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Genetic Engineering
Principles and Methods

Volume 27
 GENETIC ENGINEERING
Principles and Methods

Advisory Board

Carl W. Anderson
Peter Day
Donald R. Helinski
Maynard V. Olson
John Shanklin

A Continuation Order Plan is available for this series. A continuation order will bring deliv-
ery of each new volume immediately upon publication. Volumes are billed only upon actual
shipment. For further information please contact the publisher.
Genetic Engineering
Principles and Methods

Volume 27
Edited by

Jane K. Setlow
Brookhaven National Laboratory
Upton, New York
The Library of Congress cataloged the first volume of this title as follows:
Genetic engineering: principles and methods. V. 1–
New York, Plenum Press. (1979–
v. ill. 26 cm.
Editors: J. K. Setlow and A. Hollaender
Key title: Genetic engineering. ISSN 0196-3716
1. Genetic engineering—Collected works. I. Setlow, Jane K. (1979–) II. Hollaender,
Alexander, (1979–1986).

QH442.G454 575.1 76-644807

MARC-S

ISBN-10: 0-387-25855-8 e-ISBN 0-387-25856-6

ISBN-13: 978-0387-25855-3

Printed on acid-free paper.

© 2006 Springer Science+Business Media, Inc.

All rights reserved. This work may not be translated or copied in whole or in part without the writ-
ten permission of the publisher (Springer Science+Business Media, Inc., 233 Spring Street, New
York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analy-
sis. Use in connection with any form of information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed is
forbidden. The use in this publication of trade names, trademarks, service marks, and similar
terms, even if they are not identified as such, is not to be taken as an expression of opinion as to
whether or not they are subject to proprietary rights.

Printed in the United States of America. SPI/EB

9 8 7 6 5 4 3 2 1

springeronline.com
ACKNOWLEDGMENT

The Editor again praises Bonnie McGahern for the competent final processing,
including fixing some of the Editor’s errors
CONTENTS OF EARLIER VOLUMES
VOLUME 1 (1979)
Introduction and Historical Background • Maxine F. Singer
Cloning of Double-Stranded cDNA • Argiris Efstratiadis and Lydia Villa-Komaroff
Gene Enrichment • M. H. Edgell, S. Weaver, Nancy Haigwood, and C. A. Hutchison III
Transformation of Mammalian Cells • M. Wigler, A. Pellicer, R. Axel, and S. Silverstein
Constructed Mutants of Simian Virus 40 • D. Shortle, J. Pipas, Sondra Lazarowitz,
D. DiMaio, and D. Nathans
Structure of Cloned Genes from Xenopus: A Review • R. H. Reeder
Transformation of Yeast • Christine Ilgen, P. J. Farabaugh, A. Hinnen, Jean M. Walsh,
and G. R. Fink
The Use of Site-Directed Mutagenesis in Reversed Genetics • C. Weissmann,
S. Nagata, T. Taniguchi, H. Weber, and F. Meyer
Agrobacterium Tumor Inducing Plasmids: Potential Vectors for the Genetic Engineering
of Plants • P. J. J. Hooykaas, R. A. Schilperoot, and A. Rörsch
The Chloroplast, Its Genome and Possibilities for Genetically Manipulating Plants •
L. Bogorad
Mitochondrial DNA of Higher Plants and Genetic Engineering • C. S. Levings III
and D. R. Pring
Host-Vector Systems for Genetic Engineering of Higher Plant Cells • C. I. Kado
Soybean Urease—Potential Genetic Manipulation of Agronomic Importance •
J. C. Polacco, R. B. Sparks, Jr., and E. A. Havir

VOLUME 2 (1980)
Cloning of Repeated Sequence DNA from Cereal Plants • J. R. Bedbrook
and W. L. Gerlach
The Use of Recombinant DNA Methodology in Approaches to Crop Improvement:
The Case of Zein • Benjamin Burr
Production of Monoclonal Antibodies • Sau-Ping Kwan, Dale E. Yelton, and
Matthew D. Scharff
Measurement of Messenger RNA Concentration • S. J. Flint
DNA Cloning in Mammalian Cells with SV40 Vectors • D. H. Hamer
Adenovirus–SV40 Hybrids: A Model System for Expression of Foreign Sequences in an
Animal Virus Vector • Joseph Sambrook and Terri Grodzicker
Molecular Cloning in Bacillus subtilis • D. Dubnau, T. Gryczan, S. Contente,
and A. G. Shivakumar
Bacterial Plasmid Cloning Vehicles • H. U. Bernard and D. R. Helinski

vii
viii CONTENTS OF EARLIER VOLUMES

Cloning with Cosmids in E. coli and Yeast • Barbara Hohn and A. Hinnen
DNA Cloning with Single-Stranded Phage Vectors • W. M. Barnes
Bacteriophage Lambda Vectors for DNA Cloning • Bill G. Williams and Frederick R. Blattner

VOLUME 3 (1981)
Constructed Mutants Using Synthetic Oligodeoxyribonucleotides as Site-Specific Mutagens
• M. Smith and S. Gillam
Evolution of the Insertion Element IS1 that Causes Genetic Engineering in Bacterial
Genomes In Vivo • E. Ohtsubo, K. Nyman, K. Nakamura, and H. Ohtsubo
Applications of Molecular Cloning to Saccharomyces • M. V. Olson
Cloning Retroviruses: Retrovirus Cloning? • W. L. McClements and G. F. Vande Woude
Repeated DNA Sequences in Drosophila • M. W. Young
Microbial Surface Elements: The Case of Variant Surface Glycoprotein (VSG) Genes of
African Trypanosomes • K. B. Marcu and R. O. Williams
Mouse Immunoglobulin Genes • P. Early and L. Hood
The Use of Cloned DNA Fragments to Study Human Disease • S. H. Orkin
Physical Mapping of Plant Chromosomes by In Situ Hybridization • J. Hutchinson,
R. B. Flavell, and J. Jones
Mutants and Variants of the Alcohol Dehydrogenase-1 Gene in Maize • M. Freeling
and J. A. Birchler
Developmentally Regulated Multigene Families in Dictyostelium discoideum •
R. A. Firtel, M. McKeown, S. Poole, A. R. Kimmel, J. Brandis, and W. Rowekamp
Computer Assisted Methods for Nucleic Acid Sequencing • T. R. Gingeras
and R. J. Roberts

VOLUME 4 (1982)
New Methods for Synthesizing Deoxyoligonucleotides • M. H. Caruthers,
S. L. Beaucage, C. Becker, W. Efcavitch, E. F. Fisher, G. Galluppi, R. Goldman,
P. deHaseth, F. Martin, M. Matteucci, and Y. Stabinsky
An Integrative Strategy of DNA Sequencing and Experiments Beyond • J. Messing
Transcription of Mammalian Genes In Vitro • J. L. Manley
Transcription of Eukaryotic Genes in Soluble Cell-Free Systems • N. Heintz and
R. G. Roeder
Attachment of Nucleic Acids to Nitrocellulose and Diazonium-Substituted Supports–B. Seed
Determination of the Organization and Identity of Eukaryotic Genes Utilizing Cell-Free
Translation Systems • J. S. Miller, B. E. Roberts, and B. M. Paterson
Cloning in Streptomyces: Systems and Strategies • D. A. Hopwood and K. F. Chater
Partial Sequence Determination of Metabolically Labeled Radioactive Proteins
and Peptides • C. W. Anderson
Molecular Cloning of Nitrogen Fixation Genes from Klebsiella pneumoniae and Rhizobium
meliloti • F. M. Ausubel, S. E. Brown, F. J. deBruijn, D. W. Ow, G. E. Riedel,
G. B. Ruvkun, and V. Sandaresan
The Cloning and Expression of Human Interferon Genes • R. M. Lawn
Cloning by Complementation in Yeast: The Mating Type Genes • J. B. Hicks,
J. N. Strathern, A. J. S. Klar, and S. L. Dellaporta
Construction and Screening of Recombinant DNA Libraries with Charon Vector Phages •
B. A. Zehnbauer and F. R. Blattner

VOLUME 5 (1983)
Microcloning of Microdissected Chromosome Fragments • V. Pirrotta, H. Jackle,
and J. E. Edstrom
Transient Expression of Cloned Genes in Mammalian Cells • J. Banerji and
W. Schaffner
CONTENTS OF EARLIER VOLUMES ix

Transposable Elements in Archaebacteria • W. F. Doolittle, C. Sapienza, J. D. Hofman,

R. M. Mackay, A. Cohen, and W.-L. Xu
The Application of Restriction Fragment Length Polymorphism to Plant Breeding •
B. Burr, S. V. Evola, F. A. Burr, and J. S. Beckmann
Antibodies against Synthetic Peptides • G. Walter and R. F. Doolittle
Wheat α-Amylase Genes: Cloning of a Developmentally Regulated Gene Family •
D. Baulcombe
Yeast DNA Replication • J. L. Campbell
Chromosome Engineering in Wheat Breeding and Its Implications for Molecular Genetic
Engineering • C. N. Law
Bovine Papillomavirus Shuttle Vectors • N. Sarver, S. Miltrani-Rosenbaum, M.-F. Law,
W. T. McAllister, J. C. Byrne, and P. M. Howley
Chemical Synthesis of Oligodeoxyribonucleotides: A Simplified Procedure •
R. L. Letsinger

VOLUME 6 (1984)
Cloning of the Adeno-Associated Virus • K. I. Berns
Transformation of the Green Alga Chlamydomonas reinhardii • J.-D. Rochaix
Vectors for Expressing Open Reading Frame DNA in Escherichia coli Using lacZ
Gene Fusions • G. M. Weinstock
An Enigma of the Leghemoglobin Genes • J. S. Lee and D. P. S. Verma
Yeast Transposons • G. S. Roeder
Rearrangement and Activation of C-MYC Oncogene by Chromosome Translocation in the
B Cell Neoplasias • K. B. Marcu, L. W. Stanton, L. J. Harris, R. Watt, J. Yang,
L. Eckhardt, B. Birshtein, E. Remmers, R. Greenberg, and P. Fahrlander
Screening for and Characterizing Restriction Endonucleases • I. Schildkraut
Molecular Studies of Mouse Chromosome 17 and the T Complex • L. M. Silver,
J. I. Garrels, and H. Lehrach
Use of Synthetic Oligonucleotide Hybridization Probes for the Characterization
and Isolation of Cloned DNAs • A. A. Reyes and R. B. Wallace
Hybridization of Somatic Plant Cells: Genetic Analysis • Yu. Yu. Gleba and D. A. Evans
Genetic Analysis of Cytoskeletal Protein Function in Yeast • P. Novick, J. H. Thomas,
and D. Botstein
Use of Gene Fusions to Study Biological Problems • L. Guarente
The Use of the Ti Plasmid of Agrobacterium to Study the Transfer and Expression
of Foreign DNA in Plant Cells: New Vectors and Methods • P. Zambryski,
L. Herrera-Estrella, M. De Block, M. Van Montagu, and J. Schell
Analysis of Eukaryotic Control Proteins at Their Reception Sequences by Scanning
Transmission Electron Microscopy • P. V. C. Hough, M. N. Slmon, and
I. A. Mastrangelo
The Mass Culture of a Thermophilic Spirulina in the Desert • K. Qian, G. H. Sato,
V. Zhao, and K. Shinohara
DNA-Mediated Gene Transfer in Mammalian Gene Cloning • F. H. Ruddle,
M. E. Kamarck, A. McClelland, and L. C. Kühn

VOLUME 7 (1985)
Biochemical and Genetic Analysis of Adenovirus DNA Replication In Vitro •
B. W. Stillman
Immunoscreening λGT11 Recombinant DNA Expression Libraries • R. A. Young and
R. W. Davis
In Situ Hybridization to Cellular RNAs • R. C. Angerer, K. H. Cox, and L. M. Angerer
Computer Methods to Locate Genes and Signals in Nucleic Acid Sequences •
R. Sladen
Biochemical and Molecular Techniques in Maize Research • N. Fedoroff
x CONTENTS OF EARLIER VOLUMES

Analysis of Chromosome Replication with Eggs of Xenopus laevis • R. A. Laskey,

S. E. Kearsey, and M. Mechali
Molecular Genetic Approaches to Bacterial Pathogenicity to Plants • M. J. Daniels
and P. C. Turner
Synthesis of Hybridization Probes and RNA Substrates with SP6 RNA Polymerase •
P. A. Krieg, M. R. Rebagliati, M. R. Green, and D. A. Melton
Identification and Isolation of Clones by Immunological Screening of cDNA Expression
Libraries • D. M. Helfman, J. R. Feramisco, J. C. Fiddes, G. P. Thomas,
and S. H. Hughes
Molecular Studies on the Cytomegaloviruses of Mice and Men • D. H. Spector
Gene Transfer with Retrovirus Vectors • A. Bernstein, S. Berger, D. Huszar,
and J. Dick
HPRT Gene Transfer as a Model for Gene Therapy • T. Friedmann
Catabolic Plasmids: Their Analysis and Utilization in the Manipulation of Bacteria Metabolic
Activities • S. Harayama and R. H. Don
Transcription of Cloned Eukaryotic Ribosomal RNA Genes • B. Sollner-Webb, J. Tower,
V. Culotta, and J. Windle
DNA Markers in Huntington’s Disease • J. F. Gusella

VOLUME 8 (1986)
Regulation of Gene Activity During Conidiophore Development in Aspergillus nidulans •
W. E. Timberlake and J. E. Hamer
Regulation of Expression of Bacterial Genes for Bioluminescence • J. Engebrecht
and M. Silverman
Analysis of Genome Organization and Rearrangements by Pulse Field Gradient Gel
Electrophoresis • C. L. Smith, P. E. Warburton, A. Gaal, and C. R. Cantor
Structural Instability of Bacillus subtilis Plasmids • S. D. Ehrlich, Ph. Noirot, M. A. Petit,
L. Jannière, B. Michel, and H. te Riele
Geminiviruses, The Plant Viruses with Single-Stranded DNA Genome • A. J. Howarth
The Use of Bacterial Plasmids in the Investigation of Genetic Recombination • A. Cohen
Shuttle Mutagenesis: A Method of Introducing Transposons into Transformable Organisms
• H. S. Seifert, M. So, and F. Heffron
Genetic Advances in the Study of Rhizobium Nodulation • S. R. Long
Galactokinase Gene Fusion in the Study of Gene Regulation in E. coli, Streptomyces,
Yeast and Higher Cell Systems • M. Rosenberg, M. Brawner, J. Gorman,
and M. Reff
Structure and Function of the Signal Recognition Particle • V. Siegel and P. Walter
Alteration of the Structure and Catalytic Properties of Rubisco by Genetic Manipulation •
S. Gutteridge
Electrophoresis of DNA in Denaturing Gradient Gels • L. S. Lerman
Caulimoviruses as Potential Gene Vectors for Higher Plants • R. J. Shepherd
An Insect Baculovirus Host–Vector System for High-Level Expression of Foreign Genes •
D. W. Miller, P. Safer, and L. K. Miller
Preparation of cDNA Libraries and the Detection of Specific Gene Sequences •
J. Brandis, D. Larocca, and J. Monahan
Construction of Human Chromosome Specific DNA Libraries: The National Laboratory
of Gene Library Project • L. L. Deaven, C. E. Hildebrand, J. C. Fuscoe,
and M. A. Van Dilla
New Approaches to the Expression and Isolation of a Regulatory Protein • D. Bastia,
J. Germino, S. Mukherjee, and T. Vanaman
CONTENTS OF EARLIER VOLUMES xi

VOLUME 9 (1987)
Gene Transfer in the Sea Urchin • B. R. Hough-Evans and E. H. Davidson
Properties and Uses of Heat Shock Promoters • H. Pelham
The Expression of Introduced Genes in Regenerated Plants • D. Dunsmuir,
J. Bedbrook, D. Bond-Nutter, C. Dean, D. Gidoni, and J. Jones
Control of Maize Zein Gene Expression • R. S. Boston and B. A. Larkins
Dnase I Footprinting as an Assay for Mammalian Gene Regulatory Proteins •
W. S. Dynan
Use of Gene Transfer in the Isolation of Cell Surface Receptor Genes • D. R. Littman
and M. V. Chao
A New Method for Synthesizing RNA on Silica Supports • D. J. Dellinger and
M. H. Caruthers
Activity Gels: Reformation of Functional Proteins from SDS-Polyacrylamide Gels •
R. P. Dottin, B. Haribabu, C. W. Schweinfest, and R. E. Manrow
Plasmid Vectors Carrying the Replication Origin of Filamentous Single-Stranded Phages
• G. Cesareni and J. A. H. Murray
High Level Production of Proteins in Mammalian Cells • R. J. Kaufman
Plant Microinjection Techniques • R. J. Mathias
Genetic Transformation to Confer Resistance to Plant Virus Disease • R. N. Beachy,
S. G. Rogers, and R. T. Fraley
Alternative Splicing: Mechanistic and Biological Implications of Generating Multiple
Proteins from a Single Gene • B. Nadal-Ginard, M. E. Gallego, and
A. Andreadis

VOLUME 10 (1988)
Genomic Footprinting • P. B. Becker and G. Schütz
Theoretical and Computer Analysis of Protein Primary Sequences: Structure Comparison and
Prediction • P. Argos and P. McCaldon
Affinity Chromatography of Sequence-Specific DNA-Binding Proteins • C. Wu, C. Tsai,
and S. Wilson
Applications of the Firefly Luciferase as a Reporter Gene • S. Subramani and
M. DeLuca
Fluorescence-Based Automated DNA Sequence Analysis • L. M. Smith
Phosphorothioate-Based Oligonucleotide-Directed Mutagenesis • J. R. Sayers and
F. Eckstein
Design and Use of Agrobacterium Transformation Vectors • M. Bevan and
A. Goldsbrough
Cell Commitment and Determination in Plants • F. Meins, Jr.
Plasmids Derived from Epstein–Barr Virus: Mechanisms of Plasmid Maintenance
and Applications in Molecular Biology • J. L. Yates
Chromosome Jumping: A Long Range Cloning Technique • A. Poustka and
H. Lehrach
Isolation of Intact MRNA and Construction of Full-Length cDNA Libraries: Use of a New
Vector, λgt22, and Primer-Adapters for Directional cDNA Cloning • J. H. Han
and W. J. Rutter
The Use of Transgenic Animal Techniques for Livestock Improvement • R. M. Strojek
and T. E. Wagner
Plant Reporter Genes: The GUS Gene Fusion System • R. A. Jefferson
Structure of the Genes Encoding Proteins Involved in Blood Clotting •
R. T. A. MacGillivray, D. E. Cool, M. R. Fung, E. R. Guinto, M. L. Koschinsky, and
B. A. Van Oost
xii CONTENTS OF EARLIER VOLUMES

VOLUME 11 (1989)
DNA Methylases • A. Razin
Advances in Direct Gene Transfer into Cereals • T. M. Klein, B. A. Roth, and
M. E. Fromm
The Copy Number Control System of the 2µm Circle Plasmid of Saccharomyces
cerevisiae • B. Futcher
The Application of Antisense RNA Technology to Plants • W. R. Hiatt, M. Kramer, and
R. E. Sheehy
The Pathogenesis-Related Proteins of Plant • J. P. Carr and D. F. Klessig
The Molecular Genetics of Plasmid Partition: Special Vector Systems for the Analysis of
Plasmid Partition • A. L. Abeles and S. J. Austin
DNA-Mediated Transformation of Phytopathogenetic Fungi • J. Wang and S. A. Leong
Fate of Foreign DNA Introduced to Plant Cells • J. Paszkowski
Generation of cDNA Probes by Reverse Translation of Amino Acid Sequence • C. C. Lee
and C. T. Caskey
Molecular Genetics of Self-Incompatibility in Flowering Plants • P. R. Ebert,
M. Altschuler, and A. E. Clarke
Pulsed-Field Gel Electrophoresis • M. V. Olson

VOLUME 12 (1990)
Folding of Eukaryotic Proteins Produced in Escherichia coli • R. F. Kelley and
M. E. Winkler
Human Retinoblastoma Susceptibility Gene • C.-C. Lai and W.-H. Lee
α-Oligodeoxynucleotides (α-DNA): A New Chimeric Nucleic Acid Analog • F. Morvan,
B. Rayner, and J.-L. Imbach
The Utility of Streptomycetes and Hosts for Gene Cloning • P. K. Tomich and Y. Yagi
From Footprint to Function: An Approach to Study Gene Expression and Regulatory
Factors in Transgenic Plants • E. Lam
Purification of Recombinant Proteins with Metal Chelate Adsorbent • E. Hochuli
Determinants of Translation Efficiency of Specific mRNAs in Mammalian Cells •
D. S. Peabody
The Polymerase Chain Reaction • N. Arnheim
Regulation of Alternative Splicing • M. McKeown
Structure and Function of the Nuclear Receptor Superfamily for Steroid, Thyroid Hormone
and Retinoic Acid • V. Giguère
Identification and Functional Analysis of Mammalian Splicing Factors • A. Bindereif and
M. R. Green
The Genes Encoding Wheat Storage Proteins: Towards a Molecular Understanding of
Bread-Making Quality and Its Genetic Manipulation • V. Colot
Control of Translation Initiation in Mammalian Cells • R. J. Kaufman
Electroporation of Bacteria: A General Approach to Genetic Transformation •
W. J. Dower
The Isolation and Identification of cDNA Genes by Their Heterologous Expression
and Function • G. G. Wong
Molecular Cloning of Genes Encoding Transcription Factors with the Use of Recognition
Site Probes • H. Singh

