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Chromatin Remodeling Methods and Protocols 1st
Edition Junbiao Dai Digital Instant Download
Author(s): Junbiao Dai, Jef D. Boeke (auth.), Randall H. Morse (eds.)
ISBN(s): 9781617794773, 1617794775
Edition: 1
File Details: PDF, 8.17 MB
Year: 2012
Language: english
METHODS IN MOLECULAR BIOLOGY™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Chromatin Remodeling

Methods and Protocols

Edited by

Randall H. Morse
Wadsworth Center, New York State Department of Health, Albany, NY, USA
Editor
Randall H. Morse
Wadsworth Center
New York State Department of Health
Albany, NY, USA
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-476-6 e-ISBN 978-1-61779-477-3
DOI 10.1007/978-1-61779-477-3
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011943306

© Springer Science+Business Media, LLC 2012

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA),
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Preface

No modern molecular biologist needs convincing of the central importance of chromatin

to gene regulation in eukaryotes. From large-scale domains to individual gene promoters,
gene expression is regulated by histone modifications and histone variants, nucleosome
positioning, and nucleosome stability. A panoply of approaches has evolved in the labora-
tory over the past three decades to study chromatin structure and its alterations, and methods
of investigating chromatin remodeling – changes in nucleosome structure or position with
respect to the incorporated DNA, or in histone modifications – have progressed rapidly
over the past 10 years. Here are presented a wide array of protocols for studying chromatin
remodeling. We include methods for investigating chromatin remodeling in vitro and
in vivo, in yeast, plants, and mammalian cells, and at local and global levels. Both gene-
specific and genome-wide approaches are covered, and in recognition of the increasing
prevalence of the latter type of study, the final two chapters focus on bioinformatic/
computational approaches to analyzing genome-wide data on chromatin structure.
We hope that this volume will be used by readers in more than way. The obvious utility
is as a direct guide to a multitude of techniques, served up as recipes with introductions and
special “Notes” sections. These latter sections, which provide extra tips and background for
the user, have been an especially appreciated feature of these volumes over the years. We
also hope the chapters may be read comparatively and have allowed some overlap among
approaches to this end, to permit the reader to benefit from delving into differences as to
how different authors approach similar problems. Finally, simply perusing the titles may
refresh or illuminate readers as to the variety of methods available to study chromatin
remodeling and perhaps invigorate their next grant application or project.
This volume has benefited from a group of authors with tremendous collective experi-
ence and authority, who have generously given time and effort to provide these detailed
explications of protocols. I am grateful for their terrific cooperation in this project.

Albany, NY, USA Randall H. Morse

v
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Strain Construction and Screening Methods for a Yeast Histone H3/H4

Mutant Library. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Junbiao Dai and Jef D. Boeke
2 Measuring Dynamic Changes in Histone Modifications and Nucleosome
Density during Activated Transcription in Budding Yeast . . . . . . . . . . . . . . . . . . . . 15
Chhabi K. Govind, Daniel Ginsburg, and Alan G. Hinnebusch
3 Monitoring the Effects of Chromatin Remodelers on Long-Range
Interactions In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Christine M. Kiefer and Ann Dean
4 Measuring Nucleosome Occupancy In Vivo by Micrococcal Nuclease . . . . . . . . . . . 47
Gene O. Bryant
5 Analysis of Nucleosome Positioning Using a Nucleosome-Scanning Assay. . . . . . . . 63
Juan Jose Infante, G. Lynn Law, and Elton T. Young
6 Assaying Chromatin Structure and Remodeling by Restriction Enzyme
Accessibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Kevin W. Trotter and Trevor K. Archer
7 Generation of DNA Circles in Yeast by Inducible Site-Specific
Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Marc R. Gartenberg
8 An Efficient Purification System for Native Minichromosome
from Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Ashwin Unnikrishnan, Bungo Akiyoshi, Sue Biggins, and Toshio Tsukiyama
9 Simultaneous Single-Molecule Detection of Endogenous C-5 DNA
Methylation and Chromatin Accessibility Using MAPit . . . . . . . . . . . . . . . . . . . . . . 125
Russell P. Darst, Carolina E. Pardo, Santhi Pondugula,
Vamsi K. Gangaraju, Nancy H. Nabilsi, Blaine Bartholomew,
and Michael P. Kladde
10 Analysis of Stable and Transient Protein–Protein Interactions . . . . . . . . . . . . . . . . . 143
Stephanie Byrum, Sherri K. Smart, Signe Larson, and Alan J. Tackett
11 Monitoring Dynamic Binding of Chromatin Proteins In Vivo
by Fluorescence Recovery After Photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Florian Mueller, Tatiana S. Karpova, Davide Mazza,
and James G. McNally
12 Monitoring Dynamic Binding of Chromatin Proteins In Vivo by Fluorescence
Correlation Spectroscopy and Temporal Image Correlation Spectroscopy . . . . . . . . 177
Davide Mazza, Timothy J. Stasevich, Tatiana S. Karpova,
and James G. McNally

vii
viii Contents

13 Analysis of Chromatin Structure in Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Mala Singh, Amol Ranjan, Krishan Mohan Rai, Sunil Kumar Singh,
Verandra Kumar, Ila Trivedi, Niraj Lodhi, and Samir V. Sawant
14 Analysis of Histones and Histone Variants in Plants. . . . . . . . . . . . . . . . . . . . . . . . . 225
Ila Trivedi, Krishan Mohan Rai, Sunil Kumar Singh,
Verandra Kumar, Mala Singh, Amol Ranjan, Niraj Lodhi,
and Samir V. Sawant
15 Reconstitution of Modified Chromatin Templates for In Vitro
Functional Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Miyong Yun, Chun Ruan, Jae-Wan Huh, and Bing Li
16 A Defined In Vitro System to Study ATP-Dependent Remodeling
of Short Chromatin Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Verena K. Maier and Peter B. Becker
17 In Vitro Reconstitution of In Vivo-Like Nucleosome Positioning
on Yeast DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Christian J. Wippo and Philipp Korber
18 Activator-Dependent Acetylation of Chromatin Model Systems. . . . . . . . . . . . . . . . 289
Heather J. Szerlong and Jeffrey C. Hansen
19 Mapping Assembly Favored and Remodeled Nucleosome Positions
on Polynucleosomal Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Hillel I. Sims, Chuong D. Pham, and Gavin R. Schnitzler
20 Analysis of Changes in Nucleosome Conformation Using Fluorescence
Resonance Energy Transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Tina Shahian and Geeta J. Narlikar
21 Preparation of Nucleosomes Containing a Specific H2A–H2A Cross-Link
Forming a DNA-Constraining Loop Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Ning Liu and Jeffrey J. Hayes
22 Sulfyhydryl-Reactive Site-Directed Cross-Linking as a Method for Probing
the Tetrameric Structure of Histones H3 and H4 . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Andrew Bowman and Tom Owen-Hughes
23 Genomic Approaches for Determining Nucleosome Occupancy in Yeast . . . . . . . . . 389
Kyle Tsui, Tanja Durbic, Marinella Gebbia, and Corey Nislow
24 Genome-Wide Approaches to Determining Nucleosome Occupancy
in Metazoans Using MNase-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Kairong Cui and Keji Zhao
25 Salt Fractionation of Nucleosomes for Genome-Wide Profiling . . . . . . . . . . . . . . . . 421
Sheila S. Teves and Steven Henikoff
26 Quantitative Analysis of Genome-Wide Chromatin Remodeling . . . . . . . . . . . . . . . 433
Songjoon Baek, Myong-Hee Sung, and Gordon L. Hager
27 Computational Analysis of Nucleosome Positioning . . . . . . . . . . . . . . . . . . . . . . . . 443
Itay Tirosh
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Contributors

