Non-Natural Amino Acids

491 Pages·2009·6.34 MB·English

by  Tom W. Muir

METHODS IN ENZYMOLOGY
EDITORS-IN-CHIEF
John N. Abelson Melvin I. Simon
DIVISIONOFBIOLOGY
CALIFORNIAINSTITUTEOFTECHNOLOGY
PASADENA,CALIFORNIA
FOUNDINGEDITORS
Sidney P. Colowick and Nathan O. Kaplan
Contributors to Volume 392
Articlenumbersareinparenthesesandfollowingthenamesofcontributors.
Affiliationslistedarecurrent.
Hideo Akashi (6), Department of Chem- VickiChandler(1),DepartmentofPlant
istry and Biotechnology, School of Sciences,UniversityofArizona,Tucson,
Engineering, The University of Tokyo, Arizona85721
Tokyo113-8656,Japan
Karen Cone (1), Division of Biologi-
Jose´ Alberola-Ila(12),DivisionofBiol- cal Sciences, University of Missouri,
ogy, Cal Tech, Pasadena, California Columbia,Missouri65211
91125
Bryan R. Cullen (22), Howard Hughes
SusanArmknecht(4),DepartmentofGe-
Medical Institute, Department of
netics,HarvardMedicalSchool, Droso-
Molecular Genetics and Microbiology,
phila RNAi Screening Center, Boston,
Duke University Medical Center,
Massachusetts02115
Durham,NorthCarolina27710
HyeJungBack(21),NationalCancerIn-
stitute, National Institutes of Health, Jonathan R. Davey (24), Diabetes and
Obesity Research Program, Garvan
Bethesda,Maryland20892
Institute of Medical Research, Darlin-
MaureenBarr(3),UniversityofWiscon- ghurst,SydneyNSW2010,Austalia
sinSchoolofPharmacy,Pharmaceutical
Sciences Division, Madison, Wisconsin Beverly L. Davidson (9), Department
53705-2222 of Internal Medicine, Neurology, Phy-
Queta Boese (5), Dharmacon Inc., siology and Biophysics, University of
Iowa College of Medicine, Iowa City,
Lafayette,Colorado80026
Iowa52242
Michael Boutros (4), German Can-
cer Research Center, 69120 Heidelberg, Antonin de Fougerolles (16), Alny-
Germany lam Pharmaceuticals, Cambridge, Mas-
sachusetts02142
Robert M. Brazas (7), Mirus Bio
Corporation,Madison,Wisconsin53719 David Dorris (15), Ambion Inc., Austin,
Zuezhong Cai (22), Howard Hughes Texas78744-1832
MedicalInstitute,DepartmentofMolde-
Christophe Echeverri (15), Cenix
cular Genetics and Microbiology, Duke
BioScience GmbH, 01307 Dresden,
University Medical Center, Durham,
Germany
NorthCarolina27710
Daniela Castanotto (10), City of Hope Mark Feitelson (14), Department
Medical Center, Division of Molecular of Pathology and Cell Biology, Jeffer-
Biology, Beckman Research Institute, son Medical College, Philadelphia,
Duarte,California91010-3000 Pennsylvania19107-5587
ix
x contributors to volume 392
Witold Filipowicz (19), Friedrich Kasia Jaronczyk (19), Department of
Miescher Institute for Biomedical Cell Biology, University of Alberta,
Research,4002Basel,Switzerland Edmonton,Alberta,CanadaT6G2E1
Anne Grabner (15), Cenix BioScience Heidi Kaeppler (1), Department of
GmbH,01307Dresden,Germany Agronomy, University of Wisconsin,
Madison,Wisconsin53705
Michael W. Graham (24), Benitec Ltd.,
University of Queensland, St. Lucia Shawn Kaeppler (1), Department of
QLD4072,Australia Agronomy, University of Wisconsin,
Madison,Wisconsin53705
Peter J. Goss (24), Benitec Ltd., Univer-
sityofQueensland,St.LuciaQLD4072, Andrew J. Kassianos (24), Benitec Ltd.,
Australia University of Queensland, St. Lucia
QLD4072,Australia
Dirk Grimm (23), Department of Pedia-
Yoshio Kato (6), Gene Function
trics, Stanford University, Stanford,
Research Center, National Institute
California,94305-5208
of Advanced Industrial Science and
James E. Hagstrom (7), Mirus Bio Technology (AIST), Tsukuba Science
Corporation, Madison,Wisconsin53719 City305-8562,Japan
Michael Hannus (15), Cenix BioScience MarkA.Kay(23),DepartmentofPedia-
GmbH,01307Dresden,Germany trics and Genetics, Stanford University,
Stanford,California,94305-5208
ScottQ.Harper(9),DepartmentofInter-
Arthur Kerschen (1), Department of
nalMedicine,UniversityofIowaCollege
