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Subject Index to Volumes 33 75 95 120 140 175 199 229
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 Preface

The need for a cumulative subject index was recognized by the founding editors
of Methods in Enzymology who prepared one for Volumes I through VI by weeding
and interpolating from the entries that had been indexed in the individual volumes.
As the series developed in number and complexity, different individuals with
different backgrounds served as volume indexers. Subsequently, the series was
fortunate in having Dr. Martha G. Dennis and Dr. Edward A. Dennis accept the
challenge of digitizing the data available from these indexes, and this effort resulted
in Volumes 33, 75, and 95, which cover Volumes I through 80.
Although each of these three books produced with the aid of digitization
provided an appropriate cumulative index, major problems were encountered. One
was time, both expensive computer time and lag time before such efforts resulted in
publication. The most important difficulty was that the compilers were hampered
by the lack of uniformity in the indexing of the individual volumes, resulting in
the need for much hand editing to achieve a reasonable collation. The products
were very decent, if uneven, indexes that also contributed to the methodology of
computerized indexing, albeit with much delay and great expense.
This cumulative index was produced by Keith Shostak. Following a set of
established guidelines, Dr. Shostak collected and compiled the entries from the in-
dividual volume indexes to produce the cumulative index. Since there is a tendency
to identify specific topics and methods with particular individuals, a contributor
index is included as well as the complete table of contents of each of the volumes
indexed. In addition to Volume 320, the cumulative indexes in this series include
Volumes 33, 75, 95, 120, 140, 175, 199, 229, 265,285, and 320.

vii
 C o n t e n t s of V o l u m e s 3 2 1 - 3 5 4

V O L U M E 321
NUMERICAL COMPUTER METHODS
Michael L. Johnson and Ludwig Brand

1. Physiological Modeling with Virtual Cell Frame- JAMES C. SCHAFF,

work BORIS M. SLEPCHENKO,
AND LESLIEM. LOEW 1

2. Kinetic Analysis ofDynamics of Muscle Function JUL1EN S. DAVIS 23

3. Molecular Parameters from Sedimentation BORRIES DEMELER,
Velocity Experiments: Whole Boundary Fitting JOACHIM BEHLKE,
Using Approximate and Numerical Solutions AND Oq?FORISTAU 38
of Lamm Equation

4. Sedimentation Velocity Analysis of Macromolec- LENNY M. CARRUTHERS,

ular Assemblies VIRGILR. SCHIRF,
BORRIES DEMELER,
AND JEFFREYC. nANSEN 66

5. Analysis of Weight Average Sedimentation JOHN J. CORREIA 81

Velocity Data

6. Sedimentation Equilibrium Analysis of Mixed JOHN S. PHILO 100

Associations Using Numerical Constraints to
Impose Mass or Signal Conservation

7. Optimal Data Analysis Using Transmitted Light EMILIOS K. DIMITRIADIS

Intensities in Analytical Ultracentrifuge AND MARC S. LEWIS 121

8. Ultracentrifugal Analysis of Molecular Mass MARC S. LEWISAND

of Glycoproteins of Unknown or Ill-Defined RICHARD P. JUNGHANS
Carbohydrate Composition 136

9. Irregularity and Asynchrony in Biologic Network STEVEN M. PINCUS 149

Signals

10. Distribution Methods and Analysis of Nonlinear MICHELLELAMPLAND

Longitudinal Data MICHAELL. JOHNSON 182

11. Distinguishing Models of Growth with Approxi- MICHAELL. JOHNSON,

mate Entropy MICHELLELAMPL,
AND MARTIN STRAUME 196

ix
 CONTENTS OF VOLUMES 321-354 X

12. Approximate Entropy as Indication of Goodness- MICHAELL. JOHNSON

of-Fit AND MARTINSTRAUME 207

13. Kinetic Models and Data Analysis Methods for EDWARDL. RACHOFSKY
Fluorescence Anisotropy Decay ANDWILLIAMR. LAWS 216

14. Analysis of Nonequilibrium Facets of Pulsatile JOHANNESD. VELDHUIS

Sex-Steroid Secretion in Presence of Plasma- ANDMICHAELL. JOHNSON 239
Binding Proteins

15. Monte Carlo Simulations of Lateral Membrane MADSC. SABRA

Organization AND OLE G. MOURITSEN 263

16. Hydrodynamic Bead Modeling of Biological OLWYNBYRON 278

Macromolecules

17. Bayesian Hierarchical Models CHRISTOPHERH. SCHMID

AND EMERYN. BROWN 305

18. Monte Carlo Applications to Thermal and Chem- D. JEREMYWILLIAMS

ical DenaturationExperiments ofNucleic Acids AND KATHLEENB. HALL 330
and Proteins

19. Analysis of Drug-DNA Binding Data XIAOGANGQU AND

JONATHANB. CHAIRES 353

20. Neural Network Techniques for Informatics of WILLIAMW. VAN OSDOL,

Cancer Drug Discovery TIMOTHYG. MYERS,
AND JOHN N. WEINSTEIN 369

21. Dynamic Network Model of Glucose Counter- BORIS E KOVATCHEV,

regulation in Subjects with Insulin-Requiring MARTINSTRAUME,
Diabetes LEON S. FARHY,
AND DANIELJ. COX 396

22. Association of Self-Monitoring Blood Glucose BORIS E KOVATCHEV,

Profiles with Glycosylated Hemoglobin in DANIELJ. COX,
Patients with Insulin-DependentDiabetes MARTINSTRAUME,
AND LEON S. FARHY 410

23. Outliers and Robust Parameter Estimation MICHAEL

L. JOHNSON 417

24. Parameter Correlations while Curve Fitting MICHAEL

L. JOHNSON 424
xi CONTENTS OF VOLUMES 321-354

V O L U M E 322
APOPTOSIS
John C. Reed

S e c t i o n I. M e a s u r i n g Apoptosis and Apoptosis-lnduced

Endonucleases
1. Detection of DNA Cleavage in Apoptotic Cells SCOTT H. KAUFMANN,
PETER W. MESNER, JR.,
KUMIKO SAMEJIMA,
SHIGENOBUTONIC,
AND WILLIAMC. EARNSHAW 3
2. Detection of Apoptosis by Annexin V Labeling ELLA BOSSY-VV'ETZEL
AND DOUGLASR. GREEN 15
3. Analysis of Apoptotic Cells by Flow and Laser ZBIGNIEWDARZYNKIEWICZ
Scanning Cytometry AND ELZBIETABEDNER 18
4. Quantitative Measurement of Apoptosis Induced MICHELE BARRY,
by Cytotoxic T Lymphocytes JEFFREY HEIBEIN,
MICHAELPINKOSKI,
AND R. CHRIS BLEACKLEY 40
5. Apoptotic Nuclease Assays FRANCISM. HUGHES,JR.
AND JOHN A. CIDLOWSKI 47

S e c t i o n II. M e a s u r i n g Apoptosis in Lower Organisms

6. Analysis of Programmed Cell Death and Apop- PO CHEN AND
tosis in Drosophila JOHN M. ABRAMS 65
7. Programmed Cell Death in the Nematode DUNCANLEDWICH,
Caenorhabditis elegans YI-CHUN WU,
MONICA DRISCOLL,
AND DING XUE 76

S e c t i o n III. P r o t e a s e s Involved in Apoptosis and Their Inhibitors

8. Caspase Assays HENNING R. STENNICKE
AND GUY S. SALVESEN 91
9. Determination of Caspase Specificities Using a NANCY A. THORNBERRY,
Peptide Combinatorial Library KEVIN T. CHAPMAN,AND
DONALD W. NICHOLSON 100
10. Criteria for Identifying Authentic Caspase SOPHIE ROY AND
Substrates during Apoptosis DONALD W. NICHOLSON 110
11. Purification and Use of Granzyme B LIANFASHI, XIAOHEYANG,
CHRISTOPHER J. FROELICH,
AND ARNOLDH. GREENBURG 125
 CONTENTS OF VOLUMES 321-354 xii

12. Viral Caspase Inhibitors CrmA and p35 QIAO ZHOU

AND GUY S. SALVESEN 143

13. Purification and Use of Recombinant Inhibitor QUINN L. DEVERAUX,

of Apoptosis Proteins as Caspase Inhibitors KATEWELSH,
ANDJOHN C. REED 154

14. Monitoring Activity of Caspases and Their Reg- CHRISTINEJ. HAWKINS,

ulators in Yeast Saccharomyces cerevisiae SUSANL. WANG,
AND BRUCEA. HAY 162

Section IV. Cell-Free Systems for Monitoring Steps

in Apoptosis P a t h w a y s
15. In Vitro Assays for Caspase-3 Activationand DNA XUESONGLIU
Fragmentation AND XIAODONGWANG 177

16. Cell-Free Apoptosis in Xenopus laevis Egg OLIVERVONAHSENAND

Extracts DONALDD. NEWMEYER 183

17. Cytofluorometric Quantificationof NuclearApop- HANSK. LORENZO,

tosis Induced in a Cell-Free System SANTOSA. SUSIN,
AND GUIDOKROEMER 198

Section V. Mitochondria a n d Apoptosis

18. Purification of Mitochondria for Apoptosis SANTOSA. SUSIN,
Assays NATHANAELLAROCHETTE,
MAURICEGEUSKENS,
AND GUIDOKROEMER 205

19. Quantitation of Mitochondrial Transmembrane NAOUFALZAMZAMI,

Potential in Cells and in Isolated Mitochondria DIDIERMI~T1VIER,
AND GUIDOKROEMER 208
20. Nitrogen Cavitation for Cell Disruption to Obtain ROBERTAA. GOTTLIEB
Mitochondria from Cultured Cells AND SOUICHIADACHI 213
21. Apoptosis-Related Activities Measured with GARY FISKUM,
Isolated Mitochondria and Digitonin-Permea- ALICIAJ. KOWALTOWSKI,
bilized Cells ALEXANDERY. ANDREYEV,
YULIAE. KUSHNAREVA,
AND ANATOLYm. STARKOV 222
22. Assays for Cytochrome c Release from Mitochon- ELLA BOSSY-WETZEL
dria during Apoptosis AND DOUGLASR. GREEN 235
23. Purification and Liposomal Reconstitution of CATHERINEBRENNER,
Permeability Transition Pore Complex ISABELMARZO,
HELENAL. DE
ARAUJOVIEIRA,AND
GUIDOKROEMER 243
xiii CONTENTS OF VOLUMES 321-354

S e c t i o n VI. Bcl-2 Family Proteins

24. Monitoring Interactions of Bcl-2 Family Proteins Jos~-LuIS DIAZ,
in 96-Well Plate Assays TILMAN OLTERSDORF,
AND LAWRENCEC. FRITZ 255
25. Analysis of Dimerization of Bcl-2 Family Pro- ZH1HUAXIE AND
teins by Surface Flasmon Resonance JOHN C. REED 266

26. Measuring Pore Formation by Bcl-2 Family SHARONL. SCHENDEL

Proteins AND JOHN C. REED 274

27. Assays for Studying Bax-Induced Lethality in the QUNLI XU, NING KE,
Yeast Saccharomyces cerevisiae SHIGEMIMATSUYAMA,
AND JOHN C. REED 283
28. Exploiting the Utility of Yeast in the Context of CATHERINEN. TORGLER,
Programmed Cell Death ROBIN BROWN,
AND ERIC MELDRUM 297

S e c t i o n VII. S t u d y i n g R e c e p t o r s a n d S i g n a l T r a n s d u c t i o n Events
Implicated in Cell Survival and Cell Death
29. Production of Recombinant TRAIL and TRAIL PASCALSCHNEIDER 325
Receptor: Fc Chimeric Proteins

30. Expression of Lymphotoxins and Their Receptor- ISABELLEROONEY,

Fc Fusion Proteins by Baculovirus KRIST1NEBUTROVICH,
AND CARL E WARE 345
31. Analysis of the CD95 (APO-1/Fas) Death-Induc- CARSTEN SCAFFIDI,
ing Signaling Complex by High-Resolution FRANKC. KISCHKEL,
Two-Dimensional Gel Electrophoresis PETER H. KRAMMER,
AND MARCUS E. PETER 363

32. Measurement of Ceramide Levels by the Di- RON BOSE AND

acylglycerol Kinase Reaction and by High- RICHARD KOLESNICK 373
Performance Liquid Chromatography-Fluo-
rescence Spectrometry

33. Measurement of Ceramide Synthase Activity RON BOSE AND

RICHARD KOLESNICK 378
34. Measurement of Sphingomyelinase Activity ERICH GULBINSAND
RICHARD KOLESNICK 382
35. Assays for JNK and p38 Mitogen-Activated TATSUHIKOSUDO
Protein Kinases AND MICHAELKARIN 388
36. Assaying for IKB Kinase Activity JOSEPH A. DIDONATO 393
37. Assays for Akt THOMAS F. FRANKE 400
 CONTENTS OF VOLUMES 321-354 xiv

S e c t i o n VIII. O t h e r M e t h o d s
38. Measurement of Cellular Oxidation, Reactive LISA M. ELLERBY
Oxygen Species, and Antioxidant Enzymes AND DALEE. BREDESEN 413
during Apoptosis

39. Volume Regulation and Ion Transport during CARL D. BORTNER

Apoptosis ANDJOHN A. CIDLOWSKI 421

40. Assays for Transglutaminases in Cell Death GERRYMELINO,

ELEONORACANDI,
AND PETERM. STEINERT 433

41. Anoikis STEVENM. FRISCH 472

42. TransientTransfectionAssay of Cell Death Genes MASAYUKIMIURA

AND JUNYINGYUAN 480

43. Sindbis Virus Vector System for Functional J. MARIEHARDWICK

Analysis of Apoptosis Regulators AND BETH LEVlNE 492

44. Transduction of Full-Length Tat Fusion Proteins ADAMINAVOCERO-AKBANI,

Directly into Mammalian Cells: Analysis of NATALIEA. LISSY,
T Cell Receptor Activation-InducedCell Death AND STEVENF. DOWDY 508

VOLUME 323
ENERGETICS OF BIOLOGICALMACROMOLECULES (PART C)
Michael L. Johnson and Gary K. Ackers

1. Quantitative Dissection of Transcriptional MARCR. VAN GILST

Control System: N-Dependent Antitermination AND PETERH. VON HIPPEL 1
Complex of Phase L as Regulatory Paradigm

2. Problems and Prospects in Microcalorimetry of GEORGEP. PRIVALOV

Biological Macromolecules AND PETERL. PRIVALOV 31

3. Probing Stability of Helical Transmembrane KARENG. FLEMING 63

Proteins
4. Energetics of Vinca Alkaloid Interactions with SHARONLOBERT
Tubulin ANDJOHN J. CORREIA 77
5. KineticsandThermodynamicsofConformational CAMMONB. ARRINGTONAND
Equilibria in Native Proteins by Hydrogen ANDREWD. ROBERTSON 104
Exchange

