Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, A Coruña, Spain
Correspondence
T. G. Villa
mpvilla{at}usc.es
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ABSTRACT |
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INTRODUCTION |
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Ancient DNA although more resistant than RNA is subject to the degradative action of different exogenous agents (Cano, 1996; Henwood, 1993
; Poinar, 1994
, 2002
; Service, 1996
), water being the most important one, plus the action of free radicals and UV light; base conversion phenomena due to hydrolytic deamination are also common.
Amber is a mixture of terpenes, organic acids, alcohols and sugars secreted by higher plants (mostly conifers) and has been subject to polymerization and fossilization for millions of years, this affording what is known as amber or retinite. During this process, micro-organisms, pollen, or insects are entrapped and remain in a frozen state up until now, as widely reported in the literature (Cano et al., 1992; Cano & Poinar, 1993
; DeSalle et al., 1993
; Schultz, 2000
). The relationships between insects and micro-organisms have been also addressed (Wier et al., 2002
).
It is possible to differentiate the origin of amber stones according to their resin composition (Lambert & Poinar, 2002). Thus, conifers are the oldest amber-producing fossils found in Central Europe, whereas members of the Leguminosae produced the amber from Mexico and the Dominican Republic in the Americas (Poinar & Brown, 2002
). Recently, new techniques based on NMR have allowed the unambiguous geographic characterization of amber samples (Lambert & Poinar, 2002
). The high level of sugars in amber provides a hyperosmotic medium and hence induces sample dehydration. The development of specific procedures for molecular biology such as PCR has to a large extent facilitated studies with ancient DNA in a field already known as molecular palaeontology. As recovered from amber, ancient DNA is already degraded to a certain extent but this, in turn, facilitates the detection of contamination with current DNA because it is difficult to obtain long-chain amplicons (Handt et al., 1994
). Jumping-PCR may represent another drawback in this type of study (DeSalle et al., 1993
; Handt et al., 1994
; Taylor, 1996
). The inhibitory action of some amber compounds, such as tannic substances, may be overcome by the addition of bovine serum albumin (Pääbo, 1990
). Initial studies addressed the direct cloning of fossil DNA, but this soon fell into disuse and was replaced by PCR, thus avoiding the undesired DNA repair that occurs in direct cloning (Pääbo et al., 1989
).
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METHODS |
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Sterilization and opening of the amber.
The accomplishment of surface sterilization and the opening of the amber were generally as recommended by Lambert et al. (1998). Sterilization was accomplished by a thorough surface wash with sterile water. Then, the samples were maintained in 2 % glutaraldehyde (Merck) at 40 °C for 48 h and subjected to periodic ultrasonic treatment (30 min). Following this, the stones were incubated in the presence of 10 % CaCl2 (Merck) at 25 °C for 24 h and also ultrasonicated. Finally, they were incubated in 70 % ethanol (Merck) at 22 °C for 24 h and again subjected to ultrasonication. Once frozen in liquid N2, the stones were ground in a sterile mortar. The powder was resuspended in liquid brain heart infusion (BHI) broth (Pronadisa), aliquotted and stored at 80 °C until use.
DNA extraction.
DNA from S. cerevisiae was prepared using the Wizard Genomic DNA Purification System (Promega). DNA from Chlamydomonas reinhardtii was prepared as indicated at www.biology.duke.edu/chlamy/methods/dna.html and DNA from Pinus edulis was extracted with the DNeasy Plant Mini Kit (Qiagen). Fossil DNA from amber stones was prepared using the Ancient DNA Kit (GeneClean; Bio 101). The powdered stones were resuspended in 10 µl 0·5 M EDTA (Merck), 200 µl 10 % SDS (Merck) and 200 µl Proteinase K (20 mg ml1; Fluka) and kept at 37 °C for 15 h. After this, the kit instructions were followed.
Oligonucleotides and amplification techniques.
The oligonucleotides used to amplify ancient DNA are listed in Table 1. The amplification reaction mixture was as follows: 1 U Taq polymerase (Takara Shuto), 2 ng BSA ml1 (Promega), 0·5 µM each oligonucleotide (Invitrogen), 2 mM MgCl2 (Takara Shuto), 0·2 mM dNTPs mix (Takara Shuto), 50 ng fossil DNA, Taq polymerase buffer (Takara Shuto) and deionized sterile water to a final volume of 50 µl. When the ODA4 gene was to be amplified, the MgCl2 concentration was 1·5 mM. The reaction was carried out using a Robocycler Gradient 96 (Stratagene) and the programme was: 1 cycle of 5 min at 94 °C, 35 cycles of 1 min at 94 °C, 30 s at X °C (where X depended on the oligonucleotide pair) and 1 min at 72 °C. The process was completed with one cycle of 10 min at 72 °C. For each amplification cycle, the appropriate controls were conducted using each primer alone.
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Controls of contamination.
Working surfaces were periodically treated with ethanol (70 %) and before and after each work session they were treated with 10 % sodium hypochlorite (Merck). All culture media were maintained for 15 days at 21, 30 and 37 °C before use. All solutions used to sterilize the amber stone surfaces were previously filtered through 0·22 µm membranes (three times) that had been treated against microbial contamination. Before stone grinding, the samples were incubated in liquid BHI medium and subjected to the same temperature cycle to discard any possible contamination. After grinding, microbial contamination was investigated again. The solutions used in DNA extraction and PCR were periodically controlled for fortuitous contamination. Throughout the process, particular care was taken to not use glassware or equipment that had previously been in contact with current DNA.