BUNGO AKIYOSHI • Basic Sciences Division, Fred Hutchinson Cancer Research Center,
Seattle, WA, USA
TREVOR K. ARCHER • Laboratory of Molecular Carcinogenesis, National Institute
of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC, USA
SONGJOON BAEK • Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
BLAINE BARTHOLOMEW • Department of Biochemistry and Molecular Biology,
Southern Illinois School of Medicine, Carbondale, IL, USA
PETER B. BECKER • Adolf-Butenandt Institute, Ludwig-Maximilians-Universität,
Munich, Germany
SUE BIGGINS • Basic Sciences Division, Fred Hutchinson Cancer Research Center,
Seattle, WA, USA
JEF D. BOEKE • High Throughput Biology Center, Johns Hopkins University
School of Medicine, Baltimore, MD, USA
ANDREW BOWMAN • The Wellcome Trust Biocentre, University of Dundee,
Dundee, Scotland, UK
GENE O. BRYANT • Molecular Biology Program, Memorial Sloan Kettering
Cancer Center, New York, NY, USA
STEPHANIE BYRUM • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
KAIRONG CUI • Laboratory of Molecular Immunology, National Heart,
Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
JUNBIAO DAI • School of Life Sciences, Tsinghua University, Beijing, China
RUSSELL P. DARST • Department of Biochemistry and Molecular Biology,
University of Florida and Shands Cancer Center, University of Florida College
of Medicine, Gainesville, FL, USA
ANN DEAN • Laboratory of Cellular and Developmental Biology, National Institutes
of Health, Bethesda, MD, USA
TANJA DURBIC • The Donnelly Centre for Biomolecular Research, University of Toronto,
Toronto, ON, Canada
VAMSI K. GANGARAJU • Yale Stem Cell Institute, New Haven, CT, USA
MARC R. GARTENBERG • Department of Pharmacology, Robert Wood Johnson
Medical School, University of Medicine and Dentistry of New Jersey, Piscataway,
NJ, USA; The Cancer Institute of New Jersey, New Brunswick, NJ, USA
MARINELLA GEBBIA • The Donnelly Centre for Biomolecular Research,
University of Toronto, Toronto, ON, Canada
DANIEL GINSBURG • Department of Biomedical Sciences, Long Island University,
Brookville, NY, USA

ix
x Contributors

CHHABI K. GOVIND • Department of Biological Sciences, Oakland University,

Rochester, MI, USA
GORDON L. HAGER • Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
JEFFREY C. HANSEN • Department of Biochemistry and Molecular Biology,
Colorado State University, Fort Collins, CO, USA
JEFFREY J. HAYES • Department of Biochemistry and Biophysics, University of Rochester
Medical Center, Rochester, NY, USA
STEVEN HENIKOFF • Division of Basic Sciences, Fred Hutchinson Cancer Research
Center and Howard Hughes Medical Institute, Seattle, WA, USA
ALAN G. HINNEBUSCH • Laboratory of Gene Regulation and Development,
Eunice Kennedy Shriver National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD, USA
JAE-WAN HUH • Department of Biochemistry and Molecular Biology,
University of Ulsan College of Medicine, Seoul, South Korea
JUAN JOSE INFANTE • Bionaturis, Seville, Spain
TATIANA S. KARPOVA • Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
CHRISTINE M. KIEFER • Laboratory of Cellular and Developmental Biology,
National Institutes of Health, Bethesda, MD, USA
MICHAEL P. KLADDE • Department of Biochemistry and Molecular Biology,
University of Florida and Shands Cancer Center, University of Florida College
of Medicine, Gainesville, FL, USA
PHILIPP KORBER • Molecular Biology Unit, Adolf-Butenandt-Institut,
University of Munich, Munich, Germany
VERANDRA KUMAR • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
SIGNE LARSON • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
G. LYNN LAW • Department of Microbiology, University of Washington,
Seattle, WA, USA
BING LI • Department of Molecular Biology, UT Southwestern Medical Center,
Dallas, TX, USA
NING LIU • Department of Biochemistry and Biophysics, University of Rochester
Medical Center, Rochester, NY, USA
NIRAJ LODHI • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
VERENA K. MAIER • Department of Molecular Biology, Massachusetts General Hospital,
Department of Genetics, Harvard Medical School, Boston, USA
DAVIDE MAZZA • Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
JAMES G. MCNALLY • Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
FLORIAN MUELLER • Groupe Imagerie et Modélisation, Institut Pasteur,
CNRS, URA 2582, Paris, France
 Contributors xi

NANCY H. NABILSI • Department of Biochemistry and Molecular Biology,

University of Florida and Shands Cancer Center, University of Florida College
of Medicine, Gainesville, FL, USA
GEETA J. NARLIKAR • Department of Biochemistry and Biophysics,
University of California, San Francisco, CA, USA
COREY NISLOW • The Donnelly Centre for Biomolecular Research,
University of Toronto, Toronto, ON, Canada
TOM OWEN-HUGHES • The Wellcome Trust Biocentre, University of Dundee,
Dundee, Scotland, UK
CAROLINA E. PARDO • Department of Biochemistry and Molecular Biology,
University of Florida and Shands Cancer Center, University of Florida College
of Medicine, Gainesville, FL, USA
CHUONG D. PHAM • AstraZeneca R&D Boston, Waltham, MA, USA
SANTHI PONDUGULA • Department of Biochemistry and Molecular Biology,
University of Florida and Shands Cancer Center, University of Florida College
of Medicine, Gainesville, FL, USA
KRISHAN MOHAN RAI • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
AMOL RANJAN • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
CHUN RUAN • Department of Molecular Biology, UT Southwestern Medical Center,
Dallas, TX, USA
SAMIR V. SAWANT • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
GAVIN R. SCHNITZLER • Molecular Cardiology Research Institute, Tufts Medical Center,
Boston, MA, USA
TINA SHAHIAN • Department of Biochemistry and Biophysics, University of California,
San Francisco, CA, USA
HILLEL I. SIMS • Department of Biology, Rosenstiel Basic Medical Sciences Research
Center, Brandeis University, Waltham, MA, USA
MALA SINGH • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
SUNIL KUMAR SINGH • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
SHERRI K. SMART • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
TIMOTHY J. STASEVICH • Graduate School of Frontier Biosciences, Osaka University,
Suita, Osaka, Japan
MYONG-HEE SUNG • Laboratory of Receptor Biology and Gene Expression,
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
HEATHER J. SZERLONG • Department of Biochemistry and Molecular Biology,
Colorado State University, Fort Collins, CO, USA
ALAN J. TACKETT • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
SHEILA S. TEVES • Division of Basic Sciences, Fred Hutchinson Cancer Research
Center and Molecular and Cellular Biology Program, University of Washington,
Seattle, WA, USA
xii Contributors