6. Mathematical Modeling of Cooperative Interac- MICHAELL. JOHNSON 124

tions in Hemoglobin
7. Deriving Complex Ligand-BindingFormulas MICHAELL. JOHNSON
ANDMARTINSTRAUME 155
xv CONTENTS OF VOLUMES 321-354

8. Calculation ofEntropy Changes in Biological Pro- L. MARIOAMZEL 167

cesses: Folding, Binding, and Oligomerization

9. Evaluating Energetics of Erythropoietin Ligand PRESTON HENSLEY,

Binding to Homodimerized Receptor Extracel- MICHAELL. DOYLE,
lular Domains DAVID G. MYSZKA,
ROBERT W. WOODY,
MICHAELR. BRIGHAM-BURKE,
CONNIE L. ERICKSON-MILLER,
CHARLES A. GRIFFIN,
CHRISTOPHER S. JONES,
DEAN E. MCNULTY,
SHAWNP. O'BRIEN,
BERNARD Y. AMEGADZIE,
LAURIEMACKENZIE,
M. DOMINICRYAN,
AND PETER R. YOUNG 177

10. Measurement of Protein Interaction Bioener- MICHAELL. DOYLE,

getics: Application to Structural Variants of MICHAEL R. BRIGHAM-BURKE,
Anti-sCD4 Antibody MICHAELN. BLACKBURN,
IAN S. BROOKS,
THOMAS M. SMITH,
ROLAND NEWMAN,
MITCHELLRUFF,
WALTERE STAFFORDHI,
RAYMONDW. SWEET,
ALEMSEGEDTRUNEH,
PRESTON HENSLEY, AND
DANIELJ. O'SHANNESSY 207

11. AnalysisoflnteractionofRegulatoryProteinTyrR GEOFFREYJ. HOWLETT

with DNA AND BARRIEE. DAVlDSON 231

12. Proteolytic Footprinting Titrations for Estimat- MADELINEA. SHEA,

ing Ligand-Binding Constants and Detecting BRENDA R. SORENSEN,
Pathways of Conformational Switching of Cal- SUSANPEDIGO, AND
modulin AMY S. VERHOEVEN 254

13. Analysis °f Reversibly Interacting Macr°m°lecu- WALTERE STAFFORD 302

lar Systems by Time Derivative Sedimentation
Velocity

14. Kinetic, Equilibrium, and Thermodynamic DAVID G. MYSZKA 325

Analysis of Macromolecular Interactions with
BIACORE

15. Structure-Function Relationships in Two-Com- ISTVANE SUGAR

ponent Phospholipid Bilayers: Monte Carlo AND RODNEY L.B~TONEN 340
Simulation Approach Using a Two-State Model
 CONTENTS OF VOLUMES 321-354 xvi

16. Parsing Free Energies of Drug-DNA Interactions IHTSHAMULHAQ,

TERENCEC. JENKINS,
BABURZ. CHOWDHRY,
JINSONG REN, AND
JONATHANB. CHAIRES 373
17. Direct Measurement of Sodium Ion Release from CHARLES H. SPINK
DNA on Binding of Cationic Ligands AND THOMASP. WHITE 406
18. Enthalpy-Entropy Compensations in Nucleic LUIS A. MARKY
Acids: Contribution of Electrostriction and ANDDONALDW. KUPKE 419
Structural Hydration

19. Time-Resolved Fluorescence Methods for DAVIDP. MILLAR 442

Analysis of DNA-Protein Interactions

20. Use of Fluorescence Spectroscopy as Thermo- MAURICER. EFTINK 459

dynamics Tool
21. Microsecond Dynamics of Biological Macro- JOSEPH R. LAKOWICZ,
molecules IGNACYGRYCZYNSKI,
GRZEGORZPISZCZEK,
LEAH TOLOSA,RAJESHNAIR,
MICHAELL. JOHNSON, AND
KAZIMIERZNOWACZYK 473

VOLUME 324
BRANCHED-CHAIN AMINO ACIDS (PART B)
Robert A. Harris and John R. Sokatch

S e c t i o n I. P r e p a r a t i o n o f S u b s t r a t e s , A s s a y s o f I n t e r m e d i a t e s
and Enzymes, and Use of Enzyme Inhibitors
1. Synthesis and Gas Chromatography/Mass ORVALA. MAMER 3
Spectrometry Analysis of Stereoisomers of
2-Hydroxy-3 -methylpentanoic Acid
2. Analysis of Intracellular Metabolites as Tool for SABINEEPELBAUM,
StudyingBranched-ChainAmino Acid Biosyn- DAVIDM. CHIPMAN,
thesis and Its Inhibition in Bacteria AND ZE'EV BARAK 10
3. Determinationof Branched-ChainL-Amino-Acid PETERSCHADEWALDT 23
Aminotransferase Activity
4. Analysis of (S)- and (R)-3-Methyl-2-oxopenta- PETERSCHADEWALDT 33
noate Enantiomorphs in Body Fluids
5. Spectrophotometric Assay for Measuring PHILIPR. BECKETT 40
Branched-Chain Amino Acids
xvii CONTENTS OF VOLUMES 321-354

6. Determination of Branched-Chain c~-Keto Acid NAOYANAKAI,

Dehydrogenase Activity State and Branched- RUMI KOBAYASHI,
Chain ot-Keto Acid Dehydrogenase Kinase KIRILLM. POPOV,
Activity and Protein in Mammalian Tissues ROBERTA. HARRIS,AND
YOSHIHARUSHIMOMURA 48
7. Simultaneous Quantification of Plasma Levels MARZIAGALLIKIENLE
of t~-Ketoisocaproate and Leucine by Gas AND FULVIOMAGNI 62
Chromatography-Mass Spectrometry

8. Synthesis of Methacrylyl-CoA and (R)- and (S)- JOHN W. HAWES

3-Hydroxyisobutyryl-CoA AND EDWINT. HARPER 73

9. Pathways of Leucine and Valine Catabolism in J. RICHARDDICKINSON 80

Yeast

S e c t i o n II. C l o n i n g , E x p r e s s i o n , a n d P u r i f i c a t i o n o f E n z y m e s
of Branched-Chain Amino Acid Metabolism
10. Isolation of Subunits of Acetohydroxy Acid MARIA VYAZMENSKY,
Synthase Isozyme III and Reconstitution of TSIONAELKAYAM,
Holoenzyme DAVID M. CHIPMAN,
ANDZE'EV BARAK 95

11. Branched-Chain Amino-Acid Aminotransferase HIROYUKIKAGAMIYAMA

of Escherichia coli AND HIDEYUKIHAYASHI 103

12. Purification of Sodium-Coupled Branched- YOSHIHIKOURATANI

Chain Amino Acid Carder of Pseudomonas AND TOSHIMITSUHOSHINO 114
aeruginosa
13. ReconstitutionofPseudomonasaeruginosaHigh- TOSHIMITSUHOSHINO
Affinity Branched-Chain Amino Acid Trans- AND YOSHIHIKOURATANI 122
port System

14. Purification of Pseudomonas putida Branched- KATHRYNL. HESTER,

Chain Keto Acid Dehydrogenase E1 Com- JINHE LUO, AND
ponent JOHN R. SOKATCH 129

15. Pseudomonas mevalonii 3-Hydroxy-3-methyl- HENRY M. MIZIORKO

glutaryl-CoA Lyase ANDCHAKRAVARTHY
NARASIMHAN 139

16. Human 3-Hydroxy-3-methylglutaryl-CoALyase HENRY M. MIZIORKO,

CHAKRAVARTHY
NARASIMHAN,AND
JACQUELINER. ROBERTS 150

17. Branched-Chain a-Keto Acid Dehydrogenase KIRILLM. POPOV,

Kinase YOSHIHARUSHIMOMURA,
JOHN W. HAWES,AND
ROBERTA. HARRIS 162
 CONTENTS OF VOLUMES 321-354 xviii

18. Expression of E1 Component of Human Branched- R. MAX WYNN,

Chain a-Keto Acid Dehydrogenase Complex JAMESR. DAVIE,
in Escherichia coli by Cotransformation with JIU-LISONG,
Chaperonins GroEL and GroES JACINTAL. CHUANG,
AND DAVIDT. CHUANG 179
19. Production of Recombinant Mammalian Holo- JACINTAL. CHUANG,
E2 and E3 and Reconstitution of Functional JAMES R. DAVIE,
Branched-Chain ot-Keto Acid Dehydrog- R. MAX WYNN,
enase Complex with Recombinant E1 AND DAVIDT. CHUANG 192
20. Production of Recombinant E1 Component of JOHN W. HAWES,YU ZHAO,
Branched-Chain ot-Keto Acid Dehydrogenase KIRILLM. PoPO~
Complex YOSHIHARUSHIMOMURA,
AND ROBERTA. HARRIS 200
21. Mammalian Methylmalonate-Semialdehyde NATALIAY. KEDISHVILI,
Dehydrogenase GARY W. GOODWlN,
KIRILL M. POPOV,
AND ROBERTA. HARRIS 207
22. Mammalian 3-Hydroxyisobutyrate Dehydrog- JOHN W. HAWES,
enase DAVID W. CRABB,
REBECCA M. CHAN,
PAUL M. ROUGRAFF,
RALPH PAXTON,AND
ROBERTA. HARRIS 218
23. 3-Hydroxyisobutyryl-CoAHydrolase YOSHIHARUSHIMOMURA,
TAROMURAKAMI,
NAOYANAKAI,BOLl HUANG,
JOHN W. HAWES,
AND ROBERTA. HARRIS 229
24. Mammalian Branched-Chain Acyl-CoA Dehy- JERRY VOCKLE~
drogenases: Molecular Cloning and Charac- AL-WAMDA. MOHSEN,
terization of Recombinant Enzymes BARBARABINZAK,
JAN WlLLARD,
AND ABDULFAUQ 241

25. 3-Hydroxy-3-methylglutaryl-CoAReductase VICTORW. RODWELL,

MICHAELJ. BEACH,
KENNETHM. BISCHOFF,
DANIELA. BOCHAR,
BRYANTG. DARNAY,
JON A. FRIESEN,
JOHN E GILL,
MATIJAHEDL,
TUAJUANDAJORDAN-STARCK,
PETER J. KENNELLY,
DONGYULKIM,
AND YULIWANG 259
xix CONTENTS OF VOLUMES 321-354

26. Characterization of 3-Methylcrotonyl-CoA Car- EVE SYRKINWURTELE

boxylase from Plants AND BASILJ. NIKOLAU 280

27. Purification of D-HydroxyisovalerateDehydroge- RAINERZOCHER 293

nase from Fusarium sambucinum

28. Purification and Characterization of Recombinant YOKO HAYASHI-IWASAKI

3-Isopropylmalate Dehydrogenases from Ther- AND TAIRO OSHIMA 301
mus thermophilus and Other Microorganisms

29. Wild-Type and Hexahistidine-TaggedDerivatives ROWENA G. MATTHEWS,

of Leucine-Responsive Regulatory Protein from YUHAICHI,
Escherichia coli DEVORAHFRIEDBERG,
AND JOSEPH M. CALVO 322

30. Purification of Branched-Chain Keto Acid Dehy- KUNAPULIT. MADHUSUDHAN

drogenase Regulator from Pseudomonasputida AND JOHN R. SOKATCH 329

31. Mitochondrial Import of Mammalian Branched- DEAN J. DANNER 336

Chain a-Keto Acid Dehydrogenase Complex
Subunits

32. Cloning, Expression, and Purification of Mam- NICHOLASP. CROUCH,

malian 4-HydroxyphenylpyruvateDioxygenase/MENG-HUEE LEE, TERESA
c~-Ketoisocaproate Dioxygenase ITURRIAGAGOITIA-BUENO,
AND COLIN H. MACKINNON 342

33. Mammalian Branched-Chain Aminotransferases MYRACONWAY

AND SUSANHUTSON 355

34. Branched-Chain-Amino-Acid Transaminases of CORINNA PROHL,

Yeast Saccharomyces cerevisiae GYULA KISPAL,
AND ROLANDLILL 365

35. Purification, Properties, and Sequencing of NANAYATAMAKI,

Aminoisobutyrate Aminotransferases from SHIGEKOFUJIMOTOSAKATA,
Rat Liver AND KOICHIMATSUDA 376

36. Branched-Chain Keto Acid Dehydrogenase of J. RICHARDDICKINSON 389

Yeast

37. fl-Alanine Synthase, an Enzyme Involved in THOMASW. TRAUT 399

Catabolism of Uracil and Thymine

S e c t i o n III. D e t e c t i o n a n d C o n s e q u e n c e s o f G e n e t i c D e f e c t s
in Genes Encoding Enzymes of Branched-Chain
Amino Acid Metabolism
38. Diagnosis and Mutational Analysis of Maple JACINTAL. CHUANG
Syrup Urine Disease Using Cell Cultures AND DAVIDT. CHUANG 413
 CONTENTS OF VOLUMES 321-354 XX

39. Detection of Gene Defects in Branched-Chain CHARLESR. ROE

Amino Acid Metabolism by Tandem Mass AND DIANES. ROE 424
Spectrometry of Carnitine Esters Produced by
Cultured Fibroblasts
40. Molecular and Enzymatic Methods for Detection K. MICHAELGIBSON,
of Genetic Defects in Distal Pathways of MAGDALENAUGARTE,
Branched-Chain Amino Acid Metabolism TOSHIYUKIFUKAO,
AND GRANTA. MITCHELL 432
41. Genetic Defects in E3 Component of ot-Keto Acid MULCHANDS. PATEL,
Dehydrogenase Complexes YOUNGSOO HONG,
ANDDOUGLASS. KERR 453
42. Targeting E3 Component of ~-Keto Acid Dehy- MARK T. JOHNSON,
drogenase Complexes HSIN-SHENGYANG,
AND MULCHANDS. PATEL 465

S e c t i o n IV. R e g u l a t i o n a n d E x p r e s s i o n o f E n z y m e s o f
Branched-Chain Amino Acid Metabolism
43. Regulation of Expression of Branched-Chain JEFFREYM. CHINSKY
ot-Keto Acid Dehydrogenase Subunits in Per- ANDPAULA. COSTEAS 479
manent Cell Lines
44. Expression of Murine Branched-Chain ot-Keto CHRISTOPHERB. DOERING
Acid Dehydrogenase Kinase AND DEANJ. DANNER 491
45. Regulation of Branched-Chain ot-Keto Acid YI-SHUIANHUANG
Dehydrogenase Kinase Gene Expression by AND DAVIDT. CHUANG 498
Glucocorticoids in Hepatoma Cells and Rat
Liver

VOLUME 325
REGULATORS AND EFFECTORS OF SMALL GTPASES (PART D: RHO FAMILY)
W. E. Balch, Channing J. Der, and Alan Hall

