Molecular Plant Biology A Practical Approach Volume 2

by Gilmartin, Philip M.; Bowler, Chris

• ISBN13: 9780199638192
• ISBN10: 0199638195
• Format: Hardcover
• Copyright: 2002-08-29
• Publisher: Oxford University Press

Summary

Molecular Plant Biology is an all-new replacement for the original Practical Approach book Plant Molecular Biology that was first published in 1988. The rapid advances made in plant sciences during the past decade are reflected by the need to produce a two-volume book to cover all therelevant methodologies. The new book incorporates many of the fundamental procedures outlined in the original book, but these are fully updated to reflect advances technology and the development of new methodologies. The new books also incorporate many approaches that were not available in theearlier volume. Each chapter has been written by an international expert with current practical expertise in the topics covered. Each book is divided into three sections. The approaches outlined in Volume One cover a wide range of techniques for gene isolation, gene identification and subsequent isolation, as well as for studies of gene organization. The methods described range from classical mutagenesis through planttransformation, T-DNA and transposon tagging strategies, genomic subtraction, gene mapping, construction and screening of YAC, BAC and cosmid libraries chromosome in situ, and isolation of cDNA sequences by western and southwestern library screens, and complementation cloning. Volume Two focuses on experimental approaches for studies on gene expression, gene product analysis, with the final section devoted to emerging technologies. Topics covered include a range of techniques for transcript analysis, including In situ Hybridization and DNA microarrays. DNA-proteininteraction methods are also covered in detail. Inducible gene expression in plants as well as expression and analysis of recombinant proteins, and analysis of protein import into chloroplasts are covered as well as techniques for fractionation of plant tissue for biochemical analyses and the studyof protein-protein interactions with the yeast two-hybrid system. A range of approaches for using antibodies as tools are also described including the use of antibody phage display libraries. The final section on emerging technologies describes methodologies for calcium imaging and for the spatialand temporal analysis of reporter genes such as luciferase and green fluorescent protein. The final area covers a range of experimental procedures for moss, which is emerging as a new model organism.