ITAY TIROSH • Department of Molecular Genetics, Weizmann Institute of Science,

Rehovot, Israel
ILA TRIVEDI • National Botanical Research Institute, Council of Scientific
and Industrial Research, Rana Pratap Marg, Lucknow, UP, India
KEVIN W. TROTTER • Laboratory of Molecular Carcinogenesis, National Institute
of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC, USA
KYLE TSUI • The Donnelly Centre for Biomolecular Research, University of Toronto,
Toronto, ON, Canada
TOSHIO TSUKIYAMA • Basic Sciences Division, Fred Hutchinson Cancer Research Center,
Seattle, WA, USA
ASHWIN UNNIKRISHNAN • Basic Sciences Division, Fred Hutchinson Cancer
Research Center, Seattle, WA, USA
CHRISTIAN J. WIPPO • Molecular Biology Unit, Adolf-Butenandt-Institut,
University of Munich, Munich, Germany
ELTON T. YOUNG • Department of Biochemistry, University of Washington,
Seattle, WA, USA
MIYONG YUN • Department of Molecular Biology, UT Southwestern Medical Center,
Dallas, TX, USA
KEJI ZHAO • Laboratory of Molecular Immunology, National Heart,
Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
 Chapter 1

Strain Construction and Screening Methods for a Yeast

Histone H3/H4 Mutant Library
Junbiao Dai and Jef D. Boeke

Abstract
A mutant library consisting of hundreds of designed point and deletion mutants in the genes encoding
Saccharomyces cerevisiae histones H3 and H4 is described. Incorporation of this library into a suitably
engineered yeast strain (e.g., bearing a reporter of interest), and the validation of individual library members
is described in detail.

Key words: Histones modification, Chromatin remodeling, High-throughput mutagenesis

1. Introduction

The eukaryotic genome consists of hundreds of thousands of its

organizational building blocks, the nucleosomes, which are arrayed
in a highly condensed form to allow the genome to fit into the
nucleus. Each nucleosome is formed by wrapping a histone octamer
with 146–147 base pairs of double-strand DNA. While packaging
of DNA into chromatin is necessary to fit the genome into a
tight space, it also forms a potent obstacle for transcription, DNA
replication, recombination, repair, and other dynamic processes.
Biological evidence has shown that DNA can however be rendered
accessible by at least three basic mechanisms including posttransla-
tional modifications (PTMs) of histone proteins, incorporation of
histone variants into nucleosomes, and ATP-dependent chromatin
remodeling.
Numerous covalent PTMs have been identified at the N-terminal
flexible “tail” domains of histones and within the structured core
regions (1, 2), which can be added or removed at specific locations

Randall H. Morse (ed.), Chromatin Remodeling: Methods and Protocols, Methods in Molecular Biology, vol. 833,
DOI 10.1007/978-1-61779-477-3_1, © Springer Science+Business Media, LLC 2012

1
2 J. Dai and J.D. Boeke

in chromatin in response to biological cues. This dynamic alteration

of nucleosome composition underlies the ability of histones to
carry out specific roles, directly or indirectly, through the recruit-
ment of accessory factors, highlighting the mechanistic significance
of modifiable histone residues within the nucleosome. For example,
methylation on histone H3 lysine 79 leads to loss of Sir3p binding
to nucleosomes, leading to general transcriptional competence
(3, 4). Additionally, a growing body of evidence indicates that
cellular processes may be regulated redundantly or cooperatively
by multiple modifications. For example, the acetylation of histone
H4 tail and the trimethylation of histone H3K4 are both intimately
associated with transcription initiation (5, 6) whereas the repair
of double strand breaks requires both H4 S1 phosphorylation
and H3 K79 methylation (7, 8) indicating that combinations of
modifications on histone residues may also contribute to specific
cellular processes.
In addition to PTMs of histone proteins, histone variants can
also be incorporated into specific nucleosomes in chromatin to
replace the canonical histones to carry out specialized functions.
For example, replacement of histone H3 by its variant Cse4 marks
the centromeric Saccharomyces cerevisiae nucleosome (9), which
leads to the association of multiple proteins to form the kineto-
chore in budding yeast. In another case, studies from multiple
organisms including yeast (10), Drosophila (11), Caenorhabditis
elegans (12), and human (13) have revealed that the histone H2A
variant H2A.Z-containing nucleosomes are especially enriched at
the +1 position near the transcriptional start site (TSS). Recent work
has shown that in Arabidopsis, the H2A.Z-containing nucleosomes
have the ability to “sense” changes in temperature and allow DNA
to unwrap progressively as temperature rises, and therefore lead
to the activation and inactivation of hundreds of temperature
regulated genes (14).
Another mechanism to open chromatin and facilitate transcrip-
tion is through chromatin remodeling complexes, which utilize
the energy from hydrolyzing ATP to enhance sliding of histone
octamer along the DNA or transfer the octamer in trans. One good
example is Gal4-mediated Gal gene induction in yeast (15). Upon
galactose addition, the inhibition of the transcriptional activator
Gal4 is alleviated, resulting in the recruitment of chromatin remod-
eling complex SWI/SNF to Gal gene promoters, where it removes
the nucleosomes and facilitate the rapid activation of transcription.
PTMs, histone variants and chromatin-remodeling are not
mutually exclusive but often interdependent. For instance, at least
in vitro studies suggest that at the Gal gene promoters the histone
acetyltransferase (HAT) complex can stabilize SWI/SNF binding
to nucleosomes and that acetylated histones are preferentially
displaced by the SWI/SNF remodeling complex (16–18). In addi-
tion, HAT complexes such as NuA4 and SAGA increase the ability
 1 Strain Construction and Screening Methods for a Yeast Histone… 3

of RSC to stimulate Pol II elongation and the stimulatory effect of

SAGA on RSC is enhanced by the addition of acetyl-CoA in vitro
(19). This observation suggests that SAGA- and NuA4-mediated
histone modifications may be a direct target for binding of RSC via
its bromodomains to the nucleosomes, although further in vivo
evidence is still lacking. Similar interplay between SAGA and SWI/
SNF is observed at the yeast PHO5 gene upon phosphate starva-
tion (20, 21), suggesting the prevalence of interdependency of
PTMs of histones and the chromatin-remodeling complex.
To dissect such complex processes effectively, a combination of
biophysical, biochemical and genetic methods are essential. We
specifically focus here on genetic approaches aimed at discovering
which processes individual histone residues participate in, and even
more specifically on a recently described library of histone point
mutants in H3 and H4.
We describe here a set of relatively generic protocols to system-
atically identify new histone mutations which are closely related to
your favorite genes/phenotypes using this publicly available histone
mutant library (22).

