Lab Report #3

Forward Genetics 2:
Complementation, Epistatic Relationships,
Sequencing Analysis, & Prediction of Protein
Function in Arabidopsis thaliana with emphasis on
trichome morphogenesis
(*companion paper to Forward Genetics: An approach to the mapping and identification of a mutation in
trichome morphogenesis of Arabidopsis thaliana (4/24/2003) pgs 1-18)

Michael Wade Jackson

BICD 101: Eukaryotic Genetics
6/11/2003

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 Abstract:

Based on previous investigation1 the location of the mutation of interest (mut-1) was
mapped to chromosome 5 of Arabidopsis on the BAC K14B20. A complementation test
was preformed and the outcome was an allelic relationship between mut-1 and mut-3.
This complementation group was the focus of further investigation. The gene of interest
was discovered through the use of Agrobacterium transformation. A wild-type gene
construct was inserted to rescue the mutant phenotype. The construct that produced a
three prong trichome (wt) was the clone AT5G65930.1 which contains the ZWICHEL
gene. ZWICHEL is one of the candidate genes located on K14B20. The actual names of
mut-1 and mut-3 were determined to be zwi-3 and zwi-9311 after sequence comparison
analysis using MacVector. The mut-1 and mut-3 sequences were compared to the
Complete ZWI gene2. A double-mutant test involving an and nok displayed that AN is
epistatic to NOK. Final analysis was performed on the possible role of the functional
domains and structural motifs of the ZWICHEL protein in relation to its role as a kinesin-
like calmodulin-binding protein (KCBP) involved in trichome morphogenesis3.
________________________________________________________________________

INTRODUCTION: were allelic. The interim time was spent

The goal of a forward genetics approach
is to: 1) choose a biological process ->
2) isolate a mutant -> 3) map the gene of
interest -> 4) study the protein -> 5)
evaluate the function. This process was
initiated during the first three weeks of
this course to identify and map a
mutation of interest. The mutation
selected was mut-1 which produced a
reduction of the number of branches on
the trichomes of Arabidopsis thaliana.
The Arabidopsis ecotype of interest was
Columbia (Col). The first three weeks
of experimentation provided the
mapping and localization of the mut-1
on chromosome 5 in the BAC K14B20.
This information along with the TAIR
database produced an assortment of
approximately 11 candidate genes that
might be the target of the mutation on
K14B20. This was the extent of the
knowledge in relation to mut-1 at the
end of the three week period. A
complementation test was setup in order
to determine if mutations 1, 2, 3, 5, or 6
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learning a reverse genetics approach
involving mouse ES and EF cell
cultures. Upon return to the process of
forward genetics the plants that were the
result of the complementation crosses
provided the insight that mut-1 and mut-
3 were allelic (i.e. in the same
complementation group). Based on this
knowledge the next logical step was to
try and rescue the mutant phenotype (2
prongs) by transformation4 with
Agrobacterium containing a wild-type
construct of each of the 11 candidate
genes. This facilitated the identification
of the clone AT5G65930.1 (ZWICHEL,
ZWI) as the gene of interest. ZWI gene
encodes KCBP which plays an integral
role in trichome development. The gene
once identified was then subjected to
sequence comparison analysis using the
TAIR database along with MacVector to
elucidate variance in the ZWI sequence
that would account for the phenotypic
mutation. The outcome of these
sequence comparisons yielded the
locations of mut-1 and mut-3 within
ZWI. The identification of the mutations
was then provided and mut-1 and mut-3
were zwi-3 allele and zwi-9311 allele,
respectively.
DOUBLE-MUTANT ANALYSIS:
In conjunction with the investigation of
mutations in ZWI, trichome
morphogenesis as a process was
analyzed involving two other mutations.
