Raul J. Cano and Monica K. Borucki have discovered and have actually revived (brought back to life!) over 1,000 types of bacteria and other microorganisms. Some of the life-forms date as far back as 135 million years which was the time of the dinosaurs. Can DNA survive that long? Many point to the plain physics of degrading DNA over time and state that organisms cannot survive for millions of years without having the bases of the DNA, which constitutes the genetic code, degrade to such an extent that the organisms would no longer be viable. So, is the presence of amber preserved DNA that is still capable of producing viable bacteria and other microorganisms, evidence that the specimen is in fact very young? That is hard to say. There are many who dismiss all claims of ancient bacteria as modern contamination. Others, however seem to dismiss the problems that the effect of time has on DNA and say, 'It must have survived, because here it is'. Neither group entertains the thought that the time factor might be off several orders of magnitude. Of course the story of Noah in the Bible suggests that these organisms were buried during the global flood, less than 6000 years ago. The young age of the specimens would allow the DNA to still have its original code largely unaffected by time. For an interesting introduction to this topic read the news article "Ancient Bacteria Brought Back to Life" by R. Monastersky in Science News Volume 147, Number 20, May 20, 1995, p. 308. Another interesting article: "Prehistoric bacteria revived from buried salt" by J. Travis in Science News Volume 155, June 12, 1999, p. 373. In this article, J. Travis has interviewed such men as William D. Rosenzweig and Russell H. Vreeland of Penn. University who have now announced to have isolated and revived bacteria from salt deposits that is 250 million years old. Also in the paper, a researcher is mentioned, who is said to have been ahead of his time claimed, back in the 1960s, to have revived bacillus and other bacteria from salt deposits more than 500 million years old. So now we have bacterial spores lasting for 250 million years and maybe as long as 500 million years. Is that possible? I especially like what Melanie R. Mormile from the Pacific Northwest National Laboratory in Richland, Wash. said: (I must tell you that Melanie has herself reported salt-derived microbes of at least 97 million years old in age) "Whenever anyone claims they have revived organisms that are millions of years old, she says, you've got to sit back and go, 'Wow, that's incredible. How can that be?' " I think this is amazing! Because the long ages supported by the evolutionary process is not questioned, researchers are forced to try to come up with unlikely possibilities. They are forced to acknowledge that the DNA must have survived for hundreds of millions of years so now they have to come up with a reason why they are present! You might think that this debate over viable fossil DNA will persist for some time since there is yet no answer to the problem; However, this same kind of debate has occurred before. In the past, the fact that fossils still had identifiable amino acids after millions of years of being buried, and it had created quite a stir in the 1950s and 1960s. But since a reasonable answer that allows amino acids to still exist, after millions of years, was not forth coming, all research on the problem eventually stopped. Contamination and the actual age of the samples are the two main alternate explanations to the long age explanation that the fossil DNA has been buried for an extremely long period of time. The contamination aspect of the Lab work is a very real possibility and thus it is one of the major addressed issues facing any project involving PCR amplification of Fossilized DNA. However, these workers are being extremely cautious in their work and I might not expect that all these groups would have contamination as their major product. . . Although it is possible. One interesting possibility is that if these samples are truly much younger then expected, as would be expected in the Creationary model, then there should be more viable DNA then expected in the samples. That would make the contamination problem of PCR to be less of a problem. Of course, evolutionists would not expect their samples to be a younger. They would expect the contamination problem to be almost insurmountable. If DNA truly does not last for millions of years, as is expected from calculations, it would be expected that all PCR results would be that of contamination. If you look at the reference (Halobacteria: the evidence for longevity. Grant WD, et. al. ) located below on this web page I think you will find this paper especially interesting! They said that they might predict that the DNA code, when comparing similar bacteria found in the ancient evaporite with those in the surface environment, that a difference would be noted. Instead, they find essentially no difference. No difference after hundreds of millions of years? Either evolution has not occurred over millions of years or the two alternate explanations of contamination and sample age could be a very real possibility. Very interesting. It seems that there should be some change after all that time. The two heterogeneous copies of the 16S rRNA gene show, according to current thinking, the possibility of evolutionary events, rearrangements of DNA, and or gene duplication. Etc. How would this non-difference between the two populations of bacteria, separated by time, affect our views on the Biological clock rates? (The changing of DNA over time.) How might it affect our understanding of how viable the theory itself is if we keep coming up with little or no change after as much as 250 million years? We will have to see if this absence of a trend, having little or no change in the genetic code, continues. It is postulated that different species change (Biological clock rate) at different rate. The Halobacteria example might be considered to be an example where the evolutionary rate is frozen. I do not know enough about Biological clock rates to know if this is reasonable within evolutionary theory. Anyone out there know? Presently, the likelihood is very good that either we are looking at contaminants or these samples are not as old as is being reported. If researchers continue to keep coming up with little or no change after long periods of time suggesting an absence of a trend, having little or no change in the genetic code; Then the possibility that the ages of the fossilized samples must be questioned. Some of the journal articles are cited below with abstracts if available.