2. Materials

2.1. Construct the 1. 2× yeast extract/peptone (YEP) medium: 20 g/L Bacto-yeast

Reporter Strain extract, 40 g/L Bacto-peptone in water and autoclaved.
2. 4% agar: Bacto-agar: 10 g of agar is added into 250 mL of
water and autoclaved.
3. 20% (w/v) dextrose stock solution, sterile.
4. Petri plates: 90 × 15 mm.
5. Deionized (Mega-ohm resistance) water, to be used for all
media preparations.
6. 1 M LiOAc stock solution: Add 51 g LiOAc·2H2O into
400 mL ddH2O to dissolve, and fill to 500 mL final volume.
The pH should be between 8.4 and 8.9. Filter-sterilize or
autoclave. This can also be made by dissolving LiOH to 1 M
and adjusting the pH with acetic acid.
7. TE: 10 mM Tris acetate pH 8, 1 mM EDTA.
8. 0.1 M LiOAc in TE.
9. Salmon sperm DNA (sheared, 10 mg/mL): boiled for 5 min
and kept on ice before use.
10. 50% (W/V) PEG 3350 stock solution: Add 125 g PEG 3350
to 120 mL ddH2O, dissolve at low heat on a stirring plate. Fill
to 250 mL final volume and filter-sterilize.
11. 40% PEG 3350 (W/V) in 0.1 M LiOAc/TE.
Exploring the Variety of Random
Documents with Different Content
her in a thousand little ways, knowing as he did the ramifications of
life in the great house; of use also now with the boy in giving vent to
his fresh and pliable character.
A remarkable difference is found to exist between the stages of
development in the physical and moral natures. The insect passes
through three degrees, the larva, the pupa, and imago, the last
phase being the noblest, and the middle the most torpid of the three
conditions. With man and woman physically it is different. The
childhood indeed corresponds to the grub stage, but this is
immediately followed by the butterfly condition, and that of
cessation of energies and deterioration of beauty follows as the third
period. In psychical development, however, man follows the same
course as the insect. After the first voracious acquisitive period of
growth, comes the pupa condition, when the human conscience,
glutted with as much knowledge and experience as it deems
sufficient, encases itself in a chrysalis of conceit, and falls asleep in
self-sufficiency. Then, after a period of comatosity, comes a shock of
awakening life, the breath of a new spirit passes over the earth, the
sun smites with provocative ray, and the sleeping soul stretches
itself, and suddenly finds its case too strait for it. Then that horny
hide of self-conceit is riven from top to bottom, and falls away, and
at length the true, the perfect spiritual character comes forth,
flutters its wings for a moment, gains fresh courage and expands
them. It is indeed true that some insects never escape out of their
chrysalis, and some birds stifle in their shells through lack of force to
rive the encasing bound. And it is also true that there are men and
women who to the last remain hide-bound in their self-esteem; and
the moral sense, the spiritual force, the power of development
becomes extinct in them.
In our gardens the spade occasionally brings up these dead pupæ
in their horny coffins; and we are continually coming across human
beings in society, in like manner enchrysalised in conceit, in which
they remain eternally encoffined.
It must not be supposed that the transition condition is without its
throes and effort. On the contrary, the advance to the better, the
perfect life is only possible through effort, and the effort is
stimulated by the sense of oppression, through realisation of the
straitness of the shell.
Hard had been the case that enclosed Jingles, but the Giles Inglett
Saltren we now see had completely emancipated himself from it.
When he opened the door of Chillacot, his mother said—“Giles, I
have secured a servant. I have promised Tamsine Kite a place in my
establishment as lady’s-maid. She will attend me to town.”
“But, mother—”
“My dear, it is settled; and see, here is Captain Tubb.”
“Captain Tubb!”
“Yes, he has come to pay me his respects before I leave, and to
congratulate me on the disposal of Chillacot for so handsome a sum,
and to enquire what I propose doing with the money—and even to
suggest a desirable investment for it.”
 CHAPTER XLVI.

ON FLOWER-POTS.