VOLUME 13 (1991)
The Mutator Transposable Element Family of Maize • V. Walbot
Protein Phosphorylation and the Regulation of Cellular Processes by the Homologous
Two-Component Systems of Bacteria • A. J. Ninfa
The Peculiar Nature of Codon Usage in Primates • S. Zhang and G. Zubay
CONTENTS OF EARLIER VOLUMES xiii

The Role of Nodulation Gene in Bacterium–Plant Communication • A. Kondorosi,

E. Kondorosi, M. John, J. Schmidt, and J. Schell
Regulation of Gene Expression by Epidermal Growth Factor • L. G. Hudson
and G. N. Gill
Machinery of Protein Import into Chloroplasts and Mitochondria • D. Pain, D. J. Schnell,
H. Murakami, and G. Blobel
High-Level Expression of Foreign Genes in Mammalian Cells • S. E. Kane
Aromatic Hydrocarbon Degradation: A Molecular Approach • G. J. Zylstra
and D. T. Gibson
Employment of Fibroblasts for Gene Transfer Applications for Grafting into the Central
Nervous System • M. D. Kawaja, J. Ray, and F. H. Gage
The Molecular Biology of Amino Acid Biosynthesis in Plants • T. Brears
and G. M. Coruzzi
Genetic Manipulation of Bacillus thuringiensis Insecticidal Crystal Protein Genes in
Bacteria • C. Gawron-Burke and J. A. Baum
Progress Towards Gene Targeting in Plants • J. I. Yoder and E. Kmiec
Molecular Biology of Mating-Type Determination in Schizophyllum commune •
R. C. Ullrich, C. A. Specht, M. M. Stankis, H. Yang, L. Giasson, and C. P. Novotny
Functions of Intracellular Protein Degradation in Yeast • M. Hochstrasser
Transgenic Fish for Aquaculture • G. L. Fletcher and P. L. Davies

VOLUME 14 (1992)
Cleavage-Site Motifs in Protein Targeting Sequences • G. von Heijne
Complications of RNA Heterogeneity for the Engineering of Virus Vaccines and Antiviral
Agents • E. Domingo and J. J. Holland
The Quaternary Structures of SV40 Large T Antigen and Tumor Suppressor p53:
Analysis by Gel Electrophoresis • J. E. Stenger, G. A. Mayr, K. Mann, S. Ray,
M. E. Anderson, and P. Tegtmeyer
Assembly of Antibodies and Mutagenized Variants in Transgenic Plants and Plant Cell
Cultures • A. Hiatt, Y. Tang, W. Weiser, and M. B. Hein
Maize Endosperm Tissue as an Endoreduplication System • R. V. Knowles, G. L. Yerk,
F. Crienc, and R. L. Phillips
Study of Chlorate-Resistant Mutants of Aradibopsis: Insights into Nitrate Assimilation and
Ion Metabolism of Plants • N. M. Crawford
Approaches and Progress in the Molecular Cloning of Plant Disease Resistance Genes •
J. L. Bennetzen and J. D. G. Jones
Is GRP78 a Sensor of Cellular Secretory Activity? • T. Leustek
The Molecular Biology of Pathogenesis in Ustilago maydis • B. J. Saville and
S. A. Leong
Molecular Design of Oligomeric Channel Proteins • A. Grove, J. M. Tomich, and M. Montal
Regulation of Gene Expression by Thyroid Hormones and Retinoic Acids • S. M. Lipkin,
M. G. Rosenfeld, and C. K. Glass
RNA Trans-Splicing • X.-Y. Huang and D. Hirsch
Structural Constraints on Residue Substitution • J. Overington
Molecular and Functional Analysis of the A Mating Type Genes of Coprinus cinereus •
U. Kües and L. A. Casselton
Physical Mapping of Human Chromosomes • G. A. Evans and D. L. McElligott

VOLUME 15 (1993)
Application of Computational Neural Networks to the Prediction of Protein Structural
Features • S. R. Holbrook
Human Cellular Protein Patterns and Their Link to Genome Data Mapping and Sequencing
xiv CONTENTS OF EARLIER VOLUMES

Data: Towards an Integrated Approach to the Study of Gene Expression •

J. E. Celis, H. H. Rasmussen, H. Leffers, P. Madsen, B. Honore, K. Dejgaard,
P. Gromov, and E. Olsen, H. J. Hoffman, M. Nielsen, B. Gesser, M. Puype, J. Van
Damme, and J. Vandekerckhove
Regulation of Translation in Plants • A. Danon, C. B. Yohn, and S. P. Mayfield
On the Origins, Structures and Functions of Restriction-Modification Enzymes •
J. Heitman
Manipulation of Amino Acid Balance in Maize Seed • T. Ueda and J. Messing
Investigational Approaches for Studying the Structures and Biological Functions in Myeloid
Antimicrobial Peptides • M. E. Selsted
Progress in the Cloning of Genes for Plant Storage Lipid Biosynthesis • V. C. Knauf
Genes for Crop Improvement • J. Bennett
Molecular Biology and Genetics of Protective Fungal Endophytes of Grasses •
C. L. Schardl and Z. An
Prospects for Human Gene Therapy • A. B. Moseley and C. T. Caskey
The Use of Microparticle Injection to Introduce Genes into Animal Cells In Vitro and In Vivo
• S. A. Johnston and D-C. Tang

VOLUME 16 (1994)
RNA Polymerase III Transcription in the Yeast Saccharomyces cerevisiae • Stephen
Buratowski
Lens Oncogenesis and Differentiation • Heiner Westphal
Genetic Engineering of Cardiac Muscle Cells: In vitro and In vivo • Stephen J. Fuller
and Kenneth R. Chien
Genetic Control of Plant Ureases • Joseph C. Polacco and Mark A. Holland
Gene Discovery of Dictyostelium • William F. Loomis, Adam Kuspa, and Gad Shaulsky
Transfer of YACs to Mammalian Cells and Transgenic Mice • Clare Huxley
Plant Genetic Engineering and Future Agriculture • S. Riazuddin
Internal Initiation of mRNA Translation in Eukaryotes • Ann Kaminski, Sarah L. Hunt,
Catherine L. Gibbs, and Richard J. Jackson
Genetic Recombination Analysis Using Sperm Typing • Karin Schmitt and Norman
Arnheim
Genetic Regulation in Plant Pathogenic Pseudomonads • David K. Willis, Jessica J.
Rich, Thomas G. Kinscherf, and Todd Kitten
Defense-Related Gene Induction in Plants • Danny Alexander, Kay Lawton, Scott Uknes,
Eric Ward, and John Ryals
The P1 Vector System for the Preparation and Screening of Genomic Libraries •
Nancy S. Shepherd and David Smoller
The Unmasking of Maternal mRNA During Oocyte Maturation and Fertilization •
James L. Grainger
Recognizing Exons in Genomic Sequences Using Grail II • Ying Xu, Richard Mural,
Manesh Shah, and Edward Uberbacher
Gene Expression of Plant Extracellular Proteins • Beat Keller

VOLUME 17 (1995)
The Molecular Biology of Nucleotide Excision Repair and Double-Strand Break Repair
in Eukaryotes • Alan R. Lehman
Manipulating and Mapping RNA with RecA-Assisted Restriction Endonuclease (RARE)
Cleavage • Lance J. Ferrin
Molecular Studies on the Virulence of Listeria monocytogenes • Michael Kuhn
and Werner Goebel
Indirect Use of Immobilized Metal Affinity Chromatography for Isolation and Characterization
of Protein Partners • Michèle Sawadogo and Michael W. Van Dyke
Structure and Function of RNA Pseudoknots • C. W. A. Pleij
CONTENTS OF EARLIER VOLUMES xv

Role of Molecular Chaperones in the Initiation of Plasmid DNA Recognition •

Dhruba K. Chattoraj
Structure, Function and Engineering of Bacillus thuringiensis Toxins •
Mark A. Thompson, H. Ernest Schnepf, and Jerald S. Feitelson
Uses of GAL4 Expression in Mammalian Cells • Ivan Sadowski
Protein Thiol Modification of Glyceraldehyde-3-Phosphate Dehydrogenase •
Bernhard Brüne and Eduardo G. Lapetina
The Genetics of Nuclear Migration in Fungi • Susan M. Beckwith, Christian H. Roghi,
and N. Ronald Morris
Structure and Function of the Platelet-Derived Growth Factor Family and Their Receptors
• Kristen C. Hart, Brendan D. Galvin, and Daniel J. Donoghue
Recombination between Prokaryotic and Eukaryotic DNA: Integration of Agrobacterium
tumefaciens T-DNA into the Plant Genome • Bruno Tinland and Barbara Hohn
Metal Precipitation by Marine Bacteria: Potential for Biotechnological Applications •
Bradley M. Tebo

VOLUME 18 (1996)
Cloning and Characterization of DNAs with Palindromic Sequences • David R. F. Leach
DNA Isolation, Manipulation and Characterization from Old Tissues • Rob DeSalle
and Elizabeth Bonwich
Growth Factors and Neural Connectivity • Sarah McFarlane and Christine E. Holt
Gene Identification by 3′ Terminal Exon Trapping • David B. Krizman
Engineering Transgenes for Use in the Mammary Gland • Sinai Yarus, Darryl Hadsell,
and Jeffrey M. Rosen
Problems that Can Limit the Expression of Foreign Genes in Plants: Lessons to Be Learned
from B.t. Toxin Genes • Scott H. Diehn, E. Jay De Rocher, and Pamela J. Green
Renaturation and Reconstitution of Functional Holoenzyme from Recombinant Subunits
of Casein Kinase II Expressed as Inclusion Bodies in E. coli • Wey-Jinq Lin,
Rolf Jakobi, and Jolinda A. Traugh
Plant ACYL-ACP Thioesterases: Chain-Length Determining Enzymes in Plant Fatty Acid
Biosynthesis • Toni Voelker
Genetic Engineering of an Insect Parasite • Randy Gaugler and Sarwar Hashmi
The Stop Signal Controls the Efficiency of Release Factor-Mediated Translational Termination
• Warren P. Tate, Mark E. Dalphin, Herman J. Pel, and Sally A. Manning
Mechanism of Replication and Copy Number Control of Plasmids in Gram-Positive
Bacteria • Saleem A. Khan
Pathways of Protein Remodeling by Escherichia coli Molecular Chaperones •
Marie Pak and Sue H. Wickner
Pheromones and Pheromone Receptors as Mating-Type Determinants in Basidiomycetes
• Lisa J. Vaillancourt and Carlene A. Raper
Synthesis and Applications of Phosphopeptides • Kazuyasu Sakaguchi, Peter K. Roller,
and Ettore Appella

VOLUME 19 (1997)
Novel Approaches to Engineering Disease Resistance in Crops • Kathy M. M. Swords,
Jihong Liang, and Dilip M. Shah
The Structure of Plant Gene Promoters • Tom J. Guilfoyle
Plasmid Stabilization by Post-Segregational Killing • Kenn Gerdes, Jimmy Schouv
Jacobsen, and Thomas Franch
Pathways and Genes Involved in Cellulose Synthesis • Yasushi Kawagoe and Deborah
P. Delmer
Conjugative Transposons • Abigail A. Salyers and Nadja B. Shoemaker
Termination of DNA Replication in Prokaryotic Chromosomes • Deepak Bastia, Adhar
C. Manna, and Trilochan Sahoo
xvi CONTENTS OF EARLIER VOLUMES

Regulation of Protein Degradation in Plants • Judy Callis

Genetic Engineering of Oilseeds for Desired Traits • Anthony J. Kinney
Specificity of Receptor Tyrosine Kinase Signaling Pathways: Lessons from Drosophila •
Willis Li and Norbert Perrimon
Switching on Gene Expression: Analysis of the Factors that Spatially and Temporally
Regulate Plant Gene Expression • Lee Meisel and Eric Lam
Nucleic Acid Transport in Plant-Pathogen Interactions • Robert Lartey and
Vitaly Citovsky
Leaf Senescence: Gene Expression and Regulation • Louis M. Weaver,
Edward Himelblau, and Richard M. Amasino
Production and Analysis of Transgenic Mice Containing Yeast Artificial Chromosomes •
Kenneth R. Peterson
Comparative Molecular Analysis of Genes for Polycyclic Aromatic Hydrocarbon
Degradation • Gerben J. Zylstra, Eungbin Kim, and Anil K. Goyal
Recognition and Signaling in Plant-Pathogen Interactions: Implications for Genetic
Engineering • Michael Lawton

VOLUME 20 (1998)
Agrobacterium-Mediated Horizontal Gene Transfer • Clarence I. Kado
Computer-Assisted Methods for the Identification and Characterization of Polymerase II
Promoters • Ingmar Reuter, Thomas Werner, and Edgar Wingender
Retroviral cDNA Integration: Mechanism, Applications and Inhibition •
Mark S. T. Hansen, Sandrine Carteau, Christopher Hoffman, Ling Li, and
Frederic Bushman
The Signal Transduction of Motion and Antigen Recognition: Factors Affecting T Cell
Function and Differentiation • Stephen C. Bunnell and Leslie J. Berg
Synthetic DNA Arrays • Alan Blanchard
Detection of Single Nucleotide Variations • Pui-Yan Kwok and Xiangning Chen
Antisense: A Key Tool for Cell and Developmental Studies in Dictyostelium •
Richard H. Gomer
Antisense in Abundance: The Ribosome as a Vehicle for Antisense RNA •
Rosemary Sweeney, Qichaag Fan, and Meng-Chao Yao
Salinity Tolerance—Mechanisms, Models and the Metabolic Engineering of Complex Traits
• Donald E. Nelson, Bo Shen, and Hans J. Bohnert
Biochemistry, Molecular Biology and Regulation of Starch Synthesis • Jack Preiss
and Mirta N. Sivak
Genetic Engineering and the Expression of Foreign Peptides or Proteins with Plant Virus-
Based Vectors • Christophe Lacomme, Lisa Smolenska, and T. Michael A. Wilson
Cloning and Expression of Large Mammalian cDNAs: Lessons from ATM •
Yosef Shiloh, Anat Bar-Shira, Yaron Galanty, and Yael Ziv
The Use of Genetically Engineered Cells in Drug Discovery • Gerhard Loeber
and Renate Schnitzer
Molecular Engineering of Monoterpene Production • Christian D. Haudenschild
and Rodney B. Croteau

VOLUME 21 (1999)
Nuclear Plasmids of Dictyostelium • Joanne E. Hughes and Dennis L. Welker
The Translation Initiation Signal in E. coli and Its Control • Eckart Fuchs
Direct Isolation of Specific Chromosomal Regions and Entire Genes by Tar Cloning •
Vladimir Larionov
Regulation of Lysine and Threonine Metabolism in Plants • Rachel Amir and Gad Galili
Genetic Engineering of Plant Chilling Tolerance • James Tokuhisa and John Browse
Role of Bacterial Chaperones in DNA Replication • Igor Konieczny and Maciej Zylicz
CONTENTS OF EARLIER VOLUMES xvii

Transformation of Cereals • Roland Bilang, Johannes Fütterer, and Christof Sautter

Mechanisms of Initiation of Linear DNA Replication in Prokaryotes • Margarita Salas
Diverse Regulatory Mechanisms of Amino Acid Biosynthesis in Plants •
Katherine J. Denby and Robert L. Last
Forage and Turf-Grass Biotechnology: Principles, Methods, and Prospects •
John W. Forster and Germán C. Spangenberg
Informatics Needs of Plant Molecular Biology • Mary Polacco

VOLUME 22 (2000)
Post-Transcriptional Light Regulation of Nuclear-Encoded Genes • Marie E. Petracek
and William F. Thompson
Novel Methods of Introducing Pest and Disease Resistance to Crop Plants •
Jeremy Bruenn
Targeting Gene Repair in Mammalian Cells Using Chimeric Oligonucleotides •
Eric B. Kmiec, Sarah Ye, and Lan Peng
Exploring the Mechanism of Action of Insecticidal Proteins by Genetic Engineering
Methods • Jeremy L. Jenkins and Donald H. Dean
Enzyme Stabilization by Directed Evolution • Anne Gershenson and Frances H. Arnold
ET-Cloning: Think Recombination First • Joep P. P. Muyrers, Youming Zhang,
and A. Francis Stewart
Growth and Genetic Modification of Human β-Cells and β-Cell Precursors •
Gillian M. Beattie, Albert Hayek, and Fred Levine
Elucidation of Biosynthetic Pathways by Retrodictive/Predictive Comparison of Isotopomer
Patterns Determined by NMR Spectroscopy • Wolfgang Eisenreich and Adelbert
Bacher
Are Gene Silencing Mutants Good Tools for Reliable Transgene Expression or Reliable
Silencing of Endogenous Genes in Plants? • Philippe Mourrain, Christophe Béclin,
and Hervé Vaucheret
Manipulating Plant Viral RNA Transcription Signals • Cynthia L. Hemenway and
Steven A. Lommel
Genetic Engineering Strategies for Hematologic Malignancies • Thomas J. Kipps
Telomerase and Cancer • Murray O. Robinson

VOLUME 23 (2001)
Evolution of Transport Proteins • Milton H. Saier, Jr.
Mechanisms of Apoptosis Repression • Collin C. Q. Vu and John A. Cidlowski
Cytokine Activation of Transcription • Kerri A. Mowen and Michael David
Enzymatic Approaches to Glycoprotein Synthesis • Pamela Sears, Thomas Tolbert,
and Chi-Huey Wong
Vector Design and Development of Host System for Pseudomonas •
Herbert P. Schweizer, Tung T. Hoang, Katie L. Propst, Henry R. Ornelas, and
RoxAnn R. Karkhoff-Schweizer
Genetic and Biochemical Studies on the Assembly of an Enveloped Virus •
Timothy L. Tellinghuisen, Rishika Perera, and Richard J. Kuhn
Enzyme and Pathway Engineering for Suicide Gene Therapy • Margaret E. Black
Reconstructing a Conserved Protein Family: The Role of MCM Proteins in Eukaryotic DNA
Replication • Sally G. Pasion and Susan L. Forsburg
Expression of Foreign Genes in the Yeast Pichia pastoris • Geoffrey P. Lin Cereghino,
Anthony J. Sunga, Joan Lin Cereghino, and James M. Cregg
Protein Splicing and Its Applications • Izabela Giriat, Thomas W. Muir, and
Francine B. Perler
Global Transcript Expression Profiling by Serial Analysis of Gene Expression (SAGE) •
Hamish S. Scott and Roman Chrast
xviii CONTENTS OF EARLIER VOLUMES

VOLUME 24 (2002)
Application of FLP/FRT Site-Specific DNA Recombination System in Plants • Hong Luo
and Albert P. Kausch
Protein Quality Control in Bacterial Cells: Integrated Networks of Chaperones and ATP-
Dependent Proteases • John M. Flanagan and Maria C. Bewley
Regulation of the Ras-MAPK Pathway at the Level of Ras and Raf • Haris Vikis
and Kun-Liang Guan
Plant Virus Gene Vectors: Biotechnology Applications in Agriculture and Medicine •
Karen-Beth G. Scholthof, T. Erik Mirkov, and Herman B. Scholthof
Integrins and the Myocardium • Shaw-Yung Shai, Alice E. Harpf, and Robert S. Ross
Foreign DNA: Integration and Expression in Transgenic Plants • Richard M. Twyman,
Ajay Kohli, Eva Stoger, and Paul Christou
Novel Approaches to Controlling Transcription • Thomas D. Schaal, Michael C. Holmes,
Edward J. Rebar, and Casey C. Case
The Use of DNA Polymorphisms in Genetic Mapping • Christopher A. Cullis
Import of Nuclear-Encoded RNAs into Yeast and Human Mitochondria: Experimental
Approaches and Possible Biomedical Applications • N. Entelis, O. Kolesnikova,
H. Kazakova, I. Brandina, P. Kamenski, R. P. Martin, and I. Tarassov
An Introduction to 13C Metabolic Flux Analysis • Wolfgang Wiechert
Gene Silencing—Principles and Application • Cathryn Horser, David Abbott,
Varsha Wesley, Neil Smith, and Peter Waterhouse

