Scott O. Rogers

Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues

SummaryWe have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.

Extraction of DNA from plant tissues

Extraction procedures for plant DNA in general must accomplish the following. (1) The cell walls must be broken (or digested away) in order to release the cellular constituents. This is usually done by grinding the tissue in dry ice or liquid nitrogen with a mortar and pestel or a food grinder. (2) The cell membranes must be disrupted, so that the DNA is released into the extraction buffer. This is accomplished by using a detergent, usually SDS (sodium dodecyl sulfate) or CTAB (cetyltrimethylammonium bromide). (3) The DNA must be protected from the endogenous nucleases. The detergents are used for this purpose, as is EDTA (ethylenediaminetetraacetic acid). It is a chelating agent that binds magnesium ions, generally considered a necessary cofactor for most nucleases (but see note f, below). In addition, the buffer/tissue mixture is emulsified with either chloroform or phenol to denature and separate the proteins from the DNA. (4) Shearing of the DNA should be minimized. DNA in solution can be broken by exposure to turbulence (e.g., being quickly drawn through a small orifice). Typically, DNA 50–100 kb in length can be obtained without great care being taken. (5) The time between thawing of the frozen, pulverized tissue and its exposure to the extraction buffer should be minimized to avoid nucleolytic degradation of the DNA.

Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer

Ribosomal RNA genes in plants are highly variable both in copy number and in intergenic spacer (IGS) length. This variability exists not only between distantly related species, but among members of the same genus and also among members of the same population of a single species. Analysis of inheritance indicates that copy number change is rapid, occurring even among somatic cells of individual plants, and that up to 90% or more of the gene copies are superfluous. Subrepetitive sequences within the IGS appear to be changing rapidly as well. They are not only variable in sequence from one species to the next, but can vary in number between neighboring gene repeats on the chromosome. In all species examined in detail they are located in the same region of the IGS and contain sequences that can be folded into stem-loop structures flanked by a pyrimidine-rich region. It has been suggested that these subrepeats function in transcriptional enhancement, termination or processing, or in recombination events generating the high multiplicity of ribosomal genes.

Extraction of total cellular DNA from plants, algae and fungi

Historically, extraction of usable nucleic acids from plants and fungi has been difficult, in some instances notoriously so. In general, success in DNA extraction is measured by DNA yield, condition (molecular weight and color), and utility (or ease of use with restriction enzymes, polymerases, ligases, etc.). While yield is important, especially when milligram and submilligram amounts of fossil, dried or mummified tissues are used, it is less important than it once was due to the advent of PCR (polymerase chain reaction) methods. The condition of the DNA is similarly not as crucial as it once was. The utility, however, of the DNA is the paramount consideration in molecular biology manipulations. In a direct comparison [14] the DNAs (and RNAs) produced by methods employing cetyltrimethylammonium bromide (CTAB) [4, 8, 12, 15-19, 21, 23] generally exhibited lower levels of enzyme inhibition than did those by other methods [1, 3, 5, 9, 11, 13]. While the yields were lower with CTAB than for some of the other methods, the yields were still adequate for most uses in molecular biology and the condition of the DNA was above average.

Assessment of Phialophora species based on ribosomal DNA internal transcribed spacers and morphology

AbstractFifty-one isolates of Phialophora americana, P. parasitica, P. richardsiae, and P. verrucosa (deuteromycetes) were initially characterized by restriction enzyme mapping of the ribosomal DNA...