Saltren moved with his mother to London, and went with her into
lodgings. Mrs. Saltren had insisted on taking Thomasine with her,
and incurred accordingly the additional expense of maintaining her
where she was not wanted. Thomasine was not likely to be of use till
the Saltrens got a house of their own, and Giles did not choose to
take one till he had got into a situation and was able to see what his
prospects were likely to be. As lady’s-maid to Mrs. Saltren,
Thomasine was, of course, no good at all, or likely, to employ that
serviceable Yorkshire word again, “to frame” as one.
“Whatever you do,” said Mrs. Saltren, “mind that we live in the
West End. Why don’t you go to Shepherd’s Bush, near the Welshes?
A man of my brother’s political and literary position must have hosts
of distinguished acquaintances, and a woman of Tryphœna’s
accomplishments and beauty must have the entrée into the highest
circles. If we lived near them we might get good introductions. If we
don’t get settled to my liking shortly in a fashionable quarter of
town, I do not know but that I may return to Orleigh.”
“Return to Orleigh!” echoed the son, “why, mother, I thought that
your desire had been to leave it. Besides, we have not a house there
any more.”
“I know we have not,” answered his mother, “but what we may be
without, it is possible that I might secure.”
“I do not understand,” said Jingles.
“I think,” said Mrs. Saltren, “that it is proper the money paid by
the railway company for Chillacot should be put into the bank in my
name and not in yours.”
“I have already told you, mother,” said Giles, “that I will not touch
it myself. I consider it yours, not mine.”
“But I have not the disposal of it.”
“Indeed, mother, you have; it is entered in your name, not in
mine, already. I have no account at the bank at all.”
“How can you talk nonsense,” said Mrs. Saltren; “you have all your
savings—quite a fortune—which you got at the Park whilst tutor to
young Giles.”
“My dear mother, I had not the time to accumulate a fortune. I
was tutor there for eighteen months and what I saved was a
hundred and twenty-five pounds, and that sum is already disposed
of.”
“Disposed of! What have you done with it?”
“I have purchased an annuity for some one.”
“For whom? for me?”
“No, mother, not for you. You have the purchase money of
Chillacot.”
“For whom then? I insist on knowing.”
“For a man who has been crippled, and is unable to earn his
livelihood.”
“What nonsense! What absurd fit of heroic charity has come over
you? Since you went to town in that strange, hurried fashion at the
time of your father’s death, you have been altered from what you
were before, as different as canister beef from that which is fresh
from the ox.”
Giles said nothing in self-defence.
“But I insist on knowing on whom you have thrown this money
away.”
“I do not wish to tell—on a man who has the nearest of claims on
me.”
Mrs. Saltren considered, then coloured, looked mortified, and did
not prosecute her inquiries. “Well,” she said petulantly, “a fool and
his money is soon parted. I am very glad I insisted on having the
Chillacot purchase money removed from your fingering. Please to
ring for my lady’s-maid.”
 “Lady’s-maid, mother?”
“For Thomasine. I want to speak to her. You may leave the room.
Here we have been in town a week and the Welshes have not called.
If we are to be more solitary here than we were at Chillacot, I shall
go back to Orleigh. Ring for my lady’s-maid.”
Mrs. Saltren was, indeed, becoming tired of London. Her
opportunities for boasting were confined to talks with her landlady
and her landlady’s visitors.
It did her soul good, said the woman of the lodgings, to hear of
lords and ladies; it was as comforting and improving as the words
that dropped from the lips of the Reverend Hezekiah Bumpas. She
felt it down to her toes.
Mrs. Saltren indulged her in this particular to her heart’s content.
She knew many persons of distinction. Lady Hermione Woodhead,
who lived in Portland Place, had once been her intimate friend, till
they differed about Lord Lamerton’s marriage. What had made them
differ? It did not become her to speak, but his lordship had set his
affections elsewhere, she could not name in what direction, and had
been inveigled by the Woodheads into an alliance with their family. It
was a mistake, an entanglement managed by designing women.
Lord Lamerton was ill after his engagement, so was another
person who must be nameless. When Lady Lamerton died, then his
first flame had married—without love, and in his desperation he
married again. Of course after that first estrangement she and Lady
Hermione never spoke. She—Marianne Saltren—had passed the Earl
of Anstey’s family repeatedly without recognition. If her landlady
doubted her word, let her accompany her to Hyde Park, and when
the Anstey family drove by, she would see that they took no notice
of each other. After what had happened it could not be otherwise.
But though Mrs. Saltren could talk what nonsense came into her vain
head to the lodging-house keeper, she was disappointed that she
could not to a larger circle, disappointed at the little notice she
attracted in town. It was most strange that the Welshes took no
notice of her. She feared that they were going to treat her with
coldness and not introduce her to the distinguished circle of
acquaintances in which they moved.
 I knew a young girl who was given lessons in oil-painting before
she had learned how to draw, and a somewhat similar inversion of
order went on in the instruction of Thomasine Kite, whom Marianne
Saltren began to train to be a lady’s-maid before the girl knew the
elements of domestic service, having previously been a farm-maid,
feeding pigs and scouring milk-pails.
Thomasine did not take readily to instruction, least of all could she
acquire deference towards her mistress; and Mrs. Saltren was
irritated at the freedom with which the girl accosted her, and at the
laughter she provoked in Thomasine when she, Marianne, assumed
her grand manner. Moreover, she discovered that her landlady had
been questioning the girl in private as to the circumstances and
former position of her mistress, and Mrs. Saltren was afraid that the
revelations in the kitchen might cause some of her stories to be
discounted. Fortunately for her, the broad dialect of Thomasine was
almost unintelligible to the landlady, and the girl had the cunning of
the uneducated, which leads them to evade giving a direct answer to
any question put to them.
Giles Inglett Saltren was unaware till he came to town that
Arminell was settled in the house of the Welshes. He knew that his
uncle had undertaken to arrange matters of business for her, and to
look out for a house and companion for her, but he had refrained
from asking questions about her, from motives of delicacy. Indeed,
he had scarcely written to Mr. Welsh since his return to Orleigh. He
was resolved not again to seek his assistance on his own behalf, but
to find a situation for himself. When, however, he came to town, and
met his uncle at an office in the city, he learned from him where
Arminell was, and at once urged on Mr. Welsh the mischief which
would ensue should Mrs. Saltren discover that Miss Inglett was alive
and their lodger. Welsh saw that, and undertook to prevent his wife
from calling on Mrs. Saltren, and promised to keep his eye open for
an opportunity of placing Arminell elsewhere. Marianne Saltren
shared the prevailing opinion that Miss Inglett was dead and Giles
was specially anxious lest she should discover that this was not the
case. If she were to see Arminell, would it be possible to control her
tongue? Would she not be eager to publish the fact that the
Honourable Miss Inglett was a guest of her brother and sister-in-
law?
It had been Saltren’s intention to keep away from Arminell, but
under this alarm he felt it his duty to see her and precipitate her
departure from Shepherd’s Bush. His mother could not be kept
indefinitely away from her brother’s house. One word from his
mother might frustrate Arminell’s intention, upset her plans. From
Mrs. Saltren the report would rapidly spread. Mrs. Cribbage had ears
like those of the trusty servant on the Winchester escutcheon, and