DNA was extracted from the fossil termite Mastotermes electrodominicus preserved in Oligo-Miocene amber (25 million to 30 million years old). Fragments of mitochondrial [16S ribosomal DNA (rDNA)] and nuclear (18S rDNA) genes were amplified by polymerase chain reaction. Phylogenetic analysis of fossil and extant 18S rDNA confirmed morphological cladistic analyses of living dictyopterans (termites, cockroaches, and mantids). The fossil termite shares several sequence attributes with Mastotermes darwiniensis. Addition of this fossil to living-species phylogeny is required to substantiate Mastotermes monophyly and affects molecular phylogenetic hypotheses of termites in this, the oldest DNA yet characterized.
DNA has been successfully isolated from both fossilized plant and animal tissues. The oldest material, dated as 25-40 million years old (Tertiary), was obtained from amber-entombed bees and termites. Tissues from both these insects yielded DNA of good quality, which could be amplified by the polymerase chain reaction (PCR) and subsequently sequenced, including the genes encoding 18S ribosomal RNA and 16S rRNA. We report here the extraction of DNA from a 120-135-million-year-old weevil (Nemonychidae, Coleoptera) found in Lebanese amber, PCR amplification of segments of the 18S rRNA gene and the internal transcribed spacer, and the corresponding nucleotide sequences of their 315- and 226-base-pair fragments, respectively. These sequences were used for preliminary phylogenetic analysis of the nemonychid's sequence with three extant coleopterans: Lecontellus pinicola (Nemonychidae), Hypera brunneipennis (Curculionidae) and the mealworm Tenebrio molitor (Tenebrionidae), and two extant dipterans: the fruitfly Drosophila melanogaster (Drosophilidae) and mosquito Aedes albopictus (Culicidae) for the purpose of ascertaining the origin of the extracted and amplified DNA. The results revealed that the PCR-amplified material is that of the extinct nemonychid weevil. This represents the oldest fossil DNA ever extracted and sequenced, extending by 80 million years the age of any previously reported DNA.
We describe a simple process for extraction of DNA from amber-entombed fossils and museum specimens that is suitable for enzymatic amplification by PCR. Five to ten milligrams of the macerated specimen were mixed in 300 microliters of silica matrix and shaken at 55 degrees C for 1 h in a sterile, screw-capped microcentrifuge tube. After incubation, the silica matrix was transferred to the upper chamber of a Spin Filter, centrifuged at maximum speed for 1 min and then washed twice with 500 microliters of wash solution and the DNA eluted with 50 microliters of TE buffer. The elute was used as template for PCR, and the results were evaluated by electrophoresis and nucleotide sequence analysis. All samples tested yielded positive results, which were subsequently verified by sequence analysis. It appears, at least in our hands, that the procedure described here is a rapid and efficient way of obtaining small amounts of DNA for PCR in museum and fossilized specimens.
DNA from 30-million-year-old amber preserved termites (Mastotermes electrodominicus) was PCR amplified with nuclear ribosomal RNA small subunit primers and cloned into the TA vector (INVITROGEN). We obtained several classes of recombinant clones as a result. Authentic Mastotermes electrodominicus clones were identified. The source of other classes of clones was identified as contaminants of the ancient DNA template. Several of the clones appeared to be chimeric in structure with half of the clone identical to the termite sequence and the other half identical to contaminant sequences. The phenomenon of PCR jumping was identified as a possible source for the chimeric clones.