Plant Sciences, University of Arizona,
ofMedicine,IowaCity,Iowa52242
Tucson,Arizona85721
Bruce T. Harrison (24), Benitec Ltd.,
University of Queensland, St. Lucia Anastasia Khvorova (5), Dharmacon
Inc.,Lafayette,Colorado80026
QLD4072,Australia
Chris A. Helliwell (2), CSIRO Plant Amy Kiger (4), Division of Biological
Sciences, University of California, San
Industry,CanberraACT2601,Australia
Diego,LaJolla,California92093
Gabriela Herna´ndez-Hoyos (12), Divi-
FabriceA.Kolb(19),FriedrichMiescher
sion of Biology, Cal Tech, Pasadena,
Institute for Biomedical Research, 4002
California91125
Basel,Switzerland
Tom C. Hobman (19), Department of
EberhardKrausz(15),yCenixBioScience
Cell Biology, University of Alberta,
GmbH,01307Dresden,Germany
Edmonton,Alberta,CanadaT6G2E1
Andrea Kronke (15), Cenix BioScience
David E. James (24), Diabetes and
GmbH,01307Dresden,Germany
Obesity Research Program, Garvan In-
stitute of Medical Research, Darlin- Devin Leake (5), Dharmacon Inc.,
ghurst,SydneyNSW2010,Austalia Lafayette,Colorado80026
yCurrentaddress:MaxPlanckInstituteofMolecularCellBiologyandGenetics,01307Dresden,
Germany
contributors to volume392 xi
David L. Lewis (20), Mirus Corporation, DaneshMoazed(17),DepartmentofCell
Madison,Wisconsin53719 Biology, Harvard Medical School, Bos-
ton,Massachusetts02115
Ming-Jie Li (13), Division of Molecular
Biology, Beckman Research Institute of AndrewN.Muirhead(24),BenitecLtd.,
the City of Hope, Duarte, California University of Queensland, St. Lucia
91010-3000 QLD4072,Australia
Concetta Lipardi (21), National Cancer Kent Nybakken (4), Department of Ge-
Institute, National Institutes of Health,
netics,HarvardMedicalSchool,Boston,
Bethesda,Maryland20892
Massachusetts02115
MuthiahManoharan(16),AlnylamPhar-
Dmitriy Ovcharenko (15), Ambion Inc.,
maceuticals, Cambridge, Massachusetts
Texas78744-1832
02142
William S. Marshall (5), Dharmacon Kusum Pandey (23), Stanford University,
Inc.,Lafayette,Colorado80026 Department of Pediatrics, Stanford,
California,94305-5208
BernardMathey-Prevot(4),Department
of Genetics, Harvard Medical School, Jack M. Parent (11), Program in Neu-
Drosophila RNAi Screening Center, roscience,UniversityofMichigan, Ann
Boston, Massachusetts02115 Arbor,Michigan48109-0669
AlexeyA.Matskevich (14),Department BruceM.Paterson(21),NationalCancer
ofPathologyandCellBiology,Jefferson Institute, National Institutes of Health,
MedicalCollege,Philadelphia,Pennsyl- Bethesda,Maryland20892
vania19107-5587
Cynthia P. Paul (8), Department
Narelle J. Maugeri (24), Benitec Ltd., of Biological Chemistry, University of
University of Queensland, St. Lucia Michigan,AnnArbor,Michigan48109
QLD4072,Australia
RobinC.May(18),HubrechtLaboratory, NorbertPerrimon(4),DepartmentofGe-
netics,HowardHughesMedicalInstitute
3584CTUtrecht,TheNetherlands*
and Harvard Medical School, Boston,
Karen McGinnis (1), Department of Massachusetts02115
Plant Sciences, University of Arizona,
Tucson,Arizona85721 Craig Pikkard (1), Department of Biol-
ogy, Washington University, St. Louis,
RachelMeyers(16),AlnylamPharmaceu- Missouri63130
ticals,Cambridge,Massachusetts02142
Ronald H. A. Plasterk (18), Hu-
Makoto Miyagishi (6), Gene Function
brecht Laboratory, 3584 CT Utrecht,
Research Center, National Institute of
TheNetherlands
Advanced Industrial Science and Tech-
nology (AIST), Tsukuba Science City Steven Read (5), Dharmacon Inc.,
305-8562,Japan;21stCenturyCenterof Lafayette,Colorado80026
Excellence Program, Graduate School
of Medicine, The University of Tokyo, Angela Reynolds (5), Dharmacon Inc.,
Tokyo113-8656,Japan Lafayette,Colorado80026
*Current address: School of Biosciences, University of Birmingham, Birmingham B15 2TT,
UnitedKingdom
xii contributors to volume 392
RobertR.Rice(24),BenitecLtd.,Univer- Bill Sun (14), Department of Pathology
