Preface A central challenge of the post-genomic era is to understand how the 30,000 to 40,000 unique genes in the human genome are selectively expressed or silenced to coordinate cellular growth and differentiation. The packaging of eukaryotic genomes in a complex of DNA, histones, and nonhistone proteins called chromatin provides a surprisingly sophisticated system that plays a critical role in controlling the flow of genetic information. This packaging system has evolved to index our genomes such that certain genes become readily acces- sible to the transcription machinery, while other genes are reversibly silenced. Moreover, chromatin-based mechanisms of gene regulation, often involving domains of covalent modifications of DNA and histones, can be inherited from one generation to the next. The heritability of chromatin states in the absence of DNA mutation has contributed greatly to the current excitement in the field of epigenetics. The past 5 years have witnessed an explosion of new research on chroma- tin biology and biochemistry. Chromatin structure and function are now widely recognized as being critical to regulating gene expression, maintaining genomic stability, and ensuring faithful chromosome transmission. Moreover, links be- tween chromatin metabolism and disease are beginning to emerge. The identi- fication of altered DNA methylation and histone acetylase activity in human cancers, the use of histone deacetylase inhibitors in the treatment of leukemia, and the tumor suppressor activities of ATP-dependent chromatin remodeling enzymes are examples that likely represent just the tip of the iceberg. As such, the field is attracting new investigators who enter with little firsthand experience with the standard assays used to dissect chromatin struc- ture and function. In addition, even seasoned veterans are overwhelmed by the rapid introduction of new chromatin technologies. Accordingly, we sought to bring together a useful ‘‘go-to’’ set of chromatin-based methods that would update and complement two previous publications in this series, Volume 170 (Nucleosomes) and Volume 304 (Chromatin). While many of the classic proto- cols in those volumes remain as timely now as when they were written, it is our hope the present series will fill in the gaps for the next several years. This 3-volume set of Methods in Enzymology provides nearly one hundred procedures covering the full range of tools—bioinformatics, structural biology, biophysics, biochemistry, genetics, and cell biology—employed in chromatin research. Volume 375 includes a histone database, methods for preparation of histones, histone variants, modified histones and defined chromatin segments, xv protocols for nucleosome reconstitution and analysis, and cytological methods for imaging chromatin functions in vivo. Volume 376 includes electron micro- scopy and biophysical protocols for visualizing chromatin and detecting chro- matin interactions, enzymological assays for histone modifying enzymes, and immunochemical protocols for the in situ detection of histone modifications and chromatin proteins. Volume 377 includes genetic assays of histones and chromatin regulators, methods for the preparation and analysis of histone modifying and ATP-dependent chromatin remodeling enzymes, and assays for transcription and DNA repair on chromatin templates. We are exceedingly grateful to the very large number of colleagues representing the field’s leading laboratories, who have taken the time and effort to make their technical expertise available in this series. Finally, we wish to take the opportunity to remember Vincent Allfrey, Andrei Mirzabekov, Harold Weintraub, Abraham Worcel, and especially Alan Wolffe, co-editor of Volume 304 (Chromatin). All of these individuals had key roles in shaping the chromatin field into what it is today. C. David Allis Carl Wu Editors’ Note: Additional methods can be found in Methods in Enzymology, Vol. 371 (RNA Polymerases and Associated Factors, Part D) Section III Chromatin, Sankar L. Adhya and Susan Garges, Editors. xvi preface METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 376 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Rhoda M. Alani (12), Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Francisco Asturias (4),Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 Andrew J. Bannister (18), Wellcome Trust/Cancer Research, United Kingdom Institute and Department of Pathology, University of Cambridge, Cambridge CB2 1QR, United Kingdom P. B. Becker (1), Adolf Butenandt Institut, Lehrstuhl fu ¨ r Molekularbiologie, Schil- lerstr. 