We report here the isolation of DNA from abdominal tissue of four extinct stingless bees (Proplebeia dominicana) in Dominican amber, PCR amplification of a 546-bp fragment of the 16S rRNA gene from Bacillus spp., and their corresponding nucleotide sequences. These sequences were used in basic local alignment search tool searches of nonredundant nucleic acid data bases, and the highest scores were obtained with 16S rRNA sequences from Bacillus spp. Phylogenetic inference analysis by the maximum-likelihood method revealed close phylogenetic relationships of the four presumed ancient Bacillus sequences with Bacillus pumilus, B. firmus, B. subtilis, and B. circulans. These four extant Bacillus spp. are commonly isolated from abdominal tissue of stingless bees. The close phylogenetic association of the extracted DNA sequences with these bee colonizers suggests that a similar bee-Bacillus association existed in the extinct species P. dominicana.
A survey of the major fossiliferous amber deposits is provided, including ages and various categories of life forms reported from each. The frequency of occurrence of the major groups of plants and animals in these amber deposits is also given. Thus far, DNA from four insect and one plant species has been extracted from amber fossils. In the case of the stingless bee in Dominican amber, evidence of reproducibility is provided, since two independent laboratories isolated DNA from six or more different specimens of the same insect. Amber sources for DNA studies are listed together with their advantages and disadvantages. The important points are the availability of desired pieces, the proper identification of the fossil, verification of the amber deposit, the cost involved, and the feasibility of causing damage to the specimen. The availability of several types of amber (Mexican, Dominican, Baltic, Chinese, Canadian, Siberian and Lebanese) at four major sources (academic collections, commercial dealers, private collections and amber mines) is discussed. The scientific implications of obtaining DNA from amber inclusions are presented.
The utility of DNA sequence characters from fossil specimens is examined from a phylogenetic perspective. Four ways that fossil characters can alter phylogenetic hypotheses are discussed. Two empirical examples and a third hypothetical example concerning amber-preserved insects are presented to illustrate these phenomena. Fossil DNA sequences as characters will be affected by the problem of missing data and missing taxa. In general, cladogram accuracy will be more greatly affected by missing taxa and cladogram resolution will be affected more acutely by missing data. Due to these points, an examination of the importance of the phylogenetic question being addressed, the utility of the fossil DNA sequences and the rarity of the fossil should be considered before damage of a fossil is undertaken.
Ancient DNA has been discovered in many types of preserved biological material, including bones, mummies, museum skins, insects in amber and plant fossils, and has become an important research tool in disciplines as diverse as archaeology, conservation biology and forensic science. In archaeology, ancient DNA can contribute both to the interpretation of individual sites and to the development of hypotheses about past populations. Site interpretation is aided by DNA-based sex typing of fragmentary human bones, and by the use of genetic techniques to assess the degree of kinship between the remains of different individuals. On a broader scale, population migrations can be traced by studying genetic markers in ancient DNA, as in recent studies of the colonisation of the Pacific islands, while ancient DNA in preserved plant remains can provide information on the development of agriculture.
The verification of DNA sequences obtained from very old tissue sources as indeed ancient is a major point of discussion in the ancient DNA field. Proper controls and the use of the phylogenetic approach are the general methods employed for verification of the ancient DNA. Most studies have reported the recovery of extremely small amounts of nucleic acids which are sheared into rather small fragments. In addition, problems such as 'PCR jumping' can produce spurious sequence information. These observations suggest that random amplification techniques and the development of primers for highly informative but short target regions are essential for the further development of the ancient DNA field.