sity of Queensland, St. Lucia QLD and Cell Biology, Jefferson Medical
4072,Australia College, Philadelphia, Pennsylvania
19107-5587
Eric Richards (1), Department of Biol-
ogy, Washington University, St. Louis, Nasser Tahbaz (19), Department
Missouri63130 of Cell Biology, University of Alberta,
John J. Rossi (13), Division of Mole- Edmonton,Alberta,CanadaT6G2E1
cular Biology, Beckman Research In- Kazunari Taira (6), Department of
stitute of the City of Hope, Duarte,
Chemistry and Biotechnology, Univer-
California91010-3000
sity of Tokyo, Tokyo 113-8656, Japan;
Christoph Sachse (15), Cenix Bio- Gene Function Research Center, Na-
Science GmbH, 01307 Dresden, Ger- tional Institute of Advanced Industrial
many ScienceandTechnology(AIST),Tsuku-
baScienceCity305-8562,Japan
Masayuki Sano (6), Gene Function
Research Center, National Institute Claude Trudel (15), Ambion Inc.,
of Advanced Industrial Science and Austin,Texas78744-1832
Technology (AIST), Tsukuba Science
City305-8562,Japan David L. Turner (11), University
of Michigan, Mental Health Re-
Stephen A. Scaringe (5), Dharmacon search Institute, Ann Arbor, Michigan
Inc.,Lafayette,Colorado80026 48109-0669
Lisa Scherer (10), City of Hope Andre´ Verdel (17), Department of Cell
Medical Center, Division of Mole- Biology, Harvard Medical School,
cular Biology, Beckman Research Boston,Massachusetts02115
Institute,Duarte,California91010-3000
Anne B. Vojtek (11), Department of
Petra L. Sedlak (24), Benitec Ltd.,
Biological Chemistry, University of
University of Queensland, St. Lucia
Michigan, Ann Arbor, Michigan
QLD4072,Australia
48109-0669
Lyudmila Sidorenko (1), Department Hans-Peter Vornlocher (16), Alnylam
of Plant Sciences, University of
EuropeAG,95326Kulmbach,Germany
Arizona,Tucson,Arizona85721
Todd Smith (1), Department of Biology, Andrew Walsh (15), Cenix BioScience
GmbH,01307Dresden,Germany
Washington University, St. Louis,
Missouri63130 Juan Wang (3), University of Wisconsin
BirteSonnichsen(15),CenixBioScience School of Pharmacy, Pharmaceutical
GmbH,01307Dresden,Germany Sciences Division, Madison, Wisconsin
53705-2222
Nathan Springer (1), Department of
Plant Biology,UniversityofMinnesota, Tsu-Wei Wang (11), Program in Neu-
St.Paul,Minnesota55108 roscience, University of Michigan, Ann
Arbor,Michigan48109-0669
David S. Strayer (14), Department
ofPathologyandCellBiology,Jefferson Peter M. Waterhouse (2), CSIRO
MedicalCollege,Philadelphia,Pennsyl- Plant Industry, Canberra ACT 2601,
vania19107-5587 Australia
contributors to volume392 xiii
Qin Wei (21), National Cancer Institute, YanZeng(22),DepartmentofMolecular
National Institutes of Health, Bethesda, Genetics and Microbiology, Howard
Maryland20892 HughesMedicalInstitute,DukeUniver-
sity Medical Center, Durham, North
Jon A. Wolff (20), Department of
Carolina27710
Pedicatrics, Waisman Center, Univer-
sity of Wisconsin-Madison, Madison, Haidi Zhang (19), Friedrich Miescher
Wisconsin53705 Institute for Biomedical Research, 4002
Basel,Switzerland
Tuya Wulan (1), Department of Biol-
ogy, Washington University, St. Louis,
Missouri63130
Jenn-Yah Yu (11), Program in Neu-
roscience,UniversityofMichigan, Ann
Arbor,Michigan48109-0669
[1] transgene-induced RNAiinmaizeand arabidopsis 1
[1] Transgene-Induced RNA Interference
as a Tool for Plant Functional Genomics
By Karen McGinnis, Vicki Chandler, Karen Cone,
Heidi Kaeppler, Shawn Kaeppler, Arthur Kerschen,
Craig Pikaard, Eric Richards, Lyudmila Sidorenko,
Todd Smith, Nathan Springer, and Tuya Wulan
Abstract
RNAinterference(RNAi)isapowerfultoolforfunctionalgenomicsin
anumber ofspecies. The logistics and procedures fordoing high-through-
putRNAitoinvestigatethefunctionsoflargenumbersofgenesinArabi-
dopsis thaliana and in Zea mays are described. Publicly available plasmid
vectors that facilitate the stable chromosomal integration of inverted re-