These mutations were in
ANGUSTIFOLIA (AN) and NOECK
(NOK). AN is an inducer to branching
and NOK is a suppressor to branching of
trichomes. Using an and nok mutants
an;nok double mutants were created and
observed to determine if these mutations
had additive, nugatory, or epistatic
relationship to the phenotype. The
an;nok double mutants established that
AN is epistatic to NOK. The goal of this
analysis was to look at trichome
morphogenesis as a process and to
investigate possible pathways involved
in the suppression and activation of
branching in the unicellular trichome.
BIOINFORMATICS: ANALYSIS OF
ZWI PROTEIN
The final analytical tool in the arsenal is
the establishment of the function of the
ZWI protein. ZWI is a KCBP which
appears to play a role in the cell cycle
construction and differentiation of the
unicellular trichome. Using the BLAST
database the sequence of the ZWI was
analyzed yielding three functional
domains of interest and two structural
motifs that may explain the function of
the ZWI protein. This analysis also
identified the sequence similarity
between this protein and homologues in
other organisms. The goal of this
analysis is to better understand the ZWI
protein and its attributes in order to
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gauge why a mutation such as zwi-3/
zwi-9311 can cause the phenotypic
changes observed.
MATERIALS & METHODS:
PREP of PCR product for DNA
SEQUENCING:
The two essential steps in preparing a
DNA sample for sequence analysis are
amplification and purification. The
DNA region of interest is first amplified
by PCR5. Using primers selected for a
region that is approximately 700bp in
length of the ZWI gene; DNA from wt.
Col., Mut-1 (Col.), Mut-2 (RLD), Mut-3
(RLD), Mut-5 (RLD) were amplified to
produce a sample size adequate for
sequencing. This sample once amplified
was then purified through the use of
agarose gel electrophoresis in a process
of size exclusion6. Once the sample had
been run on the gel the brightest band
was selected and removed. This band
was then purified and separated from the
agarose using Qiagen kit. The sample
was then analyzed using UV
spectroscopy to determine purity and
concentration. The PCR products were
then outsourced for sequencing. The
method used was direct sequencing
which utilizes the fluorescent labeling of
the bases to produce the respective
sequence7.
Agrobacterium-Mediated
TRANSFORMATION:
Transformation via Agrobacterium
utilized a natural process to introduce
foreign DNA into a host cell via a
bacterial vector. The class introduced 11
separate T-DNA plasmids into
individual plants via spray bottle soaking
of transformed Agrobacterium in media.
The goal of these transformations was to
rescue the mutant phenotype (2 prongs) identified using the SMART database11.
by inserting a wild-type construct of the The ZWI protein was then compared to
gene into the plant in order to produce a other organisms to find structurally
wild-type (3 prongs) appearance in the similar proteins based on amino acid
F1 generation. If the wt inserted copy of sequence. This was done using a protein
the gene produced a wt appearance the BLAST12. These websites allowed for
mutation is found to be in that gene. The the analysis of the ZWI protein based on
Agrobacterium itself contained a Ti the knowledge that the complete
plasmid with the wt copy of one of Arabidopsis genome is mapped.
eleven genes of interest as the T-DNA.
The plasmid itself is non-virulent and RESULTS:
contains only the machinery necessary to Complementation Test:
introduce the T-DNA of interest into
Arabidopsis. The process of selecting Cross Phenotype Cross Phenotype
the transformed plants involved the 1x2 Wt 2x5 Wt
addition of a selectable marker to the Ti- 1x3 M 2x6 Wt (?)
plasmid. Kanamycin-resistance was 1x5 Wt 3x5 Wt
added to the plasmid to allow the 1x6 Wt (?) 3x6 Wt (?)
transformed seeds to grow on an agar 2x3 Wt 5x6 Wt (?)
plate that contained kanamycin
(antibiotic). If the seed was not The complementation test results above
transformed it did not grow on the agar allow the grouping of mutation 1 and 3
plate after an initial period of growth. into a complementation group. The
Only the healthy looking green plants relationship between 1 and 3 is then said
were transformed seeds. The healthy to be allelic. The mutations in mut-1
plants were then transferred to pots and and mut-3 are then located on the same
observed once mature trichomes gene ZWI but occur at different loci on
developed to determine which of the the gene. The complementation test did
eleven genes of interest rescued the provide some level of uncertainty as that
mutant phenotype8,9,10. the groups denoted by Wt (?) were
mixed on the phenotype as to whether
DNA & PROTEIN SEQUENCE this cross resulted in mutant or Wt
ANALYSIS: progeny. The significant result of this
The analysis and comparison of the wt data is that mut-1 and mut-3 are in the
ZWI gene sequence to the mutations same gene, ZWI.