VOLUME 25 (2003)
Genotyping by Mass Spectrometry • Molly S. Bray and Peter A. Doris
Development of Targeted Viral Vectors for Cardio-Vascular Gene Therapy •
Stuart A. Nicklin and Andrew H. Baker
Practical Applications of Rolling Circle Amplification of DNA Templates •
Paul M. Richardson, Chris Detter, Barry Schwietzer, and Paul F. Predki
Structural Analyses of Living Plant Nuclei • Naohiro Kato
Bacterial Ion Channels • Ian R. Booth
Functional Analysis of Promoter Elements in Plants • Slavko Komarnytsky and Nikolai
Borisjuk
Applications of Plant Antiviral Proteins • Melan Wang and Katalin A. Hudak
Biosynthesis and Metabolism of Glutathione in Plants • Melinda Neal Martin
Delitto Perfetto Targeted Mutagenesis in Yeast with Oligonucleotides •
Francesca Storici and Michael A. Resnick
The Bacterial Scaffoldin: Structure, Function and Potential Applications in the
Nanosciences • Shi-You Ding, Raphael Larned, Edward A. Bayer, and
Michael A. Himmel
Hybrid Peptide-Polyketide Natural Products: Biosynthesis and Prospects towards
Engineering Novel Molecules • Liangcheng Du, Yi-Qiang Cheng, Gudrun
Ingenhorst, Gong-Li Tang, Yong Huang, and Ben Shen
Characterization of Protein Structure and Function at Genome Scale Using a
Computational Prediction Pipeline • Dong Yu, Dongsup Kim, Phuongan Dam,
Manesh Shah, Edward C. Uberbacher, and Ying Xu

VOLUME 26 (2004)
Arabidopsis as a Genetic Model for Interorganellle Lipid Trafficking • Christoph
Benning, Changcheng Xu, and Koichiro Awai
Protein Sequence Database Methods • Maria Jesus Martin, Claire O’Donovan, and
Rolf Apweiler
Properties and Applications of Cell-Penetrating Peptides • A. Gräslund and
L.E.G. Eriksson
CONTENTS OF EARLIER VOLUMES xix

Detection of Topological Patterns in Protein Networks • Sergei Maslov and

Kim Sneppen
Dna Microarrays: Methodology, Data Evaluation and Applications in the Analysis of Plant
Defense Signaling • E. Kuhn and A. Schaller
Approaches for Identification of Fungal Genes Essential for Plant Disease •
Candace E. Elliott and Barbara J. Howlett
Genetic Mapping in Forest Trees: Markers, Linkage Analysis and Genomics •
Matias Kirst, Alexander Myburg, and Ronald Sederoff
The Production of Long Chain Polyunsaturated Fatty Acids in Transgenic Plants •
Johnathan A. Napier, Frédéric Beaudoin, Louis V. Michaelson, and Olga Sayanova
Investigating in situ Natural Genetic Transformation of Acinetobacter Sp. BD413 in Biofilms
with Confocal Laser Scanning Microscopy • Larissa Hendrickx and Stefan Wuertz
The Path in Fungal Plant Pathogenicity: Many Opportunities to Outwit the Intruders? •
Guus Bakkeren and Scott Gold
Analysis and Annotations of Microbial Genome Sequences • Loren Hauser,
Frank Larimer, Miriam Land, Manesh Shah, and Ed Uberbacher
Brain Plasticity and Remodeling of Ampa Receptor Properties by Calcium-Dependent
Enzymes • Guy Massicotte and Michel Baudry
Gene Regulation by Tetracyclines • Christian Berens and Wolfgang Hillen
CONTENTS

IDENTIFICATION AND ANALYSIS OF MICRORNAS ....................................1

Sveta Bagga and Amy E. Pasquinelli

DORMANCY AND THE CELL CYCLE ............................................................21

Michael A. Campbell

THE OPT FAMILY FUNCTIONS IN LONG-DISTANCE

PEPTIDE AND METAL TRANSPORT IN PLANTS ........................................35
Mark Lubkowitz

PHOSPHOLIPID-DERIVED SIGNALING IN PLANT

RESPONSE TO TEMPERATURE AND WATER STRESSES ..........................57
Xuemin Wang

ANIONIC NUTRIENT TRANSPORT IN PLANTS:

THE MOLECULAR BASIS OF THE SULFATE
TRANSPORTER GENE FAMILY........................................................................67
Hideki Takahashi, Naoko Yoshimoto, and Kazuki Saito

PURIFICATION OF PROTEIN COMPLEXES

BY IMMUNOAFFINITY CHROMATOGRAPHY:
APPLICATION TO TRANSCRIPTION MACHINERY ....................................81
Nancy E. Thompson, Debra Bridges Jensen,
Jennifer A. Lamberski, and Richard R. Burgess

BIOGENESIS OF IRON-SULFUR CLUSTER

PROTEINS IN PLASTIDS ..................................................................................101
Marinus Pilon, Salah E. Abdel-Ghany,
Douglas Van Hoewyk, Hong Ye, and Elizabeth A. H. Pilon-Smits

IRON TRANSPORT AND METABOLISM IN PLANTS ................................119

Loubna Kerkeb and Erin L. Connolly

SALT STRESS SIGNALING AND MECHANISMS

OF PLANT SALT TOLERANCE ......................................................................141
Viswanathan Chinnusamy, Jianhua Zhu, and Jian-Kang Zhu

xxi
xxii CONTENTS

STRATEGIES FOR HIGH-THROUGHPUT GENE CLONING

AND EXPRESSION ............................................................................................179
L. J. Dieckman, W. C. Hanly, and F. R. Collart

MOLECULAR ROLES OF CHAPERONES IN

ASSISTED FOLDING AND ASSEMBLY OF PROTEINS ..............................191
Mark T. Fisher

ENGINEERING PLANTS FOR INCREASED NUTRITION

AND ANTIOXIDANT CONTENT THROUGH
THE MANIPULATION OF THE VITAMIN E PATHWAY..............................231
David K. Shintani

INDEX ................................................................................................................243
IDENTIFICATION AND ANALYSIS OF MICRORNAS

Shveta Bagga and Amy E. Pasquinelli

Molecular Biology Section

Division of Biology
University of California, San Diego
La Jolla, CA 92093-0349

INTRODUCTION

The first microRNA (miRNA) gene was uncovered in 1993. After lan-
guishing in near obscurity for almost a decade, this gene is now recognized as the
founding member of a new class of regulatory RNAs that control gene expres-
sion in all multicellular organisms. MicroRNA genes express ~22 nucleotide (nt)
RNAs that regulate the expression of protein-coding genes containing sequences
of antisense complementarity. The intense interest in understanding the role of
miRNAs in regulating gene expression has fueled the development of new meth-
ods to study how these tiny RNA genes are expressed and function. In this chap-
ter, we present a brief history outlining the discovery of miRNAs and the
current model for their biogenesis and mode of action. We then describe experi-
mental approaches used to analyze miRNA expression patterns and regulatory
functions.

Genetic Engineering, Volume 27, Edited by J. K. Setlow

©Springer Science+Business Media, Inc. 2006 1
2 S. BAGGA AND A. PASQUINELLI

EXPRESSION AND FUNCTION OF miRNAS

Discovery of miRNAs Through Nematode Genetics

Forty years ago, Sydney Brenner proposed adoption of the micro-

scopic nematode Caenorhabditis elegans for studying the genetic basis of ani-
mal development and behavior. Not only has the worm proven to be a model
experimental system for identifying the genes responsible for controlling cell
fate and function, but it also enabled the discovery of an entirely unexpected
class of genes and a novel regulatory mechanism. The first microRNA gene
was uncovered through classical genetic methods to identify a mutation
responsible for abnormal development of certain worm cells. The Ambros
laboratory found that the developmental defects resulted from mutation of
the lin-4 (lin = lineage) gene, which encoded a 21 nucleotide (nt) regulatory
RNA (1). This type of gene was unprecedented, but opportune work from the
Ruvkun laboratory on another developmental gene, lin-14, provided the neces-
sary clues for predicting how a tiny RNA product might control gene
expression (1, 2). The lin-4 RNA recognizes sites of imperfect complementar-
ity in the 3′ untranslated region (UTR) of the lin-14 messenger RNA
(mRNA) and halts protein expression (Figure 1). Insufficient lin-4 RNA or
deletion of the target sites in the lin-14 3′ UTR leads to failed down-regula-
tion of LIN-14 protein expression at the appropriate time and, thus, abnor-
mal development (1-4). Although the LIN-14 protein disappears in response
to the lin-4 RNA, the lin-14 mRNA remains and continues to associate with
polysomes, indicating that translational inhibition is the mechanism at work
(2, 5).

Figure 1. Conserved sites in the 3′UTR of the C. elegans lin-14 mRNA are complementary to the lin-4
miRNA (2). Shaded blocks in the lin-14 3′UTR indicate sequences of high homology (at least 10
nucleotides of exact conservation) between the related nematodes C. elegans and C. briggsae. The
striped blocks 1-7, represent regions of partial complementarity to the lin-4 miRNA. The duplexes are
shown with the lin-14 top strand reading 5′ to 3′ base-paired with the bottom strand lin-4 miRNA.
IDENTIFICATION AND ANALYSIS OF MICRORNAS 3

Another target of lin-4 regulation, the lin-28 protein-coding gene, was

also discovered by the Ambros laboratory, providing another example of a devel-
opmental gene under post-transcriptional control by the tiny RNA (6). Yet, the
question of whether this novel mode of gene regulation was restricted to nema-
todes persisted until the turn of the century (7). The identification of the let-7 (let
= lethal) gene in C. elegans as another 22 nt RNA that regulates the expression
of protein-coding genes containing 3′ UTR target sites raised the possibility that
tiny RNA genes might abound in worms and beyond. Indeed, the remarkable
conservation of the let-7 RNA sequence enabled the Ruvkun laboratory to estab-
lish that this gene is expressed in diverse animals, including fruit flies, molluscs,
sea urchins, zebrafish, and humans (8). Moreover, temporally regulated expres-
sion of the let-7 RNA and potential target sites in lin-41 homologues in all species
assayed implied that this RNA gene may be essential for development of many
animal species (8).
Around the time let-7 RNA was discovered, another type of tiny RNA
was gaining fame. In 1998, Fire and Mello reported that injection of double-
stranded RNA (dsRNA) into C. elegans could elicit the degradation of homolo-
gous mRNA and, thus, potently inhibit gene expression in a process termed RNA
interference (RNAi) (9). Shortly thereafter, several groups found that the dsRNA
is cleaved to ~22 nt small interfering RNAs (siRNAs) that serve as the guides to
target complementary mRNA sequences for destruction (10-14). It was clear that
tiny RNAs could be powerful regulators of gene expression and soon hundreds
of ~22 nt RNA genes were uncovered in animal and plant genomes (15-20). The
small size of these regulatory RNAs inspired the name microRNA (21), and genes
encoding these RNAs appear to be present in all multicellular organisms (22).

Transcription of miRNAs

Despite our relatively brief awareness of their existence, impressive

progress has been made in understanding how miRNAs are expressed and func-
tion (22). The ~22 nt, mature forms of miRNAs arise from multiple processing
steps of longer substrate RNAs. So far, little is known about the composition of
the initial miRNA transcripts, called primary miRNAs (pri-miRNAs). Both Pol II
and Pol III promoters have been used to drive ectopic expression of pri-miRNAs
(23-25). Recently, the Kim laboratory presented direct evidence that Pol II
transcribes several mammalian miRNAs (26). Additionally, a few complete
pri-miRNAs that have been characterized show hallmarks of Pol II transcrip-
tion—they apparently undergo 5′-end capping, 3′-end polyadenylation, and
splicing (26-28). These first examples of pri-miRNAs are more than 1,000
nucleotides long—remarkably lengthy transcripts to serve as substrates for ~22 nt
RNA products!
Many miRNAs are restricted to specific developmental periods or tissue
types. At least in some cases, regulated expression of miRNAs is attributable to
transcriptional control. Predicted promoter sequences of the C. elegans let-7 gene
can confine expression of green fluorescent protein (GFP) to late larval and adult
stages—the same time in development when mature let-7 is present (29). The
transcriptional control sequences for lys-6 miRNA, which regulates neuronal
4 S. BAGGA AND A. PASQUINELLI

asymmetry in C. elegans, restricts GFP expression to a subset of neurons (30).

Although these examples of predicted miRNA promoters directing protein
expression support the likelihood that they recruit Pol II, the specialized tran-
scription factors that afford temporal or spatial control are yet to be identified for
any miRNA gene.

Processing of miRNAs

Generation of the mature miRNA form requires multiple processing

and cellular transportation events (Figure 2). In animals, the nuclear localized
ribonuclease (RNase) Drosha clips the ~65 nt hairpin miRNA precursor (pre-
miRNA) from the primary transcript (31). The pre-miRNA is shuttled by
Exportin-5 to the cytoplasm for final processing by the RNase Dicer (32-38).

Figure 2. A model of miRNA biogenesis and function. The relatively long primary transcripts,
called pri-miRNAs, are initially transcribed from miRNA genes (35). The pri-miRNAs are
processed by the RNase Drosha to hairpin precursors (25, 31, 35). The precursors are recognized
by Exportin-5 and delivered to the cytoplasm for maturation to ~22 nt RNAs by Dicer (25, 32-38).
It has been proposed that a helicase activity separates the duplex (42, 43), and typically only one
half is retained and incorporated into a multi-factor RNA-induced silencing complex (RISC) (12,
52-54). The degree of complementarity between a miRNA and its target site determines the regulat-
ory mechanism: near perfect base-pairing directs RNA degradation and bulged duplexes medi-
ate translational repression (22). This model is based primarily on work in animal systems; note
that organismal differences exist for the protein factors and subcellular location of processing
events (22).
IDENTIFICATION AND ANALYSIS OF MICRORNAS 5

This enzyme appears to measure ~22 nt from the 5′ and 3′ ends of the hairpin
to position a staggered cut through both strands of the stem (25, 31, 39-41).
Typically, only one half of the resulting duplex is retained. Thermodynamic
arguments have been made to explain the choice for which strand persists. The
5′ end that is more easily peeled away from its antisense is favored for incorpo-
ration into a stable complex and, by default, the other half is unprotected and
degraded (42, 43).
Pri-miRNAs contain sequences and structures important for processing
and generation of the functional ~22 nt form. However, truncated pri-miRNA
substrates, even the hairpin precursors, can suffice as substrates to produce
mature miRNAs when overexpressed from heterologous constructs (23-25, 31,
35). In the endogenous situation, processing may be a critical control point in
miRNA biogenesis. Deletion of cis-acting sequences in pri-miRNA transcripts or
depletion of trans-acting processing factors can inhibit miRNA maturation (27,
31-34). In some cases, the miRNA substrates accumulate in vivo, indicating that
transcription of a miRNA gene and production of the mature form are not nec-
essarily coupled.

Function of miRNAs

Mature miRNAs inhibit expression of genes containing sequences of

antisense complementarity. In animals, the primary mechanism of gene regula-
tion concurs with the original model proposed for the lin-4 miRNA and
lin-14 mRNA in C. elegans (1, 2). Imperfect base-pairing between the
miRNA and sequences in the 3′ UTR of the target mRNA results in inhibit-
ed protein expression (2, 5, 44). It remains to be determined how partial
base-pairing between miRNAs and target sequences results in blocked pro-
tein production.
In plants, many miRNAs exhibit perfect, or nearly complete, base-pair
complementarity to their target mRNAs (45, 46). Not only does this feature of
plant miRNAs make it simpler to predict specific targets, but it also results in tar-
get degradation (46-50). Animal miRNAs can also direct mRNA destabilization
if they share near-perfect complementarity with target sequences (24, 51-54). In
fact the vertebrate miRNA, miR-196, can form a complete duplex with sequences
in HOXB8 mRNA and direct degradation of this target (55). The general model
holds that miRNAs can regulate gene expression by either translational inhibi-
tion or mRNA destabilization, depending on the nature of the duplex formed
with the target sequences.

EXPERIMENTAL IDENTIFICATION OF miRNAS

The noncoding nature and the extraordinarily small size of miRNAs

make their detection challenging. For a long time, the conventional cloning and
identification techniques and the gene prediction databases were clearly biased
for long protein-coding sequences. The discovery of tiny RNAs in C. elegans and
elucidation of the RNAi mechanism led several groups to adopt novel or modi-
fied conventional methods to detect miRNAs.
6 S. BAGGA AND A. PASQUINELLI

Genetic Screening

The discovery of pioneer members of the miRNA family—lin-4 and

let-7—demonstrated the potential of classical genetic screens in detection of
miRNAs (1, 2, 56). Although time-consuming and labor-intensive, identification
of an miRNA through a genetic screen can readily give important clues about its
function and gene targets. Also, rare and nonconserved miRNAs, which usually
evade cloning and computational detection, can be identified by genetic methods.
A loss-of-function screen led to the identification of C. elegans miRNA lys-6,
which controls left/right neuronal asymmetry (30). Gain-of-function genetic
screens based on mutations in negatively regulated targets or forcing altered
expression of miRNAs have also contributed to the detection of new miRNAs
(48, 51, 57, 58). These studies emphasized an important distinction of present-
day genetic mapping—to look for noncoding, short stem-loop structures in addi-
tion to conventional open-reading frames (ORFs). Increasing efforts toward
developing full genome databases will facilitate the identification of more
miRNAs through genetic screenings. Taking into account the abundance of
miRNAs, it would not be surprising if many of the previously uncharacterized
loci in genetic screens could be ascribed to miRNAs.

Biochemical Cloning

Direct cloning of expressed miRNAs by the Ambros’, Bartel’s, and

Tuschl’s laboratories led to the identification of the first populations of miRNAs
in worms, flies, and humans (15, 16, 20). Several unique as well as highly con-
served miRNAs, like let-7, were detected in these initial cloning efforts. Northern
blot analyses of cloned miRNAs revealed both tissue-specific and stage-specific
miRNAs, emphasizing their role in developmental timing and tissue specifica-
tions. The phylogenetic distribution of miRNAs was further expanded by the
cloning of plant miRNAs (17-20, 59). Cloning of tiny RNAs from specific
ribonucleoprotein complexes also identified several novel miRNAs (60). To date,
biochemical cloning has led to the identification of hundreds of distinct miRNAs
(15-17, 19, 20, 60-64).
An important characteristic that emerged from biochemical cloning and
complied with lin-4 and let-7 sequences is the existence of animal miRNAs as a
part of ~70 nucleotide stem-loop precursors (1, 8, 56). Processing of mature
miRNAs from hairpin precursors is now considered a signature of animal
miRNA genes (21). Although plant miRNAs also derive from precursors, com-
position of these substrates is not as well defined (19). The miRNA sequence can
reside on either arm of the stem-loop structure, and hence the location on the
precursor is not a determinant of its excision by Dicer. Cloning of miRNAs that
are clustered in the genome and identification of some in expressed sequence tag
(EST) databases hinted that miRNA precursors might derive from longer pri-
mary transcripts (15, 20, 28, 35, 61, 65). After the discovery of Drosha, it was
speculated that specific cleavage of primary transcripts determines the correct
register of Dicer action and hence the mature ends of miRNAs are determined at
the level of primary transcripts (31).
IDENTIFICATION AND ANALYSIS OF MICRORNAS 7

As the result of being RNase III Dicer products, miRNAs are cloned
based on their three distinguishing features: a length of about 22 nt, a 5′-termi-
nal monophosphate, and a 3′-terminal hydroxyl group (10, 33, 66). The general
protocol for miRNA cloning involves size fractionation of an RNA population
followed by ligation with adapter molecules (Figure 3) (10, 15, 16, 20). The chimeric
RNA is then subjected to reverse-transcriptase polymerase chain reaction (RT-
PCR), cloned, and sequenced. One of the advantages of biochemical cloning of
miRNAs is that the expressed miRNA population from any tissue or at any stage
of development can be readily detected. Cloning of mouse brain-tissue miRNAs
revealed probable orthologs of C. elegans lin-4 RNA, and the mouse sequences
revealed probable Drosophila orthologs as well (61). Homologues of the lin-4
gene had, thus far, not surfaced from informatic searches of other organisms.
Although powerful in terms of revealing expressed miRNAs directly, detection
by cloning has an inevitable drawback of selecting clones of breakdown products
of abundant cellular RNAs. Hence, to qualify as an miRNA, a small cloned
RNA should be able to form a stem-loop precursor structure with its flanking
sequences and show conservation in related species (21). Endogenous siRNAs are
usually distinguished from miRNAs by extended dsRNA structure of their pre-
cursors and by displaying less sequence conservation (21, 67).
Interestingly, cloning efforts in C. elegans and Drosophila led to the iden-
tification of new categories of noncoding RNAs designated as “tiny noncoding
RNAs” (tncRNAs) and “repeat-associated small interfering RNAs” (rasiRNAs)
(63, 64). The 24-26 nt rasiRNAs apparently derive from various repetitive
sequence elements including retrotransposons, DNA transposons, satellite, and

Figure 3. miRNA cloning strategy. Typically, total RNA is fractionated to ~22 nt size forms and
miRNAs containing 5′ phosphate and 3′ hydroxyl groups are substrates for ligation to adaptor oligonu-
cleotides (10, 15, 16, 20). The chimeric RNA is subjected to RT-PCR, cloning, and sequencing.
Legitimate miRNAs match genomic sequences that support formation of a hairpin precursor (21).
8 S. BAGGA AND A. PASQUINELLI

microsatellite sequences, complex as well as vaguely characterized repetitive

sequence motifs (64). The tncRNAs are similar in size to miRNAs but are not
processed from stem-loop precursors and do not have orthologs in other species
(63). Although some of tncRNAs exhibit temporal expression patterns, their
exact role and significance await further experimentation.

