Transcript The Biotechnology Age: Issues and Impacts

ABE: Advances in Bioscience Education
Bringing together students and faculty to explore
research in the molecular and cellular biosciences.
Dr. Kabi Neupane, Coordinator (co-PI, LCC)
Faculty Partners
John Berestecky (KCC)
Ingelia White
(WCC)
Priscilla Millen (LCC)
Janice Ito
(LCC)
Catherine Unabia (HPU)
David Christopher (PI, UH, Manoa)
Summer 06
Stepping into scientific research
 From the classroom to
the lab bench with intensity.
What are our goals?
What kind of research are we doing ?
What is an Arabidopsis plant?
Research philosophy.
Goals
• Reach for new learning and
knowledge.
• To learn and grow - actively,
by doing….to change.
• Move out of the comfort zone
and into the stretch zone.
• Take risk, try something new,
unfamiliar and break thru the fear barrier.
Research = Stretch Zone
Willing to sail into
unchartered waters
To discover…
make mistakes…
…change direction.
Have FAITH that you can learn
from mistakes as you move
along.
Scientific Research:
Learn by Trial and error.
Embrace mistakes
Persistance is more important
than strength
Scientific Research:
Discover new knowledge.
Use tools of molecular and cellular biology
to figure out the underlying biochemical
processes that control how living cells
work.
RESEARCH QUESTIONS:
Plant growth…
How do leaves, roots, stems, flowers
develop from common precursor cells?
How can native and crop plants safely resist
pests and diseases that kill them ?
How can plants be protected from environmental
stress: drought, flood, salt, heat, UV, pollution ?
“Arabidopsis thaliana: Research on a
lawn mower to understand a corvette”
Research Philosophy to gain insight
into complex systems
Learn the inner workings of something
complicated, then start by studying the simpler
version.
$ 65,0000
$ 2,000
Model System - essence of an automobile
Complex fascinating plants adapt to diverse
environments
Arabidopsis thaliana Plants
simple
Complex
Economy of Size
Cornfield
Arabidopsis “field” in
4 inch petri plate
Economy of Time
Corn  100 - 140 days
to seed
Arabidopsis 
30-40 days
to seed
Economy of Genes, Genome and DNA
Arabidopsis has smallest genome, vascular plants
Corn

48,000 genes
Arabidopsis
 29,000 genes
 10 chromosomes
 5 chromosomes
 ~ 5 billion nucleotides
 125 million nucleotides
 Much junk spacer DNA
 small spacer DNA
A
G
T
C
Arabidopsis = encapsulates the essence
of all plants in a small package
 First fully sequenced genome of any plant
 All genes mapped and isolated
 Catalogue of genes can be ordered via mail
 Knockouts of genes  MUTATE THEM
 Study the activity of 1000’s of genes
simultaneously
 Easy to genetically engineer
All of the genes in Arabidopsis have
been found in other plants .
 Used as a starting point to identify genes in other
plants
 Arabidopsis has facilitated the genetics of all plants
 Answer the question: What do the genes do?
Amazing fact !
 60% genes in plants have counterparts in human DNA
 Genes for …Basic cellular processes
 Energy production, ATP
 Cell division
 Enzymes
 Protein biosynthesis
From DNA sequence (chemical) to Life
GGTTCCAAAAGTTTATTGGATGCCG
TTTCAGTACATTTATCGTTTGCTTT
GGATGCCCTAATTAAAAGTGACCCT
TTCAAACTGAAATTCATGATACACC
AATGGATATCCTTAGTCGATAAAAT
TTGCGAGTACTTTCAAAGCCAAATG
AAATTATCTATGGTAGACAAAACAT
TGACCAATTTCATATCGATCCTCCT
GAATTTATTGGCGTTAGACACAGTT
GGTATATTTCAAGTGACAAGGACAA
TTACTTGGACCGTAATAGATTTTTT
GAGGCTCAGCAAAAAAGAAAATGGA
AATACGAGATTAATAATGTCATTAA
TAAATCAATTAATTTTGAAGTGCCA
TTGTTTTAGTGTTATTGATACGCTA
ATGCTTATAAAAGAAGCATGGAGTT
ACAACCTGACAATTGGCTGTACTTC
CAATGAGCTAGTACAAGACCAATTA
TCACTGTTTGATGTTATGTCAAGTG
AACTAATGAACCATAAACTTGGTCA
Tools of molecular and cellular biology
- Recombinant DNA technology
- Biochemistry
- Microscopy
- Molecular genetics
- Computers
Use these tools from an actual research project
National Sciences Foundation (NSF) funds ABE:
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
•
Complicated name:
What does it mean?
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
Functional Genomics:
• New field of biological science
• Rooted in Genetics
• Genome: all of the genes encoded in DNA in a
living organism.
• Function: 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 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”
Making of a protein: Converting the code in
a polymer of nucleic acid to a polymer of amino acid
DNA

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 Transcribed
RNA

PROTEIN
 Translated
ENZYME: Protein Disulfide Isomerase = PDI
• Chain of amino acids representing a PDI.
• Disulfide: “Two sulfurs”
The amino acid containing sulfur is cysteine
Bond with 2 cysteines
|
SH
|
SH
|
SH
ENZYME: Protein Disulfide Isomerase
• Isomer: 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.
•Disulfide Isomerase: emzyme that alters molecular
shape by acting on the disulfide bonds.
PDI can make different protein shapes based on
altered disulfide bonding
|
SH
|
SH
2 isomers with
new activity !
OR
-S S-
|
SH
|
SH
-S-S-
|
SH
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks
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
All proteins have to fold to proper states
Chemical Mechanism REDOX
• Oxidation Remove 2 electrons and 2 H+
2 cysteine sulfhydryls --> make disulfide bridge
• Reduction --> breaks bridge -->
– Add 2 electrons and 2 H+ to the 2 sulfhydryls
In Yeast and humans - PDIs located in the
endoplasmic reticulum (ER)
• But what about plants????
Arabidopsis thaliana Plants
Primary structure of a generic PDI
ER retention motif
C--C
C--C
Signal sequence
Thioredoxin domains
Directs a protein
to a specific
location
Contains 2 cysteines and
active catalytic site for
oxidation-reduction and
folding of proteins
KDEL
Research activities of workshop
Learn some recombinant DNA methods
Map genes that have been tagged by a T-DNA
Learn PCR and RT-PCR
Isolate proteins from leaves and detect proteins
using antibodies
Use a microscope to find where PDIs and green
fluorescent protein are located in the cell.
What kind of results might you expect?
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.