Molecular pathology in clinical practice 2nd edition debra g.b. leonard (eds.) - The complete ebook
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100 Cases in Clinical Pathology and Laboratory Medicine
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To Greg With love and thanks
Preface
Today, molecular and genomic information is informing the patient care decisions in many, if not most, areas of healthcare. Clearly, cancer diagnosis, prognosis, and treatment are driven largely by the molecular variants that drive the cancer and are the targets for new therapies. Medical genetics is moving beyond the classic single gene genetic disorders as we understand the genetic risk factors that drive the common chronic diseases that are costly to our healthcare system. While the clinical relevance of all areas of the human genome is not yet understood, our knowledge is growing rapidly and expanding well beyond the protein-coding genes to include many regulatory-coding regions, such as microRNAs and long noncoding RNAs (lncRNAs), in regions of the genome which used to be considered “junk.” For infectious diseases, we are beginning to understand not only the well-known and emerging infectious agents, but that health and disease also relates to the symbiotic relationship of each patient with their microbiomes. Finally, the technologies available to the clinical molecular laboratory have advanced so the genome of individual patients can be analyzed for clinical care, even resulting in the definition of genomic critical values, which are recommended to be reported any time an exome or genome is sequenced for clinical purposes.
Molecular Pathology in Clinical Practice addresses all areas of clinical molecular pathology practice in a single textbook. This second edition has 12 new chapters, in addition to updates on the chapters from the first edition. The new chapters cover diseases not included in the first edition, plus two chapters on next-generation sequencing applications in genetics and cancer, and a proteomics chapter. The purpose of this textbook remains to provide a comprehensive reference for the practicing molecular pathologist as well as a resource for pathologists in any area of practice. The book also will continue to be used by training programs, both for Anatomic and Clinical Pathology and for Molecular Genetic Pathology trainees. This book is not meant to be a recipe book for clinical molecular tests, simply because the specifics of testing change quite rapidly in molecular pathology as new technologies emerge and are integrated into clinical molecular practice. Instead, the emphasis remains the molecular variants being detected for clinical purposes, the clinical usefulness of molecular test results, and the clinical and laboratory issues that require special attention. While this textbook focuses on molecular and genomic testing, with only a single chapter covering proteomics, the reader must understand that the genome does not drive all disease and health, but works in concert with the environment, the metabolome, the methylome, and other determinants of disease and health.
As we move toward genomic medicine, the molecular pathologist and all pathologists will play a significant role in the proper utilization of molecular and genomic tests to improve patient outcomes and the cost-effectiveness of the care we deliver. In the era of US healthcare reform, the promise of genomic medicine aligns almost perfectly with the healthcare reform goals of improving individual patient outcomes, improving the health of populations, and reducing the cost of healthcare. While much of genomic research focuses on the clinical
significance of pathogen and patient genomic variants for diagnosis and therapy, evidence of the value of genomics in clinical care also is needed, especially as we move toward population health management and global payment models.
My hope is that you apply the information in Molecular Pathology in Clinical Practice to the care you provide for your patients.
Burlington, VT, USA
Debra G.B. Leonard
Shale A.
Whitney Wooderchak-Donahue, Tracey Lewis, Kelli L. Sumner, Cecily P. Vaughn, Rong Mao, and Daniel H. Farkas
22 Inherited Breast Cancer .......................................................................................... 315
Rachel Michaelson-Cohen, Rachel Beeri, Eliahu Golomb, and Ephrat Levy-Lahad
23 Familial Adenomatous Polyposis and Turcot and Peutz–Jeghers Syndromes................................................................................ 329
Kandelaria M. Rumilla
24 Hereditary Nonpolyposis Colorectal Cancer and Lynch Syndrome ................... 339 James P. Grenert
25 Multiple Endocrine Neoplasia Syndromes ............................................................ 351 Barbara A. Zehnbauer
26 Von Hippel-Lindau Disease ..................................................................................... 365 Catherine A. Stolle
27 Hereditary Skin Cancer........................................................................................... 369 Dani Bercovich and Inbal Kedar
28 Li-Fraumeni Syndrome ........................................................................................... 377 Arupa Ganguly and Zhao Chen
29 Retinoblastoma ......................................................................................................... 385 Arupa Ganguly and Zhao Chen
30 Hereditary Paraganglioma and Pheochromocytoma ........................................... 393 Fang-Yuan Li and Lee-Jun C. Wong
Section III Solid Tumors
31 Colorectal Cancer .................................................................................................... 403
Antonia R. Sepulveda, Deqin Ma, Kathryn C. Behling, and Helen Fernandes
32 Lung Cancer ............................................................................................................. 419
Dara L. Aisner, Robert C. Doebele, Marileila Varella-Garcia, and Wilbur A. Franklin
57 Specimen Identification Through DNA Analysis .................................................. 849
Gregary Bocsi, Andrew Ricci, Gregory J. Tsongalis, and Vivianna M. Van Deerlin
Section VII HLA Typing
58 Molecular HLA Typing ........................................................................................... 867 Malek Kamoun, Jill A. Hollenbach, Steven J. Mack, and Thomas M. Williams
Section VIII Evolving Clinical Molecular Technologies
59 Next-Generation Sequencing: Principles for Clinical Application ...................... 889
Karl V. Voelkerding, Emily M. Coonrod, Jacob D. Durtschi, and Rebecca L. Margraf
60 Next-Generation Sequencing for the Analysis of Cancer Specimens .................. 911 John D. Pfeifer
61 Clinical Applications of Proteomics ....................................................................... 933
Delphine Rolland and Kojo S.J. Elenitoba-Johnson
Section IX Laboratory Management
62 Molecular Pathology Laboratory Management .................................................... 945 Hanna Rennert and Debra G.B. Leonard
Index .................................................................................................................................. 975
Contributors
Archana Agarwal, M.D. Department of Pathology, University of Utah, Salt Lake City, UT, USA
Dara L. Aisner, M.D., Ph.D. Department of Pathology, University of Colorado, Aurora, CO, USA
Kevin Alby, Ph.D. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Robert W. Allen, Ph.D. School of Forensic Sciences, Center for Health Sciences, Oklahoma State University, Tulsa, OK, USA
Daniel A. Arber, M.D. Department of Pathology, Stanford University Medical Center, Stanford, CA, USA
Aneel A. Ashrani, M.D., M.S. Division of Hematology, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN, USA; Special Coagulation Laboratory, Mayo Comprehensive Hemophilia Center, Rochester, MN, USA; Department of Laboratory Medicine and Pathology, Division of Hematopathology and Laboratory Genetics, Mayo Clinic Rochester, Rochester, MN, USA
Renkui Bai, M.D., Ph.D., F.A.C.M.G. Genetic Testing for Mitochondrial Disorders, GeneDx, Gaithersburg, MD, USA
Monica J. Basehore, Ph.D. Greenwood Genetic Center, Greenwood, SC, USA
Samantha M. Baxter, M.S., C.G.C. Laboratory for Molecular Medicine, Partners HealthCare, Cambridge, MA, USA
Lora J.H. Bean, Ph.D., F.A.C.M.G. Department of Human Genetics, Emory Genetics Laboratory, Emory University, Atlanta, GA, USA
Rachel Beeri, Ph.D. Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel
Kathryn C. Behling, M.D., Ph.D. Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ, USA; Department of Pathology, Cooper University Hospital, Camden, NJ, USA
Daniel B. Bellissimo, Ph.D. Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Magee-Womens Hospital, Pittsburgh, PA, USA
Dani Bercovich, Ph.D. GGA–Galil Genetic Analysis, Biotechnology Program, Tel Hai College, Kazerin, Israel
D. Hunter Best, Ph.D. Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Dario de Biase, B.Sc., Ph.D. Anatomic Pathology, Ospedale Bellaria, Bologna University School of Medicine, Bologna, Italy
Andrea Biondi, M.D. Department of Pediatrics, University of Milano-Bicocca, Monza, Italy
Jon D. Blumenfeld, M.D. The Susan R. Knafel Polycystic Kidney Disease Center, The Rogosin Institute, New York, NY, USA; Clinical Medicine, Nephrology and Hypertension, Weill Cornell Medical College, New York, NY, USA
Gregary Bocsi, D.O. Department of Pathology, University of Colorado School of Medicine, Aurora, CO, USA
Rita M. Braziel, M.D. Division of Hematopathology, Department of Pathology, Oregon Health & Science University, Portland, OR, USA
Eileen M. Burd, Ph.D. Pathology and Laboratory Medicine, Microbiology Emory University, Atlanta, GA, USA
Angela M. Caliendo, M.D., Ph.D. Department of Medicine, Alpert Medical School of Brown University, Providence, RI, USA; Rhode Island Hospital, Providence, RI, USA
Giovanni Cazzaniga, Ph.D. Pediatrics, Centro Ricerca Tettamanti, Università di Milano Bicocca, Monza, Italy