Informatics

Although biochemical cloning led to the identification of several hun-

dreds of new miRNAs, it is limited for identifying rare miRNAs or those that are
triggered by specific environmental conditions. The availability of full genome
databases of several organisms enabled the development of informatics
approaches for identification of new miRNAs.
The fortuitous discovery of the first conserved miRNA, let-7, demon-
strated the potential of simple homology searches using BLASTN (8). Homology
searches with cloned miRNAs also revealed orthologs and paralogs in various
organisms (15, 16, 20). A simple homology-based strategy originally involved the
analysis of intergenic sequences among related organisms using the RNA folding
program “mfold” (16, 68). The output was scanned by eye for miRNA charac-
teristic stem-loop structures and the expression was confirmed by Northern blot-
ting. The proximal location of several miRNA genes prompted the search for new
miRNAs adjacent to the previously identified ones (20, 64, 69, 70). This approach
is most suitable for identification of rapidly evolving miRNA genes, which are
proximal to each other but are too divergent in sequence to be detected by gen-
eral methods (22).
An important advance in detection of miRNA genes has been achieved
by development of new computational approaches (63, 67, 71-76). All the pro-
grams primarily utilize sequence conservation, presence of stem-loop structures,
and intergenic location of miRNAs as basic criteria. One of the more sensitive
programs, “MiRscan,” has been applied to vertebrate and nematode genomes to
identify new miRNA genes (67, 74). The MiRscan program was developed by
using the 50 cloned miRNAs from C. elegans as the training set (16, 20). Based
on its similarity to the training set, a score is assigned to each putative genomic
candidate that is identified by conserved stem-loop structures. The evaluation is
based on seven features: 1) base-pairing of the miRNA portion of the fold-back,
2) base-pairing of the rest of the fold-back, 3) stringent sequence conservation in
the 5′ half of the miRNA, 4) slightly less stringent sequence conservation in the
3′ half of the miRNA, 5) sequence biases in the first five bases of the miRNA, 6)
a tendency toward having symmetric internal loops and bulges in the miRNA
region, and 7) the presence of 2-9 consensus base-pairs between the miRNA and
the terminal loop region with a preference for 4-6 base-pairs. The accuracy of
MiRscan predictions has been further improved by the inclusion of conserved
elements upstream of miRNA precursors (69). The successful application of this
program to vertebrates, although developed using nematode miRNAs, demon-
strated its universal application. It also emphasized that, despite sequence varia-
tions of miRNAs among diverse animals, their generic features are broadly
conserved.
IDENTIFICATION AND ANALYSIS OF MICRORNAS 9

Using a reference set of Drosophila pre-miRNA sequences, another pro-

gram called “miRseeker” identified novel miRNA genes (73). The miRseeker
algorithm detects insect miRNA genes using a three-step filter strategy. The first
step involves extraction of candidate genes using conserved and nongenic regions
of D. melanogaster and D. pseudoobscura genomes. The next step identifies and
ranks the stem-loop structured regions based on the helical length and free ener-
gy values. Finally, high-scoring regions averaged for two genomes are evaluated
for divergence using the determinants of a reference set. In principle, miRseeker
should be applicable to analysis of other sets of sequenced genomes of related
organisms.
Recent informatics approaches specifically designed to detect plant
miRNAs identified several new candidates (71, 76). These strategies are similar to
MiRscan and miRseeker in terms of using homologous fold-back sequences con-
served between Arabidopsis and Oryza sativa. However, the parameters con-
straining the selection of fold-back structures were specifically designed for plant
miRNAs. The MIRcheck algorithm utilizes the sequences and structures of puta-
tive miRNA hairpins and 20mers within them (71). MIRcheck selects the candi-
dates by restricting the number of unpaired, bulged, or asymmetrically unpaired,
consecutive unpaired nucleotides, and the length of the hairpin. Unlike other
programs, MIRcheck does not restrict based on pattern or extent of base pairing
outside the 20mer sequence, a feature typical of plant miRNAs. Several of the
plant miRNAs identified by this approach were confirmed by expression and tar-
get mRNA degradation (71).

ANALYSIS OF miRNAS

Many miRNAs exhibit diverse temporal and spatial expression patterns.

Additionally, the relative levels of a particular miRNA can vary several orders of
magnitude among different cell types. Adaptations of traditional molecular tech-
niques as well as novel methods have been developed to analyze when, where, and
how much of a specific miRNA exists and what is its biological function.

Expression Patterns

Northern blot and RNase protection assays yielded the first molecular
evidence for the existence of a ~22 nt RNA product. A specific tiny RNA prod-
uct was present in wild-type but not lin-4 mutant worms, and this RNA reap-
peared upon rescue of the mutant with a transgene containing just 693 nt of lin-4
genomic sequence (1). Typical analyses for miRNA expression by Northern blots
utilize high percentage (10-15%) polyacrylamide gel electrophoresis (PAGE),
which enables detection of the mature and precursor forms of the miRNA
(Figure 4) (1, 77). The relative level of a mature miRNA can be readily assessed
by sampling total RNA from particular tissues, developmental time points, or
experimental conditions. However, Northern blotting to detect specific miRNAs
can be labor intensive and insensitive to low-level miRNAs.
Computational prediction of miRNA genes avoids the cloning bias of
detecting the more abundant species. Confirmation of a predicted miRNA can be
10 S. BAGGA AND A. PASQUINELLI

Figure 4. Northern analysis of miRNA expression. Total RNA from wild-type worms or worms
depleted of dicer was isolated, separated by 11% polyacrylamide gel electrophoresis and subjected to
Northern hybridization analysis to detect let-7 RNA. The ~22 nt nucleotide mature form is predomi-
nant in wild-type worms, whereas the 65 nt precursor accumulates in Dicer(–) worms (32).

experimentally challenging, though, if the gene is weakly expressed or only acti-

vated under particular conditions. PCR-based approaches were developed to
help validate the expression of miRNAs identified by informatics. Strong evi-
dence for the existence of several elusive miRNAs was provided by a PCR pro-
tocol, which involves amplifying miRNA sequences from bulk miRNA cDNA
libraries by way of the common adaptor sequences (67, 75). Real-time PCR
assays have been employed for relatively high throughput analysis of miRNA
precursor expression (78). In at least some cases, the level of precursor accu-
rately reflected that of mature, as indicated by Northern analyses. More recent-
ly, an exceptionally sensitive and quantitative method was reported for detecting
precursor or mature miRNAs (79). The Invader miRNA assay can detect as lit-
tle as 20,000 molecules of a specific miRNA and has been used to show that the
amounts of human let-7a miRNA vary over several orders of magnitude among
different tissues (79).
Microarray technology offers an efficient and sensitive method to assess
global changes in miRNA expression patterns. Microchips containing oligonu-
cleotides corresponding to miRNA sequences have been used to screen various
cell types to uncover the miRNA profile (80, 81). Additionally, this type of
miRNA profiling was used to identify distinctions between normal human B cells
and those derived from chronic lymphocytic leukemia cells (82). Since their dis-
covery, miRNA genes have been considered possible disease candidates (83).
High throughput profiling of miRNA expression patterns offers a powerful tool
for correlating specific miRNAs with altered cell biological states.
Detection of miRNAs in vivo is particularly challenging considering their
small size and potentially low abundance. Nonetheless, in situ hybridization
results have indicated localized expression for a few miRNA transcripts. In
IDENTIFICATION AND ANALYSIS OF MICRORNAS 11

plants, there is an inverse correlation between expression of specific miRNAs and

proposed targets in specific tissues, supporting the model that these RNAs nega-
tively regulate protein expression to control development (84-86). Localized
expression of miR-10 in Drosophila embryos indicates a role for this miRNA in
regulating genes in the thoracic and abdominal primordia, although specific tar-
gets of miR-10 are yet to be identified (87).
An indirect method for analyzing temporal and spatial expression of
miRNA genes is to fuse predicted miRNA promoter sequences to a reporter gene,
such as GFP. This technique revealed tissue and developmental regulation of spe-
cific miRNA promoters in C. elegans that agreed with predictions about the func-
tion of the miRNAs (29, 30, 88). The lys-6 miRNA was discovered as a gene that
controls neuronal asymmetry in C. elegans by repressing expression of a tran-
scription factor in a left taste neuron (30). Consistent with the proposed function
of lys-6, a GFP reporter fused to the promoter for this miRNA gene is expressed
in the left, but not right, neuron (30). These types of reporter experiments are
very useful for predicting when and where an miRNA promoter functions as well
as for studying its transcriptional control (29, 30, 88). However, the promoters
and functions of most miRNAs are yet to be identified and, thus, caution is war-
ranted for interpreting expression patterns based on fusions to miRNAs for
which little is known about the natural biological role.
The in situ and reporter experiments described above can be used to indi-
cate when and where an miRNA gene is active, but they do not demonstrate the
production of functional miRNAs. Regulated processing and stabilization of
some miRNAs may also influence their ability to control gene expression. An
ingenious method to detect functional miRNAs in vivo was developed by the
Cohen laboratory to show spatial and temporal expression of the Drosophila
bantam miRNA (51). The “sensor” strategy is based on the demonstration that
miRNAs will direct degradation of target mRNAs containing sites of perfect
antisense complementarity (Figure 5) (24, 48, 52–54). A GFP-reporter gene con-
taining bantam miRNA complementary sites was down-regulated in response to
bantam expression. Thus, the presence of a functional miRNA can be assayed
in vivo without the knowledge of its natural targets. Identification of specific
miRNA expression patterns will greatly facilitate determination of biological
functions.

Functional Roles

The first miRNAs, lin-4 and let-7, were initially discovered as genes essen-
tial for regulating developmental timing in C. elegans (1, 56). Since the vast
majority of RNAs to join the miRNA family were isolated by biochemical or
computational means, biological functions are yet to be assigned. Considering
their abundance, it is not surprising that miRNA genes are now being uncovered
in mutant screens. Perhaps the lack of traditional gene structure allowed
miRNAs to escape previous detection, but now mutations in miRNA genes
account for broad-ranging phenotypes, including disrupted neuronal asymmetry,
misregulated cell death, abnormal fat metabolism, and cellular patterning defects
(18, 28, 30, 51, 58, 84, 88, 89). Isolation of genetic mutations in specific miRNA
12 S. BAGGA AND A. PASQUINELLI

Figure 5. The “sensor” approach to analyze miRNA expression in vivo (51). In this example, con-
structs expressing a reporter protein, such as GFP, fused to 3′UTR sequences +/− miRNA comple-
mentary sites are introduced into worms. A ubiquitous promoter drives reporter expression. If the
miRNA is absent, such as in early larval development (middle panels), GFP will be detected.
However, if the miRNA turns on later in development or in particular cell types, the reporter mRNA
will be specifically degraded and GFP will disappear (last panel, top row). This example shows a
predicted pattern for let-7 expression in C. elegans: early in development let-7 is absent and thus GFP
is expressed ubiquitously (gray shading of entire worm), including in the 10 hypodermal seam cells,
but later in development let-7 miRNA is produced and shuts off reporter expression, perhaps specif-
ically in the 16 seam cells of adult worms (absence of gray shading) (29). Importantly, expression of
a control reporter lacking the miRNA complementary sites is unaffected by miRNA expression
(bottom panels).

genes not only aids in determining biological function but also is valuable for
identifying direct targets of regulation. A genetic suppressor screen of the let-7
mutant revealed lin-41 as a target of negative regulation, which then led to the
recognition of let-7 complementary sites in the 3′UTR of lin-41 mRNA (56, 90).
In many systems, targeted disruption or isolation of mutations in specific
miRNAs is prohibitively laborious. Furthermore, homology among several
groups of miRNAs suggests that redundancy may obscure phenotypes resulting
from mutation of just one member. Overexpression or ectopic expression is
an efficient alternative to study the function of particular miRNAs. The validity
of this approach was established by introducing high copies of the lin-4
gene to worms and observing developmental defects opposite of the lin-4 loss-
of-function phenotypes (4). More recently, ectopic expression of miR-181 in
mouse hematopoietic stem cells biased their differentiation into B-lineage cells
(23). Thus, direct targets of miR-181 may be predicted by focusing on distinct
changes in gene expression in the B-lineage pathway.
The biological function of specific miRNAs can also be revealed by inhi-
bition with antisense oligonucleotides. Injection of antisense DNA oligonu-
cleotides corresponding to specific miRNAs into Drosophila embryos resulted in
developmental defects (91). More recently, 2′-O-methyl oligonucleotides were
shown to block potently the function of targeted miRNAs in Drosophila, human
cell, and C. elegans systems (92, 93). The 2′-O-methyl modification protects
the oligonucleotide against cellular RNases (94). Base-pairing of the oligonucleo-
tide to an miRNA titrates the miRNA from its endogenous targets, thus reveal-
ing the loss-of-function phenotype. Although delivery of the antisense
oligonucleotide can be technically prohibitive (92), this method of miRNA inhibition
IDENTIFICATION AND ANALYSIS OF MICRORNAS 13

offers an efficient means to uncover the biological roles of miRNAs for which
only the mature sequence is known.

PAIRING OF miRNAS WITH TARGETS

The combination of cloning and computational approaches has likely

enabled identification of the majority of miRNAs (22). However, as of yet, only
a few miRNAs have been paired with their bona fide targets. Identification of
direct miRNA targets is essential for understanding their diverse functions.
In addition to the discovery of pioneer miRNAs, the credit for the dis-
covery of the first miRNA targets also goes to classical genetics. Long before the
broad significance of tiny regulatory RNAs was appreciated, the functional pair-
ing of lin-4 RNA with its target lin-14 mRNA was proposed (1, 2). The 3′UTR
of lin-14 mRNA had partial complementarity to lin-4 RNA and was sufficient for
temporal regulation of a reporter gene. The let-7 target, lin-41, also supports the
model, both in terms of partial complementarity and reporter gene regulation
with the 3′UTR (56, 90, 95). The opposite phenotypes of lin-4 and lin-14 mutants
helped pinpoint lin-14 as a direct target of lin-4 mediated negative regulation (96,
97). A handful of other bona fide miRNA target genes were identified through
genetic screens (28, 30, 51, 57, 88, 89, 98, 99). However, for the majority of other
miRNAs either mutants are not known or their mutant phenotypes are not
apparent. Also, the small size and imperfect nature of base-pairing, particularly
in case of animal miRNAs, hampers straightforward prediction of miRNA
targets.

Target Identification and Validation for Plant miRNAs

Exact complementarity between miR171 and an mRNA target in

Arabidopsis indicated that target prediction might be less complicated for plant
compared with animal miRNAs (17, 19, 48). Indeed, “near perfect complemen-
tarity” appears to be a general rule for plant miRNA targets (46, 71, 76). Initially,
targets were identified by searching annotated Arabidopsis mRNAs for 0-4 mis-
matches to specific miRNAs (19, 46). Conservation of the predicted mRNA tar-
get sequences in rice and low hits with a random cohort of tiny RNA sequences
strengthened the validity of these proposed plant targets (46, 71). Plant miRNA
targets show a clear bias toward transcription factors involved in cell differentia-
tion and developmental patterning (46, 71). In comparison to sequences regulat-
ed by animal miRNAs, most plant miRNA-target interactions exhibit two
general distinctions: 1) plant miRNA target sites are primarily found within open
reading frames, and 2) multiple target sites within the same target mRNA are not
detected in plants. These features may have significant functional implications for
plant miRNAs—they favor an RNAi-like mechanism, as opposed to translation-
al control, to inhibit gene expression (45).
A sensitive computational approach identified several novel plant
miRNA targets belonging to families of transcription factors as well as other
genes like ATP sulfurylase, laccase, and superoxide dismutase (71). This approach
allowed for gaps and mismatches between mRNA:miRNA duplexes but
14 S. BAGGA AND A. PASQUINELLI

constrained the candidate targets to conservation between Arabidopsis and

Oryza. Validation of predicted target sequences is facilitated by the fact that
many plant miRNAs direct cleavage of their complementary mRNA targets. The
3′ cleavage product of the target, which maps to the 10th nucleotide of the
miRNA and has a characteristic phosphate at its 5′ end, can be cloned and
sequenced (47, 48, 50, 71, 76). Although absence of a 3′ cleavage product may
suggest a false or alternatively regulated target, its presence is a convincing con-
firmation of regulation by a specific miRNA.

Informatics Approaches for Target Identification in Animals

New computational methods have matched animal miRNAs with numer-

ous target genes, although many of the pairings still await experimental confi-
rmation (100-104). The small number of validated miRNA targets in animals
makes the development of reliable algorithms particularly challenging. As a
starting point, most computational methods rely on conserved complementary
sites within 3′ UTRs of potential target genes.
Identification of the hid gene as a bantam miRNA target exhibited the
potential of computational approaches for identifying targets in Drosophila (51,
101). This approach was based on the presence of miRNA target sites in 3′ UTRs
of target mRNAs and their relatively better complementarity to the 5′ end of
miRNAs. The first step for identifying genome-wide Drosophila miRNA targets
involved generation of a conserved database comparing 3′ UTRs of D.
melanogaster and D. pseudoobscura (101). The candidate target genes were then
scored based on their free energy of base-pairing with the miRNAs, as determined
by mfold (68, 101). The combination of sensitive sequence databases with that of
the RNA folding algorithm confirmed the known targets and identified several
new ones (101). A striking feature of predicted targets was the presence of clusters
of functionally related targets regulated by specific miRNAs. This included Notch
target genes for mir-7, proapoptotic genes for mir-2 family, and metabolic pathway
enzymes for mir-277 (101). Another computational method for target identifica-
tion, miRanda, relies on evolutionary relationships between miRNAs and their
targets using three insect genomes (104). The miRanda approach is a three-phase
method involving sequence matching of miRNA:mRNA pairs, estimating the
energetics of the physical interaction and using evolutionary conservation as an
informational filter. This method suggested both multiplicity (one miRNA targets
several genes) and cooperativity (one gene targeted by several miRNAs) as general
features of miRNA-regulated gene expression (104).
The TargetScan algorithm predicted more than 400 target genes for mam-
malian miRNAs (102). TargetScan also combines thermodynamics-based model-
ing of RNA:RNA duplex interactions with comparative sequence analysis to
predict miRNA targets conserved across multiple genomes. One of the criteria
for filtering miRNA:mRNA pairs using this algorithm is exact complementarity
between 2–8 bases of miRNAs counted from the 5′end of miRNA. The folding
energy of each pair is calculated using RNAeval (105), after extending the pair-
ing as far as possible. Each 3′ UTR is then scored based on the number of
miRNA:mRNA matches, free energy of interaction, and number and affinity of
IDENTIFICATION AND ANALYSIS OF MICRORNAS 15

complementary sites. Comparative ranking of UTRs among different organisms

sorted on the basis of this score then predicts the target. TargetScan revealed
that, in contrast to plant miRNA targets, only a small fraction of predicted mam-
malian targets participate in developmental control; they seem to regulate broad-
ly diverse biological processes (102). Another computational program,
DIANA-microT, was developed to study the rules of single miRNA:MRE (tar-
get mRNA) pairing and to predict targets containing a single complementary site
(103). Similar to other computational programs, DIANA-microT identifies the
putative targets by estimating the binding energies between conserved
miRNA:MRE pairs. A difference from other programs is that it also takes into
account the G-U wobble dinucleotide pairs for calculating binding energies (103).
Computational identification, based on favorable energy statistics and
evolutionary relationships, corroborated by experimental evidence provides rea-
sonable substantiation of miRNA target validity. An important consideration in
computational target prediction and confirmation is the use of correctly anno-
tated genes. Already, ambiguity in annotated genes misguided attempts to vali-
date a miRNA target (106, 107). Absent or incomplete annotations of 3′ UTRs
also hinder the comprehensive analysis of miRNA targets.
Heterologous reporter assays are most commonly used for validation of
miRNA targets (Figure 6). Typically, a reporter gene, such as luciferase or β-galac-
tosidase, is fused to sequences containing the miRNA complementary region from
a putative target. Expression of the reporter is observed in the presence or absence
of the proposed regulatory miRNA (6, 56, 90, 98, 99, 101-103). Down-regulation
of a reporter gene in the presence of the miRNA indicates the presence of regula-
tory sites in the fragment used for fusion. However, concerns of extraneous effects
due to multiple copies of complementary sites or very long UTR regions should be
kept in mind. Also, failure to demonstrate regulation of a heterologous reporter
may reflect factors independent of the miRNA: 1) the cell system might not express
additional co-factors or adequate levels of the miRNA to appreciably affect
reporter expression, 2) additional mRNA elements are required but not included in
the UTR segment of the reporter, and 3) steric hindrance imposed by the fusion of
reporter on the putative sites blocks interaction with the miRNA.

