Download The Biotechnology Age: Issues and Impacts

Introduction: stepping
into the science
What kind of research is being done on the project?
What is an Arabidopsis plant?
How does the ABE workshop fit in?
What kind of results might you expect?
What kind of research is being done on the project?
National Sciences Foundation (NSF) funds the ABE
workshop through a research project entitled:
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
•
What the heck does that mean?
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
Functional Genomics:
• New field of biological science
• Genetics-based
• Genome: all of the genes encoded in DNA in a
living organism.
• Function: Conduct research to figure out what the
genes are doing.
• What proteins do they encode and what jobs in the
cell are they responsible for?
What jobs do the proteins do in a cell?
• 1. Structure: hold things up
• 2. Enzymes: activity make and burn energy.
Stimulate growth and biomass production.
• 1000’s different enzymes -> unique activities
• Figure out their activities.
ENZ
A ----------->
B
• Where the enzyme is located in the cell?
• Do they need other protein partners to do their job?
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
ENZYME: Protein Disulfide Isomerase
• Protein = chain of amino acids. Results from decoding the mRNA sequence transcribed from DNA.
• Disulfide: “Two sulfurs”
The amino acid containing sulfur is cysteine
Protein with 2 cysteines
|
SH
|
SH
ENZYME: Protein Disulfide Isomerase
• Isomer: Different substances that have the same
components.
= Different molecules with same chemical formula
• Alter chemical bonding --> different “shapes” -->
activities and functions.
•Isomerase: an enzyme that can make different
molecular shapes out of the same substance.
Protein Disulfide Isomerase (PDI) can make
different protein shapes based on altered
sulfur bonding
|
SH
|
SH
An Isomer with
new activity !
-S-S-
Proteins do not do their job unless
they are folded correctly
• So, PDIs fold other proteins correctly in cells.
• A major responsibility for keeping cells normal,
development, metabolism and growth.
Protein Disulfide Isomerase (PDI)
Gene Family
• Study all the PDIs in the genome of a small plant.
•All the PDIs in the same related family.
• but they go off and have different jobs at various
locations in the cell.
PDI Protein folding- oxidoreductase
PDI
= cys
Inactive state
Active state
Chemical Mechanism
• Oxidation of 2 cysteine sulfhydryls --> disulfide
bridge
– Remove 2 electrons and 2 H+
• Reduction --> breaks bridge --> 2 sulfhydryls
– Add 2 electrons and 2 H+
All proteins have to fold to proper states
In Yeast and humans - PDIs located in the
endoplasmic reticulum (ER)
• But what about plants????
Arabidopsis thaliana Plants
Arabidopsis
• First plant to have its genome completely sequenced
– Smallest Plant Genome known
-> 26,000 genes but makes a plant!!
– 40 days from seed to seed.
– Easily genetically engineered.
– Easy to knock out genes to see what they do.
There are 11 different PDI’s in Arabidopsis plants
Primary Structure of the PDI family in Arabidopsis
pdi1
KDEL
pdi2
KDEL
pdi3
KDEL
pdi4
KDEL
pdi5
KDEL
pdi6
KDEL
pdi7
pdi8
pdi9
KDEL
pdi10
pdi11
Endo. Retic. signal peptide
thioredoxin domain =
PWCghC (dithiol)
thioredoxin domain =
PWCARS (monothiol)
mitochondrial
transit peptide
chloroplast
transit peptide
KDEL -endoplasmic reticulum retention motif
= 25 amino acids KEY
Research goals of workshop
Learn some recombinant DNA methods
Mapping genes that have been tagged by a T-DNA
Microscopy to locate where PDIs are using
PDI-seeking antibodies.
What kind of results might you expect?
Ooops, Not this….
Any kind of result is a success
Learn by doing !!!
Have fun while you learn !
Nothing has to work perfectly to be a
valuable learning experience.