S e c t i o n I. P u r i f i c a t i o n , P o s t t r a n s l a t i o n a l Modification, and
in Vitro R e g u l a t i o n
1. Purification and Biochemical Characterization of CHERYLL. NEUDAUER
TC10 AND IAN G. MACARA 3
2. Expression and Purification of Rho/RhoGDI PAULW. READ
Complexes AND ROBERTK. NAKAMOTO 15
3. Bacterial Expressed DH and DH/PH Domains KENTL. ROSSMAN
AND SHARONL. CAMPBELL 25
xxi CONTENTS OF VOLUMES 3 2 1 - 3 5 4

4. Biochemical Analysis of Regulation of Vav, a RAYMONDMOSTELLER,

Guanine-NucleotideExchange Factor for Rho JAEWONHAN, BALAKADAS,
Family of GTPases AND DANIELBROEK 38

5. Activation of Racl by Human Tiaml GIDEONBOLLAG,

ANNE M. CROMPTON,
DANIELLEPEVERLY-MITCHELL,
GASTONG. M. HABETS,
AND MARC SYMONS 51

6. Activation of Rho GEF Activity by Goq3 MATTHEWJ. HART,

WILLIAMROSCOE,
AND GIDEONBOLLAG 61
7. Purification and Characterization of Guanine JON P. HUTCHINSON,
Nucleotide Dissociation Stimulator Protein KATRINRITTINGER,
ANDJOHN E ECCLESTON 71
8. Purification and Biochemical Activity of ANDREAFRIEBELAND
Salmonella Exchange Factor SopE WOLF-DIETRICHHARDT 82

9. Stimulationof Rho GDI Release by ERM Proteins AKIKOMAMMOTO,

KAZUOTAKAHASHI,
TAKUYASASAKI,
AND YOSHIMITAKAI 91

10. Expression and Activity of Human Prenylcysteine- EDWIN CHOY

Directed Carboxyl Methyltransferase AND MARKPHILIPS 101

11. Purification and Evaluation of Large Clostridial MICHAELMOOS

Cytotoxins That Inhibit Small GTPases of Rho AND CHRISTOPHVON
and Ras Subfamilies EICHEL-STRE1BER 114

12. Rho GTPase-ActivatingToxins:CytotoxicNecro- GUDULASCHMIDT

tizing Factors and Dermonecrotic Toxin AND KLAUSAKTORIES 125

Section II. Purification a n d Activity of GTPase Targets

13. Measurement of GTPase-Effector Affinities DANNYMANOR 139

14. Purification and in Vitro Activity of Rho-Associ- MUTSUKIAMANO,

ated Kinase YUKOFUKATA,
HIROAKISHIMOKAWA,
AND KOZOKAIBUCHI 149

15. Purification and in Vitro Activities of p21-Acti- CHARLESC. KING,

vated Kinases ABINAM. REILLY,
AND ULLA G. KNAUS 155
 CONTENTS OF VOLUMES 321-354 xxii

16. Stimulation of Phospholipase C-fl2 by Rho DARIA ILLENBERGER,

GTPases ILONA STEPHAN,
PETER GIERSCHIK,
AND FRIEDER SCHWALD 167

17. Regulation of Phospholipase D1 Activity by Rho MICHAEL A. FROHMAN,

GTPases YASUNORIKANAHO,
YUE ZHANG,
AND ANDREWJ. MORRIS 177

18. In Vitro Interaction of Phosphoinositide-4- KIMBERLEY TOLIAS AND

phosphate 5-Kinases with Rac CHRISTOPHER L. CARPENTER 190

19. Activity and Regulation of p35/Cdk5 Kinase MARGARETA NIKOLIC

Complex AND LI-HUEI TSAI 200

20. Actin Assembly Mediated by Arp2/3 Complex R. DYCHE MULLINS AND

and WASP Family Proteins LAURAM. MACHESKY 214

21. In Vitro Actin Polymerization Using Polymorpho- SALLY H. ZIGMOND 237

nuclear Leukocyte Extracts

S e c t i o n III. A n a l y s i s o f R h o G T P a s e F u n c t i o n
22. Determination of GTP Loading on Rac and Cdc42 ANSER C. AZIM,
in Platelets and Fibroblasts KURT L. BARKALOW,
AND JOHN H. HARTWIG 257

23. Determination of GTP Loading on Rho XIANG-DONG REN

AND MARTIN ALEXANDER
SCHWARTZ 264
24. Use and Properties of ROCK-Specific Inhibitor SHUH NARUMIYA,
Y-27632 TOSHIMASA ISHIZAKI,
AND MASAYOSHI UEHATA 273

25. Inducible Membrane Recruitment of Small GTP- FLAVIA CASTELLANO

Binding Proteins by Rapamycin-Based System AND PHILIPPE CHAVRIER 285
in Living Cells
26. Expression of Rho GTPases Using Retroviral FRITS MICHIELS,
Vectors ROB A. VAN DER KAMMEN,
LENNERT JANSSEN,
GARRY NOLAN,
AND JOHN G. COLLARD 295
27. Expression of Rho GTPases Using Adenovirus DANIEL M. SULLIVAN
Vectors AND TOREN FINKEL 303
28. In Vivo Activity of Wild-Type and Mutant PAKs CHARLES C. KING,
LURAYNNE C. SANDERS,
AND GARY M. BOKOCH 315
xxiii CONTENTS OF VOLUMES 321-354

29. Single Cell Assays for Rac Activity LAURAJ. TAYLOR,

AMY B. WALSH,
PATRICKHEARING,
AND DAFNABAR-SAGI 327

30. Effect of Rho GTPases on Na-H Exchanger in SHERYLP. DENKER,

Mammalian Cells ~hrEIHONGYAN,
ANDDIANEL. BARBER 334

31. Actin Filament Assembly in Permeabilized JOHN H. HARTWIG

Platelets AND KURTL. BARKALOW 349

32. Rho GTPases: Secretion and Actin Dynamics in MARK R. HOLT

Permeabilized Mast Cells AND ANNAKOFFER 356

33. Isolation and in Vitro Contraction of Stress Fibers KAZUOKATOH,

YUMIKOKANO,
AND KEIGIFUJIWARA 369

34. Inhibitionof Rho GTPases Using Protein Geranyl- SAID M. SEBTI

geranyltransferase I Inhibitors AND ANDREWO. HAMILTON 381

35. Imaging Spatiotemporal Dynamics of Rac Activa- CHESTERE. CHAMBERLAIN,

tion in Vivo with FLAIR VADIMS. KRAYNOV,
AND KLAUSM. HAHN 389

S e c t i o n IV. B i o l o g i c a l A s s a y s o f R h o G T P a s e F u n c t i o n
36. Rho-like GTPases in Tumor Cell Invasion EVA E. EVERS,
ROB A. VAN DER KAMMEN,
JEAN P. TEN KLOOSTER,
AND JOHN G. COLLARD 403

37. Mouse Embryo Fibroblasts: A Genetic Model GIOVANNAM. D'ABACO

System for Studying Rho- and Ras-Dependent ANDMICHAELE OLSON 415
Cell Cycle Progression

38. Analyses of TransformingActivity of Rho Family PATRICIAA. SOLSKI,

Activators KARONABE,
AND CHANNINGJ. DER 425

39. Rho GTPases and Cell Migration-Fibroblast CATHERINED. NOBES 441

Wound Healing

40. Rho GTPases and Cell Migration: Measurement GARETHE. JONES,

of Macrophage Chemotaxis ANNE J. RIDLEY,
AND DANIELZICHA 449

41. Rho GTPases and Macrophage Phagocytosis JAYESH C. PATEL,

ALAN HALL, AND
EMMANUELLECARON 462
 CONTENTS OF VOLUMES 321-354 xxiv

42. Rho GTPases and Axonal Growth Cone Collapse ALYSONE. FOURNIER,
ROBERT G. KALB,AND
STEPHENM. STRITTMATTER 473
43. Study of in Situ Function of Cytoskeletal Emc V. WONG
Proteins in Lamellipodia and Filopodia Using AND DANIELG. JAY 482
Microscale Chromophore-Assisted Laser Inac-
tivation
44. Modulation of Actin Cytoskeleton by Salmonella JORGEE. GAL,~N
GTPase Activating Protein SptP AND YIXINFU 496

VOLUME 326
APPLICATIONS OF CHIMERIC GENES AND HYBRID PROTEINS (PART A:
GENE EXPRESSION AND PROTEIN PURIFICATION)
Jeremy Thomer, Scott D. Emr, and John N. Abelson

S e c t i o n I. H i s t o r i c a l O v e r v i e w
1. The All Purpose Gene Fusion JON BECKWlTH

S e c t i o n II. G e n e F u s i o n s a s R e p o r t e r s o f G e n e E x p r e s s i o n i n
Prokaryotic Cells
2. A Practical Guide to the Construction and Use of NICHOLASJ. HANDAND
lac Fusions in Escherichia coli THOMASJ. SILHAVY 11
3. TaggingExportedProteinsUsingEscherichiacoli COLINMANOIL 35
Alkaline Phosphatase Gene Fusions
4. Applications of Gene Fusions to Green Fluores- RAPHAELH. VALD1VIAAND
cent Protein and Flow Cytometry to the Study LALITARAMAKRISHNAN 47
of Bacterial Gene Expression in Host Cells
5. IVET and RIVET: Use of Gene Fusions to Iden- JAMESM. SLAUCH
tify Bacterial Virulence Factors Specifically In- AND ANDREWCAMILLI 73
duced in Host Tissues
6. Identification of Exported Bacterial Proteins via MICAHJ. WORLEY
Gene Fusions to Yersinia pseudotuberculosis ANDFREDHEFFRON 97
Invasin

S e c t i o n Ill. G e n e F u s i o n s a s R e p o r t e r s o f G e n e E x p r e s s i o n i n
Eukaryotic Cells
7. Use of Imidazoleglycerolphosphate Dehydratase JOE HORECKAAND
(His3) as a Biological Reporter in Yeast GEORGEE SPRAGUE,JR. 107
XXV CONTENTS OF VOLUMES 321-354

8. Use of Fusions to Human Thymidine Kinase as SOFIEREDA SALAMA 120

Reporters of Gene Expression and Protein
Stability in Saccharomyces cerevisiae

9. Use of Fusions to Thymidine Kinase CHRISTIAANKARREMAN 134

10. In Vivo Analysis of lacZ Fusion Genes in Trans- STEPHEN SMALL 146
genic Drosophila melanogaster

11. Utility of the Secreted Placental Alkaline Phos- BRYAN R. CULLEN 159
phatase Reporter Enzyme

12. Fusions to Imidazopyrazinone-Type Luciferases SATOSHIINOUYE 165

and Aequorin as Reporters

13. Novel Methods for ChemiluminescentDetection CORINNE E. M. OLESEN,

of Reporter Enzymes YU-XINYAN, BETI'YLIE,
DINA MARTIN,BRIAND'EON,
RAY JUDWARE,
CHRIS MARTIN,
JOHN C. VOYTA,AND
IRENABRONSTEIN 175
14. Fusions to Chloramphenicol Acetyltransferase as CLAYTONBULLOCK
a Reporter AND CORNELIAGORMAN 202

15. Fusions to ~-Lactamase as a Reporter for Gene GREGORZLOKARNIK 221

Expression in Live Mammalian Cells

S e c t i o n IV. F u n c t i o n a l T a g s f o r B i o c h e m i c a l P u r i f i c a t i o n
16. Purification of Proteins Using Polyhistidine JOSHUA A. BORNHORST
Affinity Tags ANDJOSEPHJ. FALKE 245

17. Generating Fusions to Glutathione S-Transferase DONALDB. SMITH 254

for Protein Studies

18. Use of the Strep-Tag and Streptavidin for Detec- ARNE SKERRAAND
tion and Purification of Recombinant Proteins THOMASG. M. SCHMIDT 271

19. Streptavidin-Containing Chimeric Proteins: De- TAKESHISANO

sign and Production AND CHARLESR. CANTOR 305

20. Fusions to Maltose-Binding Protein: Control of DEEPALI SACHDEV

Folding and Solubility in Protein Purification ANDJOHN M. CHIRGWIN 312

21. Thioredoxin as a Fusion Panner for Production EDWARDR. LA VALLIE,

of Soluble Recombinant Proteins in Esherichia ZHIJIANLU,
coli ELIZABETHA.
DIBLASIO-SMITH,
LISA A. COLLINS-RACIE,
ANDJOHN M. MCCOY 322
 CONTENTS OF VOLUMES 321-354 xxvi

22. Affinity Purification of Recombinant Proteins PETER VAILLANCOURT,

Fused to Calmodulin or to Calmodulin-Binding CHAO-FENG ZHENG,
Peptides DANNY Q. HOANG,
AND LISA BREISTER 340

23. The S. Tag Fusion System for Protein Purification RONALD T. RAINES,
MARK MCCORMICK,
THOMAS R. VAN OOSBREE,
AND ROBERT C. MIERENDORF 362

24. Fusions to Self-Splicing Inteins for Protein MING-QUNXU,

Purification HENRY PAULUS,
AND SHAORONGCHONG 376

25. Fusion Proteins Containing Cellulose-Binding JAE-SEON PARK,

Domains HAL-SUN SHIN,
AND ROY H. DOI 418

26. Biotinylation of Proteins in Vivo and in Vitro Us- MILLARD G. CULL

ing Small Peptide Tags AND PETER J. SCHATZ 430

27. Biotinylation of Proteins in Vivo: A Useful Post- JOHN E. CRONAN, JR.

translational Modification for Protein Analysis AND KELYNNE E. REED 440

S e c t i o n V. H y b r i d P r o t e i n s f o r D e t e c t i o n a n d P r o d u c t i o n
of Antigens and Antibodies
28. Methods for Generating Multivalent and Bi- IAN TOMLINSON
specific Antibody Fragments AND PHILIPPHOLLIGER 461

29. Design and Use of Phage Display Libraries for FRANCESCAVITI,

the Selection of Antibodies and Enzymes FREDRIK NILSSON,
SALVATOREDEMARTIS,
ADRIAN HUBER,
AND DARIO NERI 480

30. Use of an Lpp-OmpA Fusion Vehicle for Bacterial CHARLES F. EARHART 506
Surface Display

31. Identification of Bacterial Class I Accessible DOLPH ELLEFSON,

Proteins by Disseminated Insertion of Class I ADRIANUSW. M.
Epitopes VAN DER VELDEN,
DAVID PARKER,
AND FRED HEFFRON 516

32. Bordetella pertussis Adenylate Cyclase Toxin: A PIERRE GUERMONPREZ,

Vehicle to Deliver CD8-Positive T-Cell Epi- CATHERINE FAYOLLE,
topes into Antigen-Presenting Cells GOUZEL KARIMOVA,
AGNES ULLMANN,
CLAUDE LECLERC,
AND DANIEL LADANT 527
xxvii CONTENTS OF VOLUMES 321-354