without the trusty servant’s padlock on the tongue. If once the truth
got wind, to what difficulties would the Lamerton family be put, now
that they had accepted and published the death of the girl!
The author of this novel was involved many years ago in an
amateur performance of “Macbeth,” but the sole part he took in the
tragedy was to sit in the midst of the witches’ cauldron, and ignite
the several coloured fires which were destined to flame, as scale of
dragon, tooth of wolf, liver of blaspheming Jew, were cast in. But
when, to Locke’s lovely music, the imps and witches danced around
the vessel, then it was his function to explode a so-called flower-pot,
which is a roaring, spirting composition of fire-work. Unfortunately,
at the first chorus and circular dance, the blazing flower-pot tumbled
back upon the author, concealed within the depths of the cauldron,
and, to save himself from an auto-da-fé end, he enveloped the
flower-pot in a rug, and screwed it up tight and sat on it. So the
scene ended, and, believing that the fire-work was completely
extinguished, he then unfolded the rug. No sooner, however, did the
air reach the smothered fire-work, than it bounced, and roared, and
blazed with doubled vigour. It threw out sheaths of flame, it shot off
Roman candles, it ejected a score of crackers, and filled the entire
stage with smoke, and very nearly burnt down the theatre.
Saltren dreaded something of this sort happening now. The fire-
work of scandal had, indeed, been muffled up and smothered, when
first it began to fizz; but—who could tell?—if it got air again, even
through a pin-hole, it would burst into furious conflagration and defy
all efforts made to suppress it.
 The writer of this story takes this occasion of apologising—if
apology be necessary—for the introduction, on more than one
occasion, of his own adventures, his own opinions, and, if you will it,
his own prejudices into the course of his narrative. He will be told
that the author should disappear as a personality, just as the actor
merges his individuality in that of the character he represents. He
must treat himself as a flower-pot and wrap himself up in the garde-
robe of his dramatis personæ. I might, of course, have told that
story of the flower-pot in the cauldron as having happened to Jingles
at Orleigh, but then I could never have told that story again at a
dinner-party, for my guest, next but one, would say, “Ah! that
happened to my brother, or to my uncle, or to an intimate friend;”
and how can I deny that Jingles did not stand in one of these
relations to him?
Montaigne, the essayist, was a sad sinner in the introduction of
himself into his prose. The essay on which he was engaged might be
on the history of Virgil, or Julius Cæsar, but there was certain to
creep into it more of Montaigne than of either. The younger Scaliger
rebuked him for it, and, after having acquainted the world with the
ancestry of Montaigne, he adds, “His great fault is this, that he must
needs inform you, ‘For my part I am a lover of white wines or red
wines.’ What the Devil signifies it to the public,” adds Scaliger,
“whether he is a lover of white wines or red wines?” So, but with
more delicacy, and without the introduction of that personage whose
name has been written with a capital D, the reader may say to the
author, What the blank does it signify what you think, what you like,
what you did, whether you ever sat in a cauldron, whether you ever
had a flower-pot fall on your head, whether you sought to extinguish
it by sitting on it?—go on with your story.
But a man’s personality—I mean my own—is like that piece of
pyrotechnic contrivance, a flower-pot. He wraps it up, he smothers it
under fold after fold of fiction; but, fizz! fizz! out it comes at last—
here, there, on all sides, and cannot be disguised. There is, to be
sure, that subterfuge, the use of the first person plural in place of
the first person singular, but is it not more vainglorious to talk of We,
as if we were royalties, instead of plain and modest I?
 When Giles Saltren arrived at the house in the Avenue, Shepherd’s
Bush, Arminell flushed with pleasure, sprang from her seat, and with
outstretched hand started to receive him; then she checked herself,
and said, “I am glad to see you. Oh, Mr. Saltren, I hear nothing of
Orleigh, of dear, dear Orleigh! I have the heartache for news. I want
to hear my own tongue wag on the subject nearest my heart, and to
listen to tidings about the people I knew there. I am like a departed
soul looking back on familiar scenes, and unable to visit them and
old friends, and unable to communicate with them. I am Dives, and
Orleigh is to me Paradise. You have come thence with a drop of
fresh news wherewith to cool my thirsty tongue.”
“I am Lazarus indeed,” said Saltren, “but out of Paradise. Ask me
what you will about Orleigh, and I will answer what I can.”
“There is one matter that teases me,” she said; “I promised a poor
fellow, before I left, that he should have employment at a small
wage, and I do not suppose he has had what I undertook to give
him.”
“Do you mean Samuel Ceely? He is provided for.”
“How so?”
“He has come in, unexpectedly, for a little money, wherewith an
annuity has been purchased.”
“I am glad of that. And—my mother and Giles, have you seen
them?”
“Yes, I called to say farewell to both. Lady Lamerton looks worn
and sad, and your dear brother is out of spirits; but this could not be
otherwise.”
Arminell’s eyes filled, and she went to the window and dried her
tears.
“Miss Inglett,” said the young man, after she had been given time
to recover herself, “I have only ventured to call on you for one
reason, that I might impress on you the necessity of leaving this
house. My mother is in town, and she must not be allowed to know
or even suspect that you are alive and here.”
Arminell did not speak for some time. Presently she said, “Do not
let us talk about anything at present but Orleigh. I am parched for
news. I daresay there is nothing of tremendous importance to relate,
but I care for little details. How was the house looking? Were the
trees turning to their autumn tints? The Virginian creeper, was that
touched with crimson? How are Mr. and Mrs. Macduff? I could not
abide them when I was at Orleigh; I could be thankful now for a
sound of their delightful Scotch brogue. What is Giles going to do?
dear little boy! I would give a week’s sunlight for a kiss from his
moist lips—which formerly I objected to. And mamma—has she been
to the Sunday School since—since—?”
Then Arminell’s tears flowed again.
After another pause, during which the young man looked through
the photographic album on the table, Arminell recovered herself, and
said, “Do not suppose for a moment that I regret my decision. My
conscience is relieved. I am beginning to acquire fresh interests. I
am now making a frock for baby. I am godmother to Mrs. Welsh’s
child, and have come to be very fond of him. But there—tell me
something about Orleigh, and Giles, and my mother—about any
person or animal, or shrub or tree there. And, oh! can you obtain for
me some photographs of the place? I should cherish them above
everything I have. I dream of Orleigh. I think of Orleigh, and—I shall
never see dear Orleigh again.”
“I will come another day, Miss Inglett, and tell you all that I can,
but to-day I must urge on you the vital necessity of at once leaving
this house.”
“Your aunt can hardly get on without me.”
“She managed formerly without you, she must do the same
again.”
“But there was no baby in the house then. And, besides, the new
cook who was to have come has failed. The last went up a ladder
sixty feet high, and it took several constables and a sergeant to get
her down.”
Arminell laughed through her tears.
“Miss Inglett, consider what the difficulty would be in which her
ladyship would be placed should it become known—”
“Mrs. Saltren and her lady’s-maid!”
The door was thrown open by the maid of-all-work, and she
ushered into the drawing-room the person of all others—except
perhaps Mrs. Cribbage—whom it was desired to keep from the
house, and she was followed by Thomasine Kite.
Verily, the flower-pot was not smothered. It was about to fizz and
puff again.
 CHAPTER XLVII.