Table of Contents

List of protocols	xvii
Abbreviations page	xxiii
SECTION 1 GENE EXPRESSION
Transcript analysis
Graham R. Teakle
Charles P. Scutt
Philip M. Gilmartin
Introduction	3	(1)
Handling RNA	4	(1)
Quantification of RNA	5	(2)
Spectrophotometric quantification	5	(2)
Isolation of RNA	7	(5)
Isolation of total RNA using LiCl precipitation	7	(2)
RNA extraction using guanidinium hydrochloride	9	(1)
Isolation of mRNA	10	(2)
Blotting and hybridization analysis of RNA	12	(13)
Northern analysis	13	(4)
Reverse northern blotting	17	(4)
Virtual northern blotting	21	(4)
Nuclease protection assays	25	(10)
RNase protection assays	25	(7)
S1 nuclease protection assays	32	(3)
Primer extension analysis	35	(6)
References	40	(1)
In situ hybridization	41	(24)
Sabine Zachgo
Introduction	41	(1)
Fixation of tissue	42	(3)
Embedding and sectioning of tissue	45	(1)
Sample embedding	45	(1)
Sectioning of tissue	46	(1)
Probe preparation	46	(5)
Probe labelling	46	(1)
Template preparation for probe production	47	(1)
Quantification of DIG-labelled probe	47	(1)
Precautions working with RNA	47	(4)
Hydrolysis of RNA probe	51	(1)
Prehybridization treatment	52	(1)
Hybridization	52	(4)
Hybridization parameters	52	(4)
Post-hybridization washes	56	(1)
Signal detection	57	(4)
Confirmation of signals and trouble-shooting	61	(4)
Controls	61	(1)
Trouble shooting	61	(2)
References	63	(2)
DNA micro-arrays for gene expression analysis	65	(12)
Shanna Moore
Paxton Payton
Jim Giovannoni
Introduction	65	(1)
Types of arrays	65	(2)
Target preparation	67	(1)
PCR	67	(1)
Substrate/support	68	(3)
Probe synthesis and hybridization	71	(1)
Scanning	72	(1)
Data analysis/data management	73	(1)
Challenges facing the array community	74	(3)
Acknowledgements	75	(1)
References	75	(2)
DNA-protein interactions	77	(20)
Paul J. Rushton
Bernd Weisshaar
Introduction	77	(1)
Preparation of nuclear extracts	77	(3)
Preparation of nuclear extracts from protoplasts	78	(1)
Preparation of nuclear extracts from cultured cells	79	(1)
Electrophoretic mobility shift assay (EMSA)	80	(2)
DNA probes for use in EMSAs	81	(1)
EMSA reactions	82	(1)
DNase I footprinting	82	(5)
DNA probes for DNase I footprinting	83	(2)
DNase I footprinting reactions	85	(2)
In vivo footprinting	87	(8)
In vivo methylation of plant cells	87	(1)
Preparation of genomic DNA	88	(1)
Production and enrichment of promoter fragments	88	(1)
Preparation of fractionated DNA for gel electrophoresis	89	(1)
Preparation of cloned promoter fragment DNA for gel electrophoresis	90	(1)
Preparation of a genomic filter	91	(1)
Probe synthesis	92	(1)
Probe elution	93	(1)
Hybridization	94	(1)
Concluding remarks	95	(2)
Acknowledgements	95	(1)
References	95	(2)
Inducible gene expression in plants	97	(14)
Maki Ohgishi
Takashi Aoyama
Introduction	97	(1)
Inducible systems using plant promoters	98	(1)
General characteristics	98	(1)
Heat-inducible systems using plant promoters	98	(1)
Chemically-inducible systems using plant promoters	99	(1)
Other induction systems using plant promoters	99	(1)
Inducible systems composed of heterologous elements	99	(5)
General characteristics	99	(1)
Inducible systems using prokaryotic repressors	100	(2)
Inducible systems using steroid-hormone receptors	102	(1)
Other heterologous inducible systems	103	(1)
Practical tips for induction experiments	104	(7)
Acknowledgements	106	(1)
References	106	(5)
SECTION 2 GENE PRODUCT ANALYSIS
Expression of recombinant proteins	111	(12)
Avital Yahalom
Daniel A. Chamovitz
Introduction	111	(1)
Preliminary considerations	111	(1)
Does the protein need to be soluble?	111	(1)
Does the protein have to be in its native state?	112	(1)
How pure does the protein need to be?	112	(1)
Vector choices	112	(2)
Considerations for construction of plasmids	113	(1)
Host cell choices	114	(1)
E. coli	114	(1)
Eukaryotic expression systems	114	(1)
Protocols	115	(8)
References	121	(2)
Import of proteins into isolated chloroplasts and thylakoid membranes	123	(24)
Colin Robinson
Alexandra Mant
Introduction	123	(2)
Choice of plants	124	(1)
Growth conditions	125	(1)
Isolation of intact chloroplasts	125	(4)
In vitro synthesis of nuclear-encoded chloroplast proteins	129	(10)
Analysis of proteins by SDS-PAGE	133	(6)
Import of proteins into isolated chloroplasts	139	(4)
Import of proteins into isolated thylakoid membranes	143	(4)
The import pathway for thylakoid lumen proteins	143	(1)
The basic import assay	143	(2)
Some variations on the basic assay	145	(1)
References	145	(2)
Fractionation of plant tissue for biochemical analyses	147	(26)
Matthew J. Terry
Lorraine E. Williams
Introduction	147	(1)
Homogenization of plant tissue	147	(5)
General considerations	147	(1)
Homogenization procedures and conditions	148	(4)
Biochemical analysis of organelles	152	(5)
Etioplasts and developing chloroplasts	152	(4)
Mitochondria	156	(1)
Nuclei	157	(1)
Biochemical analysis of plasma membranes and intracellular membranes	157	(4)