SUMMARY AND OUTLOOK

Genetics introduced us to the existence of miRNAs. Biochemical and

molecular methods were essential for establishing the existence of vast numbers
of miRNA genes in diverse organisms. Bioinformatic approaches contributed to
the identification of additional, elusive, miRNAs as well as to the prediction of
miRNA target genes. Combined experimental and computational methods will
be required to advance our rudimentary understanding of miRNA expression
and function. Central questions remain: How are transcription and processing of
miRNAs regulated? How do miRNAs find their appropriate targets? What is the
mechanism by which miRNAs regulate expression of their targets? The discovery
of miRNAs established a new paradigm for gene regulation and understanding
the biological roles of these abundant RNA genes undoubtedly will be a chal-
lenging endeavor.
16 S. BAGGA AND A. PASQUINELLI

Figure 6. Validation of miRNA target predictions. Multiple targets with sites of partial complemen-
tarity are often identified for a single miRNA. To test the function of such predictions, sequences con-
taining the complementary elements are fused to reporter genes, such as luciferase. The reporter is
assayed for expression in the presence and absence of the candidate miRNA partner. In the depicted
example, an miRNA is predicted to recognize sites in the 3′ UTRs of three different genes. Each UTR
is fused to the reporter and the constructs are introduced to cells expressing the miRNA of interest.
Only the site from gene “1” mediates reporter repression via interaction with the miRNA.

ACKNOWLEDGMENTS

We are grateful to members of the Pasquinelli laboratory for comments

and inspiration. Research support is provided by the National Institutes of
Health (GM071654-01) and the Searle Scholar, Peter Gruber, and V
Foundations.

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DORMANCY AND THE CELL CYCLE

Michael A. Campbell

School of Science
Penn State Erie—The Behrend College
Erie, Pennsylvania 16563

INTRODUCTION

Dormancy can be viewed as a complex trait that encompasses physio-

logical and developmental responses to environmental signals. There has been con-
siderable research and emphasis in the past on the dormancy of seeds, which is to
be expected in view of the significant impact to agricultural yields, but it has only
been in the last few decades that significant research success has yielded infor-
mation on the topic of bud dormancy. Thus, this review will focus on the recent
results regarding bud dormancy and the interaction of dormancy to issues relat-
ed to phytohormones and cell division.

TYPES OF DORMANCY AND THE CELL CYCLE

Biologically, dormancy is an avoidance response to drought, cold, or

shortening days. The dormant situation is a complex set of physiological states
and conditions in which plants respond to a series of stresses such as drought and

Genetic Engineering, Volume 27, Edited by J. K. Setlow

©Springer Science+Business Media, Inc. 2006 21
22 M. CAMPBELL

over-wintering by entering a state of growth suspension. This state of growth sus-

pension can be exhibited by degrees of dormancy. The classification and degrees
of dormancy found in plant vegetative structures have been defined as endodor-
mancy, paradormancy, and ecodormancy (Figure 1) (1). Ecodormancy is a reduced
growth response to an external stimulus such as drought or cold. Removal of the
stimulus results in a resumption of growth. Paradormancy is a reduced growth
response induced by a biochemical signal that is transported to a target tissue.
Removal of the signal results in a resumption of growth. A good example of
paradormancy is apical dominance, where auxin transported from an apical
shoot suppresses the growth of lateral buds. Endodormancy is due to an endoge-
nous signal that results in growth suppression. Older references often used the
term “deep dormancy” to describe this phenomenon. In some situations, time is
all that is required for endodormancy to terminate (e.g., the potato) and in others
there is a need for a cold treatment in order to break the dormant state (such
as most flowering temperate trees and shrubs).
Defining a dormant state in a plant can be difficult because a tissue may
progress from one dormant condition to another without any phenotypic change.
For example, a meristem may enter a phase of reduced growth in late summer in
response to day-length or drought. Thus, the meristem could be considered to be
in a state of ecodormancy. This situation could be followed by a physiological
shift and a transition into a state of endodormancy. As winter progresses the
endodormant state may terminate but growth will not resume due to unfavorable
conditions and the meristem is now again in an ecodormant state. This shift
between dormant states suggests that control of the growth cycle is a complex
interaction between endogenous and environmental factors, and there is no sim-
ple genetic solution to increasing yields by manipulating dormancy. This concept
is supported by breeding experiments and quantitative trait analysis, which is
demonstrated by the significant genetic complexity controlling the onset and
breakage of dormancy in poplar (2).

Figure 1. Diagram of dormancy states in a typical perennial bud. As the season progresses, dorman-
cy types may shift between ecodormancy and endodormancy and back to ecodormancy. Redrawn from
M. Lang et al. (1).
DORMANCY AND THE CELL CYCLE 23

The variations and changes in the types of dormancy suggest that differ-
ent biological and physiological mechanisms are involved with the changing of
dormant states. As this pertains to cell cycle regulation, it is unclear whether dif-
ferent states of dormancy are associated with variations in cell cycle control. The
cell cycle is regulated by a set of protein interactions between cell division kinases
(CDKs), cyclins (CYCs), and inhibitors or regulators of the CDK/CYC com-
plex [reviewed in (3)]. The activity of the CDK/CYC complex is associated with
the establishment of restriction, or control, points throughout the cell cycle.
These control points are positioned in the gap phases (G1 or G2) of the cell cycle
(Figure 2). Thus, dormancy, which is characterized by very low or absent rates of
cell division, must function by the established control points found in the G1 and
G2 phases of the cell cycle (4). The arrest of cell division in endodormant ash
buds (5), endodormant Helianthus buds (6), endodormant potato meristems (7),
and paradormant axillary buds of pea (8) appear to be predominantly at the
G1/S-phase of the cell cycle. This commonality of regulation, despite the specif-
ic type of dormancy, suggests that attempts to alter or control the dormant state
in plants, with respect to the cell cycle state, would probably require manipulation
of the factors or signal transduction mechanisms that interact with the G1/S
restriction point. The interaction of various types of dormancy with the G1/S
portion of the cell cycle demonstrates that artificial manipulation dormancy, by
alteration of cell division or growth, will most likely involve genes or proteins that
regulate the transition from the G1 to the S-phase of the cell cycle. At this point
I do not suggest that direct alteration of genes regulating the G1/S restriction
point is a practical solution to dormancy manipulation. Cell cycle control is

Figure 2. The major regulatory positions of plant cells.

24 M. CAMPBELL

fundamental to cell proliferation and survival. However, recent analysis of plant

dormancy has narrowed the field to a number of potential targets that might
impact dormancy onset and length without directly altering basic cell cycle
machinery.

THE PARADORMANT STATE

Paradormancy, specifically apical arrest of lateral buds, is regulated by

auxin but it is not clear whether this response is directly or indirectly associ-
ated with that phytohormone [reviewed by Horvath et al., 2003 (9, 10)].
Shimizu-Sato and Mori (11) recently reviewed the topic of dormancy regula-
tion in lateral meristems and they proposed a scheme for hormonal regulation
of growth (Figure 3). In this model, auxin, produced in the apical regions of
the plant, does not directly inhibit cell division in the lateral meritems, but
does result in abscisic acid (ABA)-induced genes in the node region and the
lateral meristem. The model is supported by the work of Gocal et al. (12),
who demonstrated that auxin levels in dormant meristems are low and they
increase after release of apical repression. This suggests that, inasmuch as
high levels of auxin are not present in paradormant lateral meristems, growth
inhibition must result from some other signal. The increase in auxin as later-
al buds are released from apical repression may have some association with
entry into the cell cycle, since it has been shown that tobacco BY2 cells
express D-type cyclins in response to auxin (13). Thus, a potential working
model for paradormancy may be lateral meristem arrest by nodal ABA sig-
nals. Entry into the cell cycle begins following removal of the auxin/ABA
inhibition and production of a phytohormonal signal, such as cytokinin, that
initiates cell division.

Figure 3. The mechanism of action in a paradormant axillary bud induced by apical dominance.
Redrawn from Shimizu-Sato and Mori (11).
DORMANCY AND THE CELL CYCLE 25

Transgenic plants that overproduce cytokinins exhibit a reduced level of

lateral bud arrest (14, 15). Thus, it can be concluded that the interaction of
cytokinins and auxins regulate lateral bud growth and paradormancy. These two
hormones are probably functioning at two levels, an inhibitor process controlled
by apically produced auxin/ABA and a growth-promoting process regulated by
cytokinin.

THE ENDODORMANT STATE

In many perennial woody plants the breakage of the endodormant state

requires chilling. It has been found that chilling results in a rearrangement of
symplastic connections between cells of the apical meristem by the formation of
1,3-beta-D-glucan blockages (16). Thus, chilling induced capacity for cell division
may be a result of removal of symplastic blockage by 1,3-beta-D-glucanase
resulting in increased intercellular communication. It is possible that this
increased communication results in the transport of hormonal signals through-
out the meristem. However, it has not been shown that alterations of intercellu-
lar connections directly change hormonal transport or metabolism in meristems.
There has been recent research focused on the subject of vernalization,
which is a requirement of some species for a cold treatment for the induction of
flower development. Some parallels exist between the process of vernalization
and cold-induced breakage of endodormancy. Vernalization has been shown to
be an epigenetic process that requires prolonged exposure to cold temperatures,
resulting in a developmental shift from vegetative to floral development [(17),
reviewed in (18)]. Thus, vernalization and the breakage of endodormancy both
require a similar temporal exposure to cold. Little is known about the molecular
mechanisms associated with the breakage of endodormancy, but significant
research advances have been accomplished regarding the process of vernaliza-
tion. In Arabidopsis, vernalization has been shown to be an epigenetic process
where extended exposure to cold results in the repression of the gene FLOWER-
ING LOCUS C (FLC) with the use of chromatin remodeling induced by the
genes VRN1, VRN2, and VIN3 (19).
Exposure to cold can induce cold acclimation, vernalization, and the
breakage of endodormancy in some species. Is there any similarity in the cold-
induced regulation of these three processes? Cold treatment also induces elevat-
ed ABA levels and a series of cold-regulated genes controlled by the
transcription factors CBF1, CBF2, and CBF3 (20, 21), but prolonged cold treat-
ment reduces ABA in over-wintering endodormant buds. Liu et al. (22) demon-
strated that vernalization is not regulated by ABA or the cold-induced
transcription factors. While the onset of endodormancy appears to require
ABA, it has been shown that cold is not necessary to induce the endodormant
state in grape (23). However, to break endodormancy in some species, cold is a
requirement and, in other species, such as potato, the breakage of endodorman-
cy only requires time. A commonality between potato and species that require
cold might be the reduction of ABA levels in meristems, shifting endodorman-
cy to an ecodormant state. Another possibility is that the control of endodor-
mancy is similar to vernalization, where chromatin remodeling, controlled by
26 M. CAMPBELL

ABA, time, and/or cold treatment is a requirement. Support for this hypothesis
can be found in the work of Law and Suttle (24), who showed that in potato
tubers demethylation of CCGG regions of DNA has been linked to the break-
age of endodormancy. What remains to be determined is what regions of the
genome are remodeled. Additionally, it would be important to discern whether
a similar mechanism of remodeling is occurring in species that require cold
treatment for the breakage of endodormancy and potato, which only requires a
temporal exposure for dormancy loss. Is it possible that termination of
endodormancy follows a pattern similar to that of vernalization: cold or time
results in chromatin remodeling in an area of the genome that contains genes
that suppress growth? Meristems that enter endodormancy have usually under-
gone a significant developmental shift with leaves replaced by bracts or bud
scales at nodal regions and such developmental changes can be associated with
chromatin remodeling (25).

IS THERE A UNIFYING THEORY TO DORMANCY?

Environmental stress, such as drought, induces the production of ABA,

which results, with a complex set of responses, in growth arrest (ecodormancy).
In paradormancy, in particular apical dominance, it has been demonstrated that
there is an auxin-induced ABA response at nodal regions, which results in
growth arrest in lateral meristems. Elevated ABA levels, at least in the potato
system, induce endodormancy. Thus, a common theme in a number of plant sys-
tems, regardless of the type of dormancy, appears to involve an ABA response
at some level.
However, the removal of ABA is not always sufficient to end the dormant
state and initiate growth. Additional hormones, such as cytokinin and gib-
berellins, appear to be necessary for the resumption of cell division. Thus, dor-
mancy, irrespective of the specific type, is controlled by a process of growth
suppression and growth initiation. This might explain some of the genetic com-
plexity found by breeders who are interested in the process of dormancy. The fact
that both growth inhibitors and growth promoters regulate dormancy suggests
that cell cycle control would follow a similar path; there would be cell cycle
inhibitory mechanisms as well as cell cycle promotive mechanisms associated
with dormancy. In potato endodormancy, growth arrest seems to occur upstream
of the mechanisms of direct cell cycle control (7). This situation may be a result
of endodormancy-inducing inhibitors of the cell cycle and that ABA has a cen-
tral role in maintaining the endodormant state.

REGULATION OF THE G1 TO S TRANSITION

OF THE PLANT CELL CYCLE

The cdk/cyclin protein complex regulates cell cycle transitions and,

because dormancy appears to be a G1/S arrest, it is necessary to elucidate the
components of the CDK/CYC complex associated with that arrest. Currently,
specific proteins associated with dormancy G1/S arrest have not been found. In
Arabidopsis, there are at least four different CDKs and the activity of one class
DORMANCY AND THE CELL CYCLE 27

of the A-type of CDKs increases during the G1 to S-phase transition of the cell
cycle [reviewed in (3, 26, 27)]. Potato meristems do not change in the levels of
transcript for p34cdc2 kinase as endodormancy terminates (7). Thus, a working
hypothesis is that dormancy regulates the activity, not the transcript levels, of a
class of the A-type CDKs. The activity of a CDK requires the presence of spe-
cific cyclins, a specific phosphorylation state, and the absence of active inhibitors.
This complex arrangement for CDK activity suggests that dormancy repression
of the cell cycle at the G1 to S transition may result with the regulation of a num-
ber of different targets including cyclin levels, kinase activity, phosphatase activ-
ity, and the manipulation of inhibitors.
Plant cells contain a diverse population of cyclins, including A, B, D, and
H-types (3, 28). More cyclins await description in plants, particularly in peren-
nial species, but among the classes of cyclins known, the ones associated with
G1/S cell cycle regulation are of direct interest to dormancy studies. The D-type
cyclins have been shown to be associated with G1 to S-phase transitions in yeast
(29). In Arabidopsis, genomic analysis has revealed that there are 49 different
cyclins, which can be assigned to nine different subgroups: CYCA, CYCB,
CYCC, CYCD, CYCH, CYCT, CYCL, CYCU, and SDS (30). Although func-
tion has not been determined for each of the cyclin-like genes, experimental evi-
dence strongly suggests that the CYCD and CYCA classes are associated with
the G1/S transition of the cell cycle [(27, 31), reviewed in (32-34)]. Thus, the
CYCD and CYCA class of cyclins may be directly regulated by dormancy in
plant tissues. It should be noted that cells not undergoing a cell cycle might
exhibit low levels of many different classes of cyclins but dormant tissues, arrest-
ed in the G1 position, would first need to express the CYCD and CYCA proteins
for entry into the S-phase.
The activity of the CDK/CYC complex is regulated by additional cellular
and biochemical mechanisms. A class of proteins classified as CDK inhibitors
(CKIs) interacts with the CDK/CYC complex and prevents cell cycle progression
[reviewed in (33, 34)]. These inhibitors are interesting targets for investigating the
interaction of dormancy and the cell cycle. In mammalian systems, G1/S-specif-
ic CKIs are represented by p21Cip1, p27Kip1, and p57Kip2 (17, 35-37). De
Veylder et al. (38) have examined the activity of five Kip-related proteins (KRPs)
in Arabidopsis thaliana. Thus, in comparison to mammalian systems, plants
appear to have a greater diversity in KRP-type CDK inhibitors. Does this suggest
that plant systems utilize a greater diversity of cell division inhibitors for spatial
or temporal regulation of cell division? The results of De Veylder et al. (38)
demonstrated more of a structural relationship between the KRPs and regulation
of cell division. In Arabidopsis there are at least seven KRPs that appear to have
diverse functions temporally and spatially in the shoot apex (39), but it is not
clear how KRPs are associated with meristem activity and the process of dor-
mancy. An additional class of cell division inhibitors called ICK1 and ICK2 has
been identified in plants (40, 41). ICK1 has been shown to be induced by ABA
(40), suggesting a relationship between a phytohormone associated with the dor-
mancy response and a protein preventing entry into the cell cycle. The direct con-
nection between ABA-induced dormancy and cell cycle inhibitors has yet to be
adequately demonstrated.
28 M. CAMPBELL

THE PHYTOHORMONE CONNECTION

ABA has been shown to regulate response to drought, cold, salt stress,
and seed dormancy through a complex set of fast and slow responses [reviewed
in (42-45)]. The signal transduction mechanisms associated with ABA exposure
in plants have recently been reviewed (42, 46), and currently there are about
50 genes associated with ABA responses in Arabidopsis (43), affecting more than
1,300 different transcripts (47). The interaction of the ABA signal transduction
mechanism with genes or proteins that directly affect the dormancy response,
which is a slow response, is not clear, and it has been difficult to separate ABA
responses associated with stress and cold from those that are directly related to
dormancy. ABA has been implicated with the onset and maintenance of
endodormancy in potato (48), white birch (49), and lily (50). The interaction of
ABA with the process of cell division is still not clear. Application or inhibition
of ABA to meristematic tissues may alter the onset of endodormancy but it also
results in developmental shifts resulting in the formation of bud scales in place of
primordial leaf. Additionally, cross-talk between ABA and other hormones, par-
ticularly those that induce growth such as cytokinin, gibberellin, and auxin, com-
plicates the experimental approaches necessary to elucidate specific responses.
The role of ABA in seed dormancy and germination has progressed significantly
(51), but due to a lack of a model system, ABA control of perennial meristem
growth remains undefined. In Arabidopsis, an ABA application to germinating
embryos results in reversible growth arrest. In tomato, ABA-deficient mutants
exhibit an increase in cells arrested in G2/M, suggesting that ABA might regulate
the G1/S restriction point. The ABA regulation at the G1/S restriction point may
be due to cell cycle inhibitors such as ICK1, but there is some speculation that
seedling dormancy might be a function of a p53-regulated process (52, 53). The
idea that p53 might regulate cell division in seeds is based on the concept that
seeds can be exposed to prolonged storage, resulting in environmentally induced
DNA damage. This becomes an interesting issue in long-lived perennial species
where lateral bud arrest (paradormancy) may occur on the order of hundreds or
thousands of years and may result in significant DNA damage. However, the
connection between ABA-induced stress and DNA damage has yet to be eluci-
dated. ABA also results in reduced levels of metabolic activity, which might
reflect a lack of nutrient mobilization. In animal systems it has been shown that
serum starvation induces p53 activation and growth arrest by the ribosomal
protein L11 (54).
In addition to the production of cell cycle inhibitors, ABA appears to
interact with the phosphorylation cascade that is associated with the regula-
tion of cell division [reviewed in (43, 46)]. ABA interacts with inositol
polyphosphate 5-phosphatase (55), phospholipase C (55), cyclin-dependent
kinase (56), protein phosphatase 2C (57, 58), and mitogen-activated protein
kinase (59). Additionally, ABA is involved with the regulation of RNA
metabolism including transcript abundance, RNA stability, transport and
degradation [reviewed in (42)] and some of these transcripts may relate to cell
cycle regulation.
DORMANCY AND THE CELL CYCLE 29

Cytokinins have an important role in the breakage of dormancy (60-62).