Kristen J. Champion, Ph.D., F.A.C.M.G. Med Fusion Laboratories, Lewisville, TX, USA
Zhao Chen, Ph.D. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Christin D. Collins, Ph.D., F.A.C.M.G. Emory Genetics Laboratory, Department of Human Genetics, Emory University, Decatur, GA, USA
Emily M. Coonrod, Ph.D. Program in Personalized Health, Dean’s Office Research Unit, University of Utah, Salt Lake City, UT, USA
Shale A. Dames, M.S. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Vivianna M. Van Deerlin, M.D., Ph.D. Pathology and Laboratory Medicine, Molecular Pathology Laboratory, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
Robert C. Doebele, M.D., Ph.D. Division of Medical Oncology, University of Colorado Denver, Aurora, CO, USA
Jennifer B. Dunlap, M.D. Division of Hematopathology, Oregon Health & Sciences University, Portland, OR, USA
Jacob D. Durtschi, B.S. Bioinformatics, Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Lisa Edelmann, Ph.D. Department of Genetics and Genomic Sciences, Icahn School of Medicine, Mount Sinai Medical Center, New York, NY, USA
Kojo S.J. Elenitoba-Johnson, M.D. Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Stellar-Chance Laboratories, PA, USA
Robert W. Eppsteiner, M.D. Department of Otolaryngology—Head and Neck Surgery, University of Iowa Hospitals and Clinics, Iowa City, IA, USA
Guang Fan, M.D., Ph.D. Division of Hematopathology, Department of Pathology, Oregon Health and Science University, Portland, OR, USA
Daniel H. Farkas, Ph.D. Obstetrics, Gynecology and Reproductive Medicine, Michigan State University, Grand Rapids, MI, USA
Helen Fernandes, Ph.D. Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA
David R. Foran, Ph.D. Forensic Science Program, School of Criminal Justice, Michigan State University, East Lansing, MI, USA
Betty A. Forbes, Ph.D. Division of Clinical Pathology, Pathology Department, Virginia Commonwealth University Medical Center, Richmond, VA, USA
Wilbur A. Franklin, M.D. Department of Pathology, University of Colorado Denver, Aurora, CO, USA
Michael J. Friez, Ph.D. Med Fusion Laboratories, Lewisville, TX, USA
Birgit H. Funke, Ph.D., F.A.C.M.G. Department of Pathology, Harvard Medical School, Cambridge, MA, USA
Arupa Ganguly, Ph.D. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Christine C. Ginocchio, Ph.D., M.T.(A.S.C.P.) Department of Pathology and Laboratory Medicine, Hofstra North Shore-LIJ School of Medicine, Hempstead, NY, USA; Scientific and Medical Affairs, BioMerieux, Durham, NC, USA; Scientific and Medical Affairs, BioFire Diagnostics, Salt Lake City, UT, USA
Eliahu Golomb, M.D., Ph.D. Department of Pathology, Shaare Zedek Medical Center, Medical Genetics Institute, Jerusalem, Israel
James P. Grenert, M.D., Ph.D. University of California San Francisco, San Francisco, CA, USA
David Grimwade, Ph.D., F.R.C.Path. Department of Medical and Molecular Genetics, Cancer Genetics Laboratory, King’s College London, Faculty of Life Sciences and Medicine, London, UK
Guangyu Gu, M.D. Pathology Department, ARUP Laboratories, University of Utah, Salt Lake City, UT, USA
Kevin C. Halling, M.D., Ph.D. Department of Laboratory Medicine and Pathology, Division of Laboratory Genetics, Mayo Clinic, Rochester, MN, USA
Randall T. Hayden, M.D. Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Madhuri R. Hegde, Ph.D. Department of Human Genetics, Emory University School of Medicine, Emory University, Atlanta, GA, USA
Jaimie D. Higgs, M.S., C.G.C. GeneDx, Gaithersburg, MD, USA
David R. Hillyard, M.D. Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT, USA; Molecular Infectious Disease Testing and Infectious Disease Sequencing Core, ARUP Laboratories, Salt Lake City, UT, USA
Benjamin H. Hinrichs Pathology and Laboratory Medicine, Gastrointestinal Pathology, Emory University, Atlanta, GA, USA
Albert K. Ho, M.D., Ph.D. Quest Diagnostics, Nichols Institute, Chantilly, VA, USA
Jill A. Hollenbach, Ph.D., M.P.H. Center for Genetics, Children’s Hospital Oakland Research Institute, Oakland, CA, USA
Pei Hui, M.D., Ph.D. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
Daniel M. Jones, M.D., Ph.D. School of Health Professions, University of Texas MD Anderson Cancer Center, Houston, TX, USA
Melanie A. Jones, Ph.D. Department of Human Genetics, Emory University School of Medicine, Emory University, Atlanta, GA, USA
Jeanne A. Jordan, Ph.D. Epidemiology and Biostatistics, School of Public Health and Health Services, The George Washington University, Washington DC, USA
Malek Kamoun, M.D., Ph.D. Pathology and Laboratory Medicine, Penn Medicine, Philadelphia, PA, USA
Karen Kaul, M.D., Ph.D. Pathology and Laboratory Medicine, North Shore University Health System, Evanston, IL, USA
Inbal Kedar, M.S. Cancer Genetics, The Raphael Recanati Genetics Institute, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel
Michelle L. Kluge, M.S., C.G.C. Department of Laboratory Medicine and Pathology, Division of Hematopathology and Laboratory Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA; Mayo Comprehensive Hemophilia Center, Mayo Clinic Rochester, Rochester, MN, USA
Steven Knapper, D.M., B.M.B.Ch., F.R.C.Path. Department of Haematology, Cardiff University, Cardiff, UK
Ruth Kornreich, Ph.D. Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA
Colleen S. Kraft, M.D., M.Sc. Pathology and Laboratory Medicine, Medicine, Infectious Diseases, Emory University, Atlanta, GA, USA
Patti Krautscheid, M.S., L.C.G.C. ARUP Laboratories, Salt Lake City, UT, USA
Danielle LaGrave, M.S., L.C.G.C. ARUP Laboratories, Salt Lake City, UT, USA
Priti Lal, M.D. Pathology and Laboratory Medicine, Anatomic Pathology, University of Pennsylvania, Philadelphia, PA, USA
Ehud Lavi, M.D. Anatomic Pathology, Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA
Nicky Leeborg, M.D. Division of Hematopathology, Department of Pathology, Oregon Health and Science University, Portland, OR, USA
Debra G.B. Leonard, M.D., Ph.D. Department of Pathology and Laboratory Medicine, University of Vermont College of Medicine and University of Vermont Medical Center, Burlington, VT, USA
Ephrat Levy-Lahad, M.D. Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University Medical School, Jerusalem, Israel
Tracey Lewis, Ph.D. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Fang-Yuan Li, M.D., Ph.D. Department of Molecular and Human Genetics, Baylor Miraca Genetics Laboratory, Baylor College of Medicine, Houston, TX, USA
Elaine Lyon, Ph.D. Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA; Molecular Genetics and Genomics, ARUP Laboratories, Salt Lake City, UT, USA
Deqin Ma, M.D., Ph.D. Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA, USA
Steven J. Mack, Ph.D. Children’s Hospital Oakland Research Institute, Oakland, CA, USA
Rong Mao, M.D. Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Rebecca L. Margraf, Ph.D. ARUP Laboratories, Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Maria Martinez-Lage, M.D. Pathology and Laboratory Medicine, Neuropathology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
Jason D. Merker, M.D., Ph.D. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
Rachel Michaelson-Cohen, M.D. Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University Medical School, Jerusalem, Israel
Carolyn Mies, M.D. Genomic Health Inc., Redwood City, CA, USA
Christine E. Miller, M.S., C.L.G.C. ARUP Laboratories, Salt Lake City, UT, USA
Melissa B. Miller, Ph.D. Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC, USA
Krzysztof Mrózek, M.D., Ph.D. Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA
Duane W. Newton, Ph.D., D.(A.B.M.M.) Clinical Microbiology Laboratory, Division of Clinical Pathology, University of Michigan, Ann Arbor, MI, USA
Yuri E. Nikiforov, M.D., Ph.D. Department of Pathology and Laboratory Medicine, Division of Molecular and Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
Marina N. Nikiforova, M.D. Department of Pathology and Laboratory Medicine, Division of Molecular and Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
Frederick S. Nolte, Ph.D. Molecular Pathology, Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA
Shuji Ogino, M.D., Ph.D., M.S. Department of Pathology, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA
Nirali M. Patel, M.D. Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Molecular Genetics Laboratory, UNC Hospitals, Chapel Hill, NC, USA
Deborah Ann Payne, Ph.D. Molecular Services, American Pathology Partners, Inc., Denver, CO, USA
John D. Pfeifer, M.D., Ph.D. Department of Pathology, Washington University School of Medicine, St. Louis, MO, USA
Herbert F. Polesky, M.D. In Memoriam
V.M. Pratt, Ph.D., F.A.C.M.G. Pharmacogenomics Laboratory, Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA
Thomas W. Prior, Ph.D. Department of Pathology, Ohio State University Medical Center, Columbus, OH, USA
Rajiv K. Pruthi, M.B.B.S. Division of Hematology, Department of Internal Medicine, Mayo Clinic Rochester, Rochester, MN, USA
Raymund R. Razonable, M.D., F.I.D.S.A. Division of Infectious Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA
Hanna Rennert, Ph.D., F.A.C.M.G. Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, USA
Ran Reshef, M.D. Department of Medicine, Hematology and Oncology Division, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
Andrew Ricci, M.D. Department of Pathology, Hartford Hospital, Hartford, CT, USA
Delphine Rolland, Pharm.D., Ph.D. Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA
Kandelaria M. Rumilla, M.D. Department of Laboratory Medicine and Pathology, Laboratory Genetics, Mayo Clinic, Rochester, MN, USA
Iris Schrijver, M.D. Pathology Department, Stanford University, Stanford, CA, USA
Stuart A. Scott, Ph.D. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Mount Sinai Genetic Testing Laboratory, New York, NY, USA
Carlos Alberto Scrideli, M.D., Ph.D. Pediatrics, Pediatric Oncology and Hematology, Ribeirão Preto Medical School, University of Sao Paulo, Ribeirão Preto, Sao Paulo, Brazil
