humberto Ruz, ed. Mexico Centro de Estudios Mayas, v. 3, 1983, p.13-24, [1] leaf of plates, ill. - AUTHOR: Ahn, Y. I. TITLE: DNA polymorphisms of the apolipoprotein AI/CIII/AIV gene cluster influence plasma cholesterol and triglyceride levels in the Mayans of the Yucatan Peninsula, Mexico / Y. I. Ahn, R. Valdez, A. P. Reddy, S. A. Cole, K. M. Weiss, R. E. Ferrell. PUBLISHED IN: Human Heredity v. 41, no. 5, 1991. pp. 281-289. --- mtDNA Amerindian haplotypes 1. mtDNA haplotypes in population studies: Each mitochondria in the cell of an individual has an identical DNA molecule or mtDNA. Mitochondria are transmited only (almost!) by the mother to her children. A population harboring the same haplotype is a haplogroup. It originates from the same mother or founding mother. If a subsequent mutation arises in the haplotype of a mother in a Press RETURN for more... MAIL> #916 17-JUN-1994 10:36:43.88 MAIL haplogroup, the new haplotype will be transmitted to her children and will define a subhaplogroup and so on. This process creates a branching tree. At the base of the tree is the haplotype of Eve! In population studies, one knows only the actual branchs of the tree and tries to understand their origins. In appendix (3), are examples of Ameridians partial haplotypes. All Amerindians haplotypes originated from 4 Asian original haplotypes. 2. mtDNA haplotype definition: An mtDNA haplotype is the nucleotide sequence of a mtDNA molecule. An mtDNA molecule is a circular sequence of the nucleotides A, T, G, C in a defined order. The average molecule is 16569 nucleotides in length. 3. Each human mtDNA derived by changes from an unknown original. Changes from this unknown molecule (that of Eve's mitochondrias!) can be: a transition when a A is substituted for by a G or a C by a T. a transversion when a A or a G is substituted by a C or a T. a deletion of a part of the sequence. an addition of one or a sequence of nucleotides... 4. The description of haplotypes requires a standard: The choice of this standard is arbitrary: it is used to locate a change. Each mtDNA haplotype is described by the differences it shows with the first human mtDDNA haplotype which was sequenced, in Cambridge (UK), by Anderson S. et al. (Nature, 1981, 290: 457 - 465). The nucleotides of this standard molecule are numbered from 1 to 16569. Appendix (1) shows the sequence of the hypervariable region of mtDNA, a region which is frequently used in genetics. Appendix (2) shows how to get this standard by e-mail from Genbank 5. position of the change: For a transition, a transversion or a deletion, position is that of the modified nucleotide (s), in the standard sequence, it is the a number from 1 to 15569. For an addition, new numbers are created, for example, an addition of a C between position 301 and 302 is described at as"added C in position 301.1". When a change creates a new site, for example a site recognized by the enzyme EcoRI (this site is GAATTC), the position of the new site is given by that of its first nucleotide. Example: in the 5 nucleotide sequence GAATGC, the tranversion G-->T at np 5, creates a EcoRI site (GAATTC) at np 1. ------------------------------------------------------------------- Appendix (1): The hypervariable region of the standard mtDNA molecule (Light or L strand) sequenced by Anderson S et al. (Nature, 1981, 290: 457 - 465). It overlap the origin of the circular mtDNA molecule which arbitrarily begins at np 1 and ends at np 16569. ********** ********** ********** ********** ********** 15901 .......... .......... cggag atgaaaacct ttttccaagg 15951 acaaatcaga gaaaaagtct ttaactccac cattagcacc caaagctaag 16001 attctaattt aaactattct ctgttctttc atggggaagc agatttgggt 16051 accacccaag tattgactca cccatcaaca accgctatgt atttcgtaca 16101 ttactgccag ccaccatgaa tattgtacgg taccataaat acttgaccac MAIL 16151 ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta 16201 caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc 16251 caaagccacc cctcacccac taggatacca acaaacctac ccaccCttaa 16301 cagtacatag tacataaagc catttaccgt acatagcaca ttacagtcaa 16351 atcccttctc gtccccatgg atgacccccc tcagataggg gtcccttgac 16401 caccatcctc cgtgaaatca atatcccgca caagagtgct actctcctcg 16451 ctccgggccc ataacacttg ggggtagcta aagtgaactg tatccgacat 16501 ctggttccta cttcagggtc ataaagccta aatagcccac acgttcccct 16551 