33. Use of Anthrax Toxin Fusions to Stimulate CHRISTOPHER C. ZAROZINSKI,

Immune Responses R. JOHN COLLIER,AND
MICHAEL N. STARNBACH 542
34. Use of Fusions to Viral Coat Proteins as Antigenic PETER G. STOCKLEY
Carriers for Vaccine Development AND ROBERT A. MASTICO 551

V O L U M E 327
APPLICATIONS OF CHIMERIC GENES AND HYBRID PROTEINS (PART B: CELL
BIOLOGY AND PHYSIOLOGY)
Jeremy Thorner, Scott D. Emr, and John N. Abelson

S e c t i o n I. E p i t o p e T a g s f o r I m m u n o d e t e c t i o n
I. Epitope Tagging: General Method for Tracking CHRISTIANE. FRITZE
Recombinant Proteins AND THOMASR. ANDERSON

S e c t i o n II. M a r k e r s f o r C y t o l o g y , A n a l y s i s o f P r o t e i n
Trafficking, and Lineage Tracing
2. Alkaline Phosphatase Fusions of Ligands or Re- JOHN G. FLANAGAN,
ceptors as in Situ Probes for Staining of Cells, HWAI-JONGCHENG,
Tissues, and Embryos DAVID A. FELDHEIM,
MITSUHARUHATTORI,
QIANG LU, AND
PIERRE VANDERHAEGHEN 19
3. Chimeric Molecules Employing Horseradish COLIN HOPKINS,
Peroxidase as Reporter Enzyme for Protein ADELE GIBSON,
Localization in the Electron Microscope JANE STINCHCOMBE,
AND CLAREPJTTER 35
4. Biochemical Analyses of Trafficking with Horse- ANASTASIYAD.
radish Peroxidase-Tagged Chimeras BLAGOVESHCHENSKAYA
AND DANIELF. CUTLER 45
5. Visualizing Protein Dynamics in Yeast with Green CHRISTOPHER G. BURD 61
Fluorescent Protein
6. Kinetic Analysis of Intracellular Trafficking in KORET HIRSCHBERG,
Single Living Cells with Vesicular Stomatitis ROBERT D. PHAIR,
Virus Protein G-Green Fluorescent Protein AND JENNIFER
Hybrids LIPPINCOTT-SCHWARTZ 69
7. Dual Color Detection of Cyan and Yellow Deriva- GISELE GREEN,
fives of Green Fluorescent Protein Using Con- STEVEN R. KAIN,
ventional Fluorescence Microscopy and 35-mm AND BRIGITTEANGRES 89
Photography
 CONTENTS OF VOLUMES 321-354 xxviii

8. Invertase Fusion Proteins for Analysis of Protein TAMARADARSOW,

Trafficking in Yeast GREG ODORIZZI,
AND SCOTTD. EMR 95

9. Introduction of Kex2 Cleavage Sites in Fusion B. DIANEHOPKINS,

Proteins for MonitoringLocalization and Trans- KEN SATO,
port in Yeast Secretory Pathway AKIHIKONAKUNO,
ANDTODDR. GRAHAM 107

10. Lineage Analysis with Retroviral Vectors CONSTANCEL. CEPKO,

ELIZABETHRYDER,
CHRISTOPHERAUSTIN,
JEFFREYGOLDEN,
SHAWNFIELDS-BERRY,
AND JOHN LIN 118

11. Use of Pseudotyped Retroviruses in Zebrafish as SHAWNBURGESS

Genetic Tags ANDNANCYHOPKINS 145

S e c t i o n III. T o o l s f o r A n a l y s i s o f M e m b r a n e Proteins
12. A Gene Fusion Method for Assaying Interactions JENNIFERA. LEEDS
of Protein Transmembrane Segments in Vivo ANDJON BECKWITH 165

13. Using SUC2-HIS4C Reporter Domain to Study CHRISTIANSENGSTAG 175

Topology of Membrane Proteins in Saccharo-
myces cerevisiae

14. Detecting Interactions between Membrane Pro- IGORSTAGLJAR

teins in Vivo Using Chimeras AND STEPHAN TE HEESEN 190

15. Alkaline Phosphatase Fusion Proteins for Molec- JOHNG. FLANAGAN

ular Characterization and Cloning of Receptors AND HWAI-JONGCHENG 198
and Their Ligands

16. Surface Chimeric Receptors as Tools in Study of BRYANA. IRVING

Lymphocyte Activation ANDARTHURWEISS 210

17. Use of Chimeric Receptor Molecules to Dissect WARRENJ. LEONARD 228

Signal Transduction Mechanisms

18. Fusion Protein Toxins Based on Diphtheria Toxin: JOHANNAC. VANDERSPEK

Selective Targeting of Growth Factor Receptors AND JOHN R. MURPHY 239
of Eukaryotic Cells

19. Green Fluorescent Protein-Based Sensors for De- MICAHS. SIEGEL

tecting Signal Transductionand Monitoring Ion AND EHUD Y. ISACOFF 249
Channel Function
xxix CONTENTS OF VOLUMES 321-354

20. Metabolic Labeling of Glycoproteins with Chem- CHRISTINAL. JACOBS,

ical Tags through Unnatural Sialic Acid Biosyn- KEVINJ. YAREMA,
thesis LARA K. MAHAL,
DAVID A. NAUMAN,
NEIL W. CHARTERS,AND
CAROLYNR. BERTOZZI 260

S e c t i o n IV. S i g n a l s f o r A d d r e s s i n g P r o t e i n s t o S p e c i f i c
Subcellular Compartments
21. Using Sorting Signals to Retain Proteins in En- HUGH R. B. PELHAM 279
doplasmic Reticulum
22. Directing Proteins to Nucleus by Fusion to HEIKE KREBBER
Nuclear Localization Signal Tags AND PAMELAA. SILVER 283
23. Identification, Analysis, and Use of Nuclear KARSTENWEIS 297
Export Signals in Saccharomyces cerevisiae
24. Directing Proteins to Mitochondria by Fusion to KOSTASTOKATLIDIS 305
Mitochondrial Targeting Signals

25. Targeting Proteins to Plasma Membrane and WOUTER VAN'T HOF

Membrane Microdomains by N-Terminal AND MARILYND. RESH 317
Myristoylation and Palmitoylation

26. Analysis of Function and Regulation of Proteins GARY W. REUTHER,

That Mediate Signal Transduction by Use of JANICE E. BUSS,
Lipid-Modified Plasma Membrane-Targeting LAWRENCEm. QUILLIAM,
Sequences GEOFFREYJ. CLARK,
AND CHANNINGJ. DER 331
27. Glycerolphosphoinositide Anchors for Mem- JOHN D. FAYEN,
brane-Tethering Proteins MARK L. TYKOCINSKI,
AND M. EDWARDMEDOF 351
28. Fusions to Members of Fibroblast Growth Factor I. PRUDOVSKY,
Gene Family to Study Nuclear Translocation M. LANDRISCINA,
and Nonclassic Exocytosis R. SOLDI, S. BELLUM,
D. SMALL,W. ANDREEVA,
AND THOMASMACIAG 369

S e c t i o n V. A p p l i c a t i o n o f C h i m e r a s i n M o n i t o r i n g
and Manipulating Cell Physiology
29. Posttranslational Regulation of Proteins by Fu- DIDIERPICARD 385
sions to Steroid-Binding Domains
30. Tet Repressor-Based System for Regulated Gene UDO BARON
Expression in Eukaryotic Cells: Principles and AND HERMANNBUJARD 401
Advances
 CONTENTS OF VOLUMES 321-354 XXX

31. Coumermycin-Induced Dimerization of GyrB- MICHAELA. FARRAR,

Containing Fusion Proteins STEVENH. OLSON,AND
ROGER M. PERLMUTTER 421
32. Use ofGlutathione S-Transferase and Break Point YOSHIROMARU 429
Cluster Region Protein as Artificial Dimeriza-
tion Domains to Activate Tyrosine Kinases

33. Recombinant Aequorin as Tool for Monitoring VALERIEROBERT,

Calcium Concentrationin SubcellularCompart- PAOLOPINTON,
ments VALERIATOSELLO,
ROSARIORIZZUTO,
AND TULLIOPOZZAN 440
34. Recombinant Aequorin as Reporter of Changes JENNY STABLES,
in Intracellular Calcium in Mammalian Cells LARRY C. MATTHEAKIS,
RAY CHANG,
AND STEPHENREES 456
35. Monitoring Protein Conformations and Interac- ATSUSHIMIYAWAKI
tions by Fluorescence Resonance Energy Trans- ANDROGERY. TS1EN 472
fer between Mutants of Green Fluorescent
Protein

36. Studies of Signal Transduction Events Using TOBIASMEYER

Chimeras to Green Fluorescent Protein AND ELENAOANCEA 500
37. Use of Fusions to Green Fluorescent Protein in EVE SHINBROT,
the Detection of Apoptosis COLLIN SPENCER,
VALERIENATALE,
AND STEVENR. KAIN 513
38. Synapto-pHluorins: Chimeras between pH- RAFAELYUSTE,
Sensitive Mutants of Green Fluorescent Pro- REBECCAB. MILLER,
tein and Synaptic Vesicle Membrane Proteins KNUT HOLTHOFF,
as Reporters of Neurotransmitter Release SHIFANGZHANG,
AND GEROMIESENBOCK 522

39. Studying Organelle Physiology with Fusion MINNIE M. WU, JUANLLOPIS,

Protein-Targeted Avidin and Fluorescent Biotin STEPHENR. ADAMS,
Conjugates J. MICHAELMCCAFFERY,
KEN TETER,
MARKKUS. KULOMAA,
TERRYE. MACHEN,
HSIAO-P1NGH. MOORE,
ANDROGERY. TSIEN 546
40. Fluorescent Labeling of Recombinant Proteins in B. ALBERTGRIFFIN,
Living Cells with FlAsH STEPHENR. ADAMS,
JAY JONES,
AND ROGERY. TSIEN 565
41. Ubiquitin Fusion Technique and Its Descendants ALEXANDERVARSHAVSKY 578
xxxi CONTENTS OF VOLUMES 321-354

42. Use of Phosphorylation Site Tags in Proteins SIDNEY PESTKA,

LEI LIN, V~I WU,
AND LARA IZOTOVA 594

V O L U M E 328
APPLICATIONS OF CHIMERIC GENES AND HYBRID PROTEINS (PART C:
PROTEIN-PROTEIN INTERACTIONS AND GENOMICS)
Jeremy Thorner, Scott D. Emr, and John N. Abelson

S e c t i o n I. A p p l i c a t i o n s o f T w o - H y b r i d M e t h o d s a n d R e l a t e d
Techniques for Analysis of Protein-Protein Interactions
1. High-Throughput Screening for Protein-Protein GERARD CAGNEY,
Interactions Using Two-Hybrid Assay PETER UETZ,
AND STANLEYFIELDS

2. LexA-Based Two-Hybrid Systems SARAHJ. FASHENA,

ILYA G. SEREBRIISKII,
AND ERICA A. GOLEMIS 14

3. Interaction Mating Methods in Two-Hybrid MIKHAILG. KOLONIN,

Systems JINHUI ZHONG, AND
RUSSELLL. FINLEY,JR. 26

4. Analysis and Identification of Protein-protein In- AMI ARONHEIM

teractions Using Protein Recruitment Systems AND MICHAELKARIN 47

5. A Bacterial Two-Hybrid System that Exploits a GOUZELKARIMOVA,

cAMP Signaling Cascade in Escherichia coli AGNES ULLMANN,
AND DANIELLADANT 59

6. A Green Fluorescent Protein-Based Reverse Two- HIDEKI ENDOH,

Hybrid System: Application to the Characteri- ALBERTHAJ. M. WALHOUT,
zation of Large Numbers of Potential Protein- AND MARC VIDAL 74
Protein Interactions

7. Yeast Three-Hybrid System for Detecting ERIC C. GR1FFITH,

Ligand-Receptor Interactions EDWARDJ. LICITRA,
AND JUNO. LIU 89

8. Use of a Yeast Three-Hybrid System to Clone Jm ZHANG 103

Bridging Proteins

9. The Yeast Tribrid System: cDNA Expression JAREMAP. KOCHAN,

Cloning of Protein Interactions Dependent on CHRISTOPH VOLPERS,
Posttranslational Modifications AND MARK A. OSBORNE 111
 CONTENTS OF VOLUMES 321-354 xxxii

10. The Yeast Split-Hybrid System ANTHONYJ. DEMAGGIO,

PHYLLIS GOLDMAN,
HSIU-MINGSHIH,
RICHARDH. GOODMAN,
ANDMERLE HOEKSTRA 128

Section II. Other Approaches Using Chimeras for

Identification and Analysis of Interactions of Proteins
and Nucleic Acids
11. Mapping Protein-Protein Interaction Domains RICHARDR. BURGESS,
Using Ordered Fragment Ladder Far-Western TERRANCEM. ARTHUR,
Analysis of Hexahistidine-Tagged Fusion ANDBRADLEYC. PIETZ 141
Proteins
12. Mapping Specificity Determinants for Protein- MICHAELB. YAFFE
Protein Association Using Protein Fusions and ANDLEWISC. CANTLEY 157
Random Peptide Libraries
13. Selection of Genetic Agents from Random Pep- C. RONALDGEYER
tide Aptamer Expression Libraries AND ROGERBRENT 171
14. Detection of Protein-Protein Interactions by Pro- STEPHENW. MICHNICK,
tein Fragment Complementation Strategies INGRIDREMY, FRAN~OIS-X.
CAMPBELL-VALOIS,
ALEXIS VAEEI~E-BI~LISLE,
ANDJOELLEN. PELLETIER 208
15. Monitoring Protein-Protein Interactions in Live FABIO M. V. ROSSI,
Mammalian Cells by 13-Galactosidase Comple- BRUCE T. BEAKELY,
mentation CAROL A. CHARLTON,
ANDHELEN M. BEAU 231
16. Green Fluorescent Protein Chimeras to Probe SANG-HYUNPARK
Protein-Protein Interactions ANDRONALDT. RAINES 251
17. Protein Fusions to Coiled-Coil Domains KRISTIANM. MfJELER,
KATJAM. ARNDT,
ANDTOM AEBER 261
18. Molecular Applications of Fusions to Leucine JENNIFERD. RIEKER
Zippers ANDJAMES C. HU 282
19. Using the Yeast Three-Hybrid System to Detect BRIAN KRAEMER,
and Analyze RNA-Protein Interactions BEILIN ZHANG,
DHRUBASENGUPTA,
STANLEYFIELDS,
ANDMARVINWICKENS 297
20. Principles and Applications of a Tat-Based Assay BRYAN R. CULLEN 322
for Analyzing Specific RNA-Protein Interac-
tions in Mammalian Cells
xxxiii CONTENTS OF VOLUMES 321-354

