Introduction: stepping into the science

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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?
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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?

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Functional Genomics of Protein Disulfide
Isomerase Gene Family Unraveling Protein
Folding and Redox Regulatory Networks
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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?

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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.
• 1000s different enzymes -gt unique activities
• Figure out their activities.
• ENZ
• A -----------gt B
• Where the enzyme is located in the cell?
• Do they need other protein partners to do their
  job?

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Functional Genomics of Protein Disulfide
Isomerase Gene Family Unraveling Protein
Folding and Redox Regulatory Networks
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ENZYME Protein Disulfide Isomerase

• Protein chain of amino acids. Results from
  de-coding the mRNA sequence transcribed from DNA.

• Disulfide Two sulfurs
• The amino acid containing sulfur is cysteine
• Protein with 2 cysteines

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ENZYME Protein Disulfide Isomerase

• Isomer Different substances that have the same
  components.
• Different molecules with same chemical formula
  
• Alter chemical bonding --gt different shapes
  --gt activities and functions.
• Isomerase an enzyme that can make different
  molecular shapes out of the same substance.

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Protein Disulfide Isomerase (PDI) can make
different protein shapes based on altered sulfur
bonding
An Isomer with new activity !
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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.

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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.

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PDI Protein folding- oxidoreductase
Active state
Inactive state
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Chemical Mechanism

• Oxidation of 2 cysteine sulfhydryls --gt disulfide
  bridge
• Remove 2 electrons and 2 H
• Reduction --gt breaks bridge --gt 2 sulfhydryls
• Add 2 electrons and 2 H
• All proteins have to fold to proper states

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In Yeast and humans - PDIs located in the
endoplasmic reticulum (ER)

• But what about plants????

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Arabidopsis thaliana Plants
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Arabidopsis

• First plant to have its genome completely
  sequenced
• Smallest Plant Genome known
• -gt 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.

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There are 11 different PDIs in Arabidopsis plants
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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.
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What kind of results might you expect?
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Ooops, Not this.
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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.
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T-DNA Mapping
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PDI antisera binds to chloroplasts of Arabidopsis
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Localization to chloroplasts and cell wall
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Localization to chloroplasts Red chlorophyll
fluorescence
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Alignment of Arabidopsis PDI2 with Chlamydomonas
PDI
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PDI2 is processed by ER microsomes indicating it
has a N-terminal signal peptide destined for ER
or secrettion
0 20 60 90 min
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Transmission electron microscopy
Immunolocalization
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TEM Immunolocalization to periphery of starch in
chloroplasts
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TEM Asymmetry starch biogenesis