EQUILIBRIUM.

The story is told of a mouse having been hidden under a dish-

cover, and a married pair introduced into the dining-room and invited
to partake of every dish except that which remained covered. When
left to themselves, the woman, contrary to the advice of her
husband, raised the cover, and out ran the mouse. Blue Beard
forbade Fatima to open one door in his castle, and of course she
tried the forbidden key. There was one tree in the midst of Paradise
of which our first parents were not allowed to eat, and of course
they nibbled at the fruit to discover how it tasted. All these stories
point to the truth that nothing can be retained from human
inquisitiveness. A secret resembles a mouse more than an apple or a
dead wife of Blue Beard, for the mouse escapes when once
uncovered and can no more be hidden, whereas the apple
disappears when eaten, and the dead woman is locked up again. A
secret when once out is all over the house, and is far too wary to be
trapped again.
Who would expect to find a mouse under a dish-cover? So with
secrets, they are let loose from the most unlikely places, and many
of us know that so well that we devote our energies to, and spend
our time in lifting china cups, opening snuff-boxes, removing lids of
tea caddies, unsnapping purses, pulling out drawers, boring holes in
casks, in the hopes of letting out secrets. We suspect our
acquaintance and “visit” their goods, as if we were custom-house
officers in search of what is contraband. We know that they have a
forbidden secret somewhere, and we search and probe everywhere
to discover it.
There are mice everywhere; if we hold our breath and remain still
for two minutes we can hear them scratching and squeaking; and
there are secrets everywhere, behind the wainscot, under the floor,
in the cupboard. Once I knew of a nest of mice in a gentleman’s
boot, and once in a lady’s muff; and secrets nest and breed in quite
as extraordinary places—in a pocket, in a bunch of flowers, in
envelopes, under pillows.
Æsop tells of a beautiful cat that was transformed into a woman,
but this woman could never forget her feline instinct to run after a
mouse. A great many ladies I know have the same feline instinct to
spring out of bed, up from their sofas, to make a dart after a secret,
if they hear but the slightest footsteps, see but a whisker. I do not
blame them. Men are sportsmen, why should not women be
mousers? We find pleasure in starting a hare, why should not a
woman find as much in starting a couching secret?
I do not blame them for their love of sport, but for what they do
with their game when it is caught. We bag ours, they let theirs run.
Samson did the same. He caught foxes and tied firebrands to their
tails and sent them into the standing corn of the Philistines. Our
secret-hunters, when they have caught their game, tie brimstone
matches to their tails and send them among the stores of their
neighbours.
I do not believe in the possibility of concealing secrets, and
therefore never try to keep them. As for pursuing a secret when
once out, that is labour in vain, it changes form, it doubles, it dives,
it has as many artifices as a chased fox. As soon recover a secret as
recondense volatile essential oils that have been spilt. A secret is not
safe in our own heads, for our heads are of amber, and the secret is
visible to every one who looks at us, like a congealed fly therein.
In one of the Arabian Nights’ Tales a princess goes after a
necromancer who has transformed himself into a scorpion, and she
takes the shape of a serpent; the wizard, hard pressed, becomes a
cat, and the princess attacks him in the disguise of a wolf. Then the
cat becomes a seed, and the wolf a cock, thereat the seed falls into
a canal and is transmuted into a trout, which is at once chased by
the princess in shape of a pike. Finally both issue in flames from the
water, the wizard is reduced to ashes, but so also is the princess. If
we try to overtake and make an end of a secret, we shall meet with
less success than did this princess. She at last succeeded in
destroying her game, but we, in our efforts to catch and make an
end of an unpleasant secret, get set on flames ourselves. If we have
anything we do not want our neighbours to know, and it has got
out, we had better let it run; we cannot recover it. Indeed I believe
that the best way to conceal what we do not want to have known is
to expose it for sale, to dangle it before the eyes of every one, like
those men outside the Exchange who offer spiders at the end of
threads of elastic for one penny. Nobody buys. No one even looks at
them. But were one of these fellows to hide such a black putty
spider in his hat, up his arm, in his pocket, a crowd would collect
and pull him to pieces to find the spider.
It was not immediately that Arminell realised the serious
consequences of Mrs. Saltren’s visit, but the young man knew at
once that all chance of the secret being respected was at an end.
“I am interrupting,” said the widow, knowingly, “I am sure I hadn’t
the wish. I came to see Mrs. Welsh, and never expected to find my
son here, much less Miss Inglett.”
“Mrs. Welsh is upstairs with the baby,” said Arminell. “You have
not seen your nephew. Shall I fetch him, Mrs. Saltren?”
“Not for the world, Miss Inglett. I will run upstairs and find my
sister-in-law, who, I do say, has been negligent in calling on me. But
if the mountain won’t go to Mahomet, Mahomet must go to the
mountain. I’m sure I don’t want to intrude here. You may leave the
room, Thomasine, I don’t want you to follow me up to the nursery.
Go down to the kitchen. Every one ought to know her own place.”
When the girl had disappeared, Mrs. Saltren said confidentially,
“We brought the young person to town, and she don’t understand
how to friz the hair, and me wanting to wear a fringe. However she
could have had the face to offer for my situation as lady’s-maid,
passes my understanding. But, Miss, the conceit of the rising
generation is surprising. I want to ask Mrs. Welsh to take the
creature off my hands in any capacity she likes to name. She might
do as parlour-maid, or nurse-girl, or cook, anything but lady’s-maid.
I’ve tried to teach her to fold gowns, but folding is like music or
painting—you must be born with the gift; it cannot be learnt; and as
some have no ear for tune, and others no eye for colour, so have
some no natural gift for folding. You can’t make, as they say, a fichu
out of a bustle. I had once a red flannel coverlet, and a hole was
burnt in it, so I turned it into a petticoat. When the hot weather
came I couldn’t bear it, and as the Band of Hope wanted a banner, I
did a non-alcoholic motto on it in straw letters, and converted it into
a Temperance banner, and very inspiriting it was. It is the same with
girls. Some you can adapt to all sorts of purposes, others you can’t.”
When Mrs. Saltren had left the room in quest of her sister-in-law
and the baby, Giles said in a tone of discouragement, “I do not know
what is to be done. It is inevitable that the news of your being here
should reach Orleigh, either through my mother or the girl, probably
through both, not perhaps at once, but eventually. Then—what a
difficult position Lady Lamerton will be in!”
Arminell looked down on the carpet, and traced the pattern with
her foot. Presently she looked up and said, “I see—I never did
justice to the merits of humdrum. Even when I was shown my folly
and acknowledged my fault, I must needs still play the heroine, and
take a bold step, not altogether justifiable, because it landed me in
falsehood, and involved others in untruth. But I thought then it was
the simplest course for me to follow to escape having to equivocate
and even lie. The straight course is always the best. Now I admit
that. Short cuts do not always lead where one thinks they will. I
wish I had acted with less precipitation and more modesty, had
listened to your advice and acted without dissimulation. For myself
now I do not care, but I do not see how my mother and other
relations can extricate themselves from the dilemma in which I have
placed them.”
“Nor do I.”
“I am neither dead nor alive. The situation is almost grotesque. I
wish it were not distressing. Do not misunderstand me. It is painful
to myself only, as every sharp lesson cuts. But I am more vexed for
the sake of others than for my own. I have been a fool, an utter
fool.”
She put her hands over her eyes.
“Upon my word, Mr. Saltren,” she said after an interval, “I have
hardly an atom of self-confidence left. There never was a more
perverse girl than myself, such a profound blunderer. I make a
mistake whatever I do. What is to be done? What can I do?”
Giles Saltren was silent. The predicament was one from which
there was no escape.
“Your mother’s red coverlet was better than me,” said Arminell.
“That did serve some good purpose, to whatever end it was turned,
but I always get from one difficulty into another, and drag my friends
out of one discomfort into another still worse. Only here—here am I
of any good at all; I was born into a wrong sphere, only now have I
returned to that system in which I ought to have been planted when
called into existence. And yet even in this I produce a disturbing
effect on the system of planets I have left.”
“You cannot remain in this house, Miss Inglett, not now for the
reason I gave at first, but because too much is put upon you.”
“Nothing is put on me—I take on me what I feel qualified to
execute. Do you remember the answer made by the young Persian
to Cyrus, when the prince reprimanded him because his actions
were not in accordance with his previously expressed sentiments?
‘Sire,’ he said, ‘I perceive that I have two souls in me, one wilful and
wicked, and the other modest and righteous. Sometimes one is
awake and at other times the second.’ So it is with me. Now I trust
the nobler soul is rubbing its eyes and stretching itself, and the
sandman is scattering dust in the eyes of the baser soul. My old soul
was haughty and lived in an atmosphere of extravagance, and the
new one is humble, and delights in the breath of common-place. Do
you remember, Mr. Saltren, telling me of the effect of the contrast to
you of a return from Orleigh Park to Chillacot? You said that you
were unfitted by the grandeur of the former to endure the meanness
of the latter. At the time when you said this, I thought that such a
translation to me would be unendurable, but the translation has
been effected, and I am not miserable. On the contrary, but for my
self-reproach and looking back on lost faces and scenes, I should be
happier here; for the childlike spirit is waking in me, which is content
with trifles.”
“Happier—here! Miss Inglett, surely not.”
“Yes—happier. I am happier in helping others. I am become useful
to Mrs. Welsh, I relieve her of the baby, I can even cook fairly, I
make the glass and silver shine. The work and worry here were
more than your aunt could bear. Cooks are scarce as saints. The last
your aunt had—oh! I have already mentioned the circumstances. I
will not repeat them. I do not feel that the house is small, indeed I
am glad that it is not larger. We talk a good deal about the misdeeds
of servants, and the difficulty there is in getting cooks; in my former
world we talked a good deal about the unscrupulousness of
politicians, and the difficulty there was in getting morality among
statesmen—political morality I mean. We discuss now the humours
of the baby, what his dribbling means—whether teeth or disorder;
and we discussed then the humours of the public, and what the
dribble meant that flowed so freely at public meetings. We think now
how we may cut out and alter garments for the little creature; and
then, what adjustments and changes were needed for the
satisfaction of the public. Conversation on each subject is as
interesting and as profitless. I thought at one time that I could not
live away from rocks and trees—I hardly miss them now. I have no
time to consider whether I want them or not, because I am engaged
all day. I really believe that the servant girl, the slavey, as your uncle
calls her, is happier than your aunt or me, because she has the
fewest responsibilities and the most work.”
Arminell spoke fast, half in jest, half in tears; she spoke quickly, to
conceal the emotion she felt.
“Did you see a picture at the Royal Academy a few years ago
representing the Babylonian Marriage Market? In old Babylon all
marriageable women were sent up to auction, and the sum paid for
the pretty ones went as dower for those who were ugly. Thus was a
balance preserved. I suspect it is much the same in life. There is
equilibrium where we least expect it. The peacock has a gorgeous
plumage and a horrible voice, the nightingale the sweetest song and
the plainest feathers. Some of our most radiant flowers are without
perfume, and some that smell odoriferously have little in the way of
beauty to boast of. When I was in the aristocratic world, I had my
luxuries, intellectual, æsthetic, and physical, but somehow, I lacked
that joyousness I am finding here. In the middle class there is a
freedom from the restraints which cramped us in the class above,
and I have no doubt that there is an abandon, an insouciance in the
class below which makes up for the deficiency in the amenities,
refinements, and glow of life in higher spheres. There is a making up
of the balance, an adjustment of the equilibrium in the market-place
of modern life as in that of ancient Babylon. Those with rank and
wealth have to walk with muffled faces, only the plain and lowly may
breathe freely and let the sun kiss their cheeks.”
“Miss Inglett, I am sure, notwithstanding your efforts to make me
think the contrary, that you are not happy.”
“I tell you that I am. I say this in all sincerity. I do not deny that I
feel a heartache. That is because my conscience reproaches me, and
because I now love and regret what I once cast from me. If I had
not been born elsewhere I should be fresh and happy now, but
every plant suffers for a while when transplanted. I am throwing out
my rootlets and fastening myself into the new soil, and will soon be
firm fixed in it as if I had grown there from the beginning—my only
trouble that I have dreams of the past. A princess was once carried
off by Rübezahl, giant spirit of the mountains, to his palace of crystal
in the heart of the earth. He gave her all she could wish for, save
one thing, the sound of the cattle bells on the Alpine pastures. His
home was too far down for those sounds to reach. Whenever we are
carried away from our home, we must always carry away with us
some recollections of pleasant sounds and sights, and they linger
with us as memories over which to weep. But there—we have had
enough about myself—nay, too much. I want to hear what you are
about, and what are your prospects.”
“I am in search of occupation, and have, so far, met only with
disappointment.”
“You have been anxious. You are not looking well.”
 “Naturally, I am anxious. I, like you, have the weight of the past
oppressing me. Unlike you, I have not accommodated myself to my
transplantation, but—in fact, I have not yet found soil in which my
roots may take hold.”
“What soil do you want?”
“Any. There is a demand, I am told, for muscle; the market is
glutted with brain, or what passes for brain. As there is a deficiency
in the supply of cooks, I will mount a white cap and apron and apply
for a kitchen. But, seriously, apart from my affairs, which can wait,
yours must be attended to.”
“But nothing can be done. You propose nothing. I can suggest
nothing.”
Then in came Mrs. Welsh and Mrs. Saltren. The former was
carrying the baby.
“It is all settled,” said Tryphœna Welsh. “Rejoice with me, Miss
Inglett. I did want a cook, one not given to climbing ladders, and
now I have got one; now James will swear, for he has been spoiled
by your cookery, Miss Inglett; at last I have got a cook, the girl
Thomasine Kite. Come, kiss the baby and thank Heaven.”
 CHAPTER XLVIII.