A bacterial spore was revived, cultured, and identified from the abdominal contents of extinct bees preserved for 25 to 40 million years in buried Dominican amber. Rigorous surface decontamination of the amber and aseptic procedures were used during the recovery of the bacterium. Several lines of evidence indicated that the isolated bacterium was of ancient origin and not an extant contaminant. The characteristic enzymatic, biochemical, and 16S ribosomal DNA profiles indicated that the ancient bacterium is most closely related to extant Bacillus sphaericus. Comment in: Science 1995 May 19;268(5213):977
Much of what we know about extinct organisms comes from traits that are not preserved in the fossil record. Until recently, morphological analysis was the only tool available for scientists to determine relationships for extinct fossil organisms. We now know that "ancient' DNA can be preserved in the remains of extinct organisms. By targeting specific gene sequences, it may be possible to deduce biochemical characteristics and through sequence comparisons, to estimate the extent of evolutionary divergence. By comparing the amount and type of these changes, one could estimate how quickly some DNA 'evolves' relative to other segments, or which genes have the most flexibility or are more conserved over time. The compilation of these data would yield greater understanding of the physiology of extinct organisms and provide a much clearer picture of genetic change over time, and the mechanics behind 'evolution'.
The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.
Apparently ancient DNA has been reported from amber-preserved insects many millions of years old. Rigorous attempts to reproduce these DNA sequences from amber- and copal-preserved bees and flies have failed to detect any authentic ancient insect DNA. Lack of reproducibility suggests that DNA does not survive over millions of years even in amber, the most promising of fossil environments.
We have developed a model based on the analyses of modern and Pleistocene eggshells and mammalian bones which can be used to understand the preservation of amino acids and other important biomolecules such as DNA in fossil specimens. The model is based on the following series of diagenetic reactions and processes involving amino acids: the hydrolysis of proteins and the subsequent loss of hydrolysis products from the fossil matrix with increasing geologic age; the racemization of amino acids which produces totally racemized amino acids in 10(5)-10(6) years in most environments on the Earth; the introduction of contaminants into the fossil that lowers the enantiomeric (D:L) ratios produced via racemization; and the condensation reactions between amino acids, as well as other compounds with primary amino groups, and sugars which yield humic acid-like polymers. This model was used to evaluate whether useful amino acid and DNA sequence information is preserved in a variety of human, amber-entombed insect and dinosaur specimens. Most skeletal remains of evolutionary interest with respect to the origin of modern humans are unlikely to preserve useful biomolecular information although those from high latitude sites may be an exception. Amber-entombed insects contain well-preserved unracemized amino acids, apparently because of the anhydrous nature of the amber matrix, and thus may contain DNA fragments which have retained meaningful genetic information. Dinosaur specimens contain mainly exogenous amino acids, although traces of endogenous amino acids may be present in some cases. Future ancient biomolecule research which takes advantage of new methologies involving, for example, humic acid cleaving reagents and microchip-based DNA-protein detection and sequencing, along with investigations of very slow biomolecule diagenetic reactions such as the racemization of isoleucine at the beta-carbon, will lead to further enhancements of our understanding of biomolecule preservation in the fossil record.
Two bacterial isolates, designated AMG-D1T and AMG-D2, were recovered from 25-35-million-year-old Dominican amber. AMG-D1T and AMG-D2 biochemically most closely resemble Staphylococcus xylosus; they differ physiologically from other staphylococci. Fatty acid analysis and comparisons with extensive databases were unable to show relatedness to any specific taxon. Moreover, AMG-D1T and AMG-D2 contain tuberculostearic acid and meso-diaminopimelic acid, characteristic of the G + C-rich coryneform bacteria, as opposed to L-lysine characteristic of staphylococci. AMG-D1T and AMG-D2 have a G + C ratio of 35 mol%. Phylogenetic analysis with the 16S rRNA gene indicated that AMG-D1T and AMG-D2 were most closely related to Staphylococcus equorum, S. xylosus, Staphylococcus saprophyticus and other novobiocin-resistant staphylococci. Stringent DNA-DNA hybridization studies with AMG-D1T revealed similarities of 38% with S. equorum, 23% with S. xylosus and 6% with S. saprophyticus. The results indicate that AMG-D1T and AMG-D2 represent a novel species, which was named Staphylococcus succinus sp. nov. The type strain of the new species is AMG-D1 (ATCC 700337).
We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.