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RESULTS AND DISCUSSION |
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Six powdered aliquots were prepared from each amber stone and one of them was used to test the sterilization status by inoculation in both BHI broth and solid medium. Although Austin et al. (1997) have reported different methods suitable for amber DNA extraction, in our hands the commercial kit for fossil DNA extraction was very effective, affording samples of fair purity and also avoiding problems during amplification. Extraction efficiency was about 20 ng DNA µl1 and purity (A260/A280) was 1·6; although both these parameters varied from stone to stone, it was observed that younger samples yielded more DNA than older ones.
The approaches to sequence validity must be established a priori and must not be changed during the course of the work. In this case, the following criteria were adopted: (i) DNA was extracted only from stones that had passed all the contamination checks; (ii) samples from the same stones had to show similar results; (iii) ancestral sequences had to show homology with current ones and had to display phylogenetic coherence; and (iv) large DNA fragments (longer than 1 kb) had to be discarded to avoid either sample contamination with current DNA or jumping-PCR phenomena.
Based on direct observation of the stones employed in the present work, the entrapped insects strongly resembled ants. As happens in today's insects, yeast cells are mainly associated with the legs and possibly the lower parts of the body, hence being transported from plant to plant. Other yeast strains detected here were probably indigenous to the plant. All these yeast strains plus the insects were finally entrapped and subjected to amberization.
Genes essential for the organisms were chosen as a means to ensure pressure through natural selection, so that the amplified DNA could be easily recognized in comparison with present-day genes. It was not possible to amplify whole genes, except for ATP9 from S. cerevisiae. For the remaining genes, intra-ORF oligonucleotides had to be synthesized. Thus, for the rRNA18S gene from S. cerevisiae, the oligonucleotides flanked the coding region 1420 bp. For the ATP5 gene of this yeast, two oligonucleotide couples were synthesized flanking coding regions from nt 1 to 330 (fragment I) and from nt 331 to the end of the coding region (fragment II). For the PGU1 gene of S. cerevisiae, three oligonucleotide couples were used: fragment I (from nt 1 to 542 of the coding region), fragment II (from nt 192 to the end of the coding region) and fragment III (an internal sequence that goes from nt 192 to 542 of the coding region). The oligonucleotides for the RCBL gene (large subunit of RuBisCo) of Pinus edulis flanked nucleotides 1500 of the coding region and the oligonucleotides of the rRNA16S gene of Wolbachia sp. flanked nucleotides 5011000 of the coding region. In the case of the gene ODA4 from C. reinhardtii, the oligonucleotides were synthesized to specifically amplify the region encoding the GK4 region of the dynein heavy -chain. This is a highly conserved and small sequence (Mitchell & Brown, 1994
). The oligonucleotides reported by Kocher et al. (1989)
for mammal cytochrome c were used. All the oligonucleotides described above are shown in Table 1
.
It was possible to amplify the complete S. cerevisiae ATP9 mitochondrial gene (230 bp) in samples both from the Dominican Republic and from Poland. It was not possible, however, to amplify the whole PGU1 gene but, instead, fragments I, II and III in younger samples; older samples only allowed the amplification of fragment III. Regarding the rRNA18S genes from S. cerevisiae and RCBL from Pinus edulis (data not shown), it was only possible to amplify 500 bp and always in the youngest samples (i.e. amber from the Dominican Republic). It was not possible to amplify the nuclear ATP5 gene from S. cerevisiae, the GK region of ODA4 from C. reinhardtii, or the mammalian CYTB and rRNA16S from Wolbachia. Although we cannot categorically rule out the (remote) possibility of contamination with current fungal DNA, we are confident that the controls introduced along the sterilization process were sufficient for us to be able to disregard such a notion.
Consensus sequences were elaborated with all these ancient sequences (GenBank accession nos: AY484433, AY484434, AY484435, AY484436 and AY484437). At least six different amplicons had to be collected to obtain good sequence resolution. Thus, it was possible to establish a consensus sequence for the ATP9 gene of S. cerevisiae for both the Miocene and Oligocene. Length variation in the sequences was not taken into account because the end portions of the sequences were completely discarded, not only since they showed a high variation index but also because they showed a high sequence indetermination index. Next, the consensus sequences of ancient genes were compared with those of current genes to see the degree of similarity and coherence (Fig. 1). In this sense, ancient ATP9 from the Miocene exhibited 59 % similarity as compared to the current gene whereas the fragment from the Oligocene showed 40 % similarity. Upon comparing both ancient sequences, a similarity of 74 % was observed (Table 2a
). In the case of the ancient PGU1 gene, it was possible to rebuild almost the complete gene sequence from the Miocene samples by assembling fragments I, II and III. The sequences showed 69 % homology with the present-day one (Table 2b
). Fragment III from the Oligocene had 32 and 39 % homology with the current one and Miocene one, respectively (Table 2c
). The consensus sequences for the Miocene rRNA18S gene from S. cerevisiae showed 65 % homology with the present-day one (Table 2d
).
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Traditional studies propose either 2 % genomic variation per million years for two lineages separated by less than 10 million years, or 1x108 nucleotide substitution per nucleotide site per year for mitochondrial DNA (Avise, 1994). Thus, the tendency in the specific mutation rates for these extranuclear genomes is exponential, after which a plateau is reached (the genome is saturated in the variable substitution sites). These assumptions apply mainly for organisms with long doubling times, although they also seem to apply for S. cerevisiae, which has a short generation time.
The highest intersequence divergences occurred during the last 10 million years and the highest homology would lie among the older sequences. This phenomenon may be ascribed to the fact that the amber samples were from different geoclimatic regions of the planet. It is now quite clear that work done with fossil DNA extracted from amber, such as described here, may shed light onto the precise aspects involved in the genetic differentiation of species.
Future research will include an expansion of the number of genes studied in S. cerevisiae both nuclear and mitochondrial in more closely related (both in time and space) amber samples.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 December 2003;
revised 17 March 2004;
accepted 24 March 2004.
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