44, 80336 Munich, Germany Martin L. Bennink (6), Biophysical Tech- niques Group and MESAþ Research Institute, Department of Science Technol- ogy, University of Twente, 7500 AE Enschede, The Netherlands Bradley E. Bernstein (23), Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 Margie T. Borra (11), Department of Bio- chemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 Brent Brower-Toland (5), Biology De- partment, Washington University in St. Louis, St. Louis, Missouri 63130 Michael Bustin (14), Protein Section, National Cancer Institute, National Insti- tutes of Health, Bethesda, Maryland 20892 Juliana Callaghan (10), Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom Marek Cebrat (12), Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Julie Chaumeil (27), Mammalian Develop- mental Epigenetics Group, UMR 218-Nuclear Dynamics and Genome Plas- ticity, Curie Institute-Research Section, 75248 Paris, Cedex 05-France Dina Chaya (24), Cell and Developmental Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 Peter Cheung (15), Department of Med- ical Biophysics, University of Toronto, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada J. Chin (1), Department of Biochemistry, Northwestern University, Molecular Bio- logy and Cell Biology, Evanston, Illinois 60208-3500 David N. Ciccone (22), Department of Molecular Biology, Massachusetts Gen- eral Hospital, Boston, Massachusetts 02114 Philip A. Cole (12), Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Carlos Cordon-Cardo (13), Division of Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, New York 10021 ix Carolyn A. Craig (25), Biology Depart- ment, Washington University in St. Louis, St. Louis, Missouri 63130 John M. Denu (11), Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 Meghann K. Devlin (12), Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Marija Drobnjak (13), Division of Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, New York 10021 Brian Dynlacht (20), Department of Pathology, New York University School of Medicine, New York, New York 10016 Sarah C. R. Elgin (25), Biology De- partment, Washington University in St. Louis, St. Louis, Missouri 63130 Chukwudi Ezeokonkwo (4), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 Peggy Farnham (21), McArdle Laboratory for Cancer Research, University of Wis- consin, Madison, Wisconsin 53706 Wolfgang Fischle (9), Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908 Fred K. Friedman (14), Laboratory of Me- tabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Philippe T. Georgel (2), Department of Biological Sciences, Marshall University, Huntington, West Virginia 25755 Michael Grunstein (19), Department of Biological Chemistry, School of Medicine and Molecular Biology Institute, Univer- sity of California, Los Angeles, Los Angeles, California 90095 Jeffrey C. Hansen (2), Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 Edith Heard (27), Mammalian De- velopmental Epigenetics Group, UMR 218-Nuclear Dynamics and Genome Plas- ticity, Curie Institute-Research Section, 75248 Paris, Cedex 05, France Rachel A. Horowitz-Scherer (3), Department of Biology, University of Massachusetts, Amherst, Massachusetts 01003 Emily L. Humphrey (23), Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 Steven A. Jacobs (9), Department of Bio- chemistry and Molecular Genetics, Uni- versity of Virginia, Charlottesville, Virginia 22908 Thomas Jenuwein (16), Research Institute of Molecular Pathology (IMP), The ViennaBiocenter,Vienna,A-1030,Austria Monika Kauer (16), Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Vienna, A-1030, Austria W. Kevin Kelly (13), Genitourinary On- cology Service and Department of Medi- cine, Memorial Sloan Kettering Cancer Center, New York, New York 10021 Sepideh Khorasanizadeh (9), Depart- ment of Biochemistry and Molecular Gen- etics, University of Virginia, Charlottesville, Virginia 22908 Roger D. Kornberg (4), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 Tony Kouzarides (18), Wellcome Trust/ Cancer Research, United Kingdom Insti- tute, University of Cambridge, Cambridge CB2 1QR, United Kingdom x contributors to volume 376 Siavash K. Kurdistani (19), Department of Biological Chemistry, University of California, Los Angeles School of Medi- cine and Molecular Biology Institute, Los Angeles, California 90095 G. La ¨ ngst (1), Adolf Butenandt Institut, Lehrstuhl fu ¨ r Molekularbiologie, Schil- lerstr. 