S e c t i o n III. P h a g e D i s p l a y a n d I t s A p p l i c a t i o n s
21. Phage Display for Selection of Novel Binding SACHDEV S. SIDHU,
Peptides HENRY B. LOWMAN,
BRIAN C. CUNNINGHAM,
AND JAMES A. WELLS 333
22. Selectively Infective Phage Technology KATJA M. ARNDT,
SABINEJUNG,
CLAUS KREBBER,
AND ANDREASPLUCKTHUN 364
23. Use of Phage Display and Transition-State Ana- MIKAELWlDERSTEN,
logs to Select Enzyme Variants with Altered LARS O. HANSSON,
Catalytic Properties: Glutathione Transferase LISA TRONSTAD,
as an Example AND BENGT MANNERVIK 389
24. Selecting and Evolving Functional Proteins in JOZEF HANES,
V/tro by Ribosome Display LUTZ JERMUTUS,
AND ANDREASPLIQCKTHUN 404
25. Yeast Surface Display for Directed Evolution of ERIC T. BODER
Protein Expression, Affinity, and Stability AND K. DANE WITTRUP 430

S e c t i o n IV. C o n s t r u c t i o n o f H y b r i d M o l e c u l e s b y D N A S h u f f l i n g
and Other Methods
26. Methods for in Vitro DNA Recombination and ALEXANDERA. VOLKOV
Random Chimeragenesis AND FRANCESH. ARNOLD 447
27. Random Chimeragenesis by Heteroduplex Re- ALEXANDERA. VOLKOV,
combination ZHIXIN SHAO,
AND FRANCESH. ARNOLD 456
28. Use of Chimeras Generated by DNA Shuffling: LARS O. HANSSON
Probing Structure-Function Relationships AND BENGT MANNERVIK 463
among Glutathione Transferases
29. Protein Engineering by Expressed Protein Liga- ULRICH K. BLASCHKE,
tion JONATHAN SILBERSTEIN,
AND TOM W. MUIR 478

S e c t i o n V. A p p l i c a t i o n s of Fusions in Functional Genomics

30. A Systematic and General Proteolytic Method for JANNETTECAREY 499
Defining Structural and Functional Domains of
Proteins
31. High-Throughput Expression of Fusion Proteins MARCS. NASOFF,
MARK BERGSEID,
JAMES P. HOEFFLER,
AND JOHN A. HEYMAN 515
 CONTENTS OF VOLUMES 321-354 xxxiv

32. Rapid Construction of Recombinant DNA by the QINGHUALIU, MAMIEZ. LI,

Univector Plasmid-Fusion System DOU LIU, AND
STEPHENJ. ELLEDGE 530

33. High-Throughput Methods for the Large-Scale ANUJ KUMAR,

Analysis of Gene Function by Transposon SHELLEYANN DES ETAGES,
Tagging PAULOS. R. COELHO,
G. SHIRLEENROEDER,
ANDMICHAELSNYDER 550

34. GATEWAY Recombinational Cloning: Applica- ALBERTHAJ. M. WALHOUT,

tion to the Cloning of Large Numbers of Open GARY E TEMPLE,
Reading Frames or ORFeomes MICHAELA. BRASCH,
JAMES L. HARTLEY,
MONIQUEA. LORSON,
SANDERVAN DEN HEUVEL,
ANDMARCVIDAL 575

35. Gene Trapping Methods for the Identificationand WILLIAMC. SKARNES 592
Functional Analysis of Cell Surface Proteins in
Mice

VOLUME 329
REGULATORS AND EFFECTORS OF SMALL GTPASES (PART E: GTPASES
INVOLVED IN VESICULARTRAFFIC)
W. E. Balch, Channing J. Der, and Alan Hall

S e c t i o n I. R a b G T P a s e s
1. Structural Basis for Rab Function: An Overview BRYAND. MOYER
AND WILLIAME. BALCH

2. Expression of Wild-Type and Mutant Green BRYAND. MOVER,

Fluorescent Protein-Rabl for Fluorescence JEANNE MATrESON,
Microscopy Analysis AND WILLIAME. BALCH

3. FluorescenceMethods for MonitoringInteractions KIRILLALEXANDROV,

of Rab Proteins with Nucleotides, Rab Escort AXEL J. SCHEIDIG,
Protein, and G-eranytgeranyltransferase AND ROGERS. GOODY 14

4. Prenylation of Rab Proteins in Vitro by Geranyl- JEAN H. OVERMEYER

geranyltransferases ANDWILLIAMA. MALTESE 31

5. Antibody and Oligonucleotide Probes to Distin- ASSIA SHISHEVA 39

guish IntracellularExpression and Localization
Patterns of Rab GDP-Dissociation Inhibitor
Isoforms
xxxv CONTENTS OF VOLUMES 321-354

6. Expression, Purification, and Biochemical Prop- ELKE WILL, STEFANALBERT,

erties of Ypt/Rab GTPase-Activating Proteins AND DIETERGALLWITZ 50
of Gyp Family

7. Purification and Properties of Rab3 GDP/GTP HIROYUKINAKANISHI

Exchange Protein AND YOSHIMITAKAI 59

8. Purification and Properties of Rab3 GTPase- FUMIKONAGANO,

Activating Protein TAKUYASASAKI,
AND YOSHIMITAKAI 67

9. Rabphilin-3: A Target Molecule for Rab3 Small HIROMICHISHIRATAKI,

G Proteins TAKUYASASAKI,
AND YOSHIMITAKAI 75

10. Doc2u as Modulator of Ca2+-Dependent Exocy- SATOSHIORITA,

tosis TAKUYASASAKI,
AND YOSH1MITAKAI 83

11. Purification and Properties of Sec4p GTPase- LI-LIN DU

Activating Protein AND PETERNOVlCK 91

12. Purification and Characterization of Yeast Exo- DANIELR. TERBUSH,

cyst Complex WEI GUO,
STEVENDUNKELBARGER,
AND PETERNOVICK 100

13. Expression and Properties of Rab4 and Its Effec- PETER VAN DER SLUIJS,
tor Rabaptin-4 in Endocytic Recycling KARINMOHRMANN,
MAGDADENEKA,
AND MANDYJONGENEELEN 111

14. Purification of EEA1 from Bovine Brain Cytosol SAVVASCHRISTOFORIDIS

Using Rab5 Affinity Chromatography and Ac- AND MARINOZERIAL 120
tivity Assays
15. Expression, Purification, and Characterization of ROGER LIPPl~,
Rab5 Effector Complex, Rabaptin-5/Rabex-5 HISANORIHORIUCHI,
ANJA RUNGE,
AND MARINOZERIAL 132

16. Measurement of Rab5 Protein Kinase B/akt and MANUELA. BARBIERI,

Regulation of Ras-Activated Endocytosis A. GUMUSBOGA,
RICHARDL. ROBERTS,
AND PHILIPD. STAHL 145

17. Expression, Purification, and Biochemical Prop- ARNAUDECHARD,

erties of Rabkinesin-6 Domains and Their In- AHMEDEL MARJOU,
teractions with Rab6A AND BRUNOGOUD 157

18. Expression and Functional Analyses of Rab8 and WEI CHEN AND
Rab 11a in Exocytic Transport from trans-Golgi ANGELAWANDINGER-NESS 165
Network
 CONTENTS OF VOLUMES 321-354 xxxvi

19. Expression and Properties of Rab7 in Endosome YAN FENG,

Function BARRY PRESS, WEI CHEN,
JAY ZIMMERMAN,AND
ANGELAWANDINGER-NESS 175

20. Expression, Purification, and Properties of Rab8 JOHAN PERANEN

Function in Actin Cortical Skeleton Organiza- AND JOHANNA FURUHJELM 188
tion and Polarized Transport

21. Properties of Rabl3 Interaction with Rod cGMP ANNE-MARIEMARZESCO,

Phosphodiesterase 8 Subunit THIERRY GALLI,
DANIELLOUVARD,
AND AHMEDZAHRAOUI 197

22. Subcellular Localization of Rabl7 by Cryo- PETER J. PETERS

Immunogold Electron Microscopy in Epithe- AND WALTERHUNZIKER 210
lial Cells Grown on Polycarbonate Filters

23. Expression and Properties of Rab25 in Polarized JAMES R. GOLDENRING,

Madin-Darby Canine Kidney Cells LORRAINEM. ARON,
LYNNE A. LAPIERRE,
JENNIFER NAVARRE,
AND JAMES E. CASANOVA 225

24. Purification of TRAPP from Saccharomyces MICHAEL SACHER

cerevisiae and Identification of Its Mammalian AND SUSAN FERRO-NOVICK 234
Counterpart

S e c t i o n II. A D P - R i b o s y l a t i o n F a c t o r (ARF) G T P a s e s
25. Structural and Functional Organization of ADP- SOPHIEBF.RAUD-DUFOUR
Ribosylation Factor (ARF) Proteins AND WILLIAME. BALCH 245

26. Expression and Properties of ADP-Ribosylation JULIEG. DONALDSONAND

Factor (ARF6) in Endocytic Pathway HARISHRADHAKRISHNA 247

27. Expression and Analysis of ARNO and ARNO LORRAINEC. SANTY,

Mutants and Their Effects on ADP-Ribosyla- SCOTT R. FRANK,
tion Factor (ARF)-MediatedActin Cytoskeletal AND JAMES E. CASANOVA 256
Rearrangements

28. Expression, Purification, and Measurements of SOPHIE BI~RAUD-DUFOUR

Activity of ARNO1, a Guanine Nucleotide AND SYLVIANEROBINEAU 264
Exchange Factor for ADP-Ribosylation
Factor 1 (ARF1)

29. Expression, Purification, and Biochemical PHILIPPE CHAVRIER

Properties ofEFA6, a Sec7 Domain-Containing AND MICHEL FRANCO 272
Guanine Exchange Factor for ADP-Ribosyla-
tion Factor 6 (ARF6)
xxxvii CONTENTS OF VOLUMES 321-354

30. Isolation and Properties of GRP1, an ADP- JES K. KLARLUND

Ribosylation Factor (ARF)--Guanine Nucleo- AND MICHAELP. CZECH 279
tide Exchange Protein Regulated by Phospha-
tidylinositol 3,4,5-Trisphosphate

31. Functional Analysis of ADP-Ribosylation Factor ANNE PEYROCHE

(ARF) Guanine Nucleotide Exchange Factors AND CATHERINEL. JACKSON 290
Gealp and Gea2p in Yeast

32. Isolation, Cloning, and Characterization of GUSTAVO

Brefeldin A-Inhibited Guanine Nucleotide- PACHECO-RODRIGUEZ,
Exchange Protein for ADP-Ribosylation JOEL MOSS, AND
Factor MARTHA VAUGHAN 300

33. Expression, Purification, and Properties of ADP- IRIT HUBER,

Ribosylation Factor (ARF) GAP1 MIRIAM ROTMAN,
ELAn PICK,
VARDITMAKLER,
LILAH ROTHEM,
EDNA CUKIERMAN,
AND DAN CASSEL 307

34. Expression, Analysis, and Properties of Yeast PAK PHI POON,

ADP-Ribosylation Factor (ARF) GTPase Ac- DAN CASSEL,IRIT HUBER,
tivating Proteins (GAPs) Gcsl and Glo3 RICHARD A. SINGER,
AND GERALDC. JOHNSTON 317

35. Purification and Properties of ARD1, and ADP- NICOLAS VITALE,

Ribosylation Factor (ARF)-Related Protein JOEL MOSS,
with GTPase-Activating Domain AND MARTHAVAUGHAN 324

36. Purification and Characterization of GIT Family RICHARD T. PREMONT

of ADP-Ribosylation Factor (ARF) GTPase- AND NICOLASVITALE 335
Activating Proteins

37. Assay and Purification of Phosphoinositide- PAUL A. RANDAZZO,

Dependent ADP-Ribosylation Factor (ARF) KOICHI M1URA,
GTPase-Activating Proteins AND TREVOR R. JACKSON 343

38. Biological Properties and Measurement of Phos- SHAMSHADCOCKCROVr,

pholipase D Activation by ADP-Ribosylation KUN BI, NICHOLAS
Factor (ARF) T. KTISTAKIS,
AND MICHAELG. ROTH 355

39. Use of Arninoglycoside Antibiotics and Related ROCKFORD K. DRAPER,

Compounds to Study ADP-Ribosylation Factor ROBERT 'IDD HUDSON,
(ARF)/Coatomer Function in Golgi Traffic AND TONGHUANHU 372

40. Adaptor Protein 1-Dependent Clathrin Coat YUNXIANGZHU,

Assembly on Synthetic Liposomes and Golgi MATTHEWT. DRAKE,
Membranes AND STUARTKORNFELD 379
 CONTENTS OF VOLUMES 321-354 xxxviii

41. Receptor-Dependent Formation of COPI-Coated WALTERNICKEL

Vesicles from Chemically Defined Donor ANDFELIXT. WIELAND 388
Liposomes

42. ADP-Ribosylation Factor (ARF) as Regulator of MARIA ANTONIETTA

Spectrin Assembly at Golgi Complex DE MATTEIS
ANDJON 5. MORROW 405

43. Purification, Properties, and Analysis of yARL3 FANG-JENS. LEE

AND CHUN-FANGHUANG 417

44. Preparation and Assay of Recombinant ADP- GUSTAVO

Ribosylation Factor-Like Protein-1 (ARL1) PACHECO-RODRIGUEZ,
JOEL MOSS, AND
MARTHAVAUGHAN 424

S e c t i o n III. S a t G T P a s e s
45. Purification and Properties of Rat Liver Sec23- JACQUEST. WEISSMAN,
Sec24 Complex MEIR ARIDOR,
ANDWILLIAME. BALCH 431

46. Purification of Functional Sec13p-Sec31p Com- WILLIAMJ. BELDEN

plex, A Subunit of COPII Coat AND CHARLESBARLOWE 438

S e c t i o n IV. D y n a m i n G T P a s e s
47. Expression, Purification, and Functional Assays HANNADAMKE,
for Self-Association of Dynamin-l AMY B. MUHLBERG,
SANJA SEVER,
STEVENSHOLLY,
DALE E. WARNOCK,
AND SANDRAL. SCHMID 447

48. Analysis of Phosphoinositide Binding by Pleck- ANTHONYLEE

strin Homology Domain from Dynamin AND MARKA. LEMMON 457

49. Mapping Dynamin Interdomain Interactions ELENA SMIRNOVA,

with Yeast Two-Hybrid and Glutathione S- DIXIE-LEESHURLAND,
Transferase Pulldown Experiments AND ALEXANDER
M. VAN DER BLIEK 468