Antonia R. Sepulveda, M.D., Ph.D. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA
A. Eliot Shearer, B.Sc. Department of Otolaryngology—Head and Neck Surgery, University of Iowa Hospitals and Clinics, Iowa City, IA, USA
Patricia R. Slev, Ph.D., D.(A.B.C.C) Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT, USA; Serologic Hepatitis and Retrovirus Laboratory, Immunology Core Laboratory, ARUP Laboratories, Salt Lake City, UT, USA
Richard J.H. Smith, M.D. Department of Otolaryngology—Head and Neck Surgery, University of Iowa Hospitals and Clinics, Iowa City, IA, USA
Sarah T. South, Ph.D. Pathology Department, ARUP Laboratories, University of Utah, Salt Lake City, UT, USA; Department of Pediatrics, ARUP Laboratories, University of Utah, Salt Lake City, UT, USA
Elaine Spector, Ph.D., F.A.C.M.G. Department of Pediatrics, Denver Genetics Laboratories, Children’s Hospital Colorado, Aurora, CO, USA
Steven Sperber, M.S., Ph.D., F.A.C.M.G. Department of Genetic and Genomic Sciences, Mount Sinai Genetic Testing Laboratory, Icahn School of Medicine at Mount Sinai, New York, NY, USA
Catherine A. Stolle, Ph.D. Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA
Kelli L. Sumner, B.S. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Giovanni Tallini, M.D. Anatomic Pathology, Ospedale Bellaria, Bologna University School of Medicine, Bologna, Italy
Adrian Y. Tan, Ph.D. Department of Pathology, Weill Cornell Medical College, New York, NY, USA
Reha Toydemir, M.D., Ph.D. Pathology Department, ARUP Laboratories, University of Utah, Salt Lake City, UT, USA
Gregory J. Tsongalis, Ph.D. Department of Pathology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA; Norris Cotton Cancer Center, Lebanon, NH, USA
Marileila Varella-Garcia, Ph.D. Division of Medical Oncology, University of Colorado Denver, Aurora, CO, USA
Cecily P. Vaughn, M.S. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Karl V. Voelkerding, M.D. Department of Pathology, ARUP Institute for Clinical and Experimental Pathology, University of Utah, Salt Lake City, UT, USA
Victor W. Weedn, M.D., J.D. Department of Forensic Sciences, George Washington University, Washington, DC, USA
Thomas M. Williams, M.D. In Memoriam
Robert B. Wilson, M.D., Ph.D. Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
Lee-Jun C. Wong, Ph.D., F.A.C.M.G. Department of Molecular and Human Genetics, Baylor Miraca Genetics Laboratory, Baylor College of Medicine, Houston, TX, USA
Whitney Wooderchak-Donahue, Ph.D. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
Amy C. Yang, M.D. Department of Genetics and Genomic Sciences, Division of Medical Genetics, Mount Sinai School of Medicine, New York, NY, USA
Yaping Yang, Ph.D. Department of Molecular and Human Genetics, Medical Genetics Laboratory and Whole Genome Laboratory, Baylor College of Medicine, Houston, TX, USA
Barbara A. Zehnbauer, Ph.D., F.A.C.M.G., F.A.C.B. Centers for Disease Control and Prevention, Atlanta, GA, USA
Basics of Molecular Biology
Deborah Ann Payne
Abstract
Molecular biology entails the analysis and study of the chemical organization of the cell. Molecules comprise the smallest chemical component capable of performing all the activities (structural or catalytic) of a substance. One or more atoms constitute each molecule. Many molecules comprise the various cellular and subcellular components of an organism. Molecules form not only the physical structure of the organism but communicate information between the various compartments of the cell. This communication can be the transfer of information from DNA to RNA and finally to protein or the subtle regulation of the cell’s internal homeostatic processes. This communication relies on the interaction of various molecules to insure the fidelity of the message or cellular regulation. This chapter describes the physical organization of cells, cellular organelles, and molecules important in cell division, inheritance, and protein synthesis and describes how genetic information is communicated within the cell.
Keywords
Introduction
Molecular biology entails the analysis and study of the chemical organization of the cell. Molecules comprise the smallest chemical component capable of performing all the activities (structural or catalytic) of a substance. One or more atoms constitute each molecule. Many molecules comprise the various cellular and subcellular components of an organism. Molecules form not only the physical structure of the organism but communicate information between the various compartments of the cell. This communication can be the transfer of information from DNA to RNA and finally
to protein or the subtle regulation of the cell’s internal homeostatic processes. This communication relies on the interaction of various molecules to insure the fidelity of the message or cellular regulation. This chapter describes the physical organization of cells, cellular organelles, and molecules important in cell division, inheritance, and protein synthesis and describes how genetic information is communicated within the cell.
Organization of the Cell
The cell is a mass of protoplasm surrounded by a semipermeable membrane [1 of living matter capable of functioning independently; howwith other cells. To function independently, cells must pro-
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organisms, these organic processes form and maintain tissues and the organism as a whole.
encode proteins, and control the function of the cell. comprise the two types of nucleic acids found in all cells. live and function.
Prokaryotic Cells
6 base pairs (bp) (Table 1.1) [mosomal genetic elements consist of circular plasmids alsoria [3, 4]. Transposons also may confer antibiotic resistance contact with the bacteria’s cytoplasm.
Eukaryotic Cells
Cytoplasm
highly compartmentalized structures. The cytoplasm conorganelles. The cellular membrane separates the cellular consist of hydrophobic lipid bilayers. The lipid bilayer contains proteins that serve as receptors and channels.
Nucleus and Nucleolus
The nucleus of the cell contains the cell’s linear chromosomes and serves as the primary locus of inherited genetic the nucleus and separate the chromosomes from the surrounding cytoplasm. Further partitioning occurs within the nucleus to generate the nucleolus, which functions as the-
lus organizer (a specific part of a chromosome containing the genes that encode ribosomal RNAs) interacts with other
molecules to form immature large and small ribosomal subunits. Following processing, immature subunits depart thethrough the nuclear pores and enter the cytoplasm.
Mitochondria
Mitochondria are membrane-bound organelles within the cytoplasm of cells that have several cellular functions. chromosomes, resides in mitochondria. These maternally derived organelles contain their own circular chromosome another. As a result, not all mitochondria in a given cell have diversity of these organelles within and between differentent per cell, and this number may vary with different disease states [6, 7]. Mitochondrial genes encode mitochondria-spe-various subunits of NAD dehydrogenase. Other components nuclear genes. For this reason, not all mitochondrial genetic diseases demonstrate maternal transmission. Mutations asso-
( ). The higher copy number per cell of mtDNA compared with genomic DNAacterization of mtDNA from severely degraded samples and scant samples. For this reason, mtDNA is suitable for paleontological, medical, and forensic genetic investigations. Analysis of mtDNA has applications for diagnosis of mitochondrial-inherited genetic diseases, disease prognosis, as well as forensic identification of severely decomposed bodies [6 9].
Other Cellular Organelles
Membranes not only segregate heritable genetic molecules into the nucleus and mitochondria, but also separate various cellular functions into distinct areas of the cell. The compartmentalization of cellular functions (such as molecular synthesis, modification, and catabolism) increases the local concentration of reactive molecules and improves the biochemical efficiency of the cell. This partitioning also protects inappropriate molecules from becoming substrates for these
to lipids and proteins. The basic unit of a carbohydrate consists of the simple sugars or monosaccharides. These
lysosomes segregate digestive and reactive molecules from the remainder of the cellular contents to prevent damage to the cell’s internal molecules and infrastructure. The pathologic accumulation of large molecules within lysosomes occurs when enzymes cannot chemically cleave or modify the large -
age diseases are associated with a variety of genetic variants 1].
Biological Molecules
the scaffold for all biomolecules. Basic subunit biomolecules
bohydrates, nucleic acids, and amino acids.
Carbohydrates
form a chain. As a result, the formula for a simple sugar is O)n, where n
the sugar element of DNA and RNA molecules, respectively.
Nucleic Acids
4) group, and a purine or pyrimidine base. The nucleotides are joined into a DNA or RNA strand
ring molecules, which form N-glycosidic bonds with ribose 4N4), while 4 N ).
two DNA molecules (Fig. 1.1). The additional hydrogen hydrogen bonds) dramatically enhances the strength of this interaction compared to the two hydrogen bonds present between A and T nucleotides. This hydrogen-bonding capac-
tion for all nucleic acids and assures the passage of genetic information during DNA replication, RNA synthesis from DNA (transcription), and the transfer of genetic information from nucleic acids to the amino acids of proteins.
Numerous types of base modifications increase the num-
hydrogen bonding characteristics, modified nucleotides serve various functions in the cell including (1) regulating reactivation, (3) identifying DNA damage, and (4) facilitat-in normal tissue but hypomethylated in cancer tissue [ ].
