Downloaded from www.microbiologyresearch.org by IP: 54.157.140.51 On: Thu, 24 Mar 2016 19:27:20 Ancient genes of Saccharomyces cerevisiae P. Veiga-Crespo, M. Poza, M. Prieto-Alcedo and T. G. Villa Correspondence T. G. Villa mpvilla@usc.es Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, A Corun ˜ a, Spain Received 22 December 2003 Revised 17 March 2004 Accepted 24 March 2004 Amber is a plant resin mainly produced by coniferous trees that, after entrapping a variety of living beings, was subjected to a process of fossilization until it turned into yellowish, translucent stones. It is also one of the best sources of ancient DNA on which to perform studies on evolution. Here a method for the sterilization of amber that allows reliable ancient DNA extraction with no actual DNA contamination is described. Working with insects taken from amber, it was possible to amplify the ATP9, PGU1 and rRNA18S ancient genes of Saccharomyces cerevisiae corresponding to samples from the Miocene and Oligocene. After comparison of the current genes with their ancient (up to 35–40 million years) counterparts it was concluded that essential genes such as rRNA18S are highly conserved and that even normal ‘house-keeping’ genes, such as PGU1, are strikingly conserved along the millions of years that S. cerevisiae has evolved. INTRODUCTION In the last two decades, studies with fossil DNA have increased considerably as molecular biology techniques have become available in the different fields of enquiry. These studies, however, are subject to serious potential drawbacks because ancient DNA may very easily become contaminated with current DNA and because it is normally degraded. Moreover, studies are often hampered by the uniqueness of the specimens, which evidently cannot be destroyed by DNA extraction. 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 (Pa ¨a ¨bo, 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 (Pa ¨a ¨bo et al., 1989). METHODS Amber samples. The present work was carried out with two types of amber stones: (i) amber collected in the Santiago de Los Caballeros Mountains (Dominican Republic), stratigraphically dated as ~15–30 million years old, and (ii) amber collected in Poland and dated as ~40–50 million years old (Poinar, 1992). The GenBank accession numbers for the sequences reported in this paper are AY484433, AY484434, AY484435, AY484436 and AY484437. 0002-7000 G 2004 SGM Printed in Great Britain 2221 Microbiology (2004), 150, 2221–2227 DOI 10.1099/mic.0.27000-0