1. Characterization of microorganisms and nucleic acids from ancient ice.

2. Characterization of DNA from ancient samples.

3. Investigations of introns in fungi and fish.

4. Extraction and preservation of nucleic acids.

5. Influenza A in lake ice.

6. Plant shoot apical development.

7. Phylogenetics of fungi.

8. Novel evolutionary strategies of microorganisms.






PROJECTS

1 (O). Biodiversity of microorganisms in ancient polar ice: (V. Theraisnathan, S. Shin, G. Zhang, L. Harris, Zeynep Koçer, Ram Veerapaneni, Tom D'Elia, BGSU; J.D. Castello, C.J.K. Wang, C. Catranis, Li-Jun Ma, Yinghau Zhao, Shuang Zhou, and Hua Fan, SUNY ESF; W.T. Starmer, J. Smith, Syracuse University): We have isolated ancient fungi, bacteria and viruses from ice collected in Greenland and Antarctica that is from 100 to over 250,000 years old. Older ice (between 500,000 and 1 million years old) and more extensive isolations from both Arctic and Antarctic regions are now underway. Some plant and animal (including human) pathogens are of special interest in this project. The organisms have been cultured and/or subjected to DNA sequence analyses for identification. To date, we have over 500 cultured microorganisms, and over 100 DNA sequences from these organisms. Some of the organisms found appear to be new species, while others are common in today's environment. We plan to use the microorganism profiles to examine environmental and evolutionary trends and episodes. We have also assayed ice from subarctic lakes for the presence of viruses, such as influenza A. We organized a workshop to be held in July of 2001 on life in ancient ice, the proceedings to be published by Princeton University Press in 2005. Funded by NSF, 9/1998-8/2001.

2. (O). Identification of fungi in hospital patient blood using molecular methods (F. Sadiq). Fungal infections in hospital patients has risen dramatically over the past two decades. Unfortunately, the primary clinical method to identify fungi is culturing. However, the vast majority of fungi cannot be cultured using current methods (estimates range from 83% to more than 99% that cannot be cultured). We have obtained blood samples from 200 patients that were determined to be negative for fungi in clinical culture tests. To date, we have found in 4% of these samples, including the blood of one patient who died from an undetermined infection. We use a polymerase chain reaction method to amplify a part of the fungal genomes that are species-specific. The method is rapid, in that results can be obtained in less than one day in a clinical setting. The method has the potential to save livs since it can identify pathogenic fungi so that effective treatments can be provided to the patients.

3. (O). Comprenehsive study of microbes in Lake Vostok accretion ice (T. D’Elia, V. Theraisnathan, R. Veerapaneni, G. Zhang, Z. Koçer, BGSU; S. Bulat, St. Petersburg, Russia; J-R. Petit, D. Prieur, Grenoble, France). We are assaying ice from the accretion ice above Lake Vostok, Antarctica. This is a huge lake, approximately the size of Lake Ontario, North America, which is completely covered by 3600 m of glacial ice. As the glacier moves over the lake, water from the lake freezes (or accretes) to the bottom of the glacier. This process essentially samples lake water and its inclusions. By studying the microbes in the ice we will draw conclusions about the characteristics of this lake. Funded by NSF, 2005-2007.

4. (O). Surveillance of Siberian lakes for Influenza A virus (G. Zhang, Z. Koçer, BGSU; D. Causey, U. of Alaska, Anchorage; D. Gilichinsky, S. Davydov, Moscow, Russia; Dany Shoham, Bet Dagan, Israel). We are searching for the presence of strains of influenza A virus in several Siberian lakes that are frequented by migratory waterfowl. The biotic reservoirs for these viruses are known to be birds and mammals. However, it is thought that abiotic reservoirs also exist. We have tested lake water and ice and found that the virus can be found in both. However, we found higher concentrations in ice than it water, indicating that the virus survives through the winters, and may be capable of reinfecting migratory waterfowl that arrive the following spring. This could have major implications in influenza A epidemiology. Funded by NIH NIAID, 2005-2007.

5. (O). Determination of splicing mechanisms of a small group I intron from fungi (L. Harris, BGSU; N. Walker, SUNY ESF). We have been characterizing splicing in a very small (68 nucleotides) group I intron. We are to selectively mutagenizing specific nucleotide sites (and regions) to determine their effects on splicing. In effect we are dissecting group I intron splicing using molecular biology tools. We also plan to partner with another lab to determine the structure of the intron using X-ray crystallographic methods.

6. (O). Characterization of a novel set of introns from sharks (A. Vicol, BGSU; M. Shivji, Nova Oceanographic University; M. Stanhope, M. Doasay, Queen's College, Belfast, UK, C.H. Tagliaro, Universidade Federal do Para, Brazil): We have found a novel set of introns within a diverse sample of sharks (seven Orders). The introns are novel because of their positions in the genome and the fact that this is the first report of finding this type of intron in any metazoan. The results have information regarding shark evolution, as well as intron evolution.