(1,2,3,5) was carried out using the
sequence alignment program TRANSFORMATION: Agrobacterium
MacVector. The sequences once aligned
were analyzed for polymorphisms which Clone Phenotype Clone Phenotype
might be mutations that could cause 880 M 930 Wt
developmental problems in trichomes. 890 M 940 M
The ZWI gene once identified was then 900 M 950 M
analyzed according to the protein ZWI it 910 M 960 M
produced. The structural motifs and
920 M 970 M
functional domains of interest were
925 M
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 Frequency of Transformation: of significance and require review. In
mut-1 the change at 2238 in an Intron is
32/4000=0.8% significant as it occurs near the start of
an Intron. The change from a guanine to
The transformation by Agrobacterium an adenine causes an incorrect splicing
resulted in a 0.8% transformation rate. event that gives rise to a shift in the
This means that 32 plants out of 4000 reading frame producing a stop codon
integrated the T-DNA of interest into the around 2080. This mutation is identified
host genome. as zwi-3. The second mutation of
interest is in mut-3 at position 1932 in an
The clone of interest that rescued the Exon which changes the triplet code of
mutant phenotype was AT5G65930.1. the corresponding amino acid from TTG
The gene located on this clone as = Leu to TAG = Stop. This mutation is
provided by the TAIR database is known as zwi-9311. This mutation
ZWICHEL (ZWI). occurs in an Exon and causes the
termination of transcription (RNA ->
DNA SEQUENCE ANALYSIS: Protein). These two mutations further
establish the allelic relationship of mut-1
Mutation Location Base Exon Intron and mut-3. The mutations occur in the
Mut 1 2238 G/A X same gene ZWI and are at different loci.
zwi-3
BIOINFORMATICS EXERCISE:
Mut 2 1768 A/G X
1984 G/T X
Results of protein BLAST:
Mut 3 1768 A/G X
zwi- 1932 T/A X
Species Common Percent %
9311 1984 G/T X
Name ID
Mut 5 1768 A/G X
Gossypium Cotton 939/1206
1984 G/T X
hirsutum 77%
The DNA sequence comparisons Solanum Potato 912/1256
tuberosum 72%
between the complete ZWI and the four
mutations yielded the above results. The Nicotiona Tobacco 906/1256
mutations of interest are: mut-1, 2238, tabacum 72%
G/A, Intron, and mut-3, 1933, T/A, Oryza Rice 895/1223
Exon. The other mutations occurring in sativa 73%
mut-2, 3, and 5 are due to the difference Zea mays Corn 858/1192
in ecotype. Mut-2, 3, & 5 are of the 71%
RLD ecotype while mut-1 and Complete
ZWI gene are of Col. This means that The protein BLAST analysis provides
the mutations at 1768 and 1984 are information as to the similarity of
polymorphisms that occur in non-coding proteins within other organisms. These
regions that are specific to the RLD five candidates contain a protein similar
ecotype. These polymorphisms do not to the ZWI protein. The ZWI protein is
give rise to phenotypic mutation. The a KCBP which is involved in trichome
mutations that occur in mut-1 and 3 are morphogenesis. The correlation of this
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protein to five other plants with a 70% or and plasma membrane. KISc is a
greater similarity shows the conservation kinesin motor protein which occurs at
of protein motifs and structure within the C terminal end of the protein. These
organisms. The sixth organism on the structural and functional features allow
list is the purple sea urchin for a more complete understanding of
(Strongylocentrotus purpuratus) with a ZWI protein.
