Download
1 / 34

RNA molecules stimulate prion protein conversion - PowerPoint PPT Presentation


  • 275 Views
  • Updated On :

Proteasome. WHAT WOULD YOU SEE IF YOU RAN THE THE WHOLE PROTEIN EXTRACT FROM A CELL?. WHAT INFORMATION DOES THIS GIVE YOU?. MUTATIONS

Related searches for RNA molecules stimulate prion protein conversion

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'RNA molecules stimulate prion protein conversion' - Michelle


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

Slide8 l.jpg

WHAT WOULD YOU SEE IF YOU RAN THE THE WHOLE

PROTEIN EXTRACT FROM A CELL?



Slide10 l.jpg

  • MUTATIONS

  • Given a eukaryotic gene with one intron. You isolate mutations that change the sequence of the transcript in introns or exons. For each of these mutations, indicate what you expect to see in terms of the levels and sizes of the corresponding mRNAs and the levels and size of the encoded proteins.

  • - Missense mutation that results in an amino acid change in the protein without affecting the structure of the protein.

  • Missense mutation that results in an amino acid change in the protein that destabilizes the protein (conformational change).

  • - Mutation that inserts an earlier stop codon.

  • - Mutation that eliminates stop codon.

  • - Mutation that eliminates splicing of intron (there are many outcomes here).


Slide12 l.jpg

Consequences of protein misfloding-disease

-Alzheimer's

-Huntigton's

-Prion Diseases


Slide13 l.jpg

Aggregate Fiber

PrPc

PrPSc


Slide14 l.jpg

RNA molecules stimulate prion protein conversionNATHAN R. DELEAULT, RALF W. LUCASSEN & SURACHAI SUPATTAPONENature 2003 Oct16 2003 Vol. 425: 717-720

  • Prions ProteinaceousInfectious particle

    • An infectious agent composed of protein and devoid of nucleic acid

    • Suspected cause of fatal human and animal diseases

  • Prions are chemically identical to cellular protein (PrPc), but conformationaly different

    • Different solubility, structure,and stability

  • Prions are able to convert PrPc to the prion form PrPSc (scrapie)


Slide15 l.jpg

PrPc = The cellular form of the prion protein

PrPSc = The prion (or Scrapie) form of the prion protein

+

=

Pure PrPSc can convert PrPc but it requires 50 fold molar excess PrPSc


Slide16 l.jpg

PrPc = The cellular form of the prion protein

PrPSc = The prion (or Scrapie) form of the prion protein

+

=

+

=

Cellular

cofactors

+

What is the specific cellular cofactor??


Slide17 l.jpg

Tissue from PrPSc hamster

infected brain (0.1%w/v)

+

Tissue from uninfected

hamster brain (5% w/v)

incubate

treat with protease (should remove all PrPc)

Probe for PrP via western blot

starting PrPSc

PrPres

(protease-resistant

PrPSc-like protein)

PrPres amplification assay


Slide18 l.jpg

DRAW EXPECTED BANDS ON THE GEL

Tissue from PrPSc hamster

infected brain- With Protease

Tissue from uninfected

hamster brain -With Protease

Tissue from PrPSc hamster

infected brain - No Protease

Tissue from uninfected

hamster brain - No Protease


Slide19 l.jpg

PrPres amplification assay

Tissue from PrPSc hamster

infected brain (0.1%w/v)

+

Tissue from uninfected

hamster brain (5% w/v)

incubate

treat with protease (should remove all PrPc)

Probe for PrP via western blot

starting PrPSc

PrPres

(protease-resistant

PrPSc-like protein)

DESCRIBE ALL THE STEPS

OF AN EXPERIMENT THAT

SHOWS THAT THE COFACTOR

IS RNA.


Slide20 l.jpg

Cellular co-factor appears to be RNA

IS RNA NECESSARY OR SUFFICIENT?


Slide21 l.jpg

It looks like it is ssRNA,

not dsRNA, RNA:DNA, DNA, ATP or HSPG


Slide22 l.jpg

ASSUMING THAT THE CO-FACTOR IS NOT RNA,WHAT WOULD THE AUTHORS SEE IF THEY ADD Rnase THAT HAS SOME PROTEASE IN IT (BAD BATCH OF ENZYME) versus GOOD Rnase (GOOD BATCH)? MAP OUT THE EXPERIMENT IN DETAIL AND SHOW THE GEL WITH BOTH RESULTS.

M

SC

RNase

RNase (BAD)

HOW CAN YOU MAKE SURE YOUR RNase

IS NOT CONTAMINATED WITH PROTEASE?

DETAIL THE EXPERIMENT.


Slide23 l.jpg

Effects are not due to protease contamination AUTHORS SEE IF THEY ADD Rnase THAT HAS SOME PROTEASE IN IT (BAD BATCH OF ENZYME)

in enzyme mixtures


Slide24 l.jpg

Enzyme activity is required AUTHORS SEE IF THEY ADD Rnase THAT HAS SOME PROTEASE IN IT (BAD BATCH OF ENZYME)

+EDTA

+EDTA

+DEPC


Slide25 l.jpg

THE END PRODUCTS OF RNA DIGESTION BY RNase ARE AUTHORS SEE IF THEY ADD Rnase THAT HAS SOME PROTEASE IN IT (BAD BATCH OF ENZYME)

CYCLIC 2', 3'-NUCLEOTIDES. DO YOU THINK THERE IS

ANOTHER INTERPRETATION WHY THE RNase IS INHIBITING

PrPres FORMATION? DETAIL THE EXPERIMENT.

HINT: YOU CAN BUY SOLUTIONS OF CYCLIC 2', 3'-NUCLEOTIDES.


Slide26 l.jpg

End products of RNA digestion not inhibiting AUTHORS SEE IF THEY ADD Rnase THAT HAS SOME PROTEASE IN IT (BAD BATCH OF ENZYME)

PrPres amplification in RNase treated samples


Slide27 l.jpg

1) Do the Results so far show that RNA is necessary or sufficient

For the amplification?

How can you prove the other (assume you can isolate

purified molecules in a test tube.


Slide28 l.jpg

Total RNA from hamsters can reconstruct amplification sufficient

in nuclease treated extracts


Slide29 l.jpg

Cellular cofactor for PrPres amplification: sufficient

  • is not sensitive to RNase V1 (dsRNA)

  • is not sensitive to RNase H (DNA:RNA)

  • is not sensitive to DNase

  • is not sensitive to apyrase (ATP)

  • is not sensitive to inactive RNase

  • is not sensitive to byproducts of RNA digestion

  • is not sensitive to heparinase III

  • is sensitive to RNase

  • can be reconstituted in RNase treated samples by the addition of purified total RNA

Looks like it might be the RNA


Slide30 l.jpg

What size RNA is required for this reaction? sufficient

T: Total RNA

R: Retentate RNA (>300 nt)

Size Filtration Column

F: Filtrate RNA (<300 nt)


Slide31 l.jpg

Active RNA is >300 nucleotides in length sufficient

activity remains in retentate


Slide32 l.jpg

Will just any RNA do? sufficient

Specific RNA molecules are need for PrPres amplification


Slide33 l.jpg

To sum up sufficient

  • This does not change the protein only hypothesis

  • Potentially useful in making new and better diagnostic tools

  • Suggest mechanism for strain diversity and species barriers


ad