44, 80336 Munich, Germany Ernest Laue (10), Department of Bio- chemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom Sanford H. Leuba (6), Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Hill- man Cancer Center, UPCI Research Pavilion, Pittsburgh, Pennsylvania 15213-1863 Yuhong Li (25), University of Iowa, De- partment of Biochemistry, Iowa City, Iowa 52242 John Lis (26), Cornell University, Ithaca, New York 14853 Chih Long Liu (23), Department of Chem- istry and Chemical Biology, HarvardUni- versity, Cambridge, Massachusetts 02138 Yahli Lorch (4), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 Paul A. Marks (13), Cell Biology Pro- gram, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Ronen Marmorstein (7), Structural Biol- ogy Program, The Wistar Institute, Philadelphia, Pennsylvania 19104-4268 Karl Mechtler (16), Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Vienna, A-1030, Austria Katrina B. Morshead (22), Massachusetts General Hospital, Department of MolecularBiology,Boston, Massachusetts 02114 Shiraz Mujtaba (8), Department of Physi- ology and Biophysics, Structural Biology Program, Mt. Sinai School of Medicine, New York University, New York, New York 10029 Alexey G. Murzin (10), MRC Centre for Protein Engineering, Cambridge, CB2 2QH United Kingdom Natalia V. Murzina (10), Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom Peter R. Nielsen (10), Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom Kenichi Nishioka (17), Department of De- velopmental Genetics, National Institute of Genetics, Shizuoka, Japan, 411-8540 Matthew J. Oberley (21), McArdle Laboratory for Cancer Research, Univer- sity of Wisconsin, Madison, Wisconsin 53706 Marjorie A. Oettinger (22), Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 Ikuhiro Okamoto (27), Mammalian Devel- opmental Epigenetics Group, UMR 218 – Nuclear Dynamics and Genome Plasti- city, Curie Institute-Research Section, 75248 Paris, Cedex 05, France Susanne Opravil (16), Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Vienna, A-1030, Austria Barbara Panning (28), Department of Biochemistry and Biophysics, University of California, San Francisco, San Fran- cisco, California 94143-0448 Laura Perez-Burgos (16), Research Insti- tute of Molecular Pathology (IMP), The Vienna Biocenter, Vienna, A-1030, Austria contributors to volume 376 xi Antoine H. F. M. Peters (16), Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Vienna, A-1030 Austria Danny Reinberg (17), Department of Biol- ogy, Howard Hughes Medical Institute, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854-5635 Bing Ren (20), San Diego Branch and De- partment of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, La Jolla, California 92093-0653 Victoria M. Richon (13), Discovery Biol- ogy, Aton Pharma, Inc., Tarrytown, New York 10591 Richard C. Robinson (14), Laboratory of Metabolism, National Institutes of Health, National Cancer Institute, Bethesda, Maryland 20892 Daniel Robyr (19), Department of Biol- ogical Chemistry, University of California, Los Angeles, School of Medicine and Molecular Biology Institute, Los Angeles, California 90095 Kavitha Sarma (17), Department of Biol- ogy, Howard Hughes Medical Institute, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854-5635 Stuart Schreiber (23), Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 BrianE.Schwartz(26),CornellUniversity, Ithaca,NewYork14853 J. Paul Secrist (13), Discovery Biology, Aton Pharma, Inc., Tarrytown, New York 10591 Gena E. Stephens (25), Biology Depart- ment, Washington University in St. Louis, St. Louis, Missouri 63130 Paul R. Thompson (12), Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Julissa Tsao (21), Microarray Centre, University Health Network, Toronto, Ontario M5G 2C4, Canada Lori L. Wallrath (25), Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 Michelle D. Wang (5), Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853 Ling Wang (12), Department of Pharma- cology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Janis K. Werner (26), Cornell University, Ithaca, New York 14853 Jon Widom (1), Northwestern University, Department of Biochemistry, Molecular Biology and Cell Biology, Evanston, Illinois 60208-3500 Christopher L. Woodcock (3), Department of Biology, University of Massachusetts, Amherst, Massachusetts 01003 Patrick Yau (21), Microarray Centre, Uni- versity Health Network, Toronto, Ontario M5G 2C4, Canada Ken Zaret (24), Cell and Developmental Biology Program, W. W. Smith Chair in Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 Yujun Zheng (12), Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218 Ming-Ming Zhou (8), Structural Biology Program, Department of Physiology and Biophysics, Mt. Sinai School of Medicine, New York University, New York, New York 10029-6574 xii contributors to volume 376 Xianbo Zhou (13), Discovery Biology, Aton Pharma, Inc., Tarrytown, New York 10591 Jordanka Zlatanova (6), Department of Chemical and Biological Sciences and En- gineering, Polytechnic University, Brook- lyn, New York 11201 contributors to volume 376 xiii [1] Fluorescence Anisotropy Assays for Analysis of ISWI-DNA and ISWI-Nucleosome Interactions By J. Chin,G.La ¨ ngst,P.B.Becker, and J. Widom Fluorescence anisotropy is a rapid, sensitive, and quantitative technique that is well suited to the analysis of protein-protein and protein-DNA inter- actions in solution. Fluorescence anisotropy is a measure of the depolarization of