Abstract Claims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25-40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p58.html
Subterranean salt deposits are the remains of ancient hypersaline waters that presumably supported dense populations of halophilic microorganisms including representatives of the haloarchaea (halobacteria). Ancient subterranean salt deposits (evaporites) are common throughout the world, and the majority sampled to date appear to support diverse populations of halobacteria. The inaccessibility of deep subsurface deposits, and the special requirements of these organisms for survival, make contamination by halobacteria from surface sites unlikely. It is conceivable that these subterranean halobacteria are autochthonous, presumably relict populations derived from ancient hypersaline seas that have been revived from a state of dormancy. One would predict that halobacteria that have been insulated and isolated inside ancient evaporites would be different from comparable bacteria from surface environments, and that it might be possible to use a molecular chronometer to establish if the evolutionary position of the subsurface isolates correlated with the geological age of the evaporite. Extensive comparisons have been made between the 16S rRNA genes of surface and subsurface halobacteria without showing any conclusive differences between the two groups. A further phylogenetic comparison exploits an unusual feature of one particular group of halobacteria that possess at least two heterogeneous copies of the 16S rRNA gene, the sequences of which may have been converging or diverging over geological time. However, results to date have yet to show any gene sequence differences between surface and evaporite-derived halobacteria that might arguably be an indication of long-term dormancy. I find this paper especially interesting! They said that they might predict that the DNA code, when comparing similar bacteria found in the ancient evaporite with those in the surface environment, that a difference would be noted. Instead, they find essentially no difference. No difference after hundreds of millions of years? Either evolution has not occurred over millions of years or the two alternate explanations of contamination and sample age could be a very real possibility. Very interesting. It seems that there should be some change after all that time. The two heterogeneous copies of the 16S rRNA gene show, according to current thinking, the possibility of evolutionary events, rearrangements of DNA, and or gene duplication. Etc. How would this non-difference between the two populations of bacteria, separated by time, affect our views on the Biological clock rates? (The changing of DNA over time.) How might it affect our understanding of how viable the theory itself is if we keep coming up with little or no change after as much as 250 million years? We will have to see if this absence of a trend, having little or no change in the genetic code, continues. It is postulated that different species change (Biological clock rate) at different rate. The Halobacteria example might be considered to be an example where the evolutionary rate is frozen. I do not know enough about Biological clock rates to know if this is reasonable within evolutionary theory. Anyone out there know? Presently, the likelihood is very good that either we are looking at contaminants or these samples are not as old as is being reported. If researchers continue to keep coming up with little or no change after long periods of time suggesting an absence of a trend, having little or no change in the genetic code; Then the possibility that the ages of the fossilized samples must be questioned.
The Waste Isolation Pilot Plant (WIPP) is a salt mine constructed 650 meters below the ground surface by the United States Department of Energy. The facility will be used for permanent disposal of transuranic wastes. This underground repository has been constructed in the geologically stable Permian age Salado salt formation. Of the wastes to be placed into the facility, 85% will be biodegradable cellulose. A 3-year survey of the bacterial populations existing within the facility was conducted. Bacterial populations were found to be heterogeneously distributed throughout the mine. Populations in some mine areas reached as high as 1.0 x 10(4) colony-forming units per gram of NaCl. The heterogeneous distribution of bacteria within the mine did not follow any recognizable pattern related to either age of the workings or to human activity. A biochemical comparison between ten known species of halophilic bacteria, and strains isolated from both the mine and nearby surface hypersaline lakes, showed the presence of extreme halophiles with wide biochemical diversity, some of which could prove to represent previously undescribed groups. The halophilic bacteria isolated from the mine were found to degrade cellulose and a wide variety of other carbon compounds. When exposed to two types of common laboratory paper, the cellulose-degrading halophiles attached to the substrate within 30 minutes of inoculation. Cultures enriched directly from a brine seep in the mine easily destroyed both papers and produced detectable amounts of oxalacetic and pyruvic acids. The combination of heterogeneity in the distribution of organisms, the presence of a physiologically diverse community, and the relatively slow metabolism of cellulose may explain several long-standing debates about the existence of microorganisms in ancient underground salt formations. 
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