T-DNA Mapping
M
W.T
1-a
1-b
3-Ⅰ 3-Ⅱ 3-Ⅲ
4-Ⅰ 4-Ⅱ
4-Ⅲ
PDI antisera binds to chloroplasts of Arabidopsis
Localization to chloroplasts and cell wall
Localization to chloroplasts:
Red chlorophyll fluorescence
Alignment of Arabidopsis PDI2 with Chlamydomonas PDI
PDI2
PDI-Chly
consensus
1
1
1
-- -- -- -- -- -- -- MY KS PL TL LT LL TI CF GF FD LS SA LY GS SS P- -- -V VQ LT AS NF KS
V
M N R W N L L A L T L G L L L V A A P F T K H Q F A H A S D E Y E D D E E D D A P A A P K DDDDD D
VDVTVVTVKNWD
mn rw nl la lt lg ll m
P T
f D
dddv V l
PDI2
PDI-Chly
consensus
43
61
61
KV LN SN GV VL VE FF AP WC GH CK AL TP TW EK VA NI LK GV AT -- -V AA ID AD AH QS AA QD YG
E T V K K S K F A L V E F Y A P W C G H C K T L K P E Y A K A A T A L K A A A P DDAAL L I A K V D A T Q E E S L A Q K F G
l
L VE Ff AP WC GH CK L P w K A
LKg A dalvA iDA
S AQ yG
PDI2
PDI-Chly
consensus
100
121
121
IK GF PT IK VF VP GK AP ID YQ GA RD AK SI AN FA YK QI KG -- -- -- -- -- -- -- -- -- -- -TVEDADKLKSLEADAEVVV
V Q G Y P T L K W F V D G E L A S D Y N G P R D A D G I V G W V K K K T G P PPAAV V
TVEDADKLKSLEADAEVVV
i Gf PT iK F V G
D Yq G RD A
I
f
K
pavtvedadklksleadaevvv
PDI2
PDI-Chly
consensus
138
181
181
V
PDI2
PDI-Chly
consensus
158
241
241
AAVVLL
AA
T DTI D
D TIDDS LTTDA SF L
V KT
PDI2
PDI-Chly
consensus
200
301
301
PE WK RA AK NL QG KV KL GH VN -- CD VE QS IM SR FK VQ G- -F PT IL VF GP DK SS PY PY EG AR
T V F R E A S K K F K G Q L V F V T V N NNEE G D G A D P V T N F F G L K G AATT S P V L L G F F M E K N K K F R M E G E F
wk A K
G v
VN ne D
i
F v Gat P iL F
dK
y
EG
PDI2
PDI-Chly
consensus
256
361
361
S A S A I E S F A SGEGL V E S S A G P V E V T E L T G P D V M E K K C G S A A I C F I S - - F L P D I L D S K A E G R N
T A D N V A K F A E S V V D G T A Q A V L K S E A I P E D P Y E D G V Y K I V G K T V E SSVV V L D E T K D V L L E V Y A
sA
i
FA
l Ve s A
V
tE
D
E
i sv L d
D
E
PDI2
PDI-Chly
consensus
314
421
421
Y
K Y L E M L L S V A E K F K K Q P Y S F M W V A A V T Q M D L E K R V N V G GGYYG G
YPAMVAMNVKKGVYAPLKS
PW CG HC KK LE PI YK KL AK RF KK VD SV II AK MD GT EN EH -- -- PE IE VK GF PT IL FY PA GS
y
v
fK K
F
V
V
le
N
gygyP m
vy P
S
PDI2
PDI-Chly
consensus
374
477
481
A
A F E L Q H L L E F V K D A G T G G K G N V P M N G T P E I V K T K E W D G K D G E L I E E D EEFFS SL DLEDL E L M G GGDDD D
A
DR TP IV FE GG DR SL KS LT KF IK TN AK IP YE LP KK GS DG DE GT SD DK D- -- -- -- KP -- -k
t
K
P
v
K
DG dG
e Defsldel
gdda
PDI2
PDI-Chly
consensus
434
526
541
VGSKDEL
ASDKDEL
KDEL
MNRWNLLALTLGLL
-L LS DR LE GK SK PT GG GS KE K- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -S ATDTV S
AK
L DKAAV A
D TGVLS V
F ATGV
ES
D RVAVT K N F A G E D R A T
V G Y F K A L E G E I Y D T F K S Y A A KTTE ED VD VVFVV QFTVT Q
AADAVG A
D VAKVN D
v
LE G
T
Kt ed vv fv qt ts ad va ka ag ld av dt vs vv kn fa ge dr at
- - - - - - - - - - - - - - - - K-SKE S E P S A S V E L N A S N F D D L V I E - S N E L W I V E F F A P W C G H C K K L A
A F V KKSSE E K M P P T I E F N Q K N S D K I F N S GG I N K Q L I L W T T A D D L K A D A E I M
av la td id td sl ta fv k
s vE N
N D l
g N
Iv
A
l
PDI2 is processed by ER microsomes indicating it has a
N-terminal signal peptide destined for ER or secrettion
0
20
60
90 min
Transmission electron microscopy:
Immunolocalization
TEM: Immunolocalization to periphery of starch
in chloroplasts
TEM: Asymmetry starch biogenesis