emitted fluorescence intensity obtained after excitation by a polarized light source, and depends directly on the relative rate of fluorescence emis- sion versus the rate of tumbling in solution. The concept is simple: if a fluorescent molecule (or, more typically, a molecule to which a fluorescent probe has been attached) tumbles slowly in solution relative to the lifetime of fluorescence emission, then the light emitted in response to polarized excitation will remain highly polarized. However, if the molecules tumble rapidly in comparison to the emission lifetime, then, prior to emitting, they will have tumbled sufficiently so as to have ‘‘forgotten’’ their orientation at the moment of excitation, thus depolarizing (randomizing the polarization of) the emitted light. Fluorescence anisotropy is applicable for analysis of macromolecular interactions because there is a good match between typical fluorescence lifetimes and typical macromolecular tumbling times. For approximately spherical molecules, the tumbling time scales as the molecular volume, that is, as the molecular weight. Thus, binding of an unlabeled macromolecule can make a significant change to the tumbling time of the molecule to which the fluorescent probe is attached, and hence to the measured anisot- ropy. For the studies described in the following, we utilize DNA molecules labeled at one end with the fluorescent dye fluorescein (these DNA mol- ecules may be ‘‘naked DNA’’ or they may be incorporated into nucleo- somes), and we use fluorescence anisotropy to monitor the binding of the Drosophila ISWI chromatin remodeling protein 1–3 to the labeled DNA or nucleosomes. Fluorescence anisotropy is especially useful because of its high inherent sensitivity. Dyes such as fluorescein allow quantitative analysis of emission polarization from sub-nanomolar concentrations. Since dissociation con- stants are typically nanomolar or greater, this allows experiments to be 1 T. Tsukiyama, C. Daniel, J. Tamkun, and C. Wu, Cell 83, 1021 (1995). 2 P. D. Varga-Weisz et al., Nature 388, 598 (1997). 3 G. La ¨ ngst and P. B. Becker, J. Cell Sci. 114, 2561 (2001). [1] fluorescence anisotropy assays 3 Copyright 2004, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 376 0076-6879/04 $35.00 set up with the probe concentration (K d ; consequently the free concentra- tion of the added macromolecule (ISWI, in our case), which is generally either difficult to measure or is completely unknown, will be approximately equal to the total concentration, which can be definitively measured, thus greatly simplifying the analysis of the binding measurements. Another im- portant benefit of the sensitivity of the anisotropy measurement is that it preserves precious reagents. Measurements can be made in small volumes, and samples can be recovered and reused if desired. Finally, as discussed later, the experiment can be carried out using inex- pensive conventional fluorometers such as are found at most biochemical or chemical research laboratories, or, alternatively, using an inexpensive instrument specialized for the fluorescence anisotropy experiment. Investigators planning to carry out such studies should study two par- ticularly useful references, one on fluorescence theory and methodology in general 4 and one focused on fluorescence approaches to analysis of pro- tein-DNA interactions in particular. 5 These references nicely define and explain the set of four fluorescence intensity measurements that go into a single measurement of fluorescence anisotropy; we will not duplicate this important topic here, but rather refer readers to these other sources. Fluorescein-Labeled DNA We use DNA sequences labeled with fluorescein attached at the 5 0 -end through a C6 linker. Relatively short sequences are purchased as a pair of complementary oligonucleotides, one containing 5 0 -fluorescein. These are annealed, and the resulting duplex purified away from any remaining single strand by reverse-phase HPLC on a Zorbax-10 column using a gradient of 10–20% acetonitrille in 0.1 M triethanolamine-acetate, pH 7.0, 0.1 mM EDTA, developed over 10 min at 1 ml min À1 . When longer sequences (e.g., nucleosome-length DNAs) are required, direct synthesis is not practical. Instead we use preparative scale PCR, with one of the two primers again containing 5 0 -fluorescein. The resulting PCR product is purified by gel electrophoresis in 1% agarose gels with standard TAE buffer, and extracted from the gel using Ultra-DA (Millipore) gel extraction kits. DNA concentrations are quantified by UV absorbance. 4 J. R. Lakowicz, ‘‘Principles of Fluorescence Spectroscopy,’’ 2nd Ed. Kluwer Academic/ Plenum Press, New York, 1999. 5 J. J. Hill and C. A. Royer, Meth. Enzymol. 278, 390 (1997). 4 chromatin proteins [1] [...]