taaataagac atcacgatg 1 gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat 51 ttggtatttt cgtctggggg gtatgcacgc gatagcattg cgagacgctg 101 gagccggagc accctatgtc gcagtatctg tctttgattc ctgcctcatc 151 ctattattta tcgcacctac gttcaatatt acaggcgaac atacttacta 201 aagtgtgtta attaattaat gcttgtagga cataataata acaattgaat 251 gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 301 aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca 351 aaccccaaaa acaaagaacc ctaacaccag cctaaccaga tttcaaattt 401 tatcttttgg cggtatgcac ttttaacagt caccccccaa ctaacacatt 451 attttcccct cccactccca tactactaat ctcatcaata caacccccgc 500 ccatcctacc cagcacacac acaccgctgc taaccccata ccccgaacca 551 accaaacccc aaagacaccc cccacagttt ----------------------------------------------------------------- Appendix (2) The Standard human mtDNA sequence is deposited at the Genbank, in the NCBI at the National Library of Medecine at Bethesda MD. To get it (you will get 2 mails) send an e-mail without subject to In the body of the mail write exactly (no signature!): DATALIB GENBANK BEGIN HUMMTCG ------------------------------------------------------------------ Appendix (3) Partial haplotypes as found in the litterature (Torroni, A.,et al. American Journal of Human Genetics, 1993 3, 53: 563 - 590): : (1) 4 Maya haplotypes between np 162051 and np 16251: Maya haplotypes 1, 31 and 50 are : C --> T transition at np 16223 Maya haplotype 13 is: T --> C transition at np 16527 means: 16201 caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc 16251 maya 1 T maya13 C maya31 T maya50 T (1) "All Native American group A haplotypes (AM1 - AM12 and AM51 - AM - 63) are defined by an A-to-G transition at np 663 which creates an HaeIII site gain at that position." (2) "Group B haplotypes (AM13 - AM27 and AM65 - AM70) are distinguished by a 9 nucleotide deletion occuring between nucleotides 8272 and 8289. The deletion is always found in association with the HaeIII site gain at np 16517." (3) "Amerind group C haplotypes (AM30-AM43 and AM77-AM87) are characterized by an A-to-G transition at np 13263 which simultaneously elinminates a HincII site at np 13259 and creates an AluI site at np 13262." --------------------------------------------------------------------------- Obtaining DNA from bones ------------------------ Book: Ancient DNA Recovery and analysis of genetic material from paleontological, archeological, museum, medical and forensic specimens B. Herrmann and S. Hummel Eds. Springer-Verlag 1993 ISBN 0-387-97929-8 Articles: Those who write this paper are able to discuss the subject of DNA in SKELETAL REMAINS, and to help perhaps the archaeologists working on the field! Usually, the team of King puhblishes on other subjects (breast cancer etc...). Ginther, C.; Isseltarver, L.; King, M.C. (MC King; Univ Calif Berkeley; Dept Molec & Cell Biol; Berkeley, CA 94720). Identifying Individuals by Sequencing Mitochondrial DNA from Teeth. Nature Genetics: 2(1992)2,p.135-138 5, 23, 39 (MC King; Univ Calif Berkeley; Dept Molec & Cell Biol; Berkeley, CA 94720). Abstract: Mitochondrial DNA (mtDNA) was extracted from teeth stored from 3 months to 20 years, including teeth from the semi-skeletonized remains of a murder victim which had been buried for 10 months. Tooth donors and/or their maternal relatives provided blood or buccal cells, from which mtDNA was also extracted. Enzymatic amplification and direct sequencing of roughly 650 nucleotides from two highly polymorphic regions of mtDNA yielded identical sequences for each comparison of tooth and fresh DNA. Our results suggest that teeth provide an excellent source for high molecular weight mtDNA that can be valuable for extending the time in which decomposed human remains can be genetically identified. --- Gill, P.; Ivanov, P.L.; Kimpton, C.; Piercy, R.; Benson, N.; Tully, G.; Evett, I.; Hagelberg, E.; Sullivan, K. (P Gill; Forens Sci Serv Inc; Priory House; Gooch St N; Birmingham B5 6QQ; W Midlands, England). Identification of the Remains of the Romanov Family by DNA Analysis. Nature Genetics: 6(1994)2,p.130-135 5, 23, 39 Abstract: Nine very damaged (by chemicals) skeletons found in a shallow grave in Ekaterinburg, Russia, in July 1991, were tentatively identified by Russian forensic authorities as the remains of the last Tsar, Tsarina, three of their five children, the Royal