The resumption of cell division following dormancy is often associated with an
increase in cytokinin levels. Within the last decade, significant progress has been
made regarding the molecular and genetic mechanisms associated with cytokinin
signal transduction [reviewed in (63)]. In endodormant tissues, such as potato,
meristems change in their sensitivity to exogenous cytokinins; close to harvest, or
deep dormancy, cytokinins have little effect on sprouting, but as tubers age the
sensitivity to cytokinins increases (62). It has been suggested that endormant
tubers do not respond to exogenous cytokinin because of the lack of a cytokinin
receptor or inactivity of the signal transduction mechanism of cytokinin action
(64). In Arabidopsis, it has been determined that receptors for cytokinins
(AHK2, AHK3, CRE1/AHK4) are transmembrane histidine kinases (65-68).
The expression of the cytokinin receptors appears to occur in all tissues of
Arabidopsis (63) and the AHK receptors relay endogenous cytokinin signals
resulting in shoot and root meristem growth (69). It is not known whether dor-
mancy alters receptor levels. Interestingly, Arabidopsis cre1 mutants, which have
a decreased response to endogenous cytokinin, exhibit a slight increased sensitiv-
ity to ABA (65), suggesting a level of cross-talk between the cytokinin and ABA
signal transduction systems. Exogenous cytokinin induces D-type cyclin expres-
sion and cell division (70), and cytokinin-induced cell division can be replaced by
overexpression of D-type cyclins (71). This suggests that as meristems become
active following dormancy they enter the cell cycle by cytokinin-induced expres-
sion of D-type cyclins. Thus, entry into the cell cycle may not be controlled directly
by the dormancy process but by phytohormone production after tissues have exit-
ed the dormant state.
In addition to being associated with the breakage of seed dormancy, gib-
berellins (GAs) have been linked with dormancy release in tulips [reviewed in
(72), potato (73), and lily bulbs (74, 75)]. However, the complexity of GA types
in cells and tissues, the possible presence of inhibitors, and the difficulty assess-
ing the specific dormant state makes it unclear whether GA is involved with the
breakage of dormancy or is a postdormancy growth response. Results by
Horvath et al. (76) suggest that the application of GA3 to leafy spurge resulted in
G1 to S-phase transition in adventitious buds (Figure 4).
GA has been shown to bind to a GCR1 receptor in A. thaliana seeds (77).
Additionally, G-protein-type receptors are associated with GA perception (78).
These types of binding by GA probably result in a signal transduction cascade
that has yet to be defined in its entirety, but ultimately there must be some impact
on the cell cycle machinery. In deepwater rice, GA application induces cell division
(79, 80). The regulation of GA on rice cell division appears to be largely at the
G2/M restriction point (81). One of the responses of rice to exogenous GA is to
increase the levels of transcripts for mitotic cyclins and a specific class of cyclin-
division kinase (82, 83). In the meristems of dicots, specifically tomato, GA
induces the expression of transcripts for expansins, proteins that alter cell wall
extensibility and cell expansion (84). The change in expansion expression may sug-
gest another avenue for GA impact on cell cycle machinery, since cell size has been
associated with cell cycle regulation in a number of eukaryotic organisms.
30 M. CAMPBELL

Figure 4. Possible regulatory steps in G1 by phytohormones that regulate dormancy.

CONCLUSIONS

The localization of the Kip-related proteins to meristematic regions sug-

gests that these proteins might be associated with dormancy regulation.
Additionally, the localization of chromatin remodeling to specific genes and loci
may reveal the important regulation mechanisms for the dormant state.
Developmental mutants that fail to develop bud scales and shift meristem pro-
grams toward an over-wintering bud would be informative in identifying.
A substantial body of work has been accomplished in identifying genes
and proteins associated with cell division and the cell cycle in plants. However,
most of the recent research has focused on the annual A. thaliana as a model. In
order to progress rapidly in the area of plant meristem dormancy, a model sys-
tem has to be adopted. Many perennial species are slow growing, genetically com-
plex, and have little background genetic research that can be used to support
dormancy studies. Recent advances into the genetic structure of poplar and
recent interest in some perennial relatives of A. thaliana may create opportunities
for models systems.

REFERENCES

1 Lang, G.E., Early, J.D., Martin, G.C. and Darnell, R.L. (1987)
HortScience, 22(3), 371-377.
2 Chen, T.H.H., Davis, J., Frewen, B.E., Howe, G.T. and Bradshaw, H.D.
(2000) in Dormancy in Plants: From Whole Plant Behavior to Cellular
Control (J.-D. Viemont and J. Crabbe, eds.) CABI Publishing, New
York, NY. pp. 319-330.
3 Dewitte, W. and Murray, J.A. (2003) Ann. Rev. Plant Physiol. Plant Mol.
Biol. 54, 235-264.
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stomachic aperient.—Dose, 2 to 4, at bedtime; in torpor of the large
intestines, the dyspepsia of the debilitated, &c.
Pills of Podophyllin. Syn. Pilulæ podophyllini. Prep. Resin of
podophyllin, 1⁄ 4 gr. Extract of henbane. To make one pill. One or
two for a dose.
Pills of Prussian Blue. (Jolly.) Syn. Pilulæ ferri percyanidi
composita. Prep. Prussian blue, 18 gr,; sulphate of quinine, 12 gr,;
extract of opium, 1 gr.; conserve of roses, q. s. Mix, and make into
12 pills; 1 every three hours, in neuralgia.
Pills, Plummer’s. See Pills of Calomel (Compound).
Pills, Pu′rgative. Syn. Pilulæ purgantes, L. Prep. 1. (Dr
Robinson.) Aqueous extract of aloes, 1 dr.; powdered scammony, 1⁄ 2
dr.; balsam of Peru, 10 or 12 gr.; oil of caraway, 9 or 10 drops; mix,
and divide into 30 pills. A warm, stimulating aperient, highly
recommended to excite the peristaltic action of the bowels of the
aged, sedentary, and debilitated.—Dose, 1 to 4 pills, as required.
2. (Trousseau & Reveil.) Resin of jalap, 1 dr.; scammony, 1⁄ 2 dr.;
extract of colocynth, 6 gr.; excipient, as required. For 20 (or, better,
24) pills.—Dose, 1, “every two hours, in the morning, fasting, until
they operate.” For other formulae see Pills, Aperient and Cathartic,
and Pills of Aloes, Jalap, Colocynth, &c.
Pills of Quinine′. See Pills of Sulphate of Quinine.
Pills, Reece’s. See Pills, Chirayta.
Pills, Re′nal. Syn. Pilulæ renales, L. Prep. 1. Squills, myrrh, and
digitalis, of each (in powder) 10 gr.; extract of rhubarb and mercurial
pill, of each 15 gr.; powdered nitre, 20 gr.; oil of juniper, 10 or 12
drops. For 24 pills. Alterative, diuretic, and tonic.—Dose, 3 to 6,
thrice a day. Hunter’s Renal Purifying Pills are similar, but omitting
the mercurial pill. De Roos’ Renal Pills contain a preparation of
copaiba.
 Pills, Rhe′umatism. Syn. Pilulæ antirheumaticæ, L. Prep. 1.
Gum guaiacum, 1 dr.; nitrate of potassia, 1 1⁄ 2 dr. (both in powder);
soft soap (Ph. L.), 1⁄ 2 dr.; oil of cajeput, 16 drops. For 4 dozen pills.
—Dose, 2 to 6, night and morning; in chronic rheumatism, and
rheumatic gout. Their action is accelerated by the copious use of
lemon juice during the day.
2. (Beasley.) Extract of artichoke, 1⁄ 2 dr.; powdered sarsaparilla,
20 gr.; oil of sassafras, 1 drop. For 12 pills.—Dose, 1 pill, thrice daily.
Pills of Rh′ubarb. Syn. Pilulæ rhei (Ph. E.), L. Prep. 1. (Ph. E.)
Powdered rhubarb, 9 parts; acetate of potassa, 1 part; conserve of
red roses, 5 parts; mix, and divide into 5-gr. pills. A stomachic and
gentle aperient, particularly useful in atonic dyspepsia.—Dose, 2 to 4
pills.
2. (Ph. U. S.) Powdered rhubarb, 6 dr; Castile soap, 2 dr.;
beaten up with water, q. s., and divide into 120 pills. As the last.
Pills of Rhubarb (Compound). Syn. Aromatic pills, Balsamic
laxative p., Edinburgh p., Stomachic p.; Pilula rhei composita (B. P., Ph.
L.), Pilulæ r. compositæ (Ph. E. & D.), P. stomachicæ, P. aromaticæ, L.
Prep. 1. (Ph. L.) Powdered rhubarb, 4 dr.; powdered Socotrine aloes,
3 dr.; powdered myrrh, 2 dr.; soft soap (Ph. L.), 1⁄ 2 dr.; oil of
caraway, 15 drops; treacle, q. s. to form a mass.
2. (Ph. L. 1836.) Powdered rhubarb, 1 oz.; aloes, 6 dr.; myrrh, 4
dr.; Castile soap, 1 dr.; oil of caraway, 1⁄ 2 fl. dr.; syrup, q. s.
3. (Ph. E.) Powdered rhubarb, 12 parts; aloes, 9 parts; myrrh
and Castile soap, of each 6 parts; conserve of red roses, 5 parts; oil
of peppermint, 1 part; mix, and divide into 5-gr, pills. The oil of
peppermint may be omitted, when so preferred.
4. (Ph. D.) Rhubarb, 1 1⁄ 2 oz.; hepatic aloes, 9 dr.; myrrh and
Castile soap, of each in fine powder, 6 dr.; oil of peppermint, 1 fl. dr.;
treacle, 2 oz.; mix, and beat the whole to a uniform mass.
 5. (Ph. U. S. & Ph. E. 1817.) Rhubarb, 8 dr.; aloes, 6 dr.; myrrh,
4 dr.; oil of peppermint, 1⁄ 2 fl. dr.; syrup of orange peel, q. s.; mix,
and divide into 240 pills.
6. (B. P.) Rhubarb, in fine powder, 3 oz.; Socotrine aloes, in fine
powder (some physicians prefer the aqueous extract—Squire), 2 1⁄ 2
oz.; myrrh, in fine powder, 1 1⁄ 2 oz.; hard soap, 1 1⁄ 2 oz.; English oil
of peppermint, 1 1⁄ 2 dr.; treacle, by weight, 4 oz.; reduce the soap to
fine powder and triturate it with the rhubarb, aloes, and myrrh; add
the treacle and oil, and beat into a mass.—Dose, 5 to 10 gr.
Obs. The above are tonic, stomachic, and gently laxative;
extremely useful for obviating costiveness and giving tone to the
stomach and bowels.—Dose, 6 or 8 to 20 gr. The London pill is not
only the most agreeable, but it keeps the best.
Pills of Rhubarb and Ca′raway. See Kitchener’s Peristaltic
Persuaders (Patent medicines).
Pills of Rhubarb and Cham′omile. Syn. Speediman’s pills;
Pilulæ rhei et anthemidis, L. Prep. From aloes, myrrh, rhubarb (each in
powder), and extract of chamomile, of each 1 dr.; essential oil of
chamomile, 10 or 12 drops. For 4-gr. pills. An excellent tonic and
stomachic aperient, particularly useful in the dyspepsia and loss of
appetite of hard drinkers.—Dose, 1 to 3 pills, either before dinner or
at bedtime.
Pills of Rhubarb and Copa′iba. Syn. Pilulæ rhei et copaibæ, P.
r. balsamicæ, L. Prep. (Swediaur.) Powdered rhubarb and gum, equal
parts; balsam of copaiba, q. s.
Pills of Rhubarb and Gi′nger. Syn. Stomach pills; Pilulæ rhei et
zingiberis, L. Prep. From powdered rhubarb, 1 dr.; powdered ginger,
1⁄ 2 dr.; Castile soap, 20 gr.; tincture or essence of ginger, q. s. to
form a mass. For 30 pills.—Dose, 1 to 6.
Pills of Rhubarb and Ipeca′cuanha. Syn. Pilulæ rhei et
ipecacuanhæ, L. Prep. From rhubarb, 1⁄ 2 dr.; ipecacuanha, 15 gr.;
opium, 5 gr. (each in powder); oil of cinnamon, 6 drops; syrup, q. s.
For 18 pills.—Dose. In loss of appetite and spasmodic dyspepsia, 1
to 3 pills, twice a day; in dysentery, diarrhœa, &c., to relieve tormina
and tenesmus, 1 every two hours.
Pills of Rhubarb and I′ron. Syn. Pilulæ rhei et ferri (Ph. E.),
L. Prep. (Ph. E.) Dried sulphate of iron, 4 parts; extract of rhubarb,
10 parts; conserve of red roses, 5 parts; beat them to a proper
mass, and divide this into 5-gr. pills.—Dose, 2 to 4 pills; in the atonic
dyspepsia of debilitated subjects, in chlorosis, &c.
Pills of Rhubarb and Ox-gall. Syn. Pilulæ rhei et fellis bovini,
L. Prep. From powdered rhubarb, gum ammoniacum, and
inspissated ox-gall, equal parts; beaten up with a little tincture of
ginger or proof spirit, and the mass divided into 2 1⁄ 2-gr. pills. In
dyspepsia and constipation dependent on a torpid action of the liver.
—Dose, 2 to 6 pills.
Pills of Rhubarb and So′da. Syn. Pilulæ rhei et sodæ, P. r.
comp. cum sodâ, L. Prep. (Guy’s Hosp.) Dried carbonate of soda,
powdered rhubarb, and extract of gentian, equal parts. For 4 1⁄ 2-gr.
pills.—Dose, 2 to 4 pills; in acidity, heartburn, diarrhœa, loss of
appetite, &c.
Pills, Richter’s. See Pectoral pills.

Pills, Dr Robinson’s. See Pills, Purgative.