... pitfalls It goes without saying that both buffers and < /b> samples must be free from significant fluorescent contaminants Fluorescence from the buffer alone and < /b> from unlabeled samples should be checked and < /b> shown to be negligible in comparison to the fluorescence obtained at the desired concentration of labeled sample In our experience this has never proven to be a problem using dilute buffers supplemented with approximately... specific genomic chromatin < /b> bands by Southern blotting (see later), the gel was subsequently treated with standard denaturing and < /b> neutralizing solutions, and < /b> transferred to Hybond N or NX membranes using 20Â SSC in combination with the Schleicher and < /b> Schuell Turboblotter The use of the Turboblotter (transferring from top to bottom) avoids having to flip the multigel, and < /b> in doing so minimizes the risk of separation... bed, allow the agarose to set for 30 or 60 min (9- or 18-lane slot formers, respectively) Begin preparing the agarose running gels Start by labeling capped 15-ml Kimble borosilicate (threaded end, 20 Â 125 for 9-lane gels and < /b> 16 Â 125 for 18-lane gels) tubes with the chosen agarose concentrations (see Table I for details) Add running buffer to the tubes (see Table I for volumes)  and < /b> place the tubes... nuclear extract as described earlier, and < /b> then measuring the band migrations by both fluorescent dyes and < /b> Southern hybridization Again, the measured Re and < /b> 00 values were the same within experimental error.9 14 15 J C Hansen, J Ausio, V H Stanik, and < /b> K E van Holde, Biochemistry 28, 9129 (1989) J D Dignam, R M Lebovitz, and < /b> R G Roeder, Nucleic Acids Res 11, 1475 (1983) [2] biophysical analysis of genomic... transfer, a probe covering the desired genomic region is radioactively labeled by random priming or nick-translation The unincorporated radioactivity is removed using gel filtration (BioSpin P30, BioRad) and < /b> the recovered probe is subsequentially phenol-chloroform extracted The Hybond membrane is pre-hybridized at  42 for 1.5 h in hybridizing solution (6Â SSC, 5Â Denharts, 0.5% SDS, 50% formamide, and < /b> 0.1... structures can be ‘‘docked.’’ The range of transmission EM-based technologies available for visualizing chromatin < /b> and < /b> chromatin < /b> remodeling < /b> complexes is listed in Table I For each method, we provide experimental guidelines based on recent work in our laboratory General Considerations With any imaging technique, the information retrievable is limited by the quality of the sample, and < /b> for chromatin,< /b> the... 00 , and < /b> Re of the genomic chromatin < /b> bands The results of these control experiments have been published9 and < /b> are summarized briefly here Naked DNA or model 12-mer nucleosomal arrays were added to nuclear extracts to mimic the release of genomic chromatin < /b> fragments into the same environment Under these conditions super-shifted smears were observed for both the DNA and < /b> nucleosomal arrays on standard... wavelengths, as described later 6 K J Polach and < /b> J Widom, J Mol Biol 254, 130 (1995) P T Lowary and < /b> J Widom, J Mol Biol 276, 19 (1998) 8 ˚ ¨ J D Anderson, A Thastrom, and < /b> J Widom, Mol Cell Biol 22, 7147 (2002) 9 G Voordouw and < /b> H Eisenberg, Nature 273, 446 (1978) 7 6 chromatin < /b> proteins [1] Sample Cleanliness As with any sensitive experimental method in analytical biochemistry, care must be taken in certain... running gel through use of Southern blotting 4 Calculate the , 00 , and < /b> Re of the chromatin-< /b> band(s) of interest, and < /b> the Pe of each running gel In addition, we describe western assays for determining the composition of the genomic chromatin < /b> fragment, and < /b> biophysical assays for formation of salt-dependent secondary chromatin < /b> structure and < /b> for the extent of conformational flexibility To emphasize the potential... spectrum will generally be much broader than the Raman band, so that it will be possible to choose filters that pass either the blue-side or the red-side of the fluorescence emission while rejecting the Raman The particular situation dictates the choice of filters to be used When the Raman scatter is to the blue of the fluorescence emission, cuton filter can be chosen to reject both elastic and < /b> Raman scattered . that both buffers and samples must be free from significant fluorescent contaminants. Fluorescence from the buffer alone and from unlabeled samples should be checked and shown to be negligible in. (23), Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 Margie T. Borra (11), Department of Bio- chemistry and Molecular Biology, Oregon Health and. Health, Bethesda, Maryland 20892 Juliana Callaghan (10), Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom Marek Cebrat (12), Department of Pharmacology and Molecular