Physician and three servants. We have performed DNA based sex testing and short tandem repeat (STR) analysis and confirm that a family group was present in the grave. Analysis of mitochondrial (mt) DNA reveals an exact sequence match between the putative Tsarina and the three children with a living maternal relative. Amplified mtDNA extracted from the remains of the putative Tsar has been cloned to demonstrate heteroplasmy at a single base within the mtDNA control region. One of these sequences matches two living maternal relatives of the Tsar. We conclude that the DNA evidence supports the hypothesis that the remains are those of the Romanov family. "Sex testing. The sex of the bones was determined by amplification of a portion of the X-Y homologous gene, amylogenin, which provides a robust method for typing samples of a very degraded nature: X and Y specific products of 106 and 112 (bp), respectively were "generated from a single primer pair. DNA extracts from all nine skelettons were readily typed and the results confirmed conclusions drawn from physical examination of the bones regarding their sex. In total, four male and five female bodies were identified using this method." (page 131) In a simple way: if you use the same PCR method (which means here the 2 same primers) with either male or female DNA, the amplified sequence of nucleotide will be longer in female (112 bp). The difference in length is be easy to measure in a simple electrophoresis: the fragment are separeted according to their size. B. Reference with adress of the main laboratory heads: Gill, P.; Ivanov, P.L.; Kimpton, C.; Piercy, R.; Benson, N.; Tully, G.; Evett, I.; Hagelberg, E.; Sullivan, K. (adress: Sullivan K.; Forens Sci Serv Inc; Priory House; Gooch St N; Birmingham B5 6QQ; W Midlands, England) (adress: Hagelberg. E. University of Cambridge, Department of biological anthropology, Downing street, Cambridge CB2 3DZ, UK)). Identification of the Remains of the Romanov Family by DNA Analysis. Nature Genetics: 6(1994)2,p.130-135 5, 23, 39 - Hagelberg, E.; Clegg, J.B. (E Hagelberg; Univ Oxford; John Radcliffe Hosp; Inst Molec Med; MRC; Molec Haematol Unit; Oxford OX3 9DU, England). Genetic Polymorphisms in Prehistoric Pacific Islanders Determined by Analysis of Ancient Bone DNA. Proceedings of the Royal Society of London Series B - Biological Sciences: 252(1993)1334,p.163-170 Abstract: A previously characterized Asian-specific mitochondrial DNA (mtDNA) length mutation has been detected in DNA isolated from prehistoric human bones from Polynesia, including Hawaii, Chatham Islands and Society Islands. In contrast, the Asian mutation was absent in skeletal samples from the Melanesian archipelagos of New Britain and Vanuatu and in the oldest samples from Fiji, Tonga and Samoa in the central Pacific (2700-1600 years BP) although it was present in a more recent prehistoric sample from Tonga. These results, augmented by informative DNA sequence data from the hypervariable region of mtDNA, fail to support current views that the central Pacific was settled directly by voyagers from island Southeast Asia, the putative ancestors of modern Polynesians. An earlier occupation by peoples from the neighbouring Melanesian archipelagos seems more likely. - Hagelberg E, Sykes B, and Hedges R (Hagelberg adress: Institute of Molecular Medecine, John Radcliffe Hospital, Oxford OX3 9DU, UK). Ancient Bone DNA amplified. Nature: 30 nov 89, 342: 485 Gill, P.; Ivanov, P.L.; Kimpton, C.; Piercy, R.; Benson, N.; Tully, G.; Evett, I.; Hagelberg, E.; Sullivan, K. (P Gill; Forens Sci Serv Inc; Priory House; Gooch St N; Birmingham B5 6QQ; W Midlands, England). Identification of the Remains of the Romanov Family by DNA Analysis. Nature Genetics: 6(1994)2,p.130-135 5, 23, 39 B. Answered questions: (1) Could DNA analysis be made on very very old bones. Mitochondrial DNA sequences of 121 and 205 nucleotide long were reproducibely amplified by PCR from (a) a child skull (radiocarbon date: 4810 1 80 years BP) from Maiden Castle, Dorset. (b) a human femur (radiocarbon date: 5440 years BP) from Wadi Mamed, Judean Desert. The longest sequence obtained from old bones was 750 nucleotides in length. A 1100 nucleotide sequence failed to be amplified. (from Hagelberg et al.) (2) Minimal amount of bone material necessary for DNA analysis: 