Pills, Rudius’s. Syn. Rudius’s extract; Pilulæ rudii, Extractum
rudii, L. Prep. 1. Colocynth pulp, 6 dr.; agaric, black hellebore, and
turpethum root, of each 4 dr.; cinnamon, mace, and cloves, of each
40 gr.; rectified spirit, 1⁄ 2 pint; digest for 4 days, express the
tincture, and evaporate it to a proper consistence for making pills.
Formerly esteemed one of the most safe and certain cathartics in
troublesome constipation.—Dose, 5 to 20 gr.
2. (Ph. E. 1783.) Black hellebore and colocynth, of each 2 oz.;
water, 4 pints (o. w. m.); boil to a quart, strain, evaporate to the
consistence of honey, and add, of aloes, 2 oz.; scammony
(powdered), 1 oz.; next remove the vessel from the fire, and further
add of sulphate of potassa, 2 dr.; oil of cloves, 1 dr.; and form the
whole into a pill-mass. Resembles the last (nearly).
Pills, Rufus’s. See Pills of Aloes with Myrrh.
Pills of Sabadilla. Syn. Pilulæ cevadillæ. Prep. Equal parts of
sabadilla and honey; make into 5-gr. pills.—Dose. For an adult, 4 to
6 pills; for a child, 1 to 2. Vermifuge.
Pills of Sa′ffron. Syn. Pilulæ croci, L. Prep. 1. From hay
saffron, 1 dr.; myrrh, 1⁄ 2 dr.; oil of cajeput, 6 drops; syrup of
saffron, q. s. For 36 pills.—Dose, 1 to 3 or 4, occasionally; as a
stimulant in low spirits, hypochondriasis, &c.
2. (Phœbus.) Saffron, myrrh, and sulphur, equal parts;
inspissated bile, q. s. For 2-gr. pills.—Dose, 2 to 12 daily; as an
emmenagogue.
Pills of Sagape′num (Compound). Syn. Pilulæ sagapeni
compositæ, L. Prep. (Ph. L. 1836.) Sagapenum, 1 oz.; aloes, 1⁄ 2 dr.;
syrup of ginger, q. s.—Dose, 5 to 20 gr.; as a stimulant
antispasmodic laxative, in dyspepsia with flatulence, flatulent colic,
&c.
Pills of Sal′icin. Syn. Pilulæ salicinæ, L. Prep. From salicin, 1⁄ 2
dr.; powdered rhubarb, 20 gr.; extract of gentian, q. s. to mix. For 4-
gr. pills.—Dose, 2 to 4, every three hours, during the apyrexia of
intermittents.
Pills of Sandal Wood Oil. (Ebert.) Syn. Pilulæ olei santali.
Prep. Oil of yellow sandal wood, 1⁄ 2 oz.; yellow wax, 1⁄ 2 oz. Melt
the wax into a capsule, and weigh into it the oil of sandal wood. Mix,
and stir until cold, then roll out the mass and divide it into 80 pills,
by means of the pill machine or pill-tite, in the same manner as in
the ordinary mass, and sprinkle with marshmallow root powder. Each
pill contains about 3 gr. or about 5 drops of the oil. The excipient is
unobjectionable, as it is readily soluble in the juices of the stomach.
Pills of Scam′mony (Compound). Syn. Pilulæ scammonii
compositæ, L. Prep. 1. (St. B. Hosp.) Scammony, 24 gr.; ginger, 20 gr.;
aloes and gamboge, of each 12 gr.; treacle, q. s.; mix, and divide
into 12 pills. A powerful cathartic and vermifuge.—Dose, 1 to 3 pills.
2. (B. P.) Resin of scammony, resin of jalap, of each 1 oz.; curd
soap, in powder, 1 oz.; strong tincture of ginger, 1 fl. oz.; rectified
spirit, 2 fl. oz. Add the tincture and spirit to the soap and resins, and
dissolve by the aid of a gentle heat, then evaporate the spirit over a
water-bath until the mass has a pilular consistence.—Dose, 5 gr. to
15 gr.
Pills, Scot’s. Prep. From aloes, 9 lbs.; jalap, 3 lbs.; gamboge
and ginger, of each 1⁄ 2 lb.; beaten with treacle, q. s. See Pills,
Anderson’s Scot’s.
Pills, Dr Scott’s Bil′ious and Liver. Prep. (Cooley.)
Compound extract of colocynth (Ph. L. 1836), 8 oz.; powdered
rhubarb, 4 oz.; powdered myrrh, 2 oz.; soft soap, 1⁄ 2 oz.; oil of
caraway, 2 1⁄ 2 dr.; strong syrup of saffron, q. s. to form a pill-mass.
“There are twenty-five 3 1⁄ 2-gr. pills in each 1s. 1 1⁄ 2d. box.” “It has
been stated that these pills contain a minute portion of antimony.”
(‘Anat. of Quackery.’)
Pills, Se′dative. Syn. Pilulæ sedativæ, L. Prep. 1. Hydrochlorate
of morphia, 6 gr.; powdered sumbul, 20 gr.; alcoholic extract of
Indian hemp, 1⁄ 2 dr. For 2-gr. pills.—Dose, 1 to 3, twice or thrice
daily; in excessive nervous irritability, painful menstruation, &c.
2. (U. C. Hosp.) Camphor, 1 dr.; reduce it to powder by means
of rectified spirit, 3 or 4 drops; add of extract of henbane, 20 gr.,
and divide the mass into 20 pills. To allay pain and excitement, &c.—
Dose, 1 to 2 pills.
 3. To either of the above, add of powdered rhubarb and extract
of gentian, of each 20 gr., and divide the mass into 4-gr. pills.—Dose,
1 to 4 pills; when, besides the other symptoms, the stomach and
bowels are disordered.
Pills, Sedillot’s Febrifuge. Prep. From powdered opium, 3 gr.;
sulphate of quinine, 12 gr.; confection of opium, 10 gr., or q. s. For
12 pills.—Dose, 1 to 2, every second hour, during the intermission of
an ague.
Pills of Sen′na. Syn. Pilulæ sennæ, P. s. compositæ, L. Prep. 1.
Powdered senna, 1 dr.; extract of rhubarb, 1⁄ 2 dr.; powdered
capsicum, 4 gr.; oil of juniper, 6 or 8 drops. For 3-gr. pills. An
aperient well suited for females.—Dose, 5 to 8 pills.
2. (Hufeland.) Powdered senna, 1 dr.; extract of dandelion, q. s.
to mix. For 30 pills. As the last.
Pills, Smith’s. Prep. From powdered aloes, 4 dr.; jalap, 2 dr.;
ginger and soft soap, of each 1 dr.; oil of juniper, 1⁄ 2 dr.; emetic
tartar, 6 gr. For 120 pills. Laxative and diuretic.—Dose, 1 to 4, at
bedtime, or early in the morning.
Pills, Dr Hugh Smith’s. See Stomach Pills.
Pills of Soap. Syn. Pilulæ saponis, P. cum sapone, L. Prep. (P.
Cod.) White Castile soap, 32 parts; powdered marshmallow root, 4
parts; powdered nitrate of potassa, 1 part; beat them to a mass, and
divide this into 4-gr. pills. In habitual costiveness, calculary
affections, &c.—Dose, 1 to 6 pills, twice or thrice a day.
Pills of Soap (Compound). Syn. Pills of soap and opium,
Laudanum pills; Pilula saponis composita (Ph. L.), Pilulæ saponis cum opio,
L. Prep. 1. (Ph. L.) Opium and liquorice, of each (in powder) 2 dr.;
soft soap (Ph. L.), 6 dr.; beat them to a uniform mass.
2. (B. P. & Ph. U. S.) Opium (in fine powder), 1⁄ 2 oz.; Castile
soap, 2 oz.; distilled water, 1⁄ 2 fl. dr., or q. s.; reduce the soap to
powder, mix it with the other ingredients, and beat the whole
together, as before.—Dose, 3 gr. to 5 gr. See Pills of Opium.
Obs. The above pills contain 1-5th part of their weight in dry
opium. The dose is 3 to 10 gr., in the usual cases in which the
administration of opium is indicated. Mr Skey, the eminent surgeon
of St. Bartholomew’s Hospital, has shown the great value of this pill
in promoting the healing of obstinate ulcers, more especially those
of the legs.
Pills of Soda. Syn. Pilulæ sodæ carbonatis, L. Prep. (Ph. E.
1817.) Exsiccated carbonate of soda, 4 parts; Castile soap, 3 parts;
syrup, q. s. to form a mass. Antacid and slightly laxative.—Dose, 10
to 20 gr. This pill was a great favourite of the once celebrated Dr
Beddoes.
Pills, Soot. (Dr Neligan.) Syn. Pilulæ fuliginis. Prep. Extract of
soot, 1⁄ 2 dr.; compound galbanum pill, 1 scruple; oil of valerian, 15
minims. Make into 12 pills. Take 2 three times a day. For hysteria.
Pills, Speediman’s. Prep. (Cooley.) Aloes, 3 dr.; rhubarb,
myrrh (all in powder), and extract of chamomile, of each 1 dr.; oil of
chamomile, 20 drops. For 4-gr. pills. An excellent aperient, tonic, and
stomachic.—Dose, 2 to 4 pills, as a purgative; 1, as a stomachic or
dinner pill.
Pills, Splenet′ic. Syn. Pilulæ antispleneticæ, L. Prep. (Saunders.)
Strained aloes and gum ammoniacum, of each 3 dr.; myrrh and
bryony, of each 1⁄ 2 dr. For 4-gr. pills.—Dose, 3 to 5, “Extolled in
amenorrhœa and hypochondriasis.” (Dr R. E. Griffith.)
Pills of Squill (Compound). Syn. Cough pills, Pills of squills
and ginger; Pilula scillæ compositæ (B. P., Ph. L.), Pilulæ scillæ compositæ
(Ph. D.), P. scillæ (Ph. E.), L. Prep. 1. (Ph. L.) Freshly powdered
squills, 1 dr.; powdered ginger and powdered ammoniacum, of each
2 dr.; mix, add of soft soap (Ph. L.), 3 dr.; treacle, 1 dr.; and beat the
whole together, so that a mass may be formed.
 2. (Ph. E.) Squills, 5 parts; ammoniacum, ginger (all in fine
powder), and Spanish soap, of each 4 parts; conserve of red roses,
2 parts; mix, as before, and divide the mass into 5-gr. pills.
3. (Ph. D.) Squills (in fine powder), 2 1⁄ 2 dr.; ammoniacum,
ginger, and Castile soap, of each (in fine powder) 2 dr.; treacle, 1⁄ 2
oz.
4. (B. P.) Squill (in fine powder), 1 1⁄ 4; ginger (in fine powder),
1; ammoniacum (in powder), 1; hard soap (in powder), 1; treacle
(by weight), 2, or a sufficiency; mix the powders, add the treacle,
and beat into a mass.—Dose, 5 to 10 grains.
Obs. Compound squill pill is a most useful expectorant in chronic
coughs, asthmas, bronchial affections, difficulty of breathing, &c.;
and, combined with calomel and foxglove, and, occasionally, with
croton oil, as a diuretic, &c., in dropsies. Unfortunately, however, it
soon spoils; and, therefore, to be effective as a remedy, it must be
recently prepared. As an expectorant, it should not be administered
until the inflammatory symptoms have been subdued by purgatives
or bleeding. A little powdered opium, or extract of henbane, is
occasionally added, to allay irritation.—Dose, 5 to 20 gr., twice or
thrice a day, accompanied by an occasional aperient.
Pills, Stahl’s. See Pills, Aperient.
Pills, Starkey’s. Prep. (Original formula.) Extract of opium, 4
oz.; mineral benzoar and nutmeg, of each 2 oz.; saffron and
Virginian snake-root, of each 1 oz.; Starkey’s soap, 1⁄ 2 lb.; oil of
sassafras, 1⁄ 2 oz.; tincture of antimony (Old Ph.), 2 fl. oz. Anodyne,
diaphoretic, &c.—Dose, 3 to 10 gr. The formula already given under
Matthew’s pills is erroneously assigned to this pill by some writers.
Pills, Mrs. Stephen’s. This once celebrated remedy for stone
was prepared from the calcined shells of eggs and snails, made into
3-gr. pills with soft soap. Its active ingredients were, consequently,
lime and potash.
 Pills, Stim′ulant. Syn. Pilulæ stimulantes, L. Prep. 1. Capsicum,
1⁄ 2 dr.; nitrate of silver, 2 gr.; conserve of hips, q. s. For 12 pills.—
Dose, 2 to 4, washed down with a spoonful of warm spirit and water,
and repeated hourly until reaction ensues; in cholera, &c.
2. (A. T. Thomson.) Strychnine, 1 gr.; acetic acid, 1 drop; crum
of bread, 20 gr.; mix very carefully, and divide the mass into 10 pills.
—Dose, 1 every six hours; in paralysis arising from lead.
Pills, Stoerck’s. Syn. Pilulæ conii, P. cicutæ, L. Prep. From
extract of hemlock, 1 dr.; powdered hemlock, q. s. to make a mass.
For 2-gr. pills.—Dose, 1 to 4, twice a day; in various glandular and
visceral enlargements, pulmonary affections, cancer, scrofula,
neuralgia, &c.
Pills, Stomach. Syn. Pilulæ stomachicæ, L. Prep. 1.
Ipecacuanha, 10 gr.; sumbul and extract of rhubarb, of each 30 gr.;
powdered quassia, 20 gr.; oil of sassafras, 6 drops; beaten up with
essence of ginger (strongest), q. s. For 3-gr. pills.—Dose, 1 to 3,
thrice daily; in loss of appetite, flatulence, dyspepsia, &c.
2. (Dr Hugh Smith’s.) From aloes, rhubarb, ginger (all
powdered), and sagapenum, of each 1 dr.; oils of peppermint and
cloves, of each 10 drops; balsam of Peru, q. s. to mix. For 5-gr. pills
—Dose, 2 or 3 nightly; or 1 to 2 before dinner. For other formulæ,
see Dinner, Aperient, Compound rhubarb, Aloes and Mastic pills, &c.
Pills of Sto′rax (Compound). Syn. Storax pills; Pilula styracis
composita (Ph. L.), Pilulæ styracis (Ph. E.), L. Prep. 1. (Ph. L.)
Prepared storax, 6 dr.; saffron and powdered opium, of each 2 dr.
beat them together to a uniform mass. Contains 1-5th of its weight
of opium.
2. (Ph. E.) Opium and saffron, of each 1 part; extract of styrax,
2 parts; beat them to a uniform mass, and divide this into 4-gr. pills.
Contains 1⁄ 4th part of opium.
 Obs. The storax is here chiefly employed to disguise the odour
and taste of opium. The name of the preparation has been chosen
so that the word ‘opium’ may not appear in the prescription, a point
highly necessary with certain patients.—Dose, 3 to 10 gr.; as
compound soap pill, and as an anodyne and expectorant in chronic
coughs, &c.
Pills of Stramo′′nium. Syn. Pilulæ stramonii, L. Prep. 1.
Stramonium seeds (in powder), 12 gr. (or leaves, 25 gr.); powdered
camphor and extract of seneka root, of each 1 dr.; powdered savine,
1 1⁄ 2 dr.; oil of cajeput, 15 drops. For 2 1⁄ 2-gr. pills.—Dose, 2 to 4,
thrice daily; in rheumatism, &c.
2. (Sir H. Halford.) Extract of stramonium and liquorice powder,
of each 1 dr.; powdered Castile soap, 2 dr.; mucilage, q. s. to mix.
For 60 pills.—Dose, 1 night and morning; in asthmas, &c.
Pills of Strych′nine. Syn. Pilulæ strychniæ, L. Prep.
(Magendie.) Strychnine, 2 gr.; conserve of hips, 36 gr. (liquorice
powder, q. s.); mix very carefully, divide the mass into 24 pills, and
silver them.—Dose, 1 pill night and morning; in amaurosis,
impotence, paralysis, &c.
Pills of Sulphate of Copper. (Brande.) Syn. Pilulæ cupri
sulphatis. Prep. Sulphate of copper, 3 gr.; bread crum, 1 dr. Mix, for
24 pills; 1, three or four times a day.
Pills of Sul′phate of I′ron. Syn. Pilulæ ferri sulphatis (Ph. E.),
L. Prep. 1. (Ph. E.) Dried sulphate of iron and conserve of red roses,
of each 2 parts; extract of dandelion, 5 parts. For 5-gr. pills. A useful
chalybeate tonic. Dose, 1 to 2, twice or thrice daily; in dyspepsia,
chlorosis, amenorrhœa, &c.
2. (Ph. E. 1817.) Sulphate of iron (dried), 1 oz.; extract of
chamomile, 1 1⁄ 2 oz.; oil of peppermint, 1 dr.; syrup, q. s. As the
last.
 3. (Ph. U. S.) As No. 1, but substituting extract of gentian for
extract of dandelion. For other formulæ, see Pills, Hooper’s female,
&c.
Pills of Sulphate of Quinine′. Syn. Pilulæ quiniæ sulphatis, P. q.
disulphatis, L. Prep. 1. Sulphate of quinine, 20 gr.; extract of gentian,
40 gr. For 20 pills.
2. (Ph. U. S.) Sulphate of quinine, 2 dr.; powdered gum, 1⁄ 2 dr.;
strained honey, q. s. For 120 pills. Each pill contains 1 gr. of the
sulphate or disulphate of quinine.—Dose, 1 or 2 twice a day, as a
tonic and stomachic; 6 to 12, every two or three hours during the
remissions of agues.
3. (B. P.) Mix 60 gr. of sulphate of quinine, and 20 gr. of
confection of hips to a uniform mass.—Dose, 2 gr. to 10 gr.
Obs. Various additions are often made to the above formulæ,
according to the indications, by which numerous other useful pills
are produced. Thus, potassio-tartrate of antimony is frequently
added in obstinate intermittents; iodide of potassium, in scrofulous
affections; foxglove, in the hectic fever of phthisis; bitter tonics and
aromatics, in dyspepsia, flatulence, &c.; carbonate of soda or
magnesia, in acidity and heartburn; calomel, mercurial pill, in bilious
affections; rhubarb and aloes, in bowelly affections; sulphate of iron
and other chalybeates, in debility, amenorrhœa, and chlorosis;
calomel, as an alterative, &c., &c.
Pills of Sulphate of Zinc. Syn. Pilulæ zinci suphatis, P. z. s.
compositæ, L. Prep. 1. Sulphate of zinc, 12 gr.; extract of gentian, 1⁄ 2
dr.; liquorice powder, q. s. For 20 pills. In dyspepsia, epilepsy, and
various convulsive diseases.
2. (Dr Paris.) Sulphate of zinc, 10 gr.; powdered myrrh, 1 1⁄ 2 dr.;
conserve of roses, q. s. For 30 pills.—Dose, 1 to 2, twice or thrice
daily; in hooping-cough, &c.
 Pills of Sulphuret of Iron. (Biett.) Syn. Pilulæ ferri sulphureti.
Prep. Sulphuret of iron, 1⁄ 2 dr.; marshmallow powder, 10 gr.; syrup,
q. s. Make into 20 pills; 1 to 4 pills daily, in scrofulous eruptions.
Pills, Syph′ilis. Syn. Pilulæ antisyphiliticæ, L. See the various
pills of mercury, gold, &c. The pills of corrosive sublimate commonly
pass under this name.
Pills, Tangore. See Pills, Arsenical.
Pills of Tan′nic Acid. Syn. Pilulæ tannini, P. acidi tannici, L.
Prep. From tannic acid or tannin and powdered sugar, of each 1⁄ 2
dr.; conserve of roses, q. s. For 24 pills.—Dose, 1 or 2 pills, thrice
daily, in diarrhœa; or 2 every three hours, in internal hæmorrhages,
spitting of blood, &c.
Pills of Tar. Syn. Pilulæ picis liquidæ, L. Prep. From tar, 1 dr.;
powdered gentian, 1⁄ 2 dr., or q. s. For 24 pills. Stimulant, diuretic,
and sudorific.—Dose, 1 to 4, thrice a day; in dropsies, worms,
ichthyosis, and several other skin diseases, &c.
Pills of Tarax′acum. Syn. Pilulæ taraxaci, L. Prep. 1. Extract of
dandelion, 1 dr.; powdered rhubarb, q. s.; divide into 3 1⁄ 2-gr. pills.
In dyspepsia, &c., complicated with congestion of the liver.
2. (St Marie.) Extract of dandelion and Castile soap, equal parts;
liquid acetate of potassa, q. s. to mix. For 4-gr. pills. As a diuretic in
dropsy.
3. Extract of dandelion, 1 dr.; mercurial pill, 20 gr.; powdered
digitalis, 15 gr.; liquorice powder, q. s. For 24 pills.—Dose, 1,
afterwards increased to 2 or 3; in dropsy connected with liver
disease.
Pills, Thomson’s Stomach and Liver. Prep. From extract of
dandelion, 1 dr.; scammony and rhubarb, of each 15 gr. For 14 pills.
—Dose, 2 pills, night and morning; in hysteria, hypochondriasis, and
chronic inflammation of the liver or kidneys.
 Pills of Tobacco. (Augustin.) Syn. Pilulæ tabaci. Prep. Powder
of tobacco, 24 gr.; confection of roses, q. s. Mix, and form 72 pills.—
Dose, 2 to 4 daily, till nausea is produced. In dropsy.
Pills, Tonic. Syn. Pilulæ tonicæ, L. Prep. 1. Sulphate of iron,
ginger, and myrrh (all in powder), equal parts; conserve of roses,
q. s.; mix, and divide into 4-gr. pills.—Dose, 1, twice a day; in
debility, chlorosis, &c.
2. Powdered myrrh and sulphate of iron, of each 1 dr.;
disulphate of quinine, 1⁄ 2 dr., powdered capsicum, 15 gr.; conserve
of roses, q. s. to mix. For 60 pills.—Dose, 1 or 2, twice or thrice a
day; in debility, dyspepsia, ague, &c.
3. (Dr Collier.) Tartrate of iron and extract of gentian, of each 1
dr.; oil of cinnamon, 2 drops. For 30 pills.—Dose, 3 to 6, three or
four times a day. A good stomachic tonic.
4. (Dr Collier.) Oxide of zinc, 1⁄ 2 dr. (or sulphate of zinc, 20 gr.);
myrrh, 2 dr.; camphor, 20 gr.; confection of hips, to mix. For 40 pills.
—Dose, 1 or two pills, three times a day; in epilepsy, chorea, and
other nervous disorders, debility, &c.
5. (Dr A. T. Thomson.) Rhubarb and ginger, of each 1⁄ 2 dr.;
extract of chamomile, 1 dr.; divide into 30 pills.—Dose, 2 or 3, twice
a day; in dyspepsia and chlorosis.
6. (Dr A. T. Thomson.) Sesquioxide of iron and extract of
hemlock, of each 1 dr.; mix, and divide into 20 pills.—Dose, 1 or 2,
twice a day; in fluor albus, scrofula, &c. Several other formulæ for
tonic pills will be found under the names of the leading ingredients,
&c. (See above.)
Pills of Turpentine. (P. Cod.) Syn. Pilulæ terebinthinæ. Prep.
Venice turpentine, 1 1⁄ 2 oz.; carbonate of magnesia, 1 oz. Make into
200 pills.
Pills of Vale′′rian (Compound). Syn. Pilulæ valerianæ
compositæ, L. Prep. (Dupuytren.) Powdered valerian, 1⁄ 2 dr.; castor
and white oxide of zinc, of each 20 gr.; syrup, q. s.; to mix. For 18
pills.—Dose, 2 or 3, thrice daily; in hysteria, hypochondriasis,
chlorosis, hemicrania, &c.
Pills of Vale′′rianate of Zinc. Syn. Pilulæ valerianas, L. Prep.
From valerianate of zinc and powdered gum, of each 15 gr.;
conserve of hips, q. s. to form a mass. For 18 pills.—Dose, 1 pill,
twice daily; in nervous headache, neuralgia, hysteria, &c.
Pills, Vallet’s. See Pills of Carbonate of Iron.

Pills, Vance’s. See Pills, Aperient.

Pills of Vera′trine. Syn. Pilulæ veratrinæ, L. Prep. 1.
(Magendie.) Veratrine, 1⁄ 2 gr.; powdered gum Arabic and syrup of
gum, of each q. s. to form 6 pills. (See below.)
2. (Turnbull.) Veratrine, 1 gr.; extract of henbane and liquorice
powder, of each 12 gr.; mix, and divide into 12 pills.—Dose, 1 pill,
every 3 hours; in dropsy, epilepsy, hysteria, paralysis, nervous
palpitations, &c. This should be prepared and used with great
caution.
Pills, Ward’s Red. Syn. Ward’s antimonial pills. Prep. From glass
of antimony (finely levigated), 4 oz.; dragon’s blood, 1 oz.; mountain
wine, q. s.; to form a mass. For 1 1⁄ 2-gr. pills. Emetic. “They are
recommended in obstinate rheumatism affections, in foulness of the
stomach and bowels, &c. Their action is often of a very unpleasant
character.” (‘Anat. of Quackery.’)
Pills, Lady Webster’s. See Pills, Dinner.
Pills, Whitehead’s Essence of Mustard. Balsam of tolu, with
resin. (Dr Paris.)
Pills, Whytt’s. Prep. (Radius.) Aloes, chloride of iron, and
extract of horehound, of each 1⁄ 2 dr.; assafœtida, 1 1⁄ 2 dr. For 2-gr.
pills.—Dose, 2 to 5, thrice daily; in leucorrhœa, chlorosis, hysteria,
&c., with constipation.
 Pills, Worm. Syn. Pilulæ anthelminticæ, P. vermifugæ, L. Prep. 1.
Calomel, 1 oz.; sugar, 1 1⁄ 2 oz.; mucilage, q. s.; mix, and divide into
240 pills.—Dose, 1 to 2, overnight, followed by a strong dose of
castor oil early the next morning.
2. Gamboge, 6 gr.; calomel, 5 gr.; mucilage, q. s.; divide into 3
pills. For a morning’s dose, fasting.
3. Extract of wormwood, calomel, and powdered scammony,
equal parts. For 4-gr. pills.—Dose, 1 to 2, as the last. For ascarides,
and other small worms.
4. (Bresmer.) Powdered aloes and tansy seed, of each 1⁄ 2 dr.;
oil of rue, 9 or 10 drops. for 12 pills.—Dose, 3 to 6, in the morning,
fasting, and repeated in two or three hours.
5. (Phœbus.) Iron filings, 1⁄ 2 dr.; assafœtida, 1 1⁄ 2 dr.; essential
oil of tansy, 10 or 12 drops; extract of wormwood, q. s.; mix, and
divide into 80 pills.—Dose, 6 pills, thrice daily.
6. (Peschier.) Ethereal extract of male fern, 30 drops; extract of
dandelion, 1 dr.; powdered rhizomes of male fern, q. s. to mix. For
30 pills. In tapeworm.—Dose, 6 to 15, at bedtime; the dose being
repeated in the morning, and then followed in an hour by a strong
dose of castor oil.
Pills, Wordsell’s (Kaye’s). Prep. (Cooley.) Powdered aloes,
gamboge, and ginger, equal parts; together with a very small
quantity of diaphoretic antimony, beaten into a mass with either
syrup or treacle, and divided into 2 1⁄ 2-gr. pills. “There are about
4 1⁄ 2 dozen pills in each 1s. 1 1⁄ 2d. box.” “The dose, as given in the
directions, is from 2 to 8 pills (or even 10 to 12) daily.” (‘Anat. of
Quackery.’) They frequently operate with great violence.
Pills, Wyndham’s (Lee’s). Prep. (Cooley.) Aloes and
gamboge, of each (in powder) 3 oz.; Castile soap and extract of
cow-parsnip, of each 1 oz.; nitre, 1⁄ 2 oz. For 5-gr. pills. A powerful
drastic cathartic.—Dose, 1 to 3 pills.
 Pills of Zinc. See Pills of Oxide, Sulphate and Valerianate of Zinc,
&c.
PILOCARPINE. Prep. Exhaust the leaves or bark of Jaborandi
with 80% alcohol, to which hydrochloric acid has been added in the
proportion of 8 grains per litre; distil and evaporate to the
consistence of an extract. Redissolve the extract with a small
quantity of distilled water and filter; treat with ammonia in slight
excess, and a large quantity of chloroform. Distil off the chloroform,
dissolve the residue in distilled water acidulated with hydrochloric
acid, and filter. Treat afresh with chloroform and ammonia. The
chloroformic solution is then shaken with water, to which
hydrochloric acid is added, drop by drop, up to the quantity sufficient
to saturate the pilocarpine. The foreign matters remain in the
chloroform, and upon evaporation of the aqueous liquid the
hydrochlorate is obtained, well crystallised, in long needles radiating
from a common centre. The hydrochlorate dissolved in distilled
water, and treated with ammonia and chloroform, yields the
pilocarpine upon evaporation of the chloroform solution.
Pilocarpine appears under the form of a soft viscous substance;
it is slightly soluble in water and very soluble in alcohol, ether and
chloroform. It presents all the chemical characters of an alkaloid,
and rotates the plane of polarized light strongly to the right. (Paris
Pharmaceutical Society.)
PIMA′RIC ACID. A resin acid first obtained by Laurent from
the turpentine of Pinus maritima (Bordeaux turpentine), by the
action of hot alcohol.
PIMEN′TO. Syn. Allspice, Clove pepper, Jamaica p., Pimento berries;
Pimenta (B. P., Ph. L., E., & E.), Piper caryophyllatum, P. Jamaicense, P.
odoratum, PIMENTÆ BACCÆ, L. “The dried unripe berries of the allspice
tree, Eugenia pimenta, from the West Indies”—B. P. “The immature
fruit of Eugenia pimenta (Myrtus pimenta, Linn.)”—Ph. L. It
possesses a mixed odour of cinnamon, cloves, and nutmegs, which,
with its other properties, it for the most yields to alcohol, ether, and
water. It is a stimulant and tonic, and is much esteemed as an
adjuvant in medicines prescribed in dyspepsia, flatulence, gout,
hysteria &c.; and also to cover the taste of disagreeable medicines.—
Dose, 5 to 30 gr., bruised or in powder. See Essence, Oils (Volatile),
Spirits, and Waters.
PIM′PLES. See Eruptions (Papular).
PINCH′BECK. A gold-like alloy of copper and zinc. See Dutch
gold.