Figure 1.1 DNA base pairing. DNA nucleotides are composed of three moieties (e.g., sugar, base, and phosphate groups). The bases are either purine (adenine and guanine) or pyrimidine (thymine and cytosine). Note the difference in hydrogen bonds between adenine and thymine base pairs, with two hydrogen bonds, compared to cytosine and guanine base pairs, with three hydrogen bonds. Reprinted with permission from
prevents various insults to the host genome by inactivating the ability of these elements to transpose themselves. Methylation also regulates the phenomenon of imprinting.
wimpy testis (in Drosophila interacting noncoding RNAs (specifically, piRNA) [11].
the function of tRNAs [ ]. Some of these modifications
cation defects result in mitochondrial disease [ 13 ]. ′-O-methylation and pseudouridylation and enable rRNA folding and stability. Such modifications result from interactions of the bases with small nucleolar ribonucleoproteins and noncoding small nucleolar RNAs [ the detection of modified bases, the role of modified bases in human disease may become better understood [14].
Amino Acids
3), a
atom. The R group can be a simple hydrogen, as found in), and determine whether an amino acid has a neutral, basic, or acidic charge. The amino group of a polypeptide is considered the beginning of the protein (N-terminus), while theity to the protein.
Genetic Molecules
Nucleic acids encode genetic information but also participate in additional physiological processes ranging from metabolism to energy transfer. Nucleotides constitute the monomeric units of nucleic acids (Fig. 1.1). Nucleosides and DNA, respectively, and either a purine or pyrimidine base). A nucleotide is produced from a nucleoside by the addition of one to three phosphate groups through a covalent ′ carbon of the nucleoside’s sugar ring.
phosphodiester bonds between the 3′ carbon of the first ′ carbon of the adjacent ′ to 3′ directionality. The alternating
Deoxyribose
D.A. Payne
Amino acid Amino acid symbols
Linear structure Three letter Single letter
AlaninealaA 3
ArginineargR )3
AsparagineasnN
Aspartic acidaspD cys glu ) glnQ ) gly his N C ile 3 3 leu 3) lysK )4
MethioninemetM 3 ) pheF pro N )3 C
SerineserS
ThreoninethrT 3
Tryptophantrp Ph C
TyrosinetyrY
ValinevalV 3)
nyl ring.
sugar-phosphate chain forms a continuous molecule with ′ carbon of each sugar. For thisbone of nucleic acids (Fig. ). The phosphate groups give nucleic acids a negative charge that imparts important physiochemical properties to nucleic acids. The negative charge of DNA facilitates the binding of mammalian DNA to various proteins and allows separation of nucleic acid molecules by charge and size during gel or capillary electrophoresis.
Structure the bases of the two strands, in which case the two strands are said to be complementary. The two strands are oriented ′ to 3′ directions, such that one strand is oriented ′ to 3′ and the complementary strand is oriented 3′ ′ in
Figure 1.2 Double-stranded DNA. The two DNA strands are oriented in an antiparallel relationship, with asymmetric base pairing of two DNA strands that generates the minor and major grooves of the DNA
an antiparallel fashion (see Fig. ′ end) of one DNA strand being adjacent to the tail (or 3′ end) of the opposite strand.
The molecular curves of the two DNA strands form anti-
turn, occupying 3.4 nm. Because the bonds between the sugar and the base are not perfectly symmetrical, the strands curve slightly. The slight curve of the offset glycosidic bonds results in major and minor grooves characteristic of the B ]. Many clinical molecular tests
Table 1.2 Amino acids
and very deep grooves.
Thermodynamics of Nucleotide Base Pairing
Thermodynamics plays a major role in the structure and stability of nucleic acid molecules. The core mechanism of nucleic acid thermodynamics centers on the hydrogenbonding capabilities of the nucleotides. The stability of these
the structure and catalytic characteristics of single-strandedtion to these physiological functions, the phenomenon of complementary base pairing profoundly impacts clinical
specific antibody to identify or detect a target protein. The procedures for generating and validating diagnostic antibod-
pair as the basis for detection and characterization of target nucleic acids has greatly facilitated clinical molecular test development. The formation of hydrogen bonding between two pieces of nucleic acid is called hybridization, or annealing, and the disruption of the hydrogen bonds holding two nucleic acid molecules together is called denaturation, or melting. The fact that clinical molecular tests use hybridiza-
scores the necessity for understanding the thermodynamics of the hydrogen base pairing of nucleic acids.
Short pieces of DNA or RNA called probes, or primers,
related region of DNA or RNA from a clinical specimen are hybridization of a DNA or RNA probe to genomic DNA for a clinical molecular test, the two genomic DNA strands must be separated, or denatured, prior to probe hybridization.
nism for disrupting the hydrogen bonds between the DNA base pairs and denaturing double-stranded DNA into singlestranded DNA molecules separate into single-stranded form constitutes the melting temperature (Tm). The shorter the two complementary DNA molecules are, the easier it is to calculate the Tmhood of nonspecific intramolecular annealing or base pairing compared to inter- and intramolecular base pairing. The simplest and least accurate formula for determining the Tm for base pairs by 4 and multiplies the sum of the A:T base pairs
T m GCAT = () é ë ù û + () é ë ù û 42::
Although this is the least accurate method for calculation of the Tm of a double-stranded DNA molecule, it mathematiTm calculation for DNA and RNA is more accurate [16, 17]:
Table 1.3 demonstrates the effect of increasing the relative Tm using these formulas.
dimensional forms within single-stranded nucleic acid mol-
RNA molecules affords great structural diversity via intramolecular base pairing. These conformations strain the linear RNA molecule and produce chemically reactive RNAlar functions and in gene-targeting therapies.
affect hybridizations. Dimers, bulge loops, and hairpin loops].
Table 1.3 Melting temperature calculations for short oligomers
Total length Number of G:C Number of A:T Tm a %G:Cb A:T + G:Cc 71.6
aNearest-neighbor calculation of Tm [16] bTm ] c
These detrimental effects also may include initiation of spurious nonspecific polymerization, steric hindrance of hybridor primers), and depletion of probes or primers away from the specific target by either primer dimerization or other mechanisms. These interactions can result in poor sensitivity or specificity for clinical molecular tests.
Topology
The DNA and RNA molecules assume various geometric shapes or topologies that are independent of base pair intercontrast to the circular forms of mitochondrial and bacterial chromosomal DNA. Transposable elements within the human genome also have a linear topology. Viral genomes occur as different forms, ranging from segmented linear to circular, and can be present in the nucleus, cytoplasm, or integrated within the human genome. Although the confor-lar base pairing, the topology of messenger RNA (mRNA) molecules is primarily linear. An organism’s genomic topol-lication and the number of replication cycles a given linear genomes limit the total number of possible replication cycles due to progressive shortening of the linear chromo-mosomes, the ends of the chromosome contain tandem
Mammalian Chromosomal Organization
× 9 base 19].
the bioinformatic definition of a gene changes [ ].
RNA- and/or chromatin-based activity with many of thesenous retroviruses, a chimeric element (SVA) composed of
The ability of retrotransposons to duplicate and insert within the genome (i.e., either autonomously or with the help of
autonomous elements) has been associated with various types of genetic mutations. Mechanisms for mutations
reports associate retrotransposons with various genetic disorders ranging from hemophilia to breast cancer [ , ].
(discussed later in this chapter). Because transposable elements can replicate and cause genetic deletions with the human genome, the number of human base pairs is not static.
cleaving transposable element transcripts [ ].
The total DNA is contained in 46 double-stranded DNA
diploid human cell possesses 46 chromosomes: two of each -
males. Since the length of each helical turn of a doublestranded DNA molecule is 3.4 nm and consists of ten bases, the length of the total genomic DNA in each cell measures
For each cell to contain these long DNA molecules, the contains positively-charged amino acids that bind to 146 either partially or tightly, resulting in compression of the DNA strand. Tight folding of the DNA condenses the DNA
visualization of condensed metaphase chromosomes. million sites with less condensed DNA in the genome [ ].-
DNA regions. These proteins also may prevent access to nucleic acid probes or primers for clinical molecular tests.
between the nucleic acid and these proteins that can cause molecular testing artifacts (e.g., false-negative results). As a
digestion step to liberate the DNA from the DNA-binding proteins. Removal of the proteins facilitates hybridization with short pieces of nucleic acid, such as primers or probes.
DNA Replication
Eukaryotic DNA Replication
cific physiological temperatures and a host of proteins. As mentioned previously, clinical molecular testing methods rely on the ability to denature or melt a double-stranded -
logical conditions, dissociation of DNA strands for replication is accomplished by numerous enzymes, such as helicases and topoisomerases. The region of transition from doublestranded to separated single-stranded DNA is called the repdouble-stranded DNA molecule as replication proceeds. At polymerases bind to the original or parental DNA strands and generate two new daughter strands. Known collectively as a replisome, these enzymatic activities generate two new nucleic acid strands that are complementary to and base paired with each of the original two template or parent DNA
tive because each resulting double-stranded DNA molecule consists of one new and one old DNA strand (Fig. 1.3).
′ to 3′ direction, and the polymerase synthesizes only by reading the parent strand in a 3′ ′ direction while ′ to 3′ direction. Therefore, synthesis cannot proceed continuously along the lagging strand, which must be copied in short stretches primed from
lagging strand is formed by removal of the RNA primer regions and ligation of the short DNA fragments into a continuous daughter strand complementary to the lagging strand.
