Title: Lecture outline: MutantAnalysis
1Lecture outline MutantAnalysis (so you have
your mutants . . . NOW WHAT?)
Biochemical strategies for identifying where the
gene product is found in the cell Subcellular
fractionation Detection of proteins Organelle
import in vitro Case history of a confounding
phenotype, endosperm opacity Introduction to the
mutants Tools for analysis Allelism
tests Microarray analysis Protein
analysis Microscopy Other important tools (for
individual discussion after class)
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3Proteins can be localized in a variety of
subcellular locationsor even secreted from the
cell How do we separate the organelles to assay
for a particular protein?
Taken from Buchanan, Gruissem, and Jones (2000)
Biochemistry and molecular biology of plants.
Fig. 1-14
4Antibodies facilitate detection of
proteins Peptide antibodies can be generated
based on deduced amino acid sequences. (more in
the lab session after lecture) Antibodies can
also be generated against recombinant proteins.
Coding sequences of the gene of interest and an
affinity tag for easy purification can be
expressed as fusion proteins in bacterial or
yeast systems.
5Separation of membrane associated and
cytosolic proteins by differential centrifugation
Homogenize tissue, filter to remove debris
Adapted from Robinson DG, Hinz G and Oberbeck K
(1994) Isolation of endo- and Plasma membranes
In. Plant Cell Biology. A practical approach.
Eds. N Harris and KJ Oparka (IRL Press, Oxford)
pp245-272.
6Characteristics of homogenization
buffer Osmoticum such as sucrose or mannitol
(0.2-0.4 M) Need to keep organelles
intact Buffering agent such as TRIS, phosphate
or HEPES (20-50 mM) Salts such as KCl to disrupt
cytoskeletal structures Divalent Cations such as
Mg to keep ribosomes with ER Protease
inhibitors
7Separation of membrane associated and
cytosolic proteins by differential centrifugation
Homogenize tissue, filter to remove debris
Adapted from Robinson DG, Hinz G and Oberbeck K
(1994) Isolation of endo- and Plasma membranes
In. Plant Cell Biology. A practical approach.
Eds. N Harris and KJ Oparka (IRL Press, Oxford)
pp245-272.
8Subcellular organelles have a range of buoyant
densities that provide a means for fractionation
Robinson DG, Hinz G and Oberbeck K (1994)
Isolation of endo- and Plasma membranes In.
Plant Cell Biology. A practical approach. Eds.
N Harris and KJ Oparka (IRL Press, Oxford)
pp245-272.
9Commonly Used Organelle Markers(Enzymatic
Activities)
- Plasma membrane
- K stimulated ATPase, vanadate inhibited
- glucan synthetase II
- Mitochondria
- cytochrome c oxidase
- Endoplasmic reticulum
- NADH cytochrome c reductase, antimycin A
insensitive - Golgi Vesicles
- IDPase
- glucan synthetase I
- Tonoplast
- NO3- inhibited ATPase (still uncertain)
Adapted from Hodges and Mills (1988) Isolation of
the plasma membrane. In Methods for Plant
Molecular Biology (Academic Press, San Diego) pp
41-54.
10Linear sucrose gradients separate membranes based
upon their buoyant densities. Enzymatic
activities can be used as markers for organelles
From Hodges, TK and Leonard, RT. (1974)
Purification of a plasma membrane-bound adenosine
triphosphatase from plant roots. In Methods in
Enzymology. pp. 392-406.
11Immunoblots (western blots) allow detection of
specific proteins in a complex mixture
Fig. 3-44 Lodish et al., Molecular Cell Biology
4th Edition, W. H. Freeman Co.
12Membrane Markers(Antibodies)
- Plasma membrane
- pmATPase
- Mitochondria
- ß-ATPaseE
- Endoplasmic reticulum
- BiP
- KDEL
- Golgi Vesicles
- RGP
- JIM84
- Tonoplast
- H-PPiase
13Immunoblot detection of marker proteins reveals
differential migration of organelles through a
linear sucrose gradient
From Movafeghi A. Happel N, Pimpl P, Tai G.-H.,
and Robinson D.G (1999). Arabidopsis Sec21p and
Sec23p Homologs. Probable coat proteins of plant
COP-coated vesicles Plant Physiol 119 1437-1446
14EDTA treatment removes ribosomes from ER
membranes and causes a decrease in buoyant
density of the organelle. Because this change is
not observed for other organelles, it can be used
as a diagnostic assay for ER.
From Movafeghi A. Happel N, Pimpl P, Tai G.-H.,
and Robinson D.G (1999). Arabidopsis Sec21p and
Sec23p Homologs. Probable coat proteins of plant
COP-coated vesicles Plant Physiol 119 1437-1446
15Overview of subcellular destinations for nuclear
encoded proteins
Fig. 17-1 Lodish et al., Molecular Cell Biology
4th Edition, W. H. Freeman Co.