5 microg "Typically, we can recover 5 - 10 microg of DNA from 2 g of powdered cortical bone. Although the DNA is degraded, it is often possible to recover material of High molecular mass... Our experience indicate that the preservation of DNA in a bone depends less on the age of the specimen than on the burial conditions of the skeleton" (from Hagelberg et al.). Optimal burial conditions were note described in this paper. (3) How to collect bones on the archaeological field. As the bones are supposed to be contaminated before being collected, it seems that the collecting itself is not a problem. The laboratory of molecular biology had good technics to clean the material. (from Gill et al.) (4) Is it possible to use badly damaged bones? the Romanov bones were 75 year old when collected. "the Bolsheviks placed the victims into a hastly dug pit in a road and to hinder identification, sulphuric acid was thrown into the open grave." "Many bones were badly damaged, but it was possible to identify nine corpses" (from Gill et al.) - DNA from teeth; collected in a grave to avoid superficial contamination. The method is surely valid for old bones. A. The paper: Ginther C, Issel-Traver L and King M C (Dept of Molecular 1 Cell Biology and School of Public Health, University of California at Berkeley 94720, USA) Identifying individuals by sequencing mitochondrial DNA from teeth. Nature Genetics october 1992, 2: 135 - 138 B Answered questions: (1) No specific precaution were taken for the extraction from the grave. (2) How were the tooth treated in the laboratory of molecular biology to discard contamination? "Surface material was removed from the teeth by soaking them individually in 3% hydrogen peroxyde or by washing in dilute bleach, 90 % ethanol and sterile water for 30 min each. Each tooth was opened ..." - When an archaeologist excavates a skeletton, he or she would like to known, from the gross external appearance if it is usable for genetic studies using PCR. Here is a comparison of (1) external appearance vs. (2) Electron Microscopy, (3) DNA extract appearance and (4) succes in PCR DNA amplification, in bad (a) and good (b) samples of archaeological bones. a) samples 1 (1 2000 y BP, from Melanesia and Central Pacific Islands): (1) external appearance: dark brown, fragile and porous: (2) E. Microscopy: there was no visible cellular organization in the tissue! (3) DNA extract appearance: brown, indicating the presence of compound derived from "humic acids" in the soil. (4) DNA amplification by PCR: in only a few samples, amplified DNA could be obtained (after 2 rounds of PCR instead of 1). This indicates that either the starting DNA amount was extremely low or that the brown co-purifying contaminant is an inhibitor of the Taq DNA polymerase used in PCR. b) samples 2 (1 700 y BP, from Polynesia): (1) external appearance: very dense with the characteristic light-yellow waxy appearance associated with good preservation: (2) E. Microscopy: the bone structure was easy to recognize. (3) DNA extract appearance: transparent. (4) DNA amplification by PCR: OK, with one PCR round (30 cycles). from a paper of: Hagelberg, E.; Clegg, J.B. Genetic Polymorphisms in Prehistoric Pacific Islanders Determined by Analysis of Ancient Bone DNA. Proceedings of the Royal Society of London Series B - Biological Sciences: 252(1993)1334,p.163-170 A more recent paper describes a method which could suppress the co-purifying inhibitors of PCR Taq polymerase (see above a 4): Goodyear, P.D.; Maclaughlinblack, S.; Mason, I.J. A Reliable Method for the Removal of Co-Purifying PCR Inhibitors from Ancient DNA. Biotechniques: (1994)2,16: 232 - 235 ------------------------------------------------------------------------- DNA analysis of diseases ------------------------ Groenen, P.M.A.; Bunschoten, A.E.; Vansoolingen, D.; Vanembden, J.D.A. (JDA Vanembden: National Inst Publ Hlth & Environm Protect; Molec Microbiol Unit; POB 1; 3720 Ba Bilthoven, Netherlands). Nature of DNA Polymorphism in the Direct Repeat Cluster of Mycobacterium - tuberculosis: Application for Strain Differentiation by a Novel Typing Method. Molecular Microbiology: (1993: 5), 10: 1057 - 1065 Abstract: Mycobacterium tuberculosis DNA contains a variable region, which consists of multiple 36 nucleotide Direct Repeats (DRs), which are interspersed