Discontinuous 3′ ′ replication results in the progres-
normal cells. The guanine-rich telomeres form secondary structures (or caps) that prevent chemical processes that can damage the chromosome. Apoptosis occurs when the number of uncapped telomeres reaches a critical threshold that trig-
to telomere homeostasis by adding bases to the 3′ end.
acid molecule, and the polymerase adds nucleotides accordand A:T pairing. The new strand is antiparallel to the parent ′ to 3′ direction. Of the two parent strands of genomic DNA, one strand (called the leading strand) can be read continuously in a 3′ ′ direction by the polymerase, with the new strand generated in a continu′ to 3′ as the lagging strand) cannot be read continuously by the
Figure 1.3 depicting the leading and lagging strands and involved with replication. Reprinted with
monary fibrosis [ ]. Telomerase activity varies with cell shortening than granulocytes. Telomeres shorten with age with the most prominent shortening occurring between birth and the first year of age, followed by childhood and after puberty or adulthood [ changes, some malignant cells retain telomerase activity that the chromosomes, prolonging the life of the cell.
determines the speed and accuracy of new strand synthesis. The rate that the four nucleotides are polymerized into a nucleic acid chain defines the processivity of the enzyme. bases per minute.
Replicated DNA double helixes
Leading strand
Lagging strand
Table 1.4 Fidelity of various polymerases
PolymeraseError rate (×10−6)
The fidelity of the polymerase refers to the accuracy of the enzyme to incorporate the correct complementary bases
bases or other replication errors can result in cell death or oncogenesis. The error rate of polymerases varies widely 1.4). DNA is susceptible to base pair changes while in the single-stranded form due to the activity of various deaminating enzymes.
have been associated with somatic hypermutation of rearranged immunoglobulin genes [ ].
This DNA editing process may be a mechanism to protect the host genome from viruses replicating within the nucleus [33, 34]. To correct the erroneous incorporation of bases or
Figure 1.4 clear panels are the ordered phases of mitosis (M phase), while the gray and black panels are the ordered stages of interphase. A anaphase, G1 gap 1, G2 MET metaphase, P prophase, PM prometaphase, S DNA synthesis, T telophase
This efficient replication process depends on the circular topology of the bacterial genome.
lication is the mechanism by which bacterial chromosomes
(Fig. 1.4). Malignant cells may not pause to allow for error correction, resulting in the accumulation of damaged or mutated DNA.
high degree of regulation for generating two strands from
These multiple sites grow progressively until the newly generated strands join to form complete chromosomallength DNA.
Bacterial and Mitochondrial Replication
6 base pairs) are replicated by a simpler mechanism
origin of replication initiates the duplication of the bacterial chromosome, and replication occurs simultaneously on both strands in opposite directions from the origin of replication.
viruses (i.e., bacteriophages). As a result, many bacteria produce restriction enzymes that degrade foreign nucleic acids.
chromosomal DNA prevents most restriction enzymes from digesting the chromosomal DNA of the bacteria. Following replication, methylating enzymes add methyl groups to the new bacterial chromosomal DNA, preventing chromosomal degradation by the restriction enzymes. This methylation and restriction process functions as a primitive immune system by destroying foreign bacteriophage DNA before it can usurp the bacteria’s replication system. Bacterial restriction enzymes are used to specifically cleave DNA in clinical molecular tests and can be used to identify genetic variations.
Additional types of replication occur in some viruses and bacteria. The rolling-circle mechanism of replication
stranded circular genomes, followed by replication pro′ to 3′ direction. The new strand
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the welfare of the state, would follow his example.
The council followed his highness into the church and at his request took the oath before him; they were then introduced by him into the presence of the empress mother, who was pleased to inform them that the act and its content were known to her, and were made with her maternal consent, but that she also was enthusiastic over her son’s conduct. Confirming all his actions she requested the council by their united endeavours to preserve the tranquillity of the empire.
N I (1796-1855)
In accordance with the measures taken, by three o’clock in the afternoon the troops as well as all grades of officials in the government service had taken the oath confirming the accession to the throne of the emperor Constantine. During the whole time tranquillity and order were preserved. It is easy to imagine the astonishment and vexation of the czarevitch when, instead of receiving the expected commands of the new emperor, he was informed that all Russia had taken the oath of allegiance to him as lawful sovereign, and that the will of the late emperor had not been fulfilled.
Meanwhile early in the morning of December 15th the grand duke Michael Pavlovitch arrived in St. Petersburg with letters from the czarevitch. To the amazement of the court and the inhabitants, the grand duke did not follow the general example of swearing fidelity to the emperor Constantine. He did not conceal his regret at what had taken place in St. Petersburg, nor the apprehension with which the necessity of a new oath filled him. He dwelt on the difficulty of explaining to the public why the place of the elder brother to whom
allegiance had already been sworn should suddenly be taken by the younger. The grand duke Nicholas in answer to his brother repeated what he had already said, that he could not have acted otherwise in such a position as that in which he was placed by his ignorance of the sacred acts of the late emperor, and that neither his conscience nor his reason reproached him. “Everything, however,” added he, “might yet be amended and take a more favourable turn if the czarevitch himself were to come to St. Petersburg; his obstinacy in remaining at Warsaw may occasion disasters, the possibility of which I do not deny, but of which in all probability I shall myself be the first victim.”
After long deliberation the grand duke Nicholas decided to write a fresh persuasive letter to the emperor Constantine, in which he asked him to decide finally what his fate was to be; and in conclusion he wrote, “In God’s name, come.” The empress Marie Feodorovna added her persuasions to those of her son, and not satisfied with these measures it was decided a few days later to despatch the grand duke Michael to Warsaw to convince the czarevitch of the necessity of his presence in St. Petersburg.
An answer from the czarevitch to the grand duke Nicholas’ letter dated the 14th of December was brought to St. Petersburg by Lazarev, aid-de-camp to Nicholas: “Your aide-de-camp, dear Nicholas, on his arrival here, confided your letter to me with all exactitude. I read it with the deepest grief and sorrow. My decision is unalterable and consecrated by my late benefactor the emperor and sovereign. Your invitation to come quickly cannot be accepted by me, and I must tell you that I shall remove myself yet further away, if all is not arranged in accordance with the will of our late emperor Your faithful and sincere friend and brother for life.” But even this letter did not decide the matter; the return of Belussov from Warsaw with the answer to the grand duke Nicholas’ letter of December 15th had yet to be awaited.
A new complication remained to be added to all these difficulties. On December 24th there came to St. Petersburg and presented himself to the grand duke Nicholas, Colonel Baron Fredericks of the Izmailovski Life Guards, who had fulfilled the functions of
commandant in Taganrog. He brought to the grand duke a packet from Baron Diebitsch addressed to his imperial majesty, to be given into his own hands. To the question as to whether he knew of the contents of the packet, Fredericks replied in the negative, but added that as the place of residence of the emperor was unknown in Taganrog, exactly the same paper had been sent also to Warsaw.
Nothing therefore remained for Nicholas to do but to open the mysterious packet and “at the first rapid glance over its contents,” writes Baron Korv, “an inexpressible horror took possession of him.” It was on reading the report contained in this packet that the grand duke first learned of the existence of secret societies formed with the object of destroying to the very roots the tranquillity of the empire. The existence of these societies had been carefully hidden from him by the late emperor Alexander.
Almost immediately thereafter the courier Belussov returned from Warsaw with the czarevitch’s decisive answer, which put an end to the interregnum. Nicholas Pavlovitch was emperor. At nine o’clock in the evening the emperor sent the following postscript to Adjutantgeneral Diebitsch:
The decisive courier has returned; by the morning of the day after to-morrow I shall be emperor or else dead. I sacrifice myself for my brother; happy if as a subject I fulfil his will. But how will it be with Russia? What about the army? General Tolle is here and I shall send him to Mohilev to bear the news to Count Saken. I am looking out for a trustworthy person for the same commission to Tultchin and to Ermolov. In a word, I hope to be worthy of my calling, not in fear and mistrustfulness, but in the hope that even as I fulfil my duty so will others fulfil their duty to me. But if anywhere anything is brewing and you hear of it, I authorise you to go at once where your presence is necessary I rely entirely upon you and give you leave beforehand to take all the measures you deem necessary The day after to-morrow if I am alive I will send you, I do not know by whom, information as to how matters have passed off; on your part do not leave me without news of how everything is going on around you, especially with Ermolov I again repeat that here until now everything is incomprehensibly quiet, but calm often precedes a storm. Enough of this, God’s will be done! In me there must only be seen the vicar and executor of the late emperor’s will and therefore I am ready for everything. I shall ever be your sincere well wisher,
N .
THE ACCESSION OF NICHOLAS
The czarevitch’s decisive answer was brought by Belussov, not through Riga, but by the Brest-Lithuani road; and therefore the grand duke Michael Pavlovitch was still in ignorance of the events at Nennal. The emperor Nicholas immediately sent an express after him commanding him to hasten to St. Petersburg. The return of the grand duke to the capital where his presence was of urgent necessity was thus by chance delayed.