50. Interactions of Dynamin and Amphiphysin with KOHJITAKEI,

Liposomes VLADIMIRI. SLEPNEV,
ANDPIETRODE CAMILLI 478

51. Activation of Dynamin GTPase Activity by Phos- BARBARABARYLKO,

phoinositides and SH3 Domain-Containing DERK D. BINNS,
Proteins AND JOSEPHP. ALBANESI 486
 CONTENTS OF VOLUMES 321-354 xl

S e c t i o n II. S a c c h a r o l y t i c E n z y m e s
10. Classification of Glycoside Hydrolases and BERNARDHENRISSAT
Glycosyltransferases from Hyperthermophiles AND PEDROM. COUTINHO 183

11. /%Glycosidase from Sulfolobus solfataricus MARCO MORACCI,

MARIA CIARAMELLA,
AND MOSE ROSSI 201

12. Xylose Isomerases from Thermotoga CLAIREVIEILLE,

DINLAKASRIPRAPUNDH,
ROBERTM. KELLY,
ANDJ. GREGORYZEIKUS 215

13. /3-Mannanases from Thermotoga Species SWAPNILCHHABRA,

KIMBERLEYN. PARKER,
DAVIDLAM,
WALTERfALLEN,
MARJORY A. SNEAD,
ERIC J. MATHUR,
JAY M. SHORT,
AND ROBERTM. KELLY 224

14. /3-Mannosidase from Thermotoga Species KIMBERLEYN. PARKER,

SWAPNILCHHABRA,
DAVID LAM,
MARJORY A. SNEAD,
ERIC J. MATHUR,
ANDROBERTM. KELLY 238

15. Ot-D-Galactosidases from Thermotoga Species E. S. MILLER,J~

KIMBERLEYN. PARKER,
WOLFGANGLIEBL,
DAVID LAM,
WALTERfALLEN,
MARJORY A. SNEAD,
ERIC J. MATHUR,
JAY M. SHORT,AND
ROBERTM. KELLY 246

16. ct-Glucosidase fromPyrococcusfuriosus STEPHENT. CHANG,

KIMBERLEYN. PARKER,
MICHAELW. BAUER,
AND ROBERTM. KELLY 260

17. Amylolytic Enzymes from Hyperthermophiles COSTANZOBERTOLDOAND

GARABEDANTRANIKIAN 269

18. Cellulolytic Enzymes from Thermotoga Species WOLFGANGLIEBL 290

xli CONTENTS OF VOLUMES 321-354

19. Hyperthermophilic Xylanases PETER L. BERGQUIST,

MORELANDD. GIBBS,
DANIELD. MORRIS,
DION R. THOMPSON,
ANDREASM. UHL,
AND ROY M. DANIEL 301

20. Chitinase from Thermococcus kodakaraensis TADAYUKIIMANAKA,

KOD1 TOSHIAKIFUKUI,
AND SHINSUKEFUJIWARA 319

21. Characterization of/%Glycosylhydrolases from THUS KAPER,

Pyrococcusfuriosus CORNI~H. VERHEES,
JOYCE H. G. LEBBINK,
JOHAN E T. VAN LIESHOUT,
LEON D. KLUSKENS,
DON E. WARD,
SERVI~W. M. KENGEN,
MARKE M. BEERTHUYZEN,
WILLEM M. DE VOS,
AND JOHN VAN DER OOST 329

22. fl-Endoglucanase from Pyrococcusfuriosus SUSANG. CADY,

MICHAELW. BAUER,
WALTERCALLEN,
MARJORYA. SNEAD,
ERIC J. MATHUR,
JAY M. SHORT,
AND ROBERTM. KELLY 346

23. or-Amylases and Amylopullulanase from Pyro- ALEXEI SAVCHENKO,

coccusfuriosus CLAIRE VIEILLE,
AND J. GREGORYZEIKUS 354

24. ~-Glucosidase CelB from Pyrococcus furiosus: JOYCE H. G. LEBBINK,

Production by Escherichia coli, Purification, THUS KAPER,
and in Vitro Evolution SERVEW. M. KENGEN,
JOHN VAN DER OOST,
AND WILLEMM. DE VOS 364

S e c t i o n III. P r o t e o l y t i c E n z y m e s
25. Purification, Characterization, and Molecular WILLEM M. DE VOS,
Modeling of Pyrolysin and Other Extracellular WILFRIEDG. B. VOORHORST,
Thermostable Serine Proteases from Hyperther- MARCEL DIJKGRAAF,
mophilic Microorganisms LEON D. KLUSKENS,
JOHN VAN DER OOST,
AND ROLAND J. SIEZEN 383

26. Pyrrolidone Carboxylpeptidase from Thermo- M . R . SINGLETON

coccus litoralis AND J. m. LITTLECHILD 394
 CONTENTS OF VOLUMES 321-354 xlii

27. Protease I from Pyrococcusfuriosus LARA S. CHANG,

PAULAM. HICKS,
AND ROBERTM. KELLY 403

28. Archaeal Proteasomes ROBERTD. BARBER

AND JAMESG. FERRY 413

29. Thiol Protease from Thermococcus kodakaraen- MASAAKIMORIKAWA

sis KOD 1 AND TADAYUKIIMANAKA 424

30. Proline Dipeptidase from Pyrococcusfuriosus AMY M. GRUNDEN,

MOUSUMIGHOSH,
AND MICHAELW° W. ADAMS 433

31. Prolyl Oligopeptidase from Pyrococcusfuriosus VALERIEJ. HARWOOD

AND HAROLDJ. SCHREIER 445

32. Homomultimeric Protease and Putative Bacteri- PAULAM. HICKS,

ocin Homolog from Thermotoga maritima LARA S. CHANG,
AND ROBERTM. KELLY 455

33. Carboxylesterase from Sulfolobus solfataricus P1 A.C. SEHGAL,

WALTERCALLEN,
ERICJ. MATHUR,
JAY M. SHORT,
AND ROBERTM. KELLY 461

VOLUME 331
HYPERTHERMOPHILICENZYMES(PARTB)
Michael W. W. Adams and Robert M. Kelly

S e c t i o n I. E n z y m e s of C e n t r a l M e t a b o l i s m
1. Citrate Synthase from Hyperthermophilic Archaea MICHAELJ. DANSON
AND DAVIDW. HOUGH

2. Isocitrate Dehydrogenase, Malate Dehydro- IDA HELENESTEEN,

genase, and Glutamate Dehydrogenase from HILDE HVOSLEF,
Archaeoglobus fulgidus TORLEW LIEN,
AND NILS-KAREBIRKELAND 13

3. Glutamate Dehydrogenases from Hyperthermo- FRANK To ROBB,

philes DENNIS L. MAEDER,
JOCELYNEDIRuGGIERO,
KIM M. BORGES,
AND NICCOLATOLLIDAY 26
xliii CONTENTS OF VOLUMES 321-354

4. ADP-Dependent Glucokinase and Phosphofruc- SERVE W. M. KENGEN,

tokinase from Pyrococcusfuriosus JUDITHE. TUININGA,
CORNI~H. VERHEES,
JOHN VAN DER OOST,
ALFONS J. M. STAMS,
AND WILLEMM. DE VOS 41

5. Pyrophosphate-Dependent Phosphofructokinase BETTINA SIEBERS

from Thermoproteus tenax AND REINHARDHENSEL 54

6. Triose-Phosphate Isomerase from Pyrococcus ALEXANDERSCHRAMM,

woesei and Methanothermus fervidus MICHAEL KOHLHOFF,
AND REINHARDHENSEL 62

7. Phosphoglycerate Kinase-Triose-Phosphate Iso- JAE-SUNGYU

merase Complex from Thermotoga neapolitana AND KENNETHM. NOEL 78

8. Phosphoglycerate Kinases from Bacteria and GINA CROWHURST,

Archaea JANE MCHARG, AND
JENNIFERA. LITTLECHILD 90

9. Glyceraldehyde-3-phosphate Dehydrogenase JENNIFERA. LITTLECHILD

from Sulfolobus solfataricus AND MICHAILISUPOV 105

10. Nonphosphorylating Glyceraldehyde-3-phos- NINA A. BRUNNER

phate Dehydrogenase from Thermoproteus AND REINHARDHENSEL 117
tenax
11. Aldehyde Oxidoreductases from Pyrococcus ROOPALI ROY,
furiosus ANGELIL. MENON, AND
MICHAELW. W. ADAMS 132

12. 2-Keto Acid Oxidoreductases from Pyrococcus GERTI J. SCHUT,

furiosus and Thermococcus litoralis ANGELIt . MENON, AND
MICHAELW. W. ADAMS 144

13. Acetyl-CoA Synthetases I and II from Pyrococcus ANDREAM. HUTCHINS,

furiosus XUHONGMAI, AND
MICHAELW. W. ADAMS 158

14. Phosphate Acetyltransferase and Acetate Kinase PETER SCHONHEIT 168

from Thermotoga maritima

15. Alcohol Dehydrogenase from Sulfolobus CARLO A. RAIA,

solfataricus ANTONIETTAGIORDANO,
AND MOS~ ROSSI 176

16. Alcohol Dehydrogenases from Thermococcus KESEN MA AND

litoralis and Thermococcus Strain ES-1 MICHAELW. W. ADAMS 195

17. Alcohol Dehydrogenase from Thermococcus DONGHUILI AND

Strain ANI KENNETHJ. STEVENSON 201

18. Hydrogenases I and II from Pyrococcusfuriosus KESEN MA AND

MICHAELW. W. ADAMS 208
 CONTENTS OF VOLUMES 321-354 xliv

19. Fe-Only Hydrogenase from Thermotoga maritima MARC F. J. M. VERHAGEN

ANDMICHAELW. W. ADAMS 216
20. Ornithine Carbamoyltransferase from Pyrococ- CHRISTIANNELEGRAIN,
cus furiosus VINCENTVILLERET,
MARTINEROOVERS,
CATHERINETRICOT,
BERNARDCLANTIN,
JOZEF VAN BEEUMEN,
VICTORSTALON,
ANDNICOLASGLANSDORFF 227
21. Carbamoyl Phosphate Synthesis: Carbamate MATXALENURIARTE,
Kinase from Pyrococcusfuriosus ALBERTOMARINA,
SANTIAGORAMON=MAIQUES,
VICENTERUBIO,
VIRG1NIEDURBECQ,
CHRISTIANNELEGRAIN,
AND NICOLASGLANSDORFF 236
22. Aspartate Transcarbamoylase from Pyrococcus CRISTINAPURCAREA 248
abyssi
23. Phosphoribosylanthranilate Isomerase and REINHARDSTERNER,
Indoleglycerol-phosphate Synthase: Trypto- ASTRIDMERZ,
phan Biosynthetic Enzymes from Thermotoga RALFTHOMA,
maritima AND KASPERKIRSCHNER 270
24. Nicotinamide-mononucleotide Adenylyltrans- NADIARAFFAELLI,
ferase from Sulfolobus solfataricus TERESALORENZI,
MONICAEMANUELLI,
ADOLFOAMICI,
SILVERIORUGGIERI,
AND GIULIOMAGNI 281
25. Nicotinamide-mononucleotide Adenylyltrans- NADIA RAFFAELLI,
ferase from Methanococcus jannaschii FRANCESCAM. PISANI,
TERESALORENZI,
MONICAEMANUELLI,
ADOLFOAMICI,
SILVERIORUGGIERI,
AND GIULIOMAGNI 292
26. Alkaline Phosphatase from Thermotoga nea- ALEXEI SAVCHENKO,
politana WEI WANG,
CLAIREVIE1LLE,
ANDJ. GREGORYZEIKUS 298
27. Dihydrofolate Reductase from Thermotoga THOMASDAMS
maritima AND RAINERJAENICKE 305
28. Tetrahydromethanopterin-Specific Enzymes from SEIGO SHIMA
Methanopyrus kandleri AND RUDOLFK. THAUER 317
xlv CONTENTS OF VOLUMES 321-354

29. Ribulose-l,5-bisphosphate Carboxylase/Oxyge- HARUYUKIATOMI,

nasefromThermococcuskodakaraensisKOD1 SATOSHIEZAKI,
ANDTADAYUKIIMANAKA 353

S e c t i o n II. R e s p i r a t o r y E n z y m e s
30. Respiratory Enzymes from Sulfolobus acidocal- GONTERSCHAFER,
darius RALF MOLL,
ANDCHRISTIANL. SCHMIDT 369
31. Siroheme-Sulfite Reductase-Type Protein from CHRISTIANEDAHL,
Pyrobaculum islandicum MICHAELMOLITOR,
ANDHANSG. TROPER 410
32. Dissimilatory ATP Sulfurylase from Archaeo- DETLEFSPERLING,
globus fulgidus ULRIKEKAPPLER,
HANS G. TR/JPER,
AND CHRISTIANEDAHL 419
33. Sulfite Reductase and APS Reductase from CHRISTIANEDAHL
Archaeoglobus fulgidus AND HANS G. TRLIPER 427
34. Hydrogen-Sulfur Oxidoreductase Complex from MARTINKELLERAND
Pyrodictium abyssi REINHARDDIRMEIER 442

VOLUME 332
REGULATORS AND EFFECTORSOF SMALL GTPASES (PART F: RAS FAMILY I)
W. E. Balch, Channing J. Der, and Alan Hall

S e c t i o n I. Protein E x p r e s s i o n a n d P r o t e i n - P r o t e i n I n t e r a c t i o n s
1. Mammalian Expression Vectors for Ras Family JAMES J. FIORDALISI,
Proteins: Generation and Use of Expression RONALDL. JOHNSONII,
Constructs to Analyze Ras Family Function AYLINS. ~ILKO,
CHANNINGJ. DER,
ANDADRIENNED. COX 3
2. Protein Transduction: Delivery of Tat-GTPase ADAMINAVOCERO-AKBANI,
Fusion Proteins into Mammalian Cells MEENA A. CHELLAIAH,
KEITHA. HRUSKA,
AND STEVENF. DOWDY 36
3. Green Fluorescent Protein-Tagged Ras Proteins EDWIN CHOY AND
for Intracellular Localization MARK PHILIPS 50
4. Targeting Proteins to Membranes, Using Signal JOHN T. STICKNEY,
Sequences for Lipid Modifications MICHELLE A. BOODEN,
ANDJANICEE. BUSS 64
 CONTENTS OF VOLUMES 321-354 xlvi