peat transgenes that trigger RNAi have been used to generate more than
50independenttransgeniclineseachinArabidopsisandmaize.Analysisof
mRNAabundanceofthetargetedgenesinindependentlinestransformed
with distinct constructs indicates that the success of RNAi-induced silenc-
ingisgenedependent.mRNAlevelswerenotdetectablyreducedforsome
genes, but were dramatically reduced for a number of genes targeted. A
common pattern was that multiple independent lines transgenic for the
sameconstructshowedthesameextentofsilencing.Thischapterdescribes
theproceduresusedtogenerateandtesttransgeniclinesmediatingRNAi
inArabidopsis and maize.
Introduction
In recent years, RNA interference (RNAi) has been exploited as a
tool for investigating gene functions in numerous organisms (reviewed by
Agrawal et al., 2003; Bailis and Forsburg, 2002; Carpenter and Sabatini,
2004; Dawe, 2003; Fraser et al., 2000; Martienssen, 2003; Matzke and
Matzke, 2003; Paddison et al., 2004; Simmer et al., 2003). Gene silencing
bytransgene-inducedRNAiisusefulbecausethelossorreductionofgene
functionisdominant,circumventingtheneedtogeneratehomozygousloss-
of-function mutations. This not only saves time, which is especially useful
in plants with long generation times, but also allows gene knockdown
studies to be conducted in F hybrids that inherit the RNAi-inducing
1
transgene from only one parent. Likewise, because RNAi mechanisms
can target multiple closely related mRNAs, dominant loss-of-function
Copyright2005,ElsevierInc.
Allrightsreserved.
METHODSINENZYMOLOGY,VOL.392 0076-6879/05$35.00
2 RNA interference [1]
phenotypes can be generated for entire gene families or for multiple
orthologous genes in polyploids (Lawrence and Pikaard, 2003).
Methods for targeted RNAi-mediated gene silencing in plants have
been reviewed recently (Waterhouse and Helliwell, 2003), and RNAi has
proven to be a useful method in a number of plant studies (Ketelaar et al.,
2004; Lawrence et al., 2004; Wesley et al., 2001; Xiao et al., 2003; Zentella
et al., 2002). In this chapter, we focus on the logistics of doing high-
throughput RNAi to investigate the functions of large numbers of targeted
genes in plants, specifically in Arabidopsis thaliana and Zea mays. The
approaches summarized here stem from the authors’ communal efforts to
generate RNAi lines targeting several hundred genes. This effort has been
funded by the United States National Science Foundation as part of its
Plant Genome Research Program and represents one of the first large-scale
efforts to use RNAi as a tool for plant functional genomics. The goal of our
project is to investigate the function of chromatin-modifying genes in maize
and Arabidopsis and to provide the research community with tools to
study these genes, including RNAilinesthat knockdown their expression
(detailed information available at http://ChromDB.org/). Central to the
effort has been the design and construction of publicly available plasmid
vectors that facilitate the stable chromosomal integration of transgenes
that trigger RNAi. Each RNAi-inducing transgene consists of a portion
of a targeted gene’s cDNA cloned in inverted orientation downstream
fromastrong,widelyexpressedpromoter,adesignbasedonthepioneering
work of the Waterhouse laboratory (Waterhouse et al., 1998). When ex-
pressed, the inverted repeats cause the transcript to fold back on itself to
generateadouble-strandedRNA(dsRNA)thatactsasasubstrateforthe
DicerandRISCcomplexes,ultimatelyleadingtothereductionorelimina-
tionofhighlyhomologousmRNAsinthecell(Hannon,2002).Thesalient
featuresofthevectorsandtheapproacheswehaveusedtogeneratelarge
numbersoftransgenicRNAilinesasefficientlyaspossiblearedescribedin
the following two sections, the first focused on A. thaliana and the second
on maize.