Nicholas had now to occupy himself with the composition of his manifesto; the inexplicable had to be explained and it presented a task of no little difficulty: Karamzin and Speranski were set to work upon it. The emperor Nicholas signed the manifesto on the 25th of December, but dated it the 24th, as the day on which the question of his accession had been definitely settled by the czarevitch. It was proposed to keep the manifesto secret until the arrival of the grand duke Michael, but it was decided that the troops should take the oath of allegiance on the 26th of December; meanwhile notifications were sent to the members of the council of state, calling upon them to assemble on Sunday, December 25th, at eight in the evening, for a general secret meeting.
When the council of state had assembled at the hour designated, Prince Sopukhin announced that the grand duke Michael would be present at the sitting. The hours passed in anxious expectation; midnight approached and the expected arrival of the grand duke did not take place. Then Nicholas decided to be present at the sitting alone. Taking the place of the president, Nicholas himself began to read the manifesto announcing his acceptance of the imperial dignity in consequence of the persisted rejection of it by the czarevitch Constantine Pavlovitch. Then the emperor ordered that the czarevitch’s rescript, addressed to Prince Sopukhin, president of the council, should be read. The 26th of December, 1825, had come. Commands had been issued that on that day all persons having access to the court should assemble at the Winter Palace for a Te Deum; eleven o’clock was the hour first named, but this was afterwards changed to two. Circumstances arose, however, which postponed the Te Deum to a still later hour. The members of the secret society decided to take advantage of the end of the
interregnum and the approach of the new oath of allegiance in order to incite the troops to rebellion and to overthrow the existing order of things in Russia. The secrecy in which the negotiations with Russia had been enveloped had given occasion for various rumours and suppositions, and for the spread of false reports which occasioned alarm in society and especially in the barracks: all this favoured the undertakings and designs of the conspirators.
The only issue from the position that had been created by Nicholas in a moment of chivalrous enthusiasm “undoubtedly noble, but perhaps not entirely wise,” would have been the arrival of the grand duke Constantine in the capital with the object of publicly and solemnly proclaiming his renunciation of the throne. But the czarevitch flatly refused to employ this means of extricating his brother from the difficult position in which he placed himself; Constantine considered that it was not for him to suffer from the consequences of an imprudence which was not his, and the danger of which might have been averted if matters had not been hurried on, and if he had been previously applied to for advice and instructions. Thus led into error, some of the lower ranks of the guards’ regiments refused to take the oath of allegiance to Nicholas Pavlovitch, and assembled at the Pelrovski square, before the senate buildings, appearing as though they were the defenders of the lawful rights of the czarevitch Constantine to the throne.
Meanwhile distinguished persons of both sexes began to drive up to the Winter Palace. Amidst the general stir and movement going on in the palace, there sat isolated and immoveable three magnates, “like three monuments,” writes Karamzin: Prince Lopukhin, Count Araktcheiev, and Prince A. B. Kurakin. At the time when the military men had already gone out on the square, Count Araktcheiev, as might have been expected, preferred to remain in the palace. “It was pitiful to look at him,” writes V. R. Martchenko in his Mémoires.
The rioters were stubborn for a long time and would not yield to exhortation; Count Miloradovitch fell mortally wounded. It began to grow dusk. Then the emperor Nicholas, at last convinced of the impossibility of pacifying the rioters without bloodshed, gave orders with a breaking heart for the artillery to fire. A few grape-shot
decided the fate of the day; the rioters were dispersed, and tranquillity at once reigned in the capital.
The Te Deum announced could take place only at half past six. The troops bivouacked round the palace. “Dear, dear Constantine,” wrote the emperor the same evening to the czarevitch, “your will is fulfilled: I am emperor, but at what price, my God!—at the price of the blood of my subjects.” Arrests were made during that night and investigations pursued to discover the leaders of the revolt. And thus in the troubles of the 26th of December, the 1st of December, 1825, was terribly recalled. “The day was one of misfortune for Russia,” writes Prince Viasenski, “and the epoch which it signalised in such a bloody manner was an awful judgment for deeds, opinions, and ideas, rooted in the past and governing the present.” According to the words of Karamzin, on that day Russia was saved from a calamity “which, if it had not destroyed her, would certainly have torn her to pieces.” “If I am emperor even for an hour, I will show that I was worthy of it”; thus spoke Nicholas on the morning of December 26th to the commanders of the guard regiments assembled at the Winter Palace; and on that awful day he triumphantly justified his first and impressive words.
TRIAL OF THE CONSPIRATORS (1826 A.D.)
The emperor Nicholas gave all possible publicity to the proceedings against the secret societies, the Southern, Northern, the United Slavonians, and the Polish; then the whole matter was transferred to the supreme criminal court, which had to pronounce sentence on the principal participators in the conspiracy. Of the accused, Rileeks, Muraviev-Alostob, Bestuzhev-Riumin, Pesteb, and Kakhovski were condemned to death, and the remaining members of the secret societies brought before the court were exiled to Siberia or other places of incarceration.
[1826 ]
No one had expected such a termination to the affair. During the whole of Alexander’s reign there had not been one case of capital punishment, and it was looked upon as entirely abolished. “It is
impossible to describe in words the horror and despair which have taken possession of all,” writes a contemporary and eye witness of the events of 1826 in Moscow. This frame of mind was reflected in the coronation ceremonies. The emperor Nicholas appeared extremely gloomy; the future seemed more sad and fuller of anxiety than ever; all was in sharp contrast to the enthusiasm and hopes that had accompanied the coronation of Alexander in 1801.
THE CORONATION OF NICHOLAS (1826 A.D.)
Immediately after the termination of the trial of the Dekabrists, the court proceeded to Moscow for the approaching coronation, which took place on the 3rd of September Previously the emperor was rejoiced at the unexpected arrival of the grand duke Constantine Pavlovitch. According to Benkendorf “the czarevitch’s appearance was a brilliant public testimony of his submission to the new emperor and of his conscientious renunciation of the throne; it was at the same time a precious pledge of the harmony which bound together all the members of the reigning family, a harmony conducive to the peace of the empire. The public was delighted and the corps diplomatique completely astounded. The people expressed their satisfaction to the czarevitch by unanimous acclamations, whilst the dignitaries of the state surrounded him with marks of respectful veneration.”
The day of the coronation was signalised by an important reform in the administration of the court; the ministry of the imperial court was created, and confided to Prince P. M. Volkonski. Thus the old and tried companion of the emperor Alexander I again occupied the post of a trusty dignitary by the side of his successor. Prince Volkonski remained minister of the court until his decease, which took place in 1852. Amongst the favours and the mitigations of punishments which were granted on the 3rd of September, the state criminals who had lately been condemned were not forgotten; by special ukases the sentences of all those sent to the galleys, to penal settlements, and hard labour were mitigated. Those who had been sent to the Siberian, Orenburg, and Caucasian garrisons, both with and without
deprivation of the rights of nobility, were enrolled in the regiments of the Caucasian corps.
During the emperor’s stay in Moscow, the poet Pushkin, who had been banished to the village of Mikhailovski, was recalled. From that moment he regained his lost liberty, besides which the emperor Nicholas said to him: “In future you are to send me all you write— henceforth I will be your censor.”
CHANGES IN INTERNAL ADMINISTRATION
On the 18th of October, 1826, the emperor Nicholas returned to St. Petersburg; although his accession to the throne did not constitute the opening of a new era for Russia, yet certain changes were made in the system of administration which had prevailed during the last decade of the reign of Alexander I. After Count Araktcheiev had been relieved of the management of the general affairs of the state, it was to be foreseen that he would not remain long at the head of the direction of the military settlements. And thus it turned out. In the spring of 1826 Count Araktcheiev, on account of illness, was given leave to go abroad. In the report presented by him on this occasion to the emperor he announced to him economies of more than 32,000,000 rubles made on the military settlements, and concluded his epistle by observing, “Those impartial judges— posterity and the future—will pronounce a just sentence on all things.”
On the return of Count Araktcheiev in the autumn from his travels abroad he did not again take up his duties. In accordance with a ukase which then followed, the staff office of the military settlements was united to the general staff of his imperial majesty, under the jurisdiction of its adjutant-general Baron Diebitsch. At the same time the Novgorod military settlement passed under the entire direction of General Prince Schahovski, who was nominated commander of the grenadier corps; the Kherson and Iekaterinoslav settlements were put under the supervision of their chief, Count Vitt (who was also commander of a separate corps), while the settlements in the villages of the Ukraine and Mohilev governments remained under the
jurisdiction of their former chiefs, who bore the rank of commanders of divisions. Count Araktcheiev, when he had finally bidden adieu to his administrative career, settled on his Georgian estates, where he died in 1834.
Having delivered Russia from the administrative guardianship of Count Araktcheiev, the emperor Nicholas, in addition, delivered Russian instruction from the influence of Michael Leontievitch Magnitzki. On the 18th of May, 1826, a ukase was issued in which it was stated that “the curator of the University of Kazan and of its educational district, the actual councillor of state Magnitzki, is by our command relieved of his functions and of his position as member of the administration of schools.” But the matter was not limited to this ukase. Magnitzki continued to live in Kazan and in accordance with his character he continued to intrigue as usual and indirectly to influence the university he had left. General Jeltukhin, who had been commissioned to make a detailed revision of the Kazan University, brought this fact to the emperor’s knowledge. Nicholas’ reply was rapid and decisive; a courier was sent with orders to the governor to arrest Magnitzki and send him to Revel under the surveillance of the commandant. Magnitzki lived there six years, having given his promise not to absent himself.