5. Targeting Proteins to Specific Cellular Compart- GARABETG. TOBY

ments to Optimize Physiological Activity ANDERICAA. GOLEMIS 77
6. Mapping Protein-Protein Interactions with Alka- MONTAROPYAMABHAI
line Phosphatase Fusion Proteins AND BRIANK. KAY 88
7. Assays of Human Postprenylation Processing YUN-JUNGCHOI,
Enzymes MICHAELNIEDBALA,
MARK LYNCH,
MARC SYMONS,
GIDEONBOLLAG,
AND ANNE K. NORTH 103
8. In Vivo Prenylation Analysis of Ras and Rho PAULT. KIRSCHMEIER,
Proteins DAVIDWHYTE,
OSWALDWILSON,
W. ROBERTBISHOP,
ANDJIN-KEoN PAI 115
9. Ras Interaction with RalGDS Effector Targets SHINYAKOYAMA
AND AKIRAKIKUCHI 127
10. RAS Interaction with RIN1 Effector Target YINGWANGAND
JOHN COLICELLI 139
I 1. Ras and Rapl Interaction with AF-6 Effector BENJAMINBOETTNER,
Target CHRISTIANHERRMANN,
AND LINDAVAN AELST 151

Section II. Screening Analyses

12. Analysis of Protein Kinase Specificity by Peptide ZHOUSONGYANG 171
Libraries and Prediction of in Vivo Substrates
13. Peptide Library Screening for Determination ZHOUSONGYANG
of SH2 or Phosphotyrosine-Binding Domain ANDDAN LIU 183
Sequences
14. Expression Cloning of Farnesylated Proteins DOUGLASA. ANDRES 195

15. Expression Cloning to Identify Monomeric GTP- DOUGLASA. ANDRES 203

Binding Proteins by GTP Overlay
16. Retrovirus cDNA Expression Library Screening GWENDOLYNM. MAHON
for Oncogenes AND IAN P. WHITEHEAD 211
17. Identification of Ras-Regulated Genes by Repre- JANIELM. SHIELDS,
sentational Difference Analysis CHANNINGJ. DER,
AND SCOTTPOWERS 221
18. DifferentialDisplayAnalysisofGeneExpression HAKRYULJO,
Altered by ras Oncogene YONG-JIGCHO,
HONG ZHANG,
AND PENGLIANG 233
xlvii CONTENTS OF VOLUMES 321-354

19. cDNA Array Analyses of K-Ras-Induced Gene GASTONG. HABETS,

Transcription MARC KNEPPER,
JAINA SUMORTIN,
YUN-JUNGCHOI,
TAKEHIKOSASAZUKI,
SENJI SHIRASAWA,
ANDGIDEONBOLLAG 245

20. Ras Signaling Pathway for Analysis of Protein- AMI ARONHEIM 260
Protein Interactions

21. Isolation of Effector-Selective Ras Mutants by KIRANJ. KAUR

Yeast Two-Hybrid Screening ANDMICHAELA. WHITE 270

22. Two-Hybrid Dual Bait System to Discriminate ILYA G. SEREBRIISKII,

Specificity of Protein Interactions in Small OLGA V. MITINA,
GTPases JONATHANCHERNOFF,
ANDERICAA. GOLEMIS 277

23. Functional Proteomics Analysis of GTPase GORDONALTON,

Signaling Networks ADRIENNED. COX,
L. GERARDTOUSSAINTIll,
AND JOHN K. WESTWICK 300

S e c t i o n III. A n a l y s e s o f M i t o g e n - A c t i v a t e d P r o t e i n
Kinase Cascades
24. Analyzing JNK and p38 Mitogen-Activated ALAN J. WHITMARSH
Protein Kinase Activity AND ROGERJ. DAVIS 319

25. Phospho-Specific Mitogen-Activated Protein SAID A. GOUELI

Kinase Antibodies for ERK, JNK, and p38 ANDBRUCEW. JARVIS 337
Activation

26. Immunostaining for Activated Extracellular DANIELG. GIOELI,

Signal-Regulated Kinases in Cells and Tissues MAJA ZECEVIC,
ANDMICHAELJ. WEBER 343

27. Dominant Negative Mutants of Mitogen- M. JANEARBOLEDA,

Activated Protein Kinase Pathway DEREK EBERWEIN,
BARBARAHIBNER,
ANDJOHN E LYONS 353

28. Scaffold Protein Regulation of Mitogen-Activated ANDREWD. CATLING,

Protein Kinase Cascade SCOT]?T. EBLEN,
HANS J. SCHAEFFER,
ANDMICHAELJ. WEBER 368

29. Bacterial Expression of Activated Mitogen- JULIEL. WILSBACHER

Activated Protein Kinases AND MELANIEH. COBB 387
 CONTENTS OF VOLUMES 321-354 xlviii

30. Steroid Receptor Fusion Proteins for Conditional MARTINMCMAHON 401

Activation of Raf-MEK-ERK Signaling
Pathway
31. Pharmacologic Inhibitors of MKK1 and MKK2 NATALIEG. AHN,
THERESASTINESNAHREINI,
NICHOLASS. TOLWlNSKI,
AND KATHERYNA. RESING 417
32. Analysis of Pharmacologic Inhibitors of Jun BRIONW. MURRAY,
N-Terminal Kinases BRYDONL. BENNETT,
AND DENNIST. SASAKI 432

VOLUME 333
REGULATORSAND EFFECTORSOF SMALLGTPASES (PARTG: RAS FAMILYII)
W. E. Balch, Channing J. Der, and Alan Hall

Section I. Cytoplasmic and Nuclear Signaling Analyses

1. Determining Involvement of Shc Proteins in JOHN E O'BRYAN 3
Signaling Pathways
2. Assaying Activity of Individual Protein Kinases SAID A. GOUELI,KEVINHSIAO,
in Crude Tissue or Cellular Extracts AND BASEMS. GOUELI 16
3. Recombinant Adenoviral Expression of PAUL DENT,
Dominant-Negative Ras N17 Blocking CRAIGLOGSDON,
Radiation-Induced Activation of Mitogen- BARBARANICKE,
Activated Protein Kinase Pathway KRISTOFFERVALERIE,
JULIE FARNSWORTH,
RUPERTSCHMIDT-ULLRICH,
ANDDEANB. REARDON 28
4. Ras Activation of Phosphatidylinositol 3-Kinase PABLORODRIGUEZ-VICIANA
and Akt AND JULIANDOWNWARD 37
5. Assays for Monitoring p70 $6 Kinase and RSK JEFFREYMASUDA-ROBENS,
Activation VERA P. KRYMSKAYA,
HONGWEIQI,
AND MARGARETM. CHOU 45
6. Ras Activation of PAK Protein Kinases ALBERTCHEN,
YI TANG,YA ZHUO,
QI WANG,ALBERTPAHK,
AND JEFFREYFIELD 55
7. Ras Signaling to Transcription Activation: GABRIELEFOOS,
Analysis with GAL4 Fusion Proteins CHRISTINAK. GALANG,
CHAO-FENGZHENG,
AND CRAIGA. I'-IAUSER 61
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Title: The haunted island

A pirate romance

Author: E. H. Visiak

Illustrator: N. W. Physick

Release date: June 26, 2024 [eBook #73917]

Language: English

Original publication: London: Elkin Mathews, 1910

Credits: Susan E., David E. Brown, and the Online Distributed

Proofreading Team at https://www.pgdp.net (This book
was produced from images made available by the
HathiTrust Digital Library.)

*** START OF THE PROJECT GUTENBERG EBOOK THE HAUNTED

ISLAND ***
The Haunted Island
 THE HAUNTED
ISLAND

A PIRATE ROMANCE

By
E. H. VISIAK

LONDON
ELKIN MATHEWS, VIGO STREET
MCMX
 THE HAUNTED ISLAND
A PIRATE ROMANCE
Being the History of an Adventure to an Island in the
Remote South Sea. Of a Wizard there. Of his Pirate Gang;
His Treasure; His Combustible; His Skeleton Antic Lad. Of
his Wisdom; Of his Poesy; His Barbarous Cruelty; His
Mighty Power. Of a Volcan on the Island. And of the
Ghostly Terror.
 TO
MY MOTHER
 CONTENTS
chap. page
I. Scampering Away 11
II. Francis Dreams a Dream 19
III. The Castaways 22
IV. Villainy 27
V. the Englishman’s Relation. (The Island) 32
VI. The Englishman’s Relation Continued. (The Cell) 38
VII. A Horrible Villain 47
VIII. Thalass 51
IX. Ouvery Delivers up the Chart 56
X. We Fall in With the Fleet of Captain Morgan. The
Buccaneer’s Hut 59
XI. The Mad Maroon 75
XII. The Warning Sea 85
XIII. The Ghost 91
XIV. The Cut Cable 100
XV. The Island of the Holy 104
XVI. Spies of Canaan 106
XVII. Doctor Copicus 114
XVIII. The Running Man 120
XIX. The Cloisters. The Doctor and the Volcan 127
XX. The Phantom Voice 133
XXI. The Manuscripts 138
XXII. The Glorious Pirate 142
XXIII. Hey-diddle-diddle! 145
XXIV. A Curse Falls Upon the Wolves 148
 XXV. A Gaudy Picture in a Dark Frame 153
XXVI. The Treasure Chamber 162
XXVII. Francis Finds his Brother’s Watch 168
XXVIII. The Ghost Face 172
XXIX. The Float. The Walking Lad 176
XXX. How Now? 180
XXXI. Neither One nor the Other 184
XXXII. Hell Shore 188

Supplement 192
THE HAUNTED ISLAND
 CHAPTER I.
SCAMPERING AWAY.
On Christmas Eve, 1668, I, Francis Clayton, was with my brother
Dick at Clayton Manor, our father’s house. ’Tis seated on the bottom
of a Bay near Wembury, on the sea coast of Devon. My brother
served in quality of lieutenant on board His Majesty’s ship Tiger, and
was but late returned from a cruise off the Island of Jamaica. His
ship lay at anchor in the bay.
We sat over against one another on the hearth, in my little snug
room. The Squire and the servants were a-bed. The great old house
slept, with starting and creaking of the timbers. The fire was sunk
together, burning with a clear glow; the candles were wasted to the
snuff in their sockets.
My brother told a marvellous tale of the South Sea, of an island
there haunted by spirits, particularly by a monstrous great ghoul, or
devil.
“Many rumours,” said he, “are gotten abroad of this enchanted
island. ’Tis said that he who laid up the treasure had a truck with the
devil, and that a frightful fiend hath guard over it. I do certainly
know ’tis there. As to the rest, I am nothing concerned with such
fantastic gear. Once ashore there,” said he, “and not all the devils in
hell shall keep me from the treasure! Yet, if seamen who have seen
the Thing be not extremely out, ’tis a pretty wight! The head of it,
say they, reaches unto the clouds, and the appearance of it is
frightful out of all description.”
There came a sudden tap-tapping in the wainscoting. It was, no
doubt, but a rat; but, I must confess, I shrank with fear of it:
whereupon my brother clapped me boisterously on the shoulder,
crying:
“Hallo, my land-captain! Did you think it was the ghosts from the
Haunted Island? I’ll show you something to cheer you up.”
Hereupon, clapping hand to pocket, he plucked forth a little box, or
casket. It was of gold, very cunningly wrought with the
representation of a galleon; and her hull was of rubies, her sails of
pearls, her flag and flowing pendants of emeralds and sapphires,
and the sea of amethyst.
It was exquisite, and I said so; but my brother bade me read what
was written on a piece of parchment that he took from the box and
laid open before me.
“I know you like poesy,” said he, “read it. ’Tis better than a sonnet to
my lady’s shoe-latchet!”
The writing was old and small, and thus it was:

Go you not nigh the island, Captain;

Go you not close abroad:
For death would ride on every side,
And hell on your steerboard.

As I was sailing the sea, Captain,

Beyond the Mexique bay,
A mickle blast the good ship cast
A monstrous long south way.

Blind ran the ship as a blind antler;

And ever her beside,
There rode a drear and hooded fear,
Till we the isle espied.

We anchored off the island, Captain;

We lay off cables four:
I swear by bell, ’twas mickle well
We went not nearer shore.
 It came in the midnight hour, Captain:
As God’s eye seeth me,
I never ween’d that such a fiend
In earth or hell might be.

Praise to the Mother of God, Captain,

Be praise and gramercy:
An image stood of the blest rood
Nailed to our mainmast tree.