Isolation of plasma membrane using aqueous two-phase partitioning	157	(2)
Sub-fractionation of membranes using density gradient centrifugation	159	(2)
Biochemical analysis of soluble proteins and cytoplasmic components	161	(1)
Extraction of protein for biochemical analysis	161	(1)
Extraction of protein for immunological analysis	162	(1)
Assessment of cellular fractions and the use of marker enzymes	162	(11)
Estimating protein concentration	162	(1)
Marker analysis	163	(6)
Acknowledgements	169	(1)
References	169	(4)
Studying protein-protein interactions with the yeast two-hybrid system	173	(26)
Glaus Schwechheimer
Xing-Wang Deng
Introduction	173	(1)
The LexA- and the GAL4-based two-hybrid systems-an overview	173	(5)
The LexA-system	174	(2)
The GAL4-system	176	(1)
Constructing two-hybrid cDNA libraries	176	(2)
Practical considerations	178	(1)
Working with yeast-the techniques for the yeast two-hybrid system	178	(12)
Growth and maintenance of yeast	178	(2)
Yeast transformation	180	(2)
Measurement of LacZ reporter gene activity	182	(4)
Recovering plasmid DNA from yeast	186	(3)
Yeast mating	189	(1)
Performing two-hybrid screens	190	(9)
Performing a two-hybrid library screen with the LexA-system	190	(2)
Performing a two-hybrid screen with the GAL4-system	192	(3)
Studying interactions between two known proteins	195	(2)
Substantiating the findings from the two-hybrid assay	197	(1)
References	197	(2)
Antibody techniques	199	(22)
William G. T. Willats
Clare G. Steele-King
Susan F. Marcus
J. Paul Knox
Introduction	199	(1)
Making antibodies	199	(11)
Antisera	199	(2)
Hybridoma technology	201	(3)
Isolation of phage display antibodies from naive libraries	204	(6)
Using antibodies	210	(11)
Immunolocalization	210	(4)
Immunochemical assays	214	(5)
References	219	(2)
Antibody phage display libraries	221	(18)
Kevin C. Gough
Yi Li
Garry C. Whitelam
Introduction	221	(1)
Naive, synthetic, and immune scFv phage display libraries	222	(5)
Construction of scFv phage display libraries	223	(4)
Affinity selection (panning) of scFvs from a phage display library	227	(2)
Isolation and analysis of monoclonal phage antibodies	229	(2)
Phage ELISA	229	(2)
Assessment of antibody phage diversity	231	(1)
Expression and characterization of soluble scFvs	231	(2)
Affinity ranking of soluble scFv proteins	231	(2)
Affinity maturation of scFvs	233	(1)
The use of isolated phage antibodies as immunoreagents	233	(1)
Conclusion	234	(5)
References	234	(5)
SECTION 3 FUNCTIONAL ANALYSIS IN VIVO
Spatial and temporal measurements of calcium ions in living plant cells	239	(26)
Rui Malho
Luisa Camacho
Ana Moutinho
Introduction	239	(1)
Fluorescent probes for intracellular Ca2+	239	(1)
Choosing the correct dye	239	(1)
Single and dual wavelength dyes	240	(1)
Loading of Ca2+-sensitive dyes into cells	240	(4)
AM-ester loading	242	(1)
Acid loading	242	(1)
Electroporation	243	(1)
Micro-injection	243	(1)
Microscope and imaging settings	244	(2)
Fluorescence microscopes	244	(2)
Objectives	246	(1)
Detectors for spatial imaging	246	(3)
Choosing your own filters	248	(1)
Hardware and software	249	(1)
Optimizing imaging performance	249	(5)
Signal-to-noise ratio	249	(1)
Filter block cross-talk	250	(1)
Calibration and behaviour of intracellular fluorescent probes	251	(1)
Calibration of [Ca2+]c	251	(2)
Image acquisition	253	(1)
Bioluminescence indicators	254	(4)
Aequorin inside plant cells	254	(1)
Luminescence measurements	255	(1)
Luminescence imaging	256	(1)
Calibration of aequorin measurements	257	(1)
Data processing	258	(3)
Numerical data extraction and statistical analysis	258	(1)
Image processing and enhancement	259	(2)
New molecular indicators	261	(4)
Ion monitoring using GFP-based indicators	261	(1)
The tools of the future	262	(3)
Reporter genes and in vivo imaging	265	(20)
Angeia Falciatore
Fabio Formiggini
Chris Bowler
Introduction	265	(1)
β-glucuronidase (GUS)	265	(5)
Introduction	265	(1)
GUS assay protocols	266	(4)
Luciferase	270	(5)
Introduction	270	(3)
Assays	273	(2)
Green Fluorescent Protein (GFP)	275	(10)
Introduction	275	(1)
Advantages and disadvantages of GFP	276	(1)
GFP spectral variants	276	(2)
Hints for construction of GFP-tagged proteins	278	(1)
Applications	278	(4)
Acknowledgements	282	(1)
References	282	(3)
Moss gene technology	285	(18)
Celia D. Knight
David J. Cove
Andrew C. Cuming
Ralph S. Quatrano
Introduction	285	(1)
Moss development and tissue culture	286	(3)
PEP: the Physcomitrella EST Programme	289	(1)
PEP resources	290	(1)
Vector construction	290	(2)
Construction of a replacement vector using pMBL6: strategic considerations	291	(1)
Protoplast transformation	292	(3)
Molecular analysis	295	(5)
Nucleic acid isolation	296	(1)
DNA extraction	296	(2)
Digestion of DNA	298	(2)
PCR analysis	300	(1)
Phenotype analysis	300	(1)
Future developments	301	(2)
References	301	(2)
A1 List of suppliers	303	(6)
Index	309