percent ID of 40%. This is an organism
from a different kingdom and shows DOUBLE MUTANT ANALYSIS:
sequence conservation amongst
organisms universally. Mutant Phenotype / Phenotype /
leaves trichome
Results of SMART analysis: (Q9FHN8) an ; an narrow Less
leaves Branch
nok ; nok Glassy More
trichome Branch
an ; nok Glassy Less
an ; nok trichome + Branch
Structural Location Functional Loc. narrow 2 prong
Motif Domain leaves
Coiled- 615-673 MyTH4 115-
Coil 274 The goal of the double mutant analysis
Coiled- 749-855 B41 275- was to investigate trichome
Coil 499 morphogenesis as a process. The
KISc 886- investigation involved mutations in AN
1217 and NOK to capture the relationship
between these two genes in on of a
The Simple Modular Architecture multitude of possible pathways of
Research Tool (SMART)13 analysis trichome development. The outcome of
provides insight into the structure of the this analysis was the determination that
ZWI protein itself. The large shapes are AN is epistatic to NOK which means that
functional domains and the colored the an;an mutants have the same
bands are the structural motifs. The phenotype as the an;nok double mutants.
structural motifs of interest are two The determination of the double mutant
coiled-coil domains which occur at 615- phenotype was made possible by
673 and 749-855. These numbers associated phenotypic variance of these
correlate with the amino acids that make mutants from Wt. The double mutant
up the ZWI protein. Three functional displays glassy trichomes with narrow
domains were found within ZWI leaves. This mutant has a lower level of
(KCBP) and they were MyTH4, B41, trichome branching establishing the
and KISc. MyTH4 is a domain found in epistatic relationship.
myosin and kinesin tails further NOK –I AN -> Branch initiation
supporting that KCBP is a minus (-)
directional motor. B41 is an ERM DISCUSSION:
protein domain that is associated with The final two weeks of the experiment
the interaction between the cytoskeleton yielded data surrounding the final two
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stages of a forward genetics approach.
These stages are: 4) study the protein
and 5) evaluate the function. The first
three weeks of the experiment produced
a map to the approximate position
associated with a particular BAC clone.
K14B20 contains approximately 11
candidate genes of interest. The
determination was made that the gene
involved in this process was ZWI.
The sequence of experiments that
comprised the final two weeks are: 1)
evaluation of the progeny from
complementation test, 2) Identify which
gene is responsible for mutation #1, by
rescuing the mutant phenotype with
wild-type constructs of candidate genes,
3) Determine whether one particular
candidate gene is responsible for
mutation #1 by sequencing that gene in
wild-type and mutant plants, 4)
Complete a double mutant analysis, 5)
Computer aided analysis of the protein
responsible for mutation #1. These
activities directed the completion of the
final two goals of a forward genetics
approach. These steps provided the
necessary information concerning the
ZWI gene and the subsequent structure
and function of the ZWI protein.
Procedural Conclusions:
• The outcome of the
complementation test was the
discovery that mut-1 and mut-3
were of the same
complementation group;
therefore mut-1 and mut-3 are
allelic.
• The transformation involving
Agrobacterium identified
AT5G65930.1 as the construct
that contained the gene of
interest. The gene was then
identified as the ZWICHEL gene.
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ZWICHEL is a kinesin-like
calmodulin binding protein and is
involved in the expansion and
growth of trichome branches.
• The DNA sequence analysis
discovered two mutations that
had a significant effect on
trichome morphogenesis zwi-3
and zwi-9311 which were found
to be mut-1 and mut-3,
respectively.
• The Bioinformatics analysis
produced five closely related
proteins to ZWI and the host
organisms that contained the
proteins.
• The SMART analysis provided
insight into the structural and
functional aspects of ZWI that
are involved in the creation of
trichome branches.
• The Double mutant analysis
established that AN is epistatic to
NOK.