by unique spacers 35 to 41 nucleotides in length. In this study the nature of the DNA polymorphism of this DR cluster is studied by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed: (1) One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. (2) The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the direct repeat cluster, we developed a novel method of strain differentiation, "Direct Variable Repeat Polymerase Chain Reaction (DVR-PCR)", which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR-PCR allows the detection of M. tuberculosis and strain differentiation at the same time. --------------------------------------------------------------------------- Possible application ideas -------------------------- Mccafferty, S.D.; Mccafferty, G.G. ENGENDERING TOMB-7 AT MONTE-ALBAN - RESPINNING AN OLD YARN Current Anthropology: (1994)2,35: 143 - 166 Abstract: A contextual analysis of material culture recovered from Tomb 7 at Monte Alban suggests a radical reinterpretation of the gender identification of the tomb's principal individual. Spinning and weaving implements found with the burial, previously interpreted as a male, indicate the strong possibility that the individual was gender-female. A reinterpretation of the skeletal remains as presented in the published accounts further indicates that the osteological evidence is ambiguous at best and the skeleton may have been of a biological female. Finally, the total assemblage is considered in reference to the religious and gender ideologies of pre-Columbian Mesoamerica to suggest that Tomb 7 may have been an important shrine to Lady 9 Grass, a principal member of the Mixtec Mother Goddess complex. This paper points up the necessity of periodic reevaluations of accepted wisdom that may have been developed under theoretical paradigms that minimized cultural diversity. - Jean-Pierre Herveg requested ideas for his study group on Mesoamerican dna, and we have had some discussion of Valley of Oaxaca skeletal remains. A second assemblage that might be very interesting is from Cholula, Puebla, where a sequence of excavations have discovered over 1000 skeletons representing a range of contexts and time periods. Over 500 individuals have been recovered from around the ceremonial center, often from ritual contexts indicating among other things cannibalized remains, and from Formative through Late Postclassic times. These data are summarized in Lopez,Lagunas, and Serrano (1976) Enterra- mientos Humanos de la Zona Arqueologica de Cholula, Puebla. This population could provide information on genetic shifts through time, relative to the ethno historic accounts of population movement and ethnic "invasions." A second mass burial that was recently discovered featured over 50 individuals from the Late Postclassic period, including a principal male and a large number of sacrificed servants/slaves. This sample could relate to political control, if it could be demonstrated that the slaves were of a distinct racial type. This site is summarized in Suarez (1990) Ultimos descubrimientos de entierros postclasicos en Cholula, Puebla, and I reviewed it in American Antiquity in 1992. Another interesting burial from the ceremonial center is reported in Suarez (1985) Un entierro del clasico superior en Cholula, Puebla (also summarized in the same AA review). Based on distinctive cranial deformation, dental inlays, and grave goods Suarez postulated that this may have been a Maya priest or merchant that for some reason was buried in Cholula. Since it was contemporary with the Mayoid Cacaxtla murals, this burial holds potential for the highland/lowland interaction problem. Another huge burial population is from the San Gabriel church in the center of town, where about 700 individuals were excavated. These date to the moment of contact, 1519, and represent the result of the Cholula massacre just prior to Cortes' arrival in Tenochtitlan. Since the massacre was indiscriminant, this probably represents a cross-section of the city. Numerous other collections also exist, but this gives a sampling. Does anyone have suggestions for additional research questions that could be addressed with this data? The majority of the skeletons are in storage in PUebla, so in addition to a hypothetical study they could actually be used. Geoff McCafferty