16Organelle import assays Synthesize RNA encoding
the protein of interest from a cDNA clone in
vitro Translate the protein with a cell free
extract (commercially available from rabbit
reticulocytes or wheat germ) Carry out
translation in the absence and presence of canine
pancreatic microsomes (also can use plant
microsomes but have to make them as they arent
commercially available) Confirm size shift from
precursor to mature form Confirm resistance to
protease degradation
17Protease protection experiments demonstrate
localization of a protein within a membrane-bound
compartment
18Protease protection experiments demonstrate
import of a protein into isolated chloroplasts
Mooney B.P., Miernyk J.A., and Randall D.D.
(1999) Cloning and characerization of the
dihydrolipoamide S-acetyltransferase subunit of
the plastid pyruvate Dehydrogenase complex (E2)
from Arabidopsis. Plant Physiol 120, 443-451.
19Proteins imported into chloroplasts can stay in
stroma or undergo a second translocation across
the thylakoid membrane
Fig. 17-1 Lodish et al., Molecular Cell Biology
4th Edition, W. H. Freeman Co.
20Protease protection assays can also be used to
demonstrate import into thylakoids
Mosely JL, Page MD, Alder NP, Eriksson M, Quinn
J, Soto F, Theg SM, Hippler M, and Merchant S.
(2002) Reciprocal expression of two candidate
di-iron enzymes affecting photosystem I and
Light-harvesting complex accumulation. Plant
Cell 14 673-688
21Protease protection experiments demonstrate
import of a protein into isolated mitochondria
Kroczynska B., Zhou R., Wood C., and Miernyk
J.A. (1996) AtJ1, a mitochondrial Homologue of
the Escherichia coli DnaJ protein. Plant Mol
Biol 31, 619-629.
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23Endosperm mutants are often opaque. Compared to
normal kernels, mutants have little capacity to
transmit light
24opaque-2 (left) and normal (right) kernels
25Opacity mutants can be recessive, dominant, or
semi-dominant
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28Normal endosperm
Opacity mutants can be semi-dominant
Zein-deficient mutants
fl2
Mc1
Soft-endosperm mutants (normal zein levels)
29Opacity mutants can be recessive, dominant, or
semi-dominant Some but not all affect the major
storage proteins called zeins The dominant and
semi-dominant mutants characterized to date would
not have been discovered by gene tagging
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31A frameshift mutation alters the small ?-zein
isoelectric point in the Mc mutant
32Zeins are the most abundant maize seed proteins
Kernel maturation
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34Microarrays are very useful for recognizing
complex changes in gene expression, but do not
necessarily reveal the role of the genes in
producing a phenotype
35The opaque phenotype remains elusive
Hunter et al., (2002) Maize Opaque Endosperm
Mutations Create Extensive Changes in Patterns of
Gene Expression Plant Cell 14 2591-2612
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37Immunoblot showing changes in major storage
proteins between normal and opaque maize lines
38Decreased zein and absence of 32 kDa protein are
both downstream effects of the opaque-2 mutation
N
o2
39Correlative gene expression can be misleading
fl2
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42floury-2 protein bodies cluster near nuclei
43Electron micrographs showing abnormal protein
bodies from some of the opacity mutants of maize
44Retention of an alpha-zein at the periphery of
floury-2 protein bodies may explain the
phenotype seen in electron micrographs
Adapted from Lending and Larkins (1989) Changes
in the Zein Composition of Protein Bodies during
Maize Endosperm Development Plant Cell 1
1011-1023
45Important mutant analysis tools not covered in
lecture (by no means an inclusive list) GFP
visualization Yeast or bacterial two hybrid
analysis Hybridization in situ Immunolocalization
Analysis of post-translational modifications Promo
ter deletion analysis
46Considerations beyond the scope of this
lecture Duplicate genes Does a lack of mutant
phenotype really mean my gene isnt important?
Overlapping expression patterns Does my gene
have a redundant function? Is it a
duplicate? Maize isnt yeast Time for more
mutagenesis Get used to it
47- What should I remember from this lecture?
- Mutant analysis can be complicated
- Tools that might help are
- subcellular localization assays
- where is the protein?
- can this info help determine function?
- microscopy
- does my mutant have visible changes in organs,
tissues, cells or organelles? - protein analysis
- does my mutant have quantitative or
qualitative changes in proteins? - do these changes suggest protein function?
- microarrays
- can I detect patterns of gene expression that
give clues to the phenotype?
48Many thanks to Norma Houston for developing the
computer exercise