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					The C3HC4-Type RING Zinc Finger and
  MYB Transcription Factor Families



               Matthew Taube
                June 5, 2008
                  HC70AL
What was the first gene studied with knockout analysis?
                        AT3G23060
• Chromosome 3

• Location: 8200643-8203240 base pairs

• 2598 base pairs long

• Forward orientation
What is the gene’s structure and where is the T-DNA insert?


• 5’ UTR, 7 exons, 6 introns, 3’ UTR




                 intron
      UTR exon
What is the gene’s structure and where is the T-DNA insert?


• 5’ UTR, 7 exons, 6 introns, 3’ UTR
• SALK_148145:
   – Expected insertion site: 255 bp
What is the gene’s structure and where is the T-DNA insert?


• 5’ UTR, 7 exons, 6 introns, 3’ UTR
• SALK_148145:
   – Expected insertion site: 255 bp
   – Actual insertion site: 156 bp UPSTREAM
       What protein does this gene encode?


• C3HC4-Type RING Zinc Finger protein

• 480 amino acids

• Cys3HisCys4 amino acid motif

• Diverse functionality
   – Binds one or more zinc ions
   – DNA binding: transcription factor
   – Also RNA, protein, and lipid binding
Was T-DNA actually inserted into the plant samples?


• Multiplex PCR to test the genotypes

• What do we expect to see on the genotyping gel?
  – Two possible bands: WT (542 bp) and T-DNA (1.1 kb)
  – 1 WT to 2 hemizygote to 1 T-DNA homozygote (1:2:1)
      Was T-DNA actually inserted into the plant samples?




                           T-DNA

                             WT




                      5 hemizygous

                         9 WT

• The knockout appears to be seed lethal
 Were there any phenotypic differences in the
             siliques and seeds?
     Hemizygous
                                               WT




• No, the siliques and seeds display WT phenotype
Were there any phenotypic differences during embryo and
                  seed development?
    Hemizygous
                                         WT




• Nomarski microscopy of the mature green stage reveals no
  apparent phenotypic differences
In what tissues is the gene active?




                         Gene specific bands,
                         indicating accumulation
                         in that sample




                     The mRNA accumulation in
                     the silique suggests that the
                     gene is active in seed
                     development
In what tissues is the gene active?
             Overview of AT3G23060
           What did we learn about this gene?

• The knockout was seed lethal but did not cause any
  apparent phenotypic changes in the hemizygous
  plants

• Active in seed development, especially important
  during the globular stage

• The T-DNA insert was found in the upstream region

       What steps should be taken for further analysis?

• The upstream region should be studied and used to
  perform GUS analysis

• Genotyping more plants to verify seed lethality
What was the second gene studied with knockout analysis?
                          AT5G62470
 • Chromosome 5

 • Location: 25096217 - 25098327 bp

 • 2111 base pairs long

 • Reverse orientation
What is the gene’s structure and where is the T-DNA insert?




   • 5’ UTR, 3 exons, 2 introns, 3’ UTR




                intron
   UTR   exon
What is the gene’s structure and where is the T-DNA insert?




   • 5’ UTR, 3 exons, 2 introns, 3’ UTR
   • SALK_111645
      – Expected insertion site: 761 bp
         What protein does this gene encode?
• MYB96: MYB domain protein 96 (R2R3)

• 352 amino acids

• 203 identified MYB family transcription factors, 126 of which are R2R3 MYB

• Large sized gene family with similar binding specificity

• DNA binding transcription factor

• Response to salt stress and dehydration
                                                              QuickTime™ and a
                                                          TIFF (LZW) decompressor
                                                       are neede d to see this picture.
Was T-DNA actually inserted into the plant samples?


 • What do we expect to see on the genotyping gel?
   – Two possible bands: WT (995 bp) and T-DNA (853 bp)
   – 1:2:1 ratio
Was T-DNA actually inserted into the plant samples?




                            WT




 • No T-DNA was inserted into the sampled plants. Why?
    – SALK error
    – Sowing error
    – Dominant mutation (seed lethal)
    – Not enough plants tested
In what tissues is the gene active?



                    Gene specific bands,
                    indicating accumulation in
                    both the silique and leaf




                  Gene appears to be active
                  in both the seed and leaf
In what tissues is the gene acitve?
Why is the upstream region important and what work
                  was done with it?
• The promoter region was isolated via PCR and ligated into the
  TOPO-vector
• E. coli cells were transformed with the recombinant plasmid
• The authenticity of the promoter insert was verified by restriction
  enzyme digestion and sequencing analysis



                                          Culture harboring
                                          recombinant plasmid



                                         Expected fragment size:
                                         2.6 kb + 3.8 kb = 5.4 kb
              Overview of AT5G62470
                What did we learn about this gene?
• No T-DNA inserts were found, and not much can be said
  without performing the appropriate knockout experiments

• Active in seed and leaf tissues in roughly equal amounts

• The upstream region is amplified and ready for GUS analysis

    What further steps should be taken to complete the analysis?
• New seeds should be ordered, grown, and genotyped

• Experiments (like those for gene one) should be carried out
  accordingly

• Perform GUS analysis with upstream region
                   Conclusions

• Even though no mutant phenotypes were observed,
  that does not mean that these two genes are not
  important in seed development
   – Redundant gene function

• There is still much to learn about these genes and the
  proteins they encode

• Further experimentation and analysis might reveal
  interesting results
Acknowledgements


   I would like to thank Dr. Goldberg,
         Anhthu, Daisy, Bekah, the
       HC70AL class, and every one
     else in the Goldberg Lab for all of
          your help and guidance
           throughout the course.

				
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