High-Throughput Generation of A. thaliana RNAi Lines
Vectors for Generating Transgenic Plants and Inducing
RNAi of Targeted Genes
The method of choice for genetically engineering A. thaliana is
Agrobacterium tumefaciens-mediated genetic transformation. A. tumefa-
ciens is a common soil bacterium that can transmit a segment of DNA,
known as the T-DNA (transferred DNA), from a large resident plasmid,
[1] transgene-induced RNAiinmaizeand arabidopsis 3
called the Ti plasmid, to a recipient plant cell through a process that
closely resembles bacterial conjugation (Gelvin, 2000, 2003; Zupan et al.,
2000). The T-DNA is delimited by short direct repeats, known as the
left border (LB) and right border (RB), which are acted on by the ma-
chinery involved in generating the transferred DNA. Most of the machin-
ery required for T-DNA transfer is encoded elsewhere on the Ti plasmid
andactsonthebordersequencesintrans.Asaresult,anysegmentofDNA
flanked by LB and RB sequences can be transferred from A. tumefaciens
to a plant cell, even if the T-DNA is located on a plasmid distinct from
theTiplasmid.Infact,atwo-plasmid,orbinary,systemiscommonlyused
for A. tumefaciens-mediated plant transformation. The plasmid that
carries the T-DNA (the binary vector) can replicate in both E. coli and
A. tumefaciens, which allows all cloning steps to be conducted by using
E. coli before mobilization of the vector plasmid into A. tumefaciens for
plant transformation.
ThebinaryvectordesignedbyourFunctionalGenomicsofChromatin
(FGC) consortium, which serves as our vector of choice for generating
transgenic Arabidopsis RNAi lines, is pFGC5941 (Fig. 1). The complete
nucleotide sequence of pFGC5941 can be retrieved from GenBank
(AY310901), restriction enzyme cleavage maps can be found at the FGC
project Web site (http://ChromDB.org/), and the plasmid can be obtained
from the Arabidopsis Biological Resource Center (ABRC, stock number
CD3-447). The backbone of pFGC5941 is the pCAMBIA1300 binary
vector plasmid developed by the Center for Application of Molecular
Biology to International Agriculture (CAMBIA; http://www.cambia.org).
pCAMBIA1300 has two origins of replication, a wide-host-range origin
thatfacilitatesplasmidreplicationinA.tumefaciensandthepBR322origin
for replication in E. coli (Hajdukiewicz et al., 1994), as well as an amino-
glycoside phosphotransferase gene that confers kanamycin resistance in
both E. coli and A. tumefaciens. Within the T-DNA region of pFGC5941,
adjacenttotheLB,isaphosphinothricinacetyltransferasegenedrivenby
the mannopine synthase 20 (Mas20) promoter. This gene confers herbicide
resistance for bialaphos and phosphinothricin and is commonly known as
the BAR (Basta resistance) gene. Adjacent to the BAR gene is the cauli-
flowermosaicvirus(CaMV)35Spromoterregion,whichincludesenhancer
sequences upstreamofthegenepromoter.The 35S promoterisexpressed
in most plant cells, and this strong promoter drives the expression of the
various inverted repeat (IR) transgenes engineered into the pFGC5941
vector. The tobacco mosaic virus omega leader sequence, which is known
toactasatranslationalenhancerinplants(Gallie,2002;Sleatetal.,1987),
servesasthe50UTRforthevarioustransgenes.Theomegasequencemost
likely is irrelevant for RNAi, which depends on RNA production rather