7. (O). Determination of the effects of chemical fixatives on nucleic acids (C. Tsai, J. Zollars, BGSU; M. Douglas, CDC, Atlanta). We have investigated the effects of some common fixatives, including formaldehyde and glutaraldehyde, on nucleic acids (DNA and RNA). The ultimate goal is to determine the potential utility of using archived tissues for studies of diseases, such as cancer. The results mirror other studies that documented numerous changes caused by formaldehyde-based fixatives. Thus, the resultant sequences do not match the sequences that existed in the tissues prior to treatment with the formaldehyde. The effects were few at pH 7, but increased as pH decreased. With the other fixatives, including glutaraldehyde, few changes were observed. These results have major implications with respect to studies using fixed and sectioned tissue samples.

8. (C). Characterization of DNA from herbarium and fossilized plant and fungus specimens (Z. Kaya, METU, Turkey; A.J. Bendich, J.F. Ammirati, University of Washington). We have used PCR amplification and DNA sequencing to characterize species and DNA from herbarium specimens of plants and fungi that are from a few years old to over 200 years old, as well as mummified plants to more than 45,000 years old. We have used similar methods to characterize DNA from mummified plant specimens that were a few hundred years old to tens of thousands of years old, including a 4,000 year old wood specimen from the tomb of King Midas, located near Ankara, Turkey, and 2,000 year old specimens from Al-Aksa Mosque, Israel. Ancient preserved specimens represent a resource of potentially useful genes that have gone extinct concurrently with the species that possessed them.

9. (O). Determination of development in vegetative plant shoots: (Seung-Geuk Shin, BGSU): We are tracing histogenic cell layers from the shoot apex to fully developed leaves and correlating that with the expression of specific developmentally regulated genes, determined by microarray analyses, and comparisons to Arabidopsis thaliana developmental studies.

10. (C). Population structure and reproduction biology of the tree-root endophyte Phialocephala fortinii and Type I (O. Holdenrieder, T. Sieber, C. Grünig, Swiss Federal Institute of Technology, ETH-Zentrum): Dark septate root endophytes (DSE) are ubiquitous fungal tree root colonizers in temperate and boreal conifer forest ecosystems. Their biology and ecological significance is insufficiently known. In this project we propose to elucidate population structure and mechanisms of reproduction of the two supposedly asexual DSE Phialocephala fortinii and Type 1. The experimental programme will be multi-disciplinary in approach, utilizing classical mycological and plant pathological techniques as well as molecular genetic techniques. We intend to study in detail the population structures of these fungi in a forest ecosystem using multiple-locus DNA markers. Specific statistical methods will be employed to help to decide whether the observed intraspecific genotypic variation is the result of clonal or recombining reproduction. In addition, we envisage to examine the function of the conidia of P. fortinii, search for mating type genes (MAT) in both P. fortinii and Type 1 and – if necessary – perform mating tests to find possible explanations for the observed variation. Funded by Swiss Federal Institute of Technology, 6/2000-5/2003.

11. (C) Microevolutionary measurements in plant and plant cell populations (F. Salehi, SUNY ESF): Mutations at specific nucleotide positions are being measured in DNA extracted from ancient seeds (100 - 50,000 years old) from populations of Juniperus osteosperma and Pinus flexilis. Mutation rates will be compared to glaciation and weather patterns that were previously determined during the same times at the same geographic locations. Mutation rates within individual long-lived trees are also being measured.

12. (O). Development of molecular biology methods for use in the field and herbaria: Methods have been developed to extract, protect and utilize biological macromolecules in the field. This will allow on-site collection and storage of these molecules to avoid the degradation that normally occurs during transit of the specimens to the lab. We have also been investigating the best methods of tissue fixation and other tissue preservation techniques to preserve macromolecules for later use.

13. (O). Studies of the teleomorph/anamorph pair, Valsa and Cytospora. Holdenrieder, T. Sieber, K. Langenegger, Swiss Federal Institute of Technology, Zürich, Switzerland). We have shown, using molecular, cultural, morphological and genetic methods, that the proposed teleomorph (sexual stage) of the mitosporic fungal genus Cytospora, is the ascomycete genus Valsa (Valsaceae, Diaporthales). Various species were isolated from a wide variety of host trees and from herbaria collections. Cultural and sequence data indicated that there are probably fewer species than currently described. Additionally, there does appear to be genetic specificity based on host and geographical location.

14. (O). Study of speciation based on ecological conditions for the fungus Herpotrichia (O. Holdenrieder, Swiss Fedearl Institute of Technology, Zürich, Switzerland). Herpotrichia juniperi grows on gymnosperm needles at and above the snowline in the Alps. A morphologically identical species, H. coulteri grows on gymnospern needles below the snowline. Some have suggested that both are one species, while others have proposed that they are separate species. Our molecular evidence points to a possible separation of the two caused by adaptations to the two environments.