An equally sad fate overtook the champion and imitator of Magnitzki, Dmitri Pavlovitch Runitch, who had filled the office of curator of the St. Petersburg educational district. By a ukase of the 7th of July, 1826, Runitch was deprived of his functions and of the position of member of the chief administration of schools, for his incompetence in the matter of the direction of the St. Petersburg educational district. The requital experienced by Runitch for his educational labours was a terrible one; he languished beneath the consequences for sixteen years and died in 1860 in the conviction that he had formerly saved Russia, and was suffering for the good work he had accomplished in the University of St. Petersburg.
Reforms in the Administration of Justice
The lamentable condition of the administration of justice in Russia was one of the first subjects to which the careful attention of the emperor Nicholas was directed. In a speech pronounced by the sovereign many years later, in 1833, before the council of state, Nicholas Pavlovitch thus expressed himself:
“From my very accession to the throne I was obliged to turn my attention to various administrative matters, of which I had scarcely any notion. The chief subject that occupied me was naturally legislation. Even from my early youth I had constantly heard of our deficiencies in this respect, of chicanery, of extortion, of the insufficiency of the existing laws or of their admixture through the extraordinary number of ukases which were not infrequently in contradiction to one another. This incited me from the very first days of my reign to examine into the state of the commission appointed for the constitution of the laws. To my regret, the information presented to me proved to me that its labours had remained almost fruitless. It was not difficult to discover the cause of this: the deficient results proceeded chiefly from the fact that the commission always directed its attention to the formation of new laws, when in reality the old ones should have been established on a firm foundation. This inspired me above all with a desire to establish a definite aim towards which the government must direct its actions in the matter of legislation; from the methods proposed to me I selected one in entire opposition to the former methods of reform. Instead of drawing up new laws, I commanded that first those which already existed should be collected and set in order, whilst I took the matter itself, on account of its great importance, under my own immediate direction and closed the previous commission.”
With this object was formed and opened on the 6th of May, 1826, the “second section of his imperial majesty’s own chancery.” M. A. Balongianski was appointed chief of the second section, but in reality the work itself was confided to Speranski. The emperor’s choice rested on the latter, out of necessity, as he did not find anyone more capable around him. When Balongianski was appointed chief of the second section, the emperor, in conversing with his former tutor, said to him, speaking of Speranski: “See that he does not play any
pranks, as in 1810.” Nevertheless, in proportion to Speranski’s successful accomplishment of the work confided to him, the emperor Nicholas’ prejudices against him gradually softened and finally gave way to sincere favour and full confidence. All the accusations and calumnies directed against Speranski were, in accordance with the emperor’s own expression, “scattered like dust.”
Thus the emperor Nicholas in his almost involuntary choice was favoured by a peculiarly fortunate chance and could hardly have found a person better fitted for the accomplishment of the work he had planned. The results of Speranski’s fresh efforts, under completely different circumstances from those against which he had formerly contended, were the “complete collection of laws,” and a systematic code.
Even before the termination of the trial of the Dekabrists, the emperor Nicholas took another important measure, which left an imprint on all the succeeding years of his reign and is directly connected with the events of the 26th of December On the 15th of July, 1826, a supreme edict was issued in the name of the minister of the interior Lanskoi, by which the private chancery of that ministry was abolished and transformed into the third section of his imperial majesty’s own chancery. In fulfilment of this ukase, it was prescribed that the governors of provinces, in matters which entered within the sphere of the former division, should no longer present their reports to the ministry of the interior, but should submit them directly to his majesty.
M W V
A W (S ) N T
Some days before, on the emperor Nicholas’ birthday, the 6th of July, a supreme order appeared naming the chief of the first cuirassier division, Adjutant-general Benkendorf, chief of the gendarmerie and commandant of the emperor’s headquarters; to him was confided the direction of the third section. Adjutant-general Benkendorf explains in his memoirs in the following manner the reasons for establishing the institution confided to his direction: “The emperor Nicholas aimed at the extirpation of the abuses that had crept into many branches of the administration, and was convinced by the sudden discovery of the conspiracy which had stained the first moments of the new reign with blood, of the necessity of a universal and more diligent surveillance. The emperor chose me to organise a higher police, which should protect the oppressed and guard the nation against conspiracies and conspirators. Never having thought of preparing myself for this sort of service, I had hardly the most superficial understanding of it; but the noble and beneficent motives which inspired the sovereign in his creation of this institution and the desire to be of use to him, forbade me to evade the duty to which his high confidence had called me. I set to work without delay and God helped me to fulfil my new duties to the satisfaction of the emperor and without setting general opinion against me. I succeeded in showing favours to many, in discovering many conspiracies, and averting much evil.” With the creation of the
new third section, the committee of the 13th of January, 1807, established by the emperor Alexander, became superfluous; and on the 29th of January a ukase was issued closing it.
The disturbances of the year 1825 did not pass without leaving traces on the peasant population; a momentary confusion ensued, freedom was talked of, and disorders arose in some provinces—a phenomenon often seen in previous times. The movement amongst the peasants incited the emperor Nicholas to publish, on the 24th of May, 1826, a manifesto in which it was declared that all “talk of exempting the villagers in the state settlements from paying taxes and of freeing landowner’s peasants and menials from subjection to their landowners are false rumours, imagined and spread by evil intentioned persons out of mere cupidity with the object of enriching themselves through these rumours at the expense of the peasants, by taking advantage of their simplicity.” It was further said in the manifesto that all classes throughout the empire must absolutely submit to the authorities placed over them, and that disturbers of the public tranquillity would be prosecuted and punished in accordance with the full severity of the laws. It was commanded that the manifesto should be read in all the churches and at the markets and fairs during a space of six months; the governors of provinces were sternly admonished to be watchful in anticipating disorders.
If, however, the emperor Nicholas was forced by circumstances to promulgate this punitive manifesto, he also issued two rescripts in the name of the minister of the interior, enjoining upon the nobility behaviour towards their peasants, which should be in accordance with the laws of Christianity, thus clearly expressing his desire to protect the peasant against the arbitrariness and tyranny of the landowners. “In all cases,” wrote the emperor: “I find it, and shall ever find it, better to prevent evil, than to pursue it by punishment when it has already arisen.”
Finally the solicitude of the emperor Nicholas for the peasant classes manifested itself by yet another action. On the 18th of December, 1826, a special secret committee was formed to which was confided the inspection of the entire state organisation and administration, with the order to represent the conclusions it arrived
at as to the changes deemed necessary; the labours of the committee were to be directed also to the consideration of the peasant question. Besides this the emperor did not leave without attention what had been said by the Dekabrists, during the time of their examination before a committee of inquiry, in regard to the internal conditions of the state in the reign of Alexander I. The emperor ordered a separate memorandum of these opinions to be drawn up for him and often perused this curious document, from which he extracted much that was pertinent.b
WAR WITH PERSIA (1826-1828 A.D.)
The shah of Persia thought he saw in the change of rulers and the troubles by which it was accompanied circumstances favourable to the recovery of the provinces ceded to Russia by the Treaty of Gulistan. In August, 1826, he ordered his troops to move forward. The solemnity of his coronation, which was then being celebrated and whose splendour was enhanced by the presence of the czarevitch, did not prevent Nicholas from promptly organising the defence of the empire. A few weeks afterwards General Paskevitch defeated the Persians at Ielisavetpol, and in the following year, transferring the theatre of war to the enemy’s territory, he seized the celebrated convent of Etchmiadzine, the seat of the Armenian patriarch, and Erivan, one of the great towns of Armenia; he moreover penetrated as far as Tauris, capital of the Azerbaijan and residence of the prince royal, Abbas Mirza. Then the shah asked for peace. It was signed at Turkmantchaï, the 22nd of February, 1828, and advanced Russia as far as the line of the Araxes, by giving up to her the provinces of Erivan and Nakhitchevan.
[1826-1828 ]
WAR WITH TURKEY (1828-1829 A.D.)
This treaty was concluded, to the great regret of Persia, when the war with Turkey broke out. This war had been threatening for years; for, deeply affected by the violences to which the Greeks in the
Ottoman Empire had been exposed ever since the hetaerist insurrection of 1821, and by the martyrdom which the Greek patriarch had been made to suffer, Alexander left the sword in its sheath only out of deference to the members of the Holy Alliance. His successor was thoroughly determined no longer to subordinate the direction of his cabinet’s policy to the interested views of these princes and to their fears, though it is true that the latter were well founded. The Divan, by signing the Treaty of Akerman (October 6th, 1826), had momentarily averted the storm which was ready to burst; but still more irritating disputes had afterwards arisen. The conclusion of the Treaty of London of the 6th of July, 1827, in virtue of which France, England, and Russia gave existence to a Christian kingdom of Greece placed under their common protection, was shortly followed by the naval battle of Navarino, fought on the 20th of October of the same year by the combined fleets of the three powers, against Ibrahim Pasha, commander-in-chief of the Egyptian forces in the Morea; and in this memorable conflict, expected by no one, but a subject of joy to some whilst judged untoward by others, the whole of the navy which the Porte still had at its disposal was destroyed. Very soon Mahmud II, yielding to the national desire, let it be understood that he had never had any intention of lending himself to the execution of a treaty in virtue of which Moldavia, Wallachia, and Servia were almost as much the czar’s vassals as his own. This was the beginning of a rupture, and Nicholas answered it by a declaration of war, which appeared June 4th, 1828, when his army had already crossed the Pruth.