Life in ancient ice

List of Figures ix List of Tables xiii Contributors xv Acknowledgments xix CHAPTER 1: Introduction by John D. Castello and Scott O. Rogers 1 CHAPTER 2: Recommendations for Elimination of Contaminants and Authentication of Isolates in Ancient Ice Cores by Scott O. Rogers, Li-Jun Ma, Yinghao Zhao, Vincent Theraisnathan, Seung-Geuk Shin, Gang Zhang, Catherine M. Catranis, William T. Starmer, and John D. Castello 5 CHAPTER 3: Perennial Antarctic Lake Ice: A Refuge for Cyanobacteria in an Extreme Environment by John C. Priscu, Edward E. Adams, Hans W. Paerl, Christian H. Fritsen, John E. Dore, John T. Lisle, Craig F. Wolf, and Jill A. Mikucki 22 CHAPTER 4: The Growth of Prokaryotes in Antarctic Sea Ice: Implications for Ancient Ice Communities by David S. Nichols 50 CHAPTER 5: Frozen in Time: The Diatom Record in Ice Cores from Remote Drilling Sites on the Antarctic Ice Sheets by Davida E. Kellogg and Thomas B. Kellogg 69 CHAPTER 6: The Nature and Likely Sources of Biogenic Particles Found in Ancient Ice from Antarctica by Raymond Sambrotto and Lloyd Burckle 94 CHAPTER 7: Microbial Life below the Freezing Point within Permafrost by Elizaveta Rivkina, Kayastas Laurinavichyus, and David A. Gilichinsky 106 CHAPTER 8: Yeasts Isolated from Ancient Permafrost 118 by Rushaniya N. Faizutdinova, Nataliya E. Suzina, Vitalyi I. Duda, Lada E. Petrovskaya, and David A. Gilichinsky 118 CHAPTER 9: Fungi in Ancient Permafrost Sediments of the Arctic and Antarctic Regions by Nataliya E. Ivanushkina, Galina A. Kochkina, and Svetlana M. Ozerskaya 127 CHAPTER 10: Viable Phototrophs: Cyanobacteria and Green Algae from the Permafrost Darkness by Tatiana A. Vishnivetskaya, Ludmila G. Erokhina, Elena V. Spirina, Anastasia V. Shatilovich, Elena A. Vorobyova, Alexander I. Tsapin, and David A. Gilichinsky 140 CHAPTER 11: The Significance and Implications of the Discovery of Filamentous Fungi in Glacial Ice by Li-Jun Ma, Catherine M. Catranis, William T. Starmer, and Scott O. Rogers 159 CHAPTER 12: Yeasts in the Genus Rhodotorula Recovered from the Greenland Ice Sheet by William T. Starmer, Jack W. Fell, Catherine M. Catranis, Virginia Aberdeen, Li-Jun Ma, Shuang Zhou, and Scott O. Rogers 181 CHAPTER 13: Plant and Bacterial Viruses in the Greenland Ice Sheet by John D. Castello, Scott O. Rogers, James E. Smith, William T. Starmer, and Yinghao Zhao 196 CHAPTER 14: Viral Pathogens of Humans Likely to Be Preserved in Natural Ice by Dany Shoham 208 CHAPTER 15: Classification of Bacteria from Polar and Nonpolar Glacial Ice by Brent C. Christner, Ellen Mosley-Thompson, Lonnie G. Thompson, and John N. Reeve 227 CHAPTER 16: Common Features of Microorganisms in Ancient Layers of the Antarctic Ice Sheet by S.S. Abyzov, M.N. Poglazova, J.N. Mitskevich, and M.V. Ivanov 240 CHAPTER 17: Comparative Biological Analyses of Accretion Ice from Subglacial Lake Vostok by Robin Bell, Michael Studinger, Anahita Tikku, and John D. Castello 251 CHAPTER 18: Search for Microbes and Biogenic Compounds in Polar Ice Using Fluorescence by Ryan Bay, Nathan Bramall, and P. Buford Price 268 CHAPTER 19: Living Cells in Permafrost as Models for Astrobiology Research by Elena A. Vorobyova, V.S. Soina, A.G. Mamukelashvili, A. Bolshakova, I.V. Yaminsky, and A.L. Mulyukin 277 CHAPTER 20: A Synopsis of the Past, an Evaluation of the Current, and a Glance toward the Future by John D. Castello and Scott O. Rogers 289 Index 301

Variation in the ribosomal RNA genes among individuals of Vicia faba.

SummaryLength heterogeneity in the ribosomal repeat of Vicia faba is due to the presence of variable numbers of a 325 bp subrepetitive element within the nontranscribed spacer region. The distribution of size classes among 88 individuals within a population was investigated by blot-hybridization. We find that individual plants can exhibit more than 20 size classes and that hybridization patterns are highly diverse from individual to individual, more so than for any species so far investigated. In contrast, no such differences are observed in patterns for different tissues from a single plant or from parental to F1 generation. Some changes were observed in the F2 generation. We conclude that unequal recombination can give rise to the diversity that we observe for the V. faba rDNA repeats.

Evidence of Influenza A Virus RNA in Siberian Lake Ice

ABSTRACT Influenza A virus infects a large proportion of the human population annually, sometimes leading to the deaths of millions. The biotic cycles of infection are well characterized in the literature, including in studies of populations of humans, poultry, swine, and migratory waterfowl. However, there are few studies of abiotic reservoirs for this virus. Here, we report the preservation of influenza A virus genes in ice and water from high-latitude lakes that are visited by large numbers of migratory birds. The lakes are along the migratory flight paths of birds flying into Asia, North America, Europe, and Africa. The data suggest that influenza A virus, deposited as the birds begin their autumn migration, can be preserved in lake ice. As birds return in the spring, the ice melts, releasing the viruses. Therefore, temporal gene flow is facilitated between the viruses shed during the previous year and the viruses newly acquired by birds during winter months spent in the south. Above the Arctic Circle, the cycles of entrapment in the ice and release by melting can be variable in length, because some ice persists for several years, decades, or longer. This type of temporal gene flow might be a feature common to viruses that can survive entrapment in environmental ice and snow.

Detection of tomato mosaic tobamovirus RNA in ancient glacial ice

Abstract Tomato mosaic tobamovirus is a very stable plant virus with a wide host range, which has been detected in plants, soil, water, and clouds. Because of its stability and prevalence in the environment, we hypothesized that it might be preserved in ancient ice. We detected tomato mosaic tobamovirus RNA by reverse-transcription polymerase chain reaction amplification in glacial ice subcores <500 to approximately 140,000 years old from drill sites in Greenland. Subcores that contained multiple tomato mosaic tobamovirus genotypes suggest diverse atmospheric origins of the virus, whereas those containing tomato mosaic tobamovirus sequences nearly identical to contemporary ones suggest that recent tomato mosaic tobamovirus populations have an extended age structure. Detection of tomato mosaic tobamovirus in ice raises the possibilities that stable viruses of humans and other hosts might be preserved there, and that entrapped ancient viable viruses may be continually or intermittently released into the modern environment.

Spatial distribution of dark septate endophytes in a confined forest plot

In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3 x 3 m plot in a spruce stand ( Picea abies ). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR-PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m 2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.