Evaluation of Function: ZWI protein:
The SMART analysis of ZWICHEL
protein identified five regions of interest
in the protein. The structural motifs of
interest were two coiled-coil regions of
the protein sequence that allow two
different amino acid strands to wind
around each other in a helical
conformation. The functional
significance of these coiled-coil regions
is that they are an essential characteristic
of kinesin-like proteins. This type of
protein, KLP, is composed of three
domains: 1) a force generating head
domain which binds to microtubules, 2)
a coiled-coil stalk region involved in
dimmer formation, and 3) a cargo
binding tail domain14. There are two
types of KLP’s based on direction of
travel along microtubules. N-type are
(+) directed KLP’s and move towards
the plus end of MT and have there motor
domain at the N terminus. C-type KLP’s
are (-) directed and move towards the
minus end of the MT with the motor
domain at the C terminus. The
ZWICHEL protein is a C-type KLP
which moves in the minus direction
along MT. This evidence is
substantiated by the three functional
domains contained in the ZWICHEL
protein. Starting from the C-terminus
for the proper direction you encounter
the KISc which is a kinesin motor. You
then move through the coiled-coil motifs
and encounter the B41 domain which is
an ERM protein domain. The B41
domain is associated with the interaction
between the plasma membrane and the
cytoskeleton. Finally you arrive at the
MyTH4 domain which is present in both
myosin and kinesin tails. MyTH4 is the
tail tether region of the kinesin motor
that moves along cytoskeletal MT’s.
This completes the functional and
structural domains of interest on the
KCBP that is ZWICHEL. The
ZWICHEL protein is a minus directed
kinesin-like motor protein. It utilizes
bonding with calmodulin to facilitate
movement along the intact actin
cytoskeleton and MT array within the
cell15.
The results of the final two
weeks of research have provided
invaluable practice not only with
laboratory technique but as well with
diagnostic methods outside the lab that
are now readily available. The use of
websites such as www.arabidopsis.org
(TAIR database),
www.ncbi.nlm.nih.gov/BLAST/ (Protein
BLAST analysis, www.smart.ox.ac.uk
(SMART analysis tool) allow for
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analysis of a large set of data quickly
and efficiently to produce viable results.
The addition of the MacVector program
facilitated the sequence comparison
analysis that identified the mutations of
interest from ecotype polymorphisms.
The techniques and methods learned in
this laboratory series captures the
integrated and interdisciplinary nature of
genetics in today’s biology. Genetics
was once thought of as a separate entity
unto its own but the advances in
biological technology and analysis have
made it essential to be well versed in
molecular, cell, and genetic techniques
of analysis.
EXTRA CREDIT:
Q: in the original research article
presenting the identification of the ZWI
gene16, what was the evidence that the
ZWI gene had been correctly identified
(e.g. transformation? Analysis of
alleles?)
Short Answer:
Cloning by T-DNA tagging determined
that the region encoded a KCBP.
Longer Answer:
The evidence supporting the correct
identification of the ZWI gene was a
combination of techniques that centered
on transformation of Arabidopsis with
Agrobacterium vectors. The
transformed plants were transformed at a
set interval and the fragments produced
were then conducive to sequence
analysis. Using three alleles the team
confirmed that a KCBP was encoded by
the ZWI gene. These alleles were two T-
DNA insertional alleles and a third allele
induced by fast neuron mutagenesis.
These three alleles were recessive in
nature and all produced invalid to
inferior motor proteins leading to
inhibited trichome development.
 Literature Reviewed:
1. Schwab, B. Trichome morphogenesis in
Arabidopsis. Phil. Trans. R. Soc. Lond.
B (2000) 355, 879-883
2. Schnittger, A. Trichome
morphogenesis: a cell-cycle
perspective. Phil. Trans. R. Soc. Lond.
B (2002) 357, 823-826
3. Mathur, J. The actin cytoskeleton is
required to elaborate and maintain
spatial patterning during trichome cell
morphogenesis in Arabidopsis thaliana.