L’ALLEMANDE.

“Why, blessings on me!” exclaimed Mrs. Saltren, on her return to

the lodgings in Bloomsbury. “Whoever expected the pleasure! And—I
am sorry that you should see us here, Captain Tubb; not settled into
our West-End house. Me and my son are looking about for a suitable
residence, genteel and commodious, and with a W. to the address;
but there is that run on the West End, and it is almost impossible,
without interest, to get a house. My brother, however, who is like to
be an M.P., is using his influence. But, captain, you see that every
house won’t suit me; I’m not going to be in the shade any more.
Well, it is a pleasure to see an Orleigh face here; and, pray, what has
brought you to town, Captain Tubb?”
The visitor was in a black suit, that obtained for his son’s funeral;
he held his hat in one hand, with a broad black cloth band about it.
With his disengaged hand he thrust up his beard and nibbled the
ends.
Ladies play with their fans, coquette with them, talk with them,
angle with them; and an uninitiated person looking on wonders what
is the meaning of the many movements made with the fan—the
unfurling, the snapping, the half-opening. Perhaps Captain Tubb may
have been coquetting, talking with his hat, for he turned it about,
then looked into it, then smoothed it where it was ruffled, then put it
under his chair, then took it up and balanced it on his knee. I cannot
tell. If he was not speaking with his hat, what else could he have
meant by all the movements he went through with it?
 “Well, ma’am,” said the captain; “seeing as how I was in London, I
thought I’d come and inquire how you was getting along. How are
you? And how is Mr. Jingles?”
“I, myself, am but middling,” answered Mrs. Saltren, with
stateliness. “My son—Mr. Giles Inglett Saltren—is very well indeed. I
have gone through a great deal of trouble, and that takes it out of
one,” said Mrs. Saltren, “like spirits of nitre.”
“So it do, ma’am. There is a vale of misery; but the sale of
Chillacot was an elevation in the same; and bank-notes are of that
spongy nature that they sop up a lot o’ tears. How, if I may make so
bold as to ask, is your son thinking of investing the money? You see,
ma’am, poor Captain Saltren and I knowed each other that intimate,
our lines o’ business running alongside of each other, that we was
always a-hailing of each other. And now that he’s gone, it seems
natural for me to come and consult with his relict.”
“You’re flattering, Mr. Tubb. I must say, it is a pity my poor
Stephen did not oftener consult me. If he had—but there, I won’t
say what I might. About Chillacot, he was that pig-headed that—but
no, not another word. I’ve always heard say that the wife is the
better half. What a mercy it is, and how it proves the wisdom of
Providence, that the wusser half was took away first.”
“You don’t know, Mrs. Saltren, how dreadful you’re missed in
Orleigh; the place don’t seem the same without you. And folks say
such spiteful things too.”
“As what, captain?”
“As that, having sold Chillacot, you ought to spend the purchase
money there, and not be throwing it about in town.”
“Do they now? But I’m not throwing it about; it is all in the bank.”
“I reckon Mr. Jingles—I mean your son, ma’am—has it there in his
own name.”
“Not at all, cap’n. The money is mine.”
Captain Tubb whisked round the brim of his hat with both hands.
“There have been changes since you’ve gone,” he said. “For one,
there is old Sam Ceely married.”
“Sam Ceely!” echoed Mrs. Saltren, and dropped her hands in her
lap.
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