Biology bartel and szostak experiment

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  • 1. Maria Chiaffarano
    Bartel and Szostak Experiment
  • 3. Four Overlapping Stages
    Scientists used four stages to understand the origin of life
    Stage 1
    Nucleotides and amino acids were produced prior to the existence of cells
    Stage 2
    Nucleotides became polymerized to form RNA and/or DNA and amino acids become polymerized to form proteins
    Stage 3
    Polymers became enclosed in membranes
    Stage 4
    Polymers enclosed in membranes acquired cellular properties
  • 4. Chemical Selection
    What is chemical selection?
    Chemical within a mixture has special advantageous properties
    Properties allow it to increase in amount
    The cellular characteristics that exist today evolved from an “RNA world”
  • 5. RNA World
    Scientists believe that the world used to consist of RNA based organisms
    Studied the building blocks
    Amino acids and nucleotides
  • 6. Key Functions of RNA
    RNA has three key functions that encourage scientists to favor it as the first macromolecule found in protobionts
    Ability to store information in its nucleotides
    Nucleotide sequence has the capacity for self-replication
    RNA has many enzymatic functions
    Act as ribozymes
    DNA and proteins are not as versatile as RNA
    DNA has limited catalytic activity
    Proteins do not undergo self-replication
    RNA can perform functions that are characteristics of proteins while simultaneously serving as genetic material with replicative and informational functions
  • 8. Bartel and Szostak Experiment
    David Bartel and Jack Szostak
    First study that used RNA molecules with a particular function (1993)
    Synthesized a mixture of 10^15 RNA molecules also known as “long RNA”
    First region (5’ end) constant region among all “long RNAs” (identical)
    Second region, variable region (220 nucleotides)
    Hypothesized that the variable region could possibly result in long RNA with the enzymatic ability to catalyze a covalent bond between two adjacent nucleotides
  • 9. Materials
    Many copies of short RNA
    Had a tag sequence that binds tightly to “beads” (column packing material)
    Had a complementary sequence to a site in the constant region of the long RNA
    No variable region, all the same
    Long RNAs with the constant and variable regions
    Variable regions were made using a PCR step (caused mutations in the region)
  • 10. Overview of the experiment
  • 11. Step 1
    The long RNAs and short RNAs were incubated together
    Promote hydrogen bonding
    Time was given for the molecules to form covalent connections (only if the RNA had the enzymatic ability to form covalent bonds)
    The long RNA molecule variable regions may rarely have enzymatic ability to connect the 3’end of short RNA to 5’end of long RNA
  • 12.
  • 13. Step 2
    The mixture passed through a column of beads
    The beads would bind to the tag sequence of the short RNA only!
    Tag sequence promoted the binding of the short RNA to the beads
    IF the long RNA had the ability to bind to the short RNA, it would also be attached to the beads
    Additional liquid was added to filter out the long RNAs that did not covalently bond to the short RNAs
  • 14. Step 3
    The main purpose of this step was to get rid of the beads
    Low pH solution was added in order to prevent the tag sequence from binding to the beads
    The tightly bound RNAs are flushed out of the column
    They were flushed into a flask and labeled “Pool #1”
    Beads were left behind
  • 15. Step 4
    Pool #1 was used to make a second batch of long RNA molecules
    Polymerase chain reaction (PCR) was used
    Variable regions were derived from the variable regions of pool #1 RNA molecules, expected to have enzymatic activity
    Reverse transcriptase was used to make cDNA
    PCR primers recognized beginning and end of the long RNA sequence and copied only this region
    cDNA used as a template to make long RNA via RNA polymerase
  • 16. Steps 5 & 6
    The procedure was repeated in order to generate 10 consecutive pools of RNA molecules
    A sample of the original population (Pool #1) and each of the following 10 pools were collected in order to test for the enzymatic ability to catalyze a covalent bond between adjacent nucleotides
  • 17.
  • 18. Conclusion
    Chemical selection is possible
    Scientists knew this because the experiment showed that there was an increase in covalent bond formation from pool 1 to pool 10
    In each generation, the catalytic enzyme activity increased
    Pool #10’s enzymatic activity was approximately 3 million times higher than the original random pool of molecules