15. (C). Detection of viruses in fog and clouds (G. Bachand, J.D. Castello, SUNY ESF): We have demonstrated that viable tobamoviruses are transported not only in water, but also in clouds and fog. Thus, long-range transport of theses viruses (and others) is possible, including intercontinental transport. Funded by USDA, 9/1993-8/1996, $200,000, and USDA McIntire-Stennis, 9/1991-8/1997.

16. (C). Characterization of a novel family of introns in the ribosomal DNA of Hyphomycetes (G. Zhang,L. Harris, BGSU; K.F. LoBuglio, UC Berkeley; M.L. Shinohara, Harvard University, C.J.K. Wang at SUNY ESF; and Z.H. Yan, Seattle): We have characterized an intron family, members of which are in several related genera of mitosporic fungi within the 18S rDNA. While the location is invariant, the sizes of the introns ranges from 68 bp to over 800 bp. The sequences are unlike any other group of introns previously described. We are investigating the evolutionary relationships between these introns and two other types, group I and mRNA introns.

17. (C). Study of the effects of plant resins (the precursors of amber) on DNA (O. Holdenrieder, K. Langenegger, Swiss Federal Institute of Technology, Zürich, Switzerland). We have treated various tissues in plant resins (from Douglas fir) under several conditions to determine the degree of DNA preservation. The plant resins themselves appear to be non-reactive towards nucleic acids, whether the temperatures are warm or cool. However, if the resin is continually hydrated, the DNA in the tissues exhibited varying amounts of degradation, including depurination, depyrimidization and backbone breakage. Rapid desiccation appears to be the most important factor in achieving good DNA preservation in amber.

18. (C) Co-evolution of fungi and plant hosts (O. Holdenrieder, T. Sieber, K. Langenegger, Swiss Federal Institute of Technology, Zürich, Switzerland). This study was to determine whether isolates of the fungus Laetiporus sulphureus are speciating based on tree host. Results showed that while there is an indication of preference for host tree species, complete speciation has not yet occurred.

19. (C) Determination of genetic diversity in forest tree species: (J. Gibbs, D. Leopold at SUNY ESF): We are assaying the genetic diversity of maples and white pines in New York state at clear-cut versus old-growth stands. The genetic markers being used are RAPDs (random amplified polymorphic DNAs) and AFLPs (amplified fragment length polymorphisms). Assessments will be made as to the amount of genetic diversity in comparison to classical measures of forest health. Funded by USDA McIntire-Stennis, 10/1997-9/2000.

20. (C) Measurement of colonization and spread of mycorrhizal and pathogens in forest ecosystems (C.J.K. Wang, SUNY ESF): We have developed a variety of DNA primers (for PCR) and probes for identification of mycorrhizal and pathogenic fungi. We are using these probes to track the colonization and spread of these fungi in various forest ecosystems, including previously harvested and old-growth sites. Funded by USDA McIntire-Stennis, 10/1991-9/2000.

21. (C) Root disease and fungal communities in a forest ecosystem.: (J.J. Worrall, D. Raynal, SUNY ESF; Phil Wargo, U.S. Forest Service): We are examining the relative rates of colonization and spread of Armillaria and Megacollybia through tree tissues. One of the applied goals of the project is to attempt to use Megacollybia as a biocontrol of Armillaria infection. Funded by USDA Cooperative State Research Education and Extension Service, 12/1998-11/2000.

22. (C) Taxonomy and phylogenetics of Basidiomycetes (J.F. Ammirati, Y.J. Liu, University of Washington): We are looking at broad relationships among Basidiomycetes using molecular biology methods (RFLP and PCR/restriction enzyme mapping). We are also examining the several genera within the Cortinariaceae in greater detail using molecular methods (PCR mapping and DNA sequencing).

23. (C) Taxonomic and phylogenetic reassessment of Cenococcum geophilum using RFLP and DNA sequence analysis (K.F. LoBuglio, UC Berkeley; M.L. Shinohara, Harvard University; C.J.K. Wang, SUNY ESF): C. geophilum, an ecologically important and globally distributed Hyphomycete, is difficult to study and identify because of the lack of useful taxonomic characters. We are using DNA characters to investigate this "type species" to determine its genetic characters. It is clear from our results that C. geophilum is a single species that is able to exist in a wide variety of habitats worldwide.

24. (C) Identification of sharks using ribosomal DNA spacers (M. Shivji, Nova University, Florida): Performing a census for most shark species is exceedingly difficult due to their small numbers, lack of knowledge of their behavior, and the great distances that they travel. Some species may already be extinct, while others may be endangered. This study uses muscle from fishing boats as well as eggs collected at sea to begin a census of six species of sharks.

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