PINE APPLE. Syn. Ananas. The fruit of Ananassa sativa, a plant

of the natural order Bromeliaceæ. It is astringent, esculent, and
possesses a rich flavour and odour. In Europe it is chiefly used as a
delicacy for the table; but in tropical climates it is said to be valuable
in renal diseases. See Essence, &c.
PI′NEY TAL′LOW. Syn. Piney resin, P. dammar. An oleo-resinous
substance obtained from the fruit of Vateria indica, a tree common
in Malabar, by boiling it with water. It is intermediate between fat
and wax, makes good soap and excellent candles. It melts at 98°
Fahr. Sp. gr. ·9250 to ·9265.
PI′NIC ACID. The portion of common resin or colophony
which is soluble in cold alcohol of sp. gr. ·833.
PINK. A well known shade of light red. The name is also
applied to several pigments, consisting of whiting stained with liquid
dyes. See Red and Yellow pigments, &c.
PINK DYE. Prep. From washed safflower, 2 oz.; salt of tartar,
1⁄ 2 oz.; cold water, 1 quart; digest for 3 hours, express the liquor,
and strain it. Used as a cosmetic, and to dye silk stockings, &c., of a
rose colour. The colour is brought out by afterwards applying to, or
passing the articles through, water soured with lemon juice. See
Saucers (Pink).
PIP′ERIN. C 17H 19NO 3. Syn. Piperina, Piperinum, L. Prep. (P.
Cod.) Alcoholic extract of black pepper is treated with a weak
solution of caustic potassa (1 to 100), and the residuum, after being
washed with cold water, is dissolved in alcohol; the solution is next
agitated with a little animal charcoal, and the filtrate is allowed to
evaporate spontaneously; the product may be purified by the re-
solution in alcohol and re-crystallisation.
Prop., &c. Colourless, or only slightly yellow; tasteless;
inodorous; fusible; and crystallisable; insoluble in water; freely
soluble in strong spirit, and in the acids; very feebly basic; a few
definite compounds have, however, been obtained with difficulty;
reddened by oil of vitriol. It has been much employed in Italy and on
the Continent as a febrifuge.—Dose, 2 to 10 gr., frequently repeated,
during the apyrexia of intermittents.
Obs. An assay for its piperin is the only certain method of
testing the quality of either black or white pepper. For this purpose a
weighted quantity of the sample is reduced to powder, and is
exhausted with alcohol of the sp. gr. ·883; the mixed tinctures are
then evaporated to an extract, which is treated as above. See Pepper.
PIPES. (In confectionery.) These are formed from any of the
common lozenge-masses, by rolling them into cylinders of about the
thickness of a goose-quill. They are frequently medicated.
PIPETTE. A graduated glass instrument, in frequent use in the
chemical laboratory, for conveying a measured quantity of fluid from
one vessel to another. The pipette mostly consists of a bulb, from
each end of which proceeds a straight, slender hollow stem,
communicating with the bulb, and varying in length with the capacity
of the instrument. Thus constructed, the lower end of the pipette
can be dipped into a vessel with a narrow and long neck, such as a
flask, containing a fluid, the required volume of which can be
removed from it. The pipette varies in capacity from 1 to 200 cubic
centimètres.
Dr Fresenius gives the following directions for its use:—“To fill a
pipette with the fluid which it is intended to transfer from one vessel
to another, the lower part of the instrument is dipped into the fluid,
and suction applied to the upper aperture, either direct with the lips
or through a caoutchouc tube until the fluid in the pipette stands a
little above the required mark; the upper, somewhat narrowed,
ground orifice is then closed with the point of the index of the right
hand, which to that end had always better be moistened a little, and
holding the pipette in a perfectly vertical direction, the excess of
over the quantity required is made to drop out by lifting the finger a
little. When the fluid in the pipette has fallen to the required level,
the drops which may happen to adhere to the outside of the pipette
are carefully wiped off, and the contents of the tube are then fully
transferred to the other vessel. In this process it is found that the
fluid does not run out completely, but that a small portion of it
remains adhering to the glass in the point of the pipette; after a
time, as this becomes increased by other minute particles of fluid
trickling down from the upper part of the tube, a drop gathers at the
lower orifice, which may be allowed to fall from its own weight, or
may be made to drop off by a slight shake; if, after this, the point of
the pipette be laid against a moist portion of the inner side of the
vessel, another minute portion of fluid will trickle out; and lastly,
another trifling droplet or so may be got out by blowing into the
pipette through the upper orifice. Now, supposing the operator
follows no fixed rule in this respect, letting the fluid, for instance, in
one operation simply run out, whilst in another operation he lets it
drain afterwards, and in a third blows off the last particles of it from
the pipette, it is evident that the respective quantities of fluid
delivered in the several operations cannot be quite equal. I prefer in
all cases the second method, viz. to lay the point of the pipette
whilst draining finally against a moist portion of the inner side of the
vessel, which I have always found to give the most accurate
corresponding measurements.”
PISTA′CHIO NUTS. Syn. Pistacia nuts; Nuces pistaciæ, L. The
kernels of the fruit of Piscatia vera (Linn.), one of the turpentine
trees. They closely resemble almonds, but are sweeter, and form a
green emulsion with water. Used in confectionery and perfumery,
and also as a dessert fruit.
PITCH. Syn. Black pitch, Boiled p., Stone p., Wood p.; Pix (Ph. L.),
Pix nigra, L. “A dry bitumen prepared from liquid pitch.” (Ph. L.) The
residuum from boiling tar in an open iron pot, or in a still, until the
volatile and liquid portion is driven off. The volatile products
principally consist of crude pyroligneous acid and oil of tar. Pitch is
chiefly employed in ship-building. As a medicine, it is stimulant and
tonic, and has been used internally in some skin diseases, and in
piles. An ointment made of it is also extensively used in cutaneous
affections of the scaly.—Dose, 10 gr. to 1⁄ 2 dr.
Pitch, Burgundy. Syn. White pitch, Burgundy pine resin; Pix
Burgundica (B. P., Ph. L., E., & D.), L. “Impure resin prepared from the
turpentine of Abies excelsa,” or Norway spruce fir. (Ph. L.) “A
concrete resinous exudation, probably, in a great measure, from
Abies excelsa.” (Ph. E.) It is chiefly used in plasters.
Obs. The importation of this substance has for some years past
been gradually lessening in amount, in consequence of the
substitution for it of a fictitious pitch, made by melting common resin
with linseed oil, and colouring the mass with annotta or palm oil.
The physiological action of the two articles is, however, considerably
different, since Burgundy pitch acts upon the skin as a powerful local
irritant, exciting a slight degree of inflammation, and not
unfrequently producing a pimply eruption and an exudation of
purulent matter. It is celebrated for its effects when employed as a
plaster in all cases where warmth, support, and long adhesion to the
skin, are desirable; and in the latter quality no substance equals it.
The fictitious Burgundy pitch has similar properties, but in an
immensely less degree.
Prepared Burgundy Pitch (Pix Burgundica præparata—Ph. L.) is
ordered to be obtained in the same way as that adopted for strained
ammoniacum. This plan is, however, seldom, if ever, adopted in
trade.
 Pitch, Burgundy (Facti′′tious). Syn. Pix Burgundica factitia, L.
Prep. By melting good yellow resin, 1 cwt.,with linseed oil, 1 gall.,
and palm oil (bright), q. s. to colour. The mixture is allowed to cool
considerably, and is then pulled with the hands in the same way as
lead plaster is treated; after which it is placed in ‘bladders’ or
‘stands’ for sale.
Obs. The product of the above formula is the ‘Burgundy pitch’ of
the shops. The ‘pulling’ or ‘working’ destroys the translucency of the
resin, and imparts to it the peculiar semi-opacity of foreign Burgundy
pitch. Cold water is commonly employed to cool it down. Annotta is
often substituted for palm oil as a colouring substance. The addition
of some of the ‘droppings’ or ‘bottoms’ of Canada balsam, Chio
turpentine, oil of juniper, &c., renders this article nearly equal to
foreign pitch; but in commerce this is never attempted, the aim
being only the production of a lively colour with moderate
toughness. A common melting-pan and fire (if clean, and carefully
managed) will succeed sufficiently, but, both for safety and
convenience, steam is preferable, and on the large scale, almost
indispensable. A good workman can pull and put into stands or casks
about 5 cwt. daily; or from 1 1⁄ 2 cwt. to 3 cwt. in bladders, the latter
quantity depending on the size of the bladders. (See above.)
Pitch, Can′ada. Syn. Hemlock gum, H. pitch. Similar to Burgundy
pitch; but from the Abies canadenses, or hemlock spruce fir.
Pitch, Jews’. Asphaltum.
Pitch, Min′eral. Indurated mineral bitumen. See Asphaltum,
Bitumen, &c.
PIT′COAL. Syn. Coal; Houille, Fr.; Steinkohle, Ger. This article
has been truly described as the most valuable of all those mineral
substances from which Great Britain derives its prosperity, and the
one which may be regarded as the main support of the whole
system of British production. It fuses the metals, it produces the
steam which sets our machinery in motion, and, in short, it may be
said to render all the resources of this country available for use.
The more important kinds of coal may be classified as follows:—
1. Lignite or brown coal (see page 969).—2. Bituminous or caking
coals. The most widely diffused and valuable of English coals. They
are subdivided into: a. Caking coal. Splinters on heating, but the
fragments then fuse together in a semi-pasty mass. The chief
sources of this valuable variety of coal are the Newcastle and Wigan
districts, b. Cherry coal or soft coal. Lustre very bright; does not
fuse, ignites well and burns rapidly. Glasgow, Staffordshire,
Derbyshire, Nottingham, Lancashire, &c. c. Splint, rough, or hard
coal. Black and glistening; does not ignite readily, but burns up to a
clear hot fire. It constitutes the bulk of the great coal fields of North
and South Staffordshire, and occurs in the Glasgow district, in
Shropshire, Leicestershire, Warwickshire, &c. d. Cannel or parrot
coal. Dense and compact, having a shelly fracture, and taking a
polish like jet. Splinters in the fire, and burns clearly and brightly.
Wigan and other parts of Lancashire, West Glasgow, &c. The curious
deposit at Bathgate, near Edinburgh, commonly known as ‘Boghead
cannel coal,’ or ‘Torbanehill mineral,’ differs considerably from the
ordinary ‘cannels,’—3. Anthracite or stone-coal. The densest,
hardest, and most lustrous of all kinds of pitcoal. Burns with little
flame or smoke, but gives great heat. South Wales, Devonshire, &c.
—4. Steam coal. Approaches nearly to anthracite. Admirably adapted
for steam-vessels. South Wales, Tyne district, &c.
The quality of coal may be ascertained by either directly testing
its heating power or by chemical analysis. In the investigations
undertaken at the Museum of Economic Geology, under the
directions of Sir H. De la Beche, and which furnished the materials
for the celebrated ‘Admiralty Reports,’ three different methods were
adopted for this purpose.[109] These consisted in—the determination
of the quantity of water which a given weight of the coal was
capable of converting into steam, the quantity of litharge which it
was capable of reducing to the metallic state, and, lastly, its ultimate
analysis by combustion with oxide of copper. See Organic Substances.
[109] See Watt’s ‘Dict. of Chemistry,’ vol. i, page 1033.

The quantity of sulphur in coal is another matter of importance

that may be determined by chemical analysis. (See Sulphur.) The
presence of more than 1% of sulphur renders coal unfit for the
economical production of good light-gas, and more than 2% of
sulphur renders it objectionable for use as domestic fuel. In like
manner, coals containing mineral ingredients in excess are to be
avoided, not merely on account of the quantity of ashes left by
them, but for their tendency to vitrify upon the bars of the furnace,
and to produce what is technically called ‘clinkers.’ The presence of
much silica or alumina, and more particularly of any of the salts of
lime, in ‘steam coal,’ is, on this account, highly objectionable.
For some further information connected with this subject, see
Anthracite, Chimneys, Coke, Fuel, Gas, Lignite, Oils, (Mineral), Organic
substances, &c.

PLAGUE. (Pestis). “A specific fever attended with bubo of the

inguinal and other glands, and occasionally with carbuncles.”
Such is the definition of plague given in the ‘Nomenclature of
Diseases’ (published in 1869), drawn up by a joint committee
appointed by the College of Physicians.
More detailed accounts of the disease, described by other
pathologists, state that it attacks the patient with great suddeness,
or only after a few premonitory symptoms. These are:—Shivering,
extreme prostration, intense headache and giddiness, excessive
restlessness, and an overwhelming sense of anxiety. The patient’s
gait becomes uncertain, and he staggers like a drunken man. These
symptoms are more or less accompanied by nausea, bilious
vomiting, and frequently by bilious diarrhœa. As the disease
advances, delirium very frequently sets in; the nausea, vomiting, and
diarrhœa increase in intensity, the tongue becomes swollen and
covered with a dark fur, whilst the lips, teeth, and nostrils are coated
with a dry fetid incrustation. Provided the attack does not terminate
fatally, in a very rapid manner, these symptoms are accompanied by
sharp pains (increasing in intensity during the progress of the
malady) in the groin, armpits, and neck. These pains in the above
parts precede the appearance of the buboes, and in many cases, of
the carbuncles, which, associated with the fever, are so characteristic
of plague. These glandular swellings vary, in different cases, as to
the time when they make their appearance. Sometimes they do so
during the first day of the attack, at others, after two or three days
—and in others, again, not until near the close of the disease. With
the buboes and carbuncles, small red purplish spots (petechiæ),
frequently appear on the body. The carbuncle is by no means an
invariable accompaniment of the disease. Dr Russell, out of 2700
cases, found only 490 in which it showed itself. He states that when
carbuncle develops itself, it is distributed over the whole surface of
the body with the exception of the scalp, the palms of the hands,
and the soles of the feet.
“The plague may be said to assume four degrees of severity:—
1. Slight fever, without delirium or buboes. 2. Fever, delirium, and
buboes. 3. Fever, delirium, or coma, buboes, carbuncles, and
petechiæ/e. 4. Congestive fever, fatal on the first, second, or third
day, before the appearance of buboes. The fever, though usually
continued, may assume the intermittent or remittent type.”[110]
[110] Hooper’s ‘Vade Mecum,’ edited by Messrs Guy and
Harley.

There is considerable diversity of opinion as to the origin of

plague. By some pathologists it is maintained that it spreads solely
by contagion; by others the contagion theory is altogether
repudiated, and certain local and epidemic agencies are referred to
as its source; whilst others, again, adopt a medium view and, whilst
not denying its contagious origin, hold that it may also be developed
by endemic and epidemic causes. It bears a great resemblance to
typhus.
 With the exception of the outbreak of plague at Veltianka, in
Astrakan, in the beginning of the current year (1879), the pestilence
has not visited Western Europe during the present century an
exemption which, being so obviously due to the improved sanitary
and hygienic conditions of the modern European cities and towns, is
a forcible illustration of how largely the power of curtailing the
propagation and progress of the scourge is within the means of
human control. There can be little, if any, doubt that the same total
absence of drainage, and the very possible consequent
contamination of drinking water, added to the narrowness of the
streets, the overcrowded and badly ventilated state of the houses
themselves, and the dirty habits of the inmates, which are also
characteristic of those quarters of eastern cities and towns in which
plague is always more or less occasionally prevalent, obtained in the
fourteenth, fifteenth, sixteenth, and seventeenth centuries, amongst
European communities.
We learn, on the authority of Mr Marshall (who gets his figures
from the weekly bills of mortality of the period), that during the
sixteenth and seventeenth centuries London was seldom free from
the pestilence, and that in several years, not usually regarded by
historians as plague epochs, it annually slew from less than 1000 to
4000 of the inhabitants.
Between the years 1593 and 1665, five severe outbreaks of the
disease occurred in London, and the number of deaths for the
respective years were as follows:—1593, 11,503; 1603, 36,269;
1625, 35,417; 1636, 10,400; 1665, 68,596. According to Sir William
Petty, the average mortality during these several attacks amounted
to about a fifth of the population.
That insanitary surroundings and the spread of plague, whilst
sanitary ones and its decline, follow each other like cause and effect,
may be emphasised by the statement of two facts:—1. The medical
commissioner lately sent by the Russian government to the seat of
the late outbreak of the malady in Astrakan, discovered the people
dirty in their habits, living in noisome, overcrowded houses, and the
atmosphere polluted with the smell of decaying fish, added to which
the village was most miserably drained. 2. Ranken records that in
Rajpootana plague propagated by the filthy habits of the inhabitants
was for some years almost entirely obliterated by the adoption of
sanitary precautions.
It may here be noticed that the Astrakan plague was associated
with inflammation of the lungs, a feature which led an eminent
Russian physician to adopt the opinion, that the Astrakan malady is
the same as the Indian plague, which is believed to be the same
disease which, under the name of ‘The Black Death,’ committed such
appalling devastation in Europe, Asia, and Africa, in the fourteenth
century.

In his ‘Epidemics of the Middle Ages,’[111] Hecker has told of the

ravages of this ruthless pestilence, which made its appearance in
Europe in 1348. Its devastations at Florence have been very
powerfully described by Boccaccio in the introduction to his
‘Decameron.’ Boccaccio was in Naples at the time it was devastating
Italy, therefore, it is conjectured, his graphic description must have
been derived from hearsay and the reports of eyewitnesses.
[111] Published by the Sydenham Society, 1844.

In August of the same year it broke out at Dorset, from which

county it soon reached Devon and Somerset, and thence rapidly
spread throughout England, slaying its thousands in its progress. In
London alone it has been estimated that the mortality caused by it
amounted to a hundred thousand.
Hecker assumes that in Europe its victims were twenty-five
millions. These however, as well as the following figures, must only
be received as approximations to the correct numbers, which, owing
to the absence of any contemporary bills of mortality, cannot but be
very imperfect:—

In Florence there died of the black plague 60,000

 In Venice 100,000
In Marseilles, in one month 16,000
In Sienna 70,000
In Paris 50,000
In St Denis 14,000
In Avignon 60,000
In Strasbourg 16,000
In Lübeck 9,000
In Basle 14,000
In Erfurt at least 16,000
In Weimar 5,000
In Lemburg 2,500
In London at least 100,000
In Norwich 51,000

To which may be added:—

Franciscan Friars in Germany 124,434

Minorites in Italy 30,000

From the circumstance—illustrative of the religious and blind

bigotry of this period—that the Jews were brutally tortured,
massacred, and burnt, on suspicion of having poisoned the wells
from which drinking water was drawn, it may be inferred that the
wells, owing to the entire absence of drainage, which led to their
contamination by sewage matters, contributed largely to the spread
of the pestilence.
Of the potency of the contagion disseminated by the ‘Black
Death’ Hecker records:—
“Every spot which the sick had touched, their breath, their
clothes, spread the contagion; and in all other places the attendants
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