Development 126, 5559-5568 (1999)
4. Preuss, M. The Cotton Kinesin-like
Calmodulin-Binding Protein Associates
with Cortical Microtubules in Cotton
Fibers. Plant Physiology. 132. 154-160
(2003)
5. Folkers et al. The cell morphogenesis
gene AUGUSTIFOLIA encodes a
CtBP/BARS-like protein and is
involved in the control of the
microtubule cytoskeleton. EMBO
journal (2002) 21(6): 1280-1288
6. Luo and Oppenheimer. Genetic control
of trichome branch number in
Arabidopsis: the roles of the FURCA
loci. Development (1999) 126: 5547-
5557.
Special thanks:
* The utmost respect and appreciation to
Joe Pearson and Mike Hannon without
whom the BICD 101: Eukaryotic
genetics lab would have been an awful
mess. World’s #1 TA’s!!
1
Jackson, Michael: University of California,
San Diego: April 24th 2003: BICD 101:
Eukaryotic Genetics: Lab Report #1: Forward
Genetics: An approach to the mapping and
identification of a mutation in trichome
morphogenesis of Arabidopsis thaliana.
2
Complete ZWI gene provided by TAIR
database at www.arabidopsis.org which is both
the genomic DNA of the entire ZWICHEL gene.
3
Krishnakumar, S., Oppenheimer, D., (1999).
Extragenic suppressors of the Arabidopsis zwi-3
mutation identify new genes that function in
trichome branch formation and pollen tube
growth. Development 126, 3079-3088
4
Herman, P.L. & Marks, M.D. (1989) Plant
Cell 1, 1051-1055
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5
Gustafson-Brown, C. Protocol for PCR
(5/27/2003) BICD 101: Eukaryotic Genetics.
6
Gustafson-Brown, C. Gel Purification of PCR
products for direct sequencing using Oiagen kit
(5/29/2003) BICD 101: Eukaryotic Genetics.
7
Gustafson-Brown, C. Section 6.7: Terminator
sequencing of DNA. Chapter 6: Molecular
Biology of DNA replication and Recombination
(5/27/2003) BICD 101: Eukaryotic Genetics.
8
Gustafson-Brown, C. Lab protocol and TA
supplementary info. (5/28/2003)
9
Gustafson-Brown, C. Campbell et al. Biology
(1999) pp 383-384 BICD 101: Eukaryotic
Genetics.
10
Gustafson-Brown, C. Chapter 21: Responses
to Plant Pathogens. Agrobacterium Handout.
BICD 101: Eukaryotic Genetics.
11
Gustafson-Brown, C. Basic Bioinformatics –
Analysis of ZWI Protein (6/5/2003) BICD 101:
Eukaryotic Genetics. SMART database which is
used to identify functional and structural
domains of interest in ZWI protein:
www.smart.ox.ac.uk
12
Gustafson-Brown, C. Basic Bioinformatics –
Analysis of ZWI Protein (6/5/2003) BICD 101:
Eukaryotic Genetics. Protein BLAST: used to
compare protein sequence to identify 5 closely
related organisms containing proteins similar to
ZWI. www.ncbi.nlm.nih.gov/BLAST/
13
SMART analysis citation: www.smart.ox.ac.uk
Schultz et al. (1998) Proc. Natl. Acad. Sci. USA
95, 5857-5864
Letunic et al. (2002) Nucleic Acids Res 30, 242-
244
14
Krishnakumar, S., Oppenheimer, D.,
(1999). Extragenic suppressors of the
Arabidopsis zwi-3 mutation identify new genes
that function in trichome branch formation and
pollen tube growth. Development 126, 3079-
3088
15
Provided by SMART analysis of Q9FHN8:
KISc SMART accession # SM0129, B41
SMART accession # SM0295, and MyTH4
SMART accession # SM0139.
www.smart.ox.ac.uk (6/5/2003)
16
Oppenheimer et al. (1997). Essential role of
a kinesin-like protein in Arabidopsis trichome
morphogenesis. Proc. Natl. Acad. Sci. USA 94:
6261-6266