Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: A problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.Correspondence to: A.C. van der Kuyl     

Recovery and identification of DNA sequences harboured in preserved ancient human bones.

A new method is presented to extract and identify specific DNA fragments from well preserved human bones, dating from three different time periods. Bone samples were thoroughly freed from surfacial contaminating DNA. Access to the inner bone spongiosum was achieved by removing the covering bone layers of the vertebra or sternum, whereas the patella, tibia and caput of the femur or humerus were cleaved with an iron saw. After the spongiosum was taken out, extraction of nucleic acids from this "sand" like material was performed by heating at 94 degrees C during 20 min in a buffer containing essentially minor concentrations of detergent, chelating and reducing agents. The extracts were used in various Polymerase Chain Reaction (PCR) protocols to amplify different human specific DNA fragments (originating from chromosomes X and 12). From 15 out of 20 bone samples human-specific gene fragments could thus be identified.     

Non-metric cranial traits: Sex difference and age dependence

Absence of association with age and/or sex in the case of discrete traits was hypothesized by Berry & Berry (1967). The important implication that skeletal material too fragmentary for sex and age determination may also be used in discrete-trait-studies stimulated us to test this hypothesis for 49 discrete traits in 254 Amsterdam crania of known age and sex. The results were compared with those of other such studies of European skulls. No significant deviation from the hypothesis was found as far as age dependence is concerned while sex difference did occur for some traits. At the same time Corruccini's (1974) conclusion that the hypothesis fails when tested in the Terry collection was found to result largely from the fact that right and left manifestations of lateral traits were treated as being independent. The possibility of including skeletal material too fragmentary for sex and age determination in future discrete-trait-studies is still open for discussion.     

The scoring of defects of the alveolar process in human crania

Scoring methods for interalveolar and alveolar resorption, furcation involvement, fenestrations and dehiscences in the alveolar process of human skull material are presented.     

Extraction of osteocalcin from fossil bones and teeth

Osteocalcin (also called 'bone Gla-protein') was detected in fossil bovid bones ranging from 12,000 years to 13 million years old and in rodent teeth 30 million years old. Both the antigenic activity and the protein-bound Gla-residues have remained intact. The protein is indistinguishable from recent bovine osteocalcin when analyzed by HPLC using ion exchange and size exclusion columns. If sufficient amounts can be extracted and an adequate purification procedure is established, this would be the first time that amino acid sequences in a protein from fossil bones may be determined. Such sequence data could offer a new approach to the phylogenetic study of extinct taxa.