The campaign of 1828, which accomplished nothing more than the taking of Braila and Varna, did not give a high idea of the strength of Russia; and when the emperor made up his mind to take part in it in person, his presence wrought no change in the feebleness of the results. But it was not the same with the campaign which followed. Not only did the Russians again pass the Danube, but after having beaten the grand vizir, Reschid Pasha, at Koulevtcha, on the 11th of June, Diebitsch marched them across the Balkans for the first time, a feat which won him the name of Sabalkanski, and proceeded straight to Adrianople, where he was scarcely more than two hundred kilometres (about 125 miles) from the Ottoman capital. At the same
time Paskevitch took Erzerum in Asia, and the two generals would doubtless have joined hands in Constantinople but for the efforts of diplomacy and the fear of a general conflagration. For Russia was already too powerful; she had been allowed more than was compatible with the policy of the system of balance, no doubt from the fear of incurring a grave responsibility by troubling the peace of Europe. But a prospect like that of the occupation by Russia of Constantinople and the Straits silenced this fear.
Austria was ready to send her troops to the help of the Turks, and the English also seemed likely to declare for the vanquished. It was therefore necessary to come to a halt. Russia reflected that, after all, “the sultan was the least costly governor-general she could have at Constantinople,” and lent an ear to moderate conditions of peace. Nevertheless, if the Treaty of Adrianople, signed September 14th, 1829, delivered nothing to her in Europe save the mouths of the Danube, in itself a very important point, it enlarged her territories in Asia by a part of the pashalik of Akhalzikh, with the fortress of that name, besides abandoning to her those of Anapa and Pothi on the Black Sea; it considerably strengthened Muscovite influence in the principalities, and still further weakened Turkey, not only morally but also materially by the great pecuniary sacrifices to which she had to subscribe. That power, once so formidable, was henceforth at the mercy of her northern neighbour, the principal instrument of her decay.
[1829 ]
[1830 ]
THE POLISH INSURRECTION (1830-1831 A.D.)
But Russia was in her turn rudely shaken by the insurrection in Poland, always her mortal enemy after she had ceased to be her rival.c
It was in Moscow that the emperor Nicholas received news of the further progress of the Belgian revolution, in consequence of which the king of the Netherlands found himself obliged to ask for the assistance of his allies by virtue of the existing treaties. The emperor at once despatched orders to Count Tchernishev, Field-marshal
Saken, and the czarevitch to place the army on a war footing. The czarevitch was not pleased at the martial turn given to the diplomatic negotiations; still more dissatisfied was the Polish Society of that time, which sympathised with the revolution of July; neither was the army in sympathy with the approaching campaign, which would bring it into armed collision with France in the name of the principles of the Holy Alliance. Although tranquillity apparently reigned in Warsaw, yet the secret societies continued to carry on their destructive work with success.
Various ominous signs of the approaching catastrophe were not, however, wanting; but the czarevitch continued to lull himself with impossible hopes that all was peaceful and tranquil and would remain so. As to the European powers allied to Russia, they did not enter into the matter with such decided zeal. In the present case it was the Russian autocrat alone who was ready with entire disinterestedness to take up the defence of the infringed lawful order. The other powers found it incomparably more expedient to have recourse to the cooperation of diplomatic remedies; the result was that, instead of an armed intervention, a general European conference for the settlement of the Belgian question by peaceful means took place in London.
C D -S (1785-1831)
Count Diebitsch was still in Berlin awaiting the termination of the negotiations confided to him, when they were suddenly broken off by an event upon which the field-marshal had not in the least calculated at the given moment. On the 3rd of December, 1830, Diebitsch
received from the Prussian minister, Count Berastorf, news of the revolution which had taken place in Warsaw on the 29th of November: the Polish army, forming a prepared coalition, had taken up arms against Russia. There remained but one thing for Diebitsch to do and that was to hasten to St. Petersburg as quickly as possible. Meanwhile in St. Petersburg the emperor Nicholas had received only the report of the czarevitch concerning the rising of the troops and of inhabitants of Warsaw on the evening of the 7th of December, 1830.
On the next day a parade of the Preobrajenski regiment was appointed to take place, and as usual the emperor came to the riding school. At first everything proceeded in the usual manner; there were even no traces of inward agitation manifest upon the handsome face with its regular, classic profile, which preserved its habitual expression of majestic nobility. At the termination of the parade the emperor rode into the middle of the riding school, called the officers around him, and personally communicated to them the intelligence of the Warsaw rebellion: “I have already made arrangements that the troops designated by me should move on Warsaw, and if necessary you too shall go, to punish the traitors and re-establish order and the offended honour of Russia. I know that under every circumstance I can rely upon you,” said the emperor. A unanimous outburst of indignation momentarily seized upon all present and then enthusiastic cries resounded: “Lead us against the rebels: we will revenge the offended honour of Russia.” They kissed the emperor’s hands and feet and the hem of his garment with shouts and cheers. The outburst of indignation was so violent that Nicholas considered it necessary to moderate it, and with the majesty that was natural to him he reminded the officers surrounding him that not all the Poles had broken their oath; that the ringleaders of the insurrection must be punished, but that vengeance must not be taken on the people: that the repentant must be pardoned and hatred not allowed.
From the subsequent reports of the grand duke the emperor learned that the czarevitch had permitted the portion of the Polish army that remained with him to return to Warsaw; in exchange for this the deputies who came to the czarevitch promised him and the
Russian detachment a free passage to the frontiers of the empire. It was decided that a sufficient number of troops should be concentrated in the Polish frontier to allow of decisive measures being taken against the insurgents. Count Diebitsch was appointed commander-in-chief of the acting army, whilst the office of chief of the staff was filled by Count Tolle.
When the czarevitch reached the Russian frontier he wrote as follows to the emperor Nicholas: “And now the work of sixteen years is completely destroyed by a set of ensign-bearers, young officers, and students. I will not further enlarge on the matter, but duty commands me to bear witness to you that the landed proprietors, the rural population, and in general all holders of property of any kind are up in despair over this. The officers and generals as well as the soldiers are unable to keep from joining the general movement, being carried away by the young people and ensign-bearers who led everyone astray. In a word, the position of affairs is extremely bad, and I really do not know what will come of it. All my measures of surveillance have led to nothing, in spite of the fact that everything was beginning to be discovered. Here are we Russians at the frontier, but great God in what a condition!—almost barefoot, for we all came out as if at the sound of an alarm, in the hopes of returning to barracks, whilst instead awful marches have had to be made. The officers have been deprived of everything and have almost nothing with which to clothe themselves. I am broken hearted; at the age of fifty-one and a half years I never thought to finish my career in this lamentable manner after thirty-five and a half years of service. I pray to God that the army to which I have devoted sixteen years of my life may be brought to reason, and return to the path of duty and honour, acknowledging its previous errors, before coercive measures have to be taken. But this is too much to expect from the age in which we live, and I greatly doubt the realisation of my desires.”
Any agreement with Poland became daily more impossible and both sides prepared for war. On the 17th of December the emperor Nicholas’ proclamation to the Polish army and nation was issued, and on the 24th a manifesto was published offering means of reconciliation to all those who returned to their duty. Meanwhile
General Chlopicki was installed as dictator in Warsaw, but he was unable to save Poland from a rupture with Russia. Two deputies were sent to St. Petersburg to enter into negotiations with the emperor Nicholas; they were the minister of finance, Prince Lubetzki and a member of the diet, Count Ezerski. But neither could these negotiations avert the bloody events of the year 1831. “It is hard to foresee the future,” wrote the emperor to the czarevitch; “but weighing the relative probabilities of success, it is difficult to suppose that the new year will show itself more distressing for us than the year 1830; God grant that I may not be mistaken. I should like to see you peacefully settled in your Belvedere and order re-established throughout; but how much there yet remains to be accomplished before we are in a condition to attain to this! Which of the two must perish—for it appears inevitable that one must perish, Russia or Poland? Decide for yourself. I have exhausted all possible means in order to avert such a calamity—all means compatible with honour and my conscience—but they are exhausted. What remains for me to do?”
Soon the diet assembled in Warsaw took a decision which completed the rupture between Poland and Russia. On the 25th of January, 1831, the diet declared the Romanov dynasty to be deprived of the throne of Poland. The Poles themselves thus unbound the hands of the emperor, and the duel between Russia and Poland became inevitable. The emperor replied to the challenge by a manifesto in accordance with which the Russian troops crossed the Polish frontier, and on the 25th of February a decisive battle took place before Prague at Grokhov, by which the Polish army was obliged to retreat to Warsaw with a loss of twelve thousand men.
[1831 . .]
But Count Diebitsch did not recognise the possibility of taking advantage of the victory gained, and which would have been inevitably completed by the occupation of the Polish capital; and Sabalkanski was not fated to become prince of Warsaw. The Polish troops retreated unhindered across the only bridge to Warsaw; the new Polish commander-in-chief Skrjinetzski set out to reorganise the army, the rising spread even to the Russian governments, and the
