Dr. G. Ray Green
Associate Professor of Pharmaceutical Sciences
- B.S., Zoology, University of Massachusetts
- M.S., Cell Biology, University of Massachusetts
- Ph.D., Cell and Developmental Biology, Amherst College/University of Massachusetts
Research Background and Interests
Epigenetic change during nuclear reprogramming in male pronuclei. We are building on our earlier work describing histone phosphorylation in sea urchin gametes and early embryogenesis by extending these studies to a broader range of epigenetic activities, including histone acetylation, methylation and ubiquitinylation. Histones are purified from male or female gametes and monospermic or polyspermic embryos and resolved by two-dimensional polyacrylamide gel electrophoresis . The gels below show Coomassie blue-stained histones from sea urchin sperm, eggs and 90 minute polyspermic embryos
resolved by this gel system.
The two-dimensional gels serve both analytical and preparative objectives. Sperm and eggspecific histone variants and their modified derivatives are readily identified by their characteristic electrophoretic mobilities and epigenetic differences between different stages of development are apparent as changes in this pattern. Individual protein spots can be electroeluted from the fixed and stained gels and used for various proteomic determinations, including phosphoamino acid analysis, tryptic peptide mapping and analysis by mass spectrometry. The two-dimensional protein patterns also can be electrophoretically transferred to membranes for western analysis employing a wide spectrum of
modification-specific antihistone antibodies.
Epigenetic change induced by mutations, drugs and cell differentiation. The methods for proteomic analysis outlined above for sea urchin development are easily adapted to other systems where epigenetic change via histone modification is expected. For example, we have developed efficient methods to faithfully preserve and analyze histone modifications in several important plant and fungal species that are of developmental or genetic interest, including wheat and alfalfa, S. cerevisiae, S. pombe and N. crassa. We also are applying the 2D method as a screen to detect epigenetic change in mammalian cells exposed to anti-cancer drugs.
Development of DNA transfection vehicles for gene therapy and vaccine development. A major hurdle for research on gene therapy and DNA vaccines has been the very slow rate at which large hydrophilic DNA molecules are taken up by target cells. Noting similarities between DNA transfection and the natural process of fertilization, we predicted that sperm-specific sea urchin histone variants formulated as DNA-histone polyplexes would significantly enhance the rate at which cells take up foreign DNA. We are currently testing this hypothesis using sea urchin sperm-specific histone variants and natural peptides derived from these proteins to enhance transfection rates in model cell systems.
- PHA 324 Medical Immunology
- PHA 325 Principles of Pharmaceutical Sciences
- PHA 581 Genetic Disorders
- PHA 804 Methods in Molecular Biology
- PHA 814 Analytical Methods and Instrumentation
- PHA 899 Doctoral Research
Lecture courses: The guiding principle in the development of my lecture courses is to maximize student learning. Because science classes and the students that compose them change continuously, the optimal formula for maximal student learning evolves quickly, requiring continuous monitoring and adjustment. Therefore I am in the habit of regularly revising my courses, updating content and searching for fresh input before every class. I make frequent use of video clips, especially effective with our current generation of visually oriented students, to illustrate and explain important or difficult topics. I also make good use of the recent news as gleaned from the morning papers or the previous evening’s news casts to emphasize the relevance of the ongoing discussion. Full sets of lecture notes are distributed several days before class and students are encouraged to come to class prepared to use this time for discussion rather than lecture, with emphasis on assessment of student progress as measured by frequent quizzes and other in-class active learning activities.
Basic principles and concepts are introduced early in a course and revisited throughout the semester to strengthen understanding and integration through repetition. Current and evolving concepts are presented in the context of our expanding experimental knowledge, showing science as process. Methodology and interpretation of data are discussed and explained, using the most recent developments from the scientific literature. Socratic questions to encourage dialogue and provide opportunities for students to apply and expand their mastery of the subject are a central part of all class meetings. Student assessments include subjective essay questions, to measure comprehension, and objective multiple choice questions, to test critical thinking skills. Thus assessments encourage students to think critically yet creatively and always be mindful of the broader relevance of specific content.
Laboratory courses: Fundamental principles, concepts and methods in cell, molecular and developmental biology are brought to life in laboratories that emphasize investigation, experimental design, careful observation and accurate, lucid presentation of results from laboratory experiences. A red blood cell bursts and reseals itself, demonstrating the fluid dynamics of biological membranes. A spectrophotometer is used to record the rate at which a dye molecule is reduced by purified chloroplasts, illustrating how plants produce biological order from sunlight and carbon dioxide. Students use protein and DNA gel electrophoresis to do a molecular dissection of chromatin, the biological form of our genes. They mix sea urchin sperm and eggs, observe fertilization and watch as embryos emerge from rapidly dividing zygotes. Students are taught basic skills in liquid sample handling, microscopy, biochemistry, centrifugation, spectrophotometry, chromatography and electrophoresis. Then they are asked to use these tools to investigate specific questions about how cells work. They design their own experiments and use hands-on approaches to obtain data, repeating experiments as necessary. They are encouraged not to “get the right answer” but to observe, record, interpret and report the specific details that emerge from their laboratory work.
Research students: Research students working in my laboratory are mature, intelligent and scientifically sophisticated undergraduate, professional or graduate students who have an opportunity to participate in and contribute to basic research in the biological sciences. As such, I treat them as fellow researchers, remembering often my own enthusiasm for laboratory science as an undergraduate and graduate student. My research students are given access to every research tool available to me, whether methodological, technological or intellectual and my most important laboratory teaching methods are availability and teaching by example. In return, I challenge my students to work hard, to be creative and productive and to be good citizens.
- Ali, A, Burns, T., Lucrezi J., May S., Green, G.R. and Matesic, D.F. (2015). Amidation Inhibitors 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2 hexenoic acid methyl ester are Novel HDAC Inhibitors with Anti-tumorigenic Properties.” Investigational New Drugs 33: 827-834.
- Smith, K.M., Dobosy, J.R., Reifsnyder, J.E., Rountree, M.R., Anderson, D.C., Green, G.R. and Selker, E.U. (2010). H2B- and H3-specific Histone Deacetylases are Required for DNA Methylation in Neurospora crassa. Genetics 86:1207-1216.
- Anderson, D.C., Green, G.R. Smith, K. and Selker, E.U. (2010). Extensive and Varied Modifications in Histone H2B of Wild Type and Histone Deacetylase 1-mutant Neurospora crassa. Biochemistry 49: 5244-5257.
- Okoroukwu, O.N., Green, G.R. and D’Souza, M.J. (2010). Development of Albumin Microspheres Containing Sp H1-DNA Complexes – A Novel Gene Delivery System”. Journal of Microencapsulation 27:142-149.
- Green, G.R. and Do, D.P. (2009). Purification and Analysis of Variant and Modified Histones Using 2D PAGE. In: The Nucleus: Chromatin, Transcription, Envelope, Proteins, Dynamics and Imaging. Methods in Molecular Biology 464:285-302.
- Green, G.R. (2001). Phosphorylation of Histone Variant Regions in Chromatin: Unlocking the Linker? Biochemistry and Cell Biology 79:275-287.
Contact Dr. G. Ray Green