Here the ballad was broken off. I asked what it was; but had never
answer. For, on a sudden, a summons loud and long sounded upon
the Manor door.
“Hallo! What’s this?” cried I. But my brother said nothing; only got
from his chair, and stepped to the casement; as I did also. He drew
the shutter; and we peered without upon the avenue. ’Twas a wintry
and a weird prospect: the moon shone bleak through the scantly
falling snow, which was flurried about in drifts of powder on the
frozen ground. The tall elms stood gauntly up, the smaller branches
of them stirring in sudden movements, like hands and fingers. But at
the great door there was a little fat man in the uniform of the
captain of a King’s ship. He stood panting, his face fiery red; and
kept pummelling with his cane upon the flagstones.
“Why, ’tis the captain of your ship!” said I. “’Tis Captain Skinton!”
But, muttering in his teeth, Dick turned and hurried from the room. I
followed, to find him at the end of the corridor warily withdrawing
the arras. We looked through.
The Captain addressed the serving-man who had opened to him,
and was beside himself with passion. He spoke in rapid and high-
pitched tones, and with robustious gesticulation. We caught a tail-
end: “—that I see Squire Clayton this night!”
“But he’s a-bed, your honour,” says Roger.
“Then wake him! wake him! wake him!” cried the little man; and,
stamping by misadventure a gouty foot, he broke into a paroxysm of
rage, just as the Squire, our father, came forth on the gallery of the
stairs.
“What’s this? What’s this?” says he, looking down in his nightcap.
“What’s this, sir?” cries Skinton, his voice rising shrill, “what’s this,
sir? Why, your son, sir ... a conspiracy, sir, a conspiracy aboard my
ship, my ship, sir!”
“Roger,” says the Squire, “how came you to admit this neighing
rascal? Show him into the stable!”
Hereupon the Captain became perfectly furious; but the Squire
returned him word for word, as hot as he gave, so that there fell a
very great clamour. And, in the hurly-burly, Dick stole past them into
the hall, I following; and so (the door having been left open), out
into the night.
We scoured down the avenue, crossed the road beyond, and,
striking into the countryside, cut crisply across the frozen fields to
the cliff-top; then down, helter-skelter, to the shore.
A great ship lay at an anchor close in, with her sails hauled up in
brails. It was the Tiger. Her captain’s boat lay beached above the
breakers; and her crew walked up and down, to keep them warm.
My brother stepped to the coxswain, “’Tis I, John,” said he, as he
got his breath; and, to the rest, he said: “Back to the ship, men! Our
plans are at a head! All’s ready! I’ll make all your fortunes, my jolly
boys!”
“Ay, ay, sir! ay, ay!” But he bade them hush, and hasten with the
boat. Thereupon, turning to me, “Get you back to the Manor!” says
he.
“No; I go with you,” said I.
He looked me up and down: “Tut, tut, d’ye know where we’re
bound? For the South Sea, and on none of the King’s business
either!”
“You’re going to steal the ship!” said I; but they were shoving off the
boat, and, before ever he could answer, or I take thought, we found
ourselves aboard and rolling over the waves. Soon there fell a great
barking of dogs from the direction of the Manor; whereupon my
brother did urge the rowers to greater effort. A few moments after
we reached the ship.
We went aboard at the gang-way. There stood a big, black-bearded
fellow, who passed on board in the style of the Quartermaster. My
brother spoke a word in his ear, and the man nodded.
“All have joined, Ouvery?” asked Dick.
“Ay, but the master and Surgeon Burke be aboard. They went not
ashore with the rest.”
“’Sdeath!” said Dick with his teeth, “where are they, then?”
“The master’s in his cabin: Burke’s in the round-house.”
“Overboard with the master! Let him swim, the preaching swab! But
we’ll have need of Burke. Have up the men; I’ll speak to ’em. Haste,
Ouvery!”
He made to the quarter-deck as he spoke, while the other went
below. Soon was heard the boatswain’s whistle, and the mariners
began to come up on deck. Meantime there came a splashing under
the stern, and the master swam presently forth within sight. Having
gotten his breath, he began to shout threats and imprecations at my
brother. But Dick laughed, and shook his finger at him, crying:
“Nay, nay, sweet sir, be not so prodigal with your strength! I warrant
you’ll need all of it anon!”
On this, with a last imprecation, the swimmer turned, and
incontinently made towards the shore. But the mariners were now
gotten all up on deck, and my brother prepared to speak to them.
But no speech was made by him. For a musket-shot rang out on the
shore, and some half-dozen men, led by the Captain and the Squire,
came forth on the cliff—the Captain shouting and gesticulating with
the smoking musket that he held; but our father stood like a man
sore stricken, so that it went to my heart to see him. I turned to my
brother:
“Go you no further with this,” said I. “Return while yet you may.”
But he did not answer me; he stood staring to windward, and I saw
his face set hard. A big ship, flying the flag of St. George, had hove
in sight round a bend of the cliff.
Next moment he turned; and, bawling out, “In with the boat!” cried
he, “All hands make sail! Up anchor and away!”
There fell a great ado on this, as the mariners went to work to the
shrill piping whistle of the boatswain, and the word of command.
Soon the cable was hove in, while the seamen sang as they strained
at the capstan. Soon the ship began to move, stretching away under
her spritsail and topsails to the shore-breeze.
But the other vessel had hoisted up her topgallant sails, and came
on apace; and now, being but a cable’s length away, she shot off a
gun.
“Come on, ye dogs!” cried Dick. “Fire away, and split!” And he hasted
to the poop, where the gunners worked, charging of the stern-
chasers.
“Double shot ’em, and cripple her!” said he.
“Ay, ay, sir! ay, ay!” But now the pursuer fired again, and a round-
shot crashed into our round-house coach.
“Fire!” and, upon the word, the guns belched forth flame. The report
shook the ship, and a cloud of cannon-smoke rolled up like a scroll.
As it cleared, our men roared out with a great cheering; for we had
hit the King’s ship full in her fore rigging, split her spritsail, smashed
her foreyard, and handsomely mauled the shrouds. The foremast
shook with the brunt, and the topsail came clattering down.
Hereupon my brother fell to capering about like a silly schoolboy.
“Well done, master-gunner!” cried he, “well done, my brave! Give
him another cheer, my jolly boys! They’ll not take us, this bout!”
 CHAPTER II.
FRANCIS DREAMS A DREAM.

We now rapidly left the floundering and mazed man-of-war. But our
men kept firing into the medley on her decks, plying the stern-
chasers in furious haste; and, ever as he was able, the enemy did
shoot at us with muskets and pistols, and once—his ship having
slewed right round—gave us a thundering great broadside. But his
gunners did much mistake, in that they hulled us (as the saying is),
instead of aiming at our masts and tackling, whereby they might
have crippled us, and, perhaps, brought us quite to a stand.
As it was, our ship took no great hurt, though the splinters flew, and
six of her ports were battered in; yet many of our men were
wounded after a dreadful manner, and one was killed outright.
Whereupon such brutish and demoniac fury came upon the rest as
transported them like very devils.
Our decks were bloody like a slaughter-house; and from the spar-
deck the wounded men roared out very grievously as Surgeon Burke
wrought upon them, so that their groans and shrieks did mingle with
the horrid blasphemies.
Now, all this time my brother, the Captain, did frantically rage as hot
as any man; yea, so far beside himself was he, that, when he found
we drew out of gunshot and could no longer murder the King’s men,
he was for going about and returning upon them. He had even given
the order to bear up the helm; but hereupon, to my huge surprise,
the Quartermaster did interpose. ’Twas no more than a look and a
word, but it availed, and the order was immediately revoked.
We now lay right out of the bay; and, having set studding sails,
stood off to sea, whilst the decks were cleared, the broken ports
stopped, and the bolts and tackling overhauled. But I, being sick at
heart, went and shut myself in the master’s cabin, which I found
empty; and, a great weariness coming over me, I got me into a
hammock, and presently slept.
I dreamed a fearful dream; and it seemed to me that one went
before me through a gully in a terrible high cliff. And the gully
became dark and darker as we went, and its sheer sides of dank
rock towered higher yet; so that they were frightful to look on. Still
they rose and rose, until their tops might no longer be discerned;
and darkness fell, a darkness that seemed to wither my soul! Then
he who went before me did turn himself about, and lo! ’twas Ouvery,
the Quartermaster. His face gleamed ghastly white, but his eyes
were blacker than the darkness. They seized on mine, and held
them—for ever!
I woke gasping and shaking; and there were two eyes as black as
jet fixed on mine! Ouvery, the Quartermaster, gazed across at me
from the middle of the cabin, where he sat in close converse with
my brother.
He got up from his chair; and, making a sign that I was awake,
immediately went out. Hereupon my brother rose also, and stepped
to my hammock.
“Well, my heart,” said he, “hast been a-voyaging to Tophet, or is’t
the ghosts from the Haunted Island? Bless us! you screeched like a
stuck pig.”
“Scoff away! Scoff away!” cried I. “’Twill be another tale to-morrow!”
“Ay!” said he, but I thought his countenance fallen, “pieces-of-eight,
and golden bars, and jewels—jewels by the shipload—that’ll be the
tale to-morrow!”
“And a hempen cord about your neck, and iron chains to hang in,
and yokel faces a-gape at you!”
On that he flamed out in a fury, snapping his fingers, swearing and
cursing. But I let him rave, answering nothing, and soon he fell
quiet, and throwing himself into a chair, sat still, seeming to brood in
his mind.
 CHAPTER III.
THE CASTAWAYS.

After these events, there fell out nothing worth remark in many
days.
We had favourable winds and calm seas, and met with no King’s
ships. What other craft we met withal, meddled not with us, nor we
with them. Through great part of the Bay of Biscay we steered our
course close under the coast of France, until we came to an anchor
in a small bay thereby, to take in water and fresh provisions; thence
we sailed away, standing to the open sea. We passed the Cape of
Finisterre; and so onwards towards the Tropic of Cancer and the
Line.
I come to a day in the fifth week of our voyage. I got up betimes,
and went on deck. The ship went merrily along. There was not a sail
in sight, nor any glimpse of land; but from horizon to horizon spread
the dancing, shining sea, and I thought it was a gracious scene, like
a world from the hands of God.
As I looked, I spied a dark blur of a thing far out upon the waters. It
disappeared, but soon hove in sight again, this time quite clear. In
the same moment, the look-out man bawled:
“Boat adrift on the larboard bow.”
“Lay to, then, and man the jolly-boat,” cried the Captain.
The mariners went eagerly to work, snapping greedily at the exciting
chance. The jolly-boat, which was towing astern, was speedily
hauled in and manned; but the coxswain was shoved from his
wonted command of her by Ouvery, who put himself into the stern-
sheets. I, being full of curiosity, jumped into the boat as they made
ready to pull away; whereupon Ouvery did look upon me balefully,
though he spoke no word, and I knew that he hated me from the
bottom of his black heart!
“Pull away,” said the Quartermaster; and she began to move apace.
I, keeping my eyes fixed upon the boat we steered for, at length
descried a huddled heap on board of her, which was presently
discovered to be the forms of two men. We drew alongside, and lay
board on board together, gazing upon a dismal, dreadful sight.
For there in the boat were two gaunt and wasted forms—yea, the
very death’s heads did grin in the dwindled faces of those poor
castaways.
One was an Indian: a tall, well-knit figure dark copper-coloured; his
face long and hard-favoured; lank black hair.
The other was an European, and, as it should seem, an Englishman.
His yellow beard fell long and untrimmed, and his clothes were
mean and old; yet there was that in his look made me think he was
a gentleman. What, however, was remarkable: on sight of the
castaways, Ouvery gave a great start and drew sharply in his breath.
“They be dead men both,” said a man, solemnly; “rest to their
souls!”
But I had got into the boat; and, taking a mirror that I had about
me, I held it in turn to those parted and writhen lips, and lo! it twice
became clouded with breath.
“They live! they live!” cried I. But Ouvery said quickly:
“Nay, nay! You know nothing at all. Down, ye meddler, and out of
the way!” And to the mariners he said:
“Overboard with them! We’ll have no truck with the dead! And keep
your tongue quiet,” said he, glaring at me, “or you shall sweep with
them!”
“I say that they live!” I returned passionately. “Carry them to the
ship!”
On that, Ouvery raised his clenched fist to strike me down. “You
barking cur,” cried he, “do you meddle with my orders? I’ll have you
clapt in irons! I’ll flay you to the bone! I’ll——”
“Belike you’re captain of the ship,” said I, flouting him; “I thought
my brother was captain.”
Ouvery sat still, all swollen with rage; but hereupon up spoke a
seaman, saying stoutly:
“Maybe the boy’s in the right. Ho! make way here!” He climbed over
into the cock-boat as he spoke, and soon had confirmed my words.
But Ouvery rose up like one possessed; and, drawing his knife, he
reached forth to have stabbed me. But the boat began to rock nigh
foundering; and, losing his footing, he fell, knocking his head against
an oar that was outboard, and slid over into the sea. They fished
him out ere he sank, and flung him like a log in the bottom of the
boat, where he lay stunned.
Hereupon, having made the cock-boat fast to our stern, we returned
to the ship. My brother stood at the gang-way, awaiting us; I
acquainted him with what had befallen, demanding that Ouvery
should be restrained. But he regarded me not at all.
They contrived a bed for the Indian in the forecastle, but the other
was installed in the master’s cabin beneath the poop, where I slept,
some bedding being laid for me on the floor. Tended by Surgeon
Burke, and cared for by the mariners, the Indian made a quick
recovery; but the other lay as one dead, being fallen into a sort of
coma or trance.
The Indian came out of his swoon about sundown. Dick, Burke, and
I stood beside his bed, when the poor man, on a sudden, stirred and
opened his eyes. He looked from one to another of us wildly; and
moved his lips as if he would speak. At this juncture, heavy and
uncertain footfalls were heard without, and Ouvery came lumbering
in, his face mottled red and purple. He approached; but, on sight of
him, the Indian stared aghast, stretched forth an arm with a fierce,
frenzied gesture, uttered a cry, and sank back beside his senses.
“Why, he knoweth him!” cried I. “He hath seen you before, Ouvery!
If he could speak, I warrant you’d stand proclaimed a black villain!”
I was out of myself with passion, and I knew not what I said. But
the Quartermaster was transported with fury, and, rasping out horrid
oaths, he drew a pistol from his belt and let fly at me. The ball
missed my head by a hair’s breadth, and was buried in the wooden
wall. Ouvery made a dash towards me, reeled, and lurched forward
upon the floor. There he lay stunned, having knocked his head
against the table-leg.
The episode had passed in a moment, and Dick and the surgeon
stood like men dumbfounded. Burke recovered himself:
“You’ve had a narrow squeak, my lad,” said he. “The man’s a maniac
—look you, Captain!”
But Dick dashed his fist against the wall for mad.
 CHAPTER IV.
VILLAINY.

I went on deck, to behold a night of calm and beauty. There was no

moon; but the hosts of the stars burned in a clear element, upon a
solemn ground, and I thought they were like gems in the mantle of
God.
But, as I stood, leaning on the bulwarks, dreaming in the blue
starlight, on a sudden a hand was laid on my shoulder, and a hearty
voice cried:
“Wake up, young star-gazer! What are you dreaming of now?
Wouldst know:
What worlds or what vast regions hold
The immortal mind that hath forsook
Her mansion in this fleshy nook?”
“What, old physicker!” (for ’twas Surgeon Burke), “‘Throw physic to
the dogs, I’ll none of it!’”
He laughed boisterously at the return; then bade me follow him
below, saying that the English castaway was coming out of his
swoon. We went quickly to the master’s cabin, where the
Englishman lay stretched upon a day-bed or settee. But, instead of
the stark and deadly look he had before, his breast heaved to a
gentle respiration, his eyes were closed, and there was even a trace
of colour in his hollow cheeks.
“When he wakes,” said Burke, “he’ll be recovered, and that will be
no great while either.”
But in this point he was out; for the exhausted man continued in his
babe-like sleep. At last, being perfectly tired out, I cast myself down
upon the bedding on the floor, and took up my repose. Yet I could
not presently sleep; for my thoughts were tumultuous and uneasy to
the last degree, running continually upon those strange happenings
—more especially upon the desire manifested by Ouvery to make
away with the castaways and strange recognition of him by the
Mosquito Indian. However, at last I slept.
It must have been upon the stroke of midnight that I woke up
suddenly from an unquiet sleep, to hear the sound of stealthy
footsteps in the gang-way without.
I listened, my heart beginning to thump upon my sides, my eyes
upon the door. The handle turned slowly; the door was softly
opened. Then, half-closing my eyes, I feigned slumber, as, vivid in
moonshine cast through the port, the face of Ouvery appeared in the
chink.
The Quartermaster stood peering and listening. He entered, and
advanced on me; and there was a knife in his hand. Yet I continued
to feign slumber, and, though near dead with terror, to make my
breathing appear regular and slow.
At last he was at my side. I opened my eyes then, ready to evade a
deadly thrust, and lo! the man was passed by me and moving
towards the Englishman on the bed. On that my nerve came back to
me; the spell was broken.
I made one leap of it half across the cabin floor; and, as the
Quartermaster, his face set in savage lines, his arm drawn back to
thrust, bent over the slumbering Englishman, quick as light I
snatched the pistol from his belt and set it close and firm to his ear.
“Drop your blade,” said I, “or I’ll fire.”
Ouvery was no craven, but, on touch of the iron muzzle, he started
and dropped his knife. He held perfectly still, muttering something
that I had no care to hear; and I bawled loud and repeatedly, “Help!
Help!”
It was then that the Englishman came out of his swoon; and, rising
up in his bed, “Where am I?” cried he. “What is this?”
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