Genotypic and phenotypic analysis of Arabidopsis thaliana S15aE rprotein genes that contain insertio

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Genotypic and phenotypic analysis of Arabidopsis
thaliana S15aE r-protein genes that contain
insertional mutations

• Stacey Abidayo
• Research Headed by Dr. Szick-Miranda

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I. Why Arabidopsis thaliana is an Ideal Model
for Analysis

• Genome sequenced
• Rapid life cycle
• Easily grown
• Many seeds
• Many mutants
• Genetic tools available

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Arabidopsis thaliana
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II. Importance of Ribosomes and Translation

• Translation is polypeptide synthesis
• Polypeptides are large molecules made of many
  amino acids
• large polypeptides are called proteins
• Ribosomes consist of a large and small subunit
• Distinct structure allows translation to occur

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tRNA
Amino Acids
Small Ribosome
mRNA
Polypeptide Product
Large Ribosome
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III. Purpose of Our Research

• Group looking at a specific ribosomal protein
  gene, S15a, which is considered to be part of the
  divergent proteins
• Many genes encode this ribosomal protein gene and
  Im working with a specific one which is the
  S15aE
• Earlier research show that the S15a protein gene
  is part of the cytosolic ribosome
• Research suggests that S15a is also incorporated
  into the mitochondria
• Asking whether or not this gene is found in
  cytocol and mitochondria or cytocol alone because
  there is evidence that it is incorporated into
  the mitochondria
• Analyzing insertional mutations in genes

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IV. Approach

• Looking at mutations that encode for those genes
  that are available through the Salk Institute
  Genome Analysis Laboratory
• We cannot assume that the seeds have an
  insertional mutation without genotypic analysis
• Polymerase Chain Reaction (PCR)
• Electrophoresis
• Then we genotype to assure whether or not the
  mutation that is Homozygous wild type,
  Heterozygous, or homozygous mutation
• Hope to find a homozygous mutation then we can
  phenotypically analyze how the mutation affects
  the phenotype, its outward appearance of the plant

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V. Methods Utilized in the Lab

• DNA extractions
• PCR
• Electrophoresis

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VI. DNA Extractions

• Purpose Extract DNA
• Used mortar and pestle to grind leaves of
  Arabidopsis thaliana to get DNA from tissue

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VII. Polymerase Chain Reaction (PCR)

• Importance of primers
• Enables production of large quantities of
  specified DNA

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VIII. Electrophoresis

• Gel composed of agarose, buffer, and ethidium
  bromide
• Agarose and buffer work as a matrix that allows
  DNA to be separated
• Ethidium bromide binds to DNA which makes if
  florescent when UV light is applied to it
• Purpose is to allow us to visualize the DNA
• Applying an electrical current to DNA, which is
  negatively charged, pulls DNA towards the
  positive end
• Distance traveled with relation to our size
  marker helps estimate how many base pairs are in
  the strand of DNA in which was extracted

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Examples of Electrophoresis