Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Save to My Library
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
Lab #3 BICD 101 6-11-2003

Lab #3 BICD 101 6-11-2003

Ratings:

5.0

(2)
|Views: 60 |Likes:
Published by mwjackso
This was the final lab report in the lab class for forward and reverse genetics.
This was the final lab report in the lab class for forward and reverse genetics.

More info:

Published by: mwjackso on Apr 04, 2009
Copyright:Traditional Copyright: All rights reserved

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF or read online from Scribd
See more
See less

05/10/2014

pdf

 
Lab #3A02-92-6779Group #6- 1 -
Lab Report #3Forward Genetics 2:
Complementation, Epistatic Relationships,Sequencing Analysis, & Prediction of ProteinFunction in
 Arabidopsis thaliana
with emphasis ontrichome morphogenesis
(*companion paper to
Forward Genetics:
An approach to the mapping and identification of a mutation intrichome morphogenesis of 
 Arabidopsis thaliana
(4/24/2003) pgs 1-18)
Michael Wade JacksonBICD 101: Eukaryotic Genetics6/11/2003
 
Lab #3A02-92-6779Group #6- 2 -
Abstract:
Based on previous investigation
1
the location of the mutation of interest (mut-1) wasmapped to chromosome 5 of 
 Arabidopsis
on the BAC K14B20. A complementation testwas 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 interestwas discovered through the use of 
 Agrobacterium
transformation. A wild-type geneconstruct was inserted to rescue the mutant phenotype. The construct that produced athree 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 comparisonanalysis using MacVector. The mut-1 and mut-3 sequences were compared to theComplete
 ZWI 
gene
2
. A double-mutant test involving
an
and
nok 
displayed that
 AN 
isepistatic to
 NOK 
. Final analysis was performed on the possible role of the functionaldomains and structural motifs of the ZWICHEL protein in relation to its role as a kinesin-like calmodulin-binding protein (KCBP) involved in trichome morphogenesis
3
. ________________________________________________________________________ 
INTRODUCTION:
The goal of a forward genetics approachis 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 wasinitiated during the first three weeks of this course to identify and map amutation of interest. The mutationselected was mut-1 which produced areduction of the number of branches onthe trichomes of 
 Arabidopsis thaliana
.The Arabidopsis ecotype of interest wasColumbia (Col). The first three weeksof experimentation provided themapping and localization of the mut-1on chromosome 5 in the BAC K14B20.This information along with the TAIR database produced an assortment of approximately 11 candidate genes thatmight be the target of the mutation onK14B20. This was the extent of theknowledge in relation to mut-1 at theend of the three week period. Acomplementation test was setup in order to determine if mutations 1, 2, 3, 5, or 6were allelic. The interim time was spentlearning a reverse genetics approachinvolving mouse ES and EF cellcultures. Upon return to the process of forward genetics the plants that were theresult of the complementation crosses provided the insight that mut-1 and mut-3 were allelic (i.e. in the samecomplementation group). Based on thisknowledge the next logical step was totry and rescue the mutant phenotype (2 prongs) by transformation
4
with
 Agrobacterium
containing a wild-typeconstruct of each of the 11 candidategenes. This facilitated the identificationof the clone AT5G65930.1 (
 ZWICHEL, ZWI 
) as the gene of interest.
 ZWI 
geneencodes KCBP which plays an integralrole in trichome development. The geneonce identified was then subjected tosequence comparison analysis using theTAIR database along with MacVector toelucidate variance in the
 ZWI 
sequencethat would account for the phenotypicmutation. The outcome of thesesequence comparisons yielded thelocations of mut-1 and mut-3 within
 
Lab #3A02-92-6779Group #6- 3 -
 ZWI 
. The identification of the mutationswas then provided and mut-1 and mut-3were
 zwi-3
allele and
 zwi-9311
allele,respectively.DOUBLE-MUTANT ANALYSIS:In conjunction with the investigation of mutations in
 ZWI 
, trichomemorphogenesis as a process wasanalyzed involving two other mutations.These mutations were in
 ANGUSTIFOLIA
(
 AN 
) and
 NOECK 
 (
 NOK 
).
 AN 
is an inducer to branchingand
 NOK 
is a suppressor to branching of trichomes. Using
an
and
nok 
mutants
an;nok 
double mutants were created andobserved to determine if these mutationshad additive, nugatory, or epistaticrelationship to the phenotype. The
an;nok 
double mutants established that
 AN 
is epistatic to
 NOK 
. The goal of thisanalysis was to look at trichomemorphogenesis as a process and toinvestigate possible pathways involvedin the suppression and activation of  branching in the unicellular trichome.BIOINFORMATICS: ANALYSIS OFZWI PROTEINThe final analytical tool in the arsenal isthe establishment of the function of theZWI protein. ZWI is a KCBP whichappears to play a role in the cell cycleconstruction and differentiation of theunicellular trichome. Using the BLASTdatabase the sequence of the ZWI wasanalyzed yielding three functionaldomains of interest and two structuralmotifs that may explain the function of the ZWI protein. This analysis alsoidentified the sequence similarity between this protein and homologues inother organisms. The goal of thisanalysis is to better understand the ZWI protein and its attributes in order togauge why a mutation such as
 zwi-3/  zwi-9311
can cause the phenotypicchanges observed.
MATERIALS & METHODS:
 PREP of PCR product for DNASEQUENCING:The two essential steps in preparing aDNA sample for sequence analysis areamplification and purification. TheDNA region of interest is first amplified by PCR 
5
. Using primers selected for aregion that is approximately 700bp inlength 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 amplifiedwas then purified through the use of agarose gel electrophoresis in a processof size exclusion
6
. Once the sample had been run on the gel the brightest bandwas selected and removed. This bandwas then purified and separated from theagarose using Qiagen kit. The samplewas then analyzed using UVspectroscopy to determine purity andconcentration. The PCR products werethen outsourced for sequencing. Themethod used was direct sequencingwhich utilizes the fluorescent labeling of the bases to produce the respectivesequence
7
.
 Agrobacterium
-MediatedTRANSFORMATION:Transformation via
 Agrobacterium
 utilized a natural process to introduceforeign DNA into a host cell via a bacterial vector. The class introduced 11separate T-DNA plasmids intoindividual plants via spray bottle soakingof transformed
 Agrobacterium
in media.The goal of these transformations was to

You're Reading a Free Preview

Download
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->