FBox Containing Tubby Transcription Factor Family

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F-Box Containing Tubby Transcription Factor Family

• Daisy Robinton
• Goldberg Lab
• Spring 2006

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What is the TUBBY Protein Gene Family?

• Studies have shown TUBBY-like proteins to play a
  vital role in the maitenance and function of
  neuronal cells during postdifferentiation and
  development in mammals
• Members of this family have been identified in
  many multicellular organisms, but no
  single-celled organisms
• The tubby domain is highly conserved
• This data suggests that tubby proteins have
  fundamental biological functions
• A conserved F-box-containing domain is present in
  plant TUBBY-like protein members

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What is this F-box Protein?

• Most F-box domains followed by specific amino
  acid sequences which are protein-protein
  interacting domains for recruiting specific
  proteins and targeting them for ubiquitin
  mediated proteolysis
• F-box proteins regulate cellular processes
  (cell-cycle transition, transcriptional
  regulation and signal transduction)
• F-box proteins regulate plant growth and
  development
• Serve vital functions in plants as indicated by
  multiple TLPs, however this may mask the effects
  of an individual loss-of-function allele

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How is this Family Related to Arabidopsis?

• In the Arabidopsis plant there is a TUBBY-like
  protein gene family with 11 members (named
  AtTLP1-11)
• All AtTLP members (except 8) have an F-box domain
  (researchers are not sure, this data was obtained
  solely via sequencing
• Seven of the AtTLP genes are on the first
  chromosome
• Two genes, one gene and one gene are on
  chromosomes II, III and V, respectively
• All members of this family except AtTLP8 contain
  a conserved F-box domain
• F-box protein has been identified to play an
  important role during seed development

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What Gene am I Working With?
AT1G25280

• This gene is 3,029 base pairs long
• It is located on the first chromosome
• It encodes the F-Box protein
• The size of the encoded protein is 446 amino acids

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Where is my Gene Active?

• Microarray data indicates that this gene is
  active in all of the above organs during seed
  development
• However, microarrays are not as accurate as the
  results obtained in RT-PCR because in RT-PCR we
  amplify the DNA so that the reaction is more
  sensitive to the presence of smaller DNA
  fragments
• My RT-PCR results confirm and agree with the
  indications from the genechip data. How do I
  know?

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What do my RT-PCR Results Mean?

• The gene-specific bands indicate that mRNA was
  made in those organs during seed development, and
  so we know that our gene is being transcribed
  during that stage in that organ

Control Bands
Gene-Specific Expected size 160 bp
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Do I Have any Mutant Plants?

• 19 Plants genotyped
• 0 Heterozygous mutants
• 2 Homozygous mutants
• There are three bands in the positive control
  lane because Melissas primers and my primers are
  present.

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Where is the Insert?
Actual Insertion Site Via Sequencing Nucleotide
642
SALK prediction insertion site at nucleotide
659 (difference of 17 nucleotides)
LBb1

• The T-DNA is inserted in the forward direction,
  interacting with the reverse primer
• The LBb1sequencing reaction begins matching with
  my WT gene DNA at nucleotide 115 (of the
  sequencing reaction)

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Does the Insert Disrupt Seed Development?
Because I found homozygous mutants it is
indicated that this knockout does not cause seed
lethality. However, upon observation of seeds
using the Nomarski microscope, interesting
phenotypes were found.
Wild Type
Heart Stage
Torpedo Stage
Globular
Mutant
NONE FOUND
There were no other apparent phenotypic
differences upon observing the actual plant.
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Wild Type v.s. MutantTorpedo Stage
Wild Type
Mutant
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When and Where does the Promoter Regulate the
Transcription of my Gene?

• Predicted size of the cloned region 3,166 base
  pairs

Compare to Sequencing

• The T7 and SP6 sequencing seem to be reliable

• Of approxiamately 97 bacteria colonies, zero were
  found to be blue
• Verification of colonies containing recombinant
  plasmids by digestion with Eco R1 yielded
  confirming results

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What is my Second Gene?AT1G76900
5---
---3

• This gene is 2,506 base pairs long
• It is located on the first chromosome
• It encodes a Tubby-Like Protein
• The size of the encoded protein is 456 amino acids

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Where is This Gene Active?

• Microarray data indicates that this gene is
  active in all of the above organs during seed
  development
• However, microarrays are not as accurate as the
  results obtained in RT-PCR because in RT-PCR we
  amplify the DNA so that the reaction is more
  sensitive to the presence of smaller DNA
  fragments
• My RT-PCR results confirm agree with the data
  from the genechip for the Silique, but not the
  Infloresence. How do I know?

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What do my RT-PCR Results Mean?

• Note the absence of bands in the -RT lanes (2
  and 4)
• There is no gene-specific band in the
  Infloresence RT. This indicates that there was
  no mRNA transcribed during the seed development
  stage in this organ.
• There is a gene-specific band in the Silique -RT.
  This indicates that mRNA is transcribed in this
  organ during seed development.

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Are there any knockouts?
Separating Primers
Multiplex

• 28 Plants genotyped
• 0 Heterozygous Mutants
• 0 Homozygous Mutants
• The separating primers genotyping shows that the
  LB primer was working (This DNA from her first
  gene SAIL line was used)
• Because there are no bands in the other LB
  reactions, no mutants of any kind were
  identified.
• Expected Wild Type Size 2,479 base pairs
• Expected Mutant Size 2, 495 base pairs
• The separating primers attends to the problem of
  the similar size of expected mutant and WT bands

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What is the Reason for the Absence of Knockouts?

• SALK sent all Wild-Type seeds
• The knockout causes seed lethality, and no seeds
  survived that contained mutant alleles (dominant
  mutation)
• More plants need to be grown and genotyped
• The LB was ineffective (for the last batch of
  genotyping)

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Where was the Insert Predicted to Be?
LB
5---
---3

• SALK predicts the insert is in the third exon, at
  nucleotide 2,151
• The insert is in the reverse direction of my
  gene, interacting with my forward primer
• The lack of identified mutant plants may indicate
  disruption in seed development

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When and Where does the Promoter Regulate the
Transcription of my Gene?

• Predicted Size of Clone Region 2,997 base pairs

• EcoR1 Digestion gel photo unavailable
• Results showed three bands in each lane one
  about 3.5kB, one about 1.3kB and one about 1.05kB
• Verification of colonies containing recombinant
  plasmids by digestion with Eco R1 yielded
  confirming results
• Of approxiamately 200 bacteria colonies, approx.
  75 were found to be blue

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What is the significance of these results?

• Studies of the first gene, AT1G25280, show a very
  interesting seed phenotype at the torpedo stage
• This phenotype may be an important clue as to the
  role this gene plays in the Arabidopsis plant
• Studies of the second gene, AT1G76900, have not
  yet provided any significant results.

What further research should be carried out?
AT1G25280
AT1G76900

• More plants need to be grown and genotyped
• More siliques and seeds should be studied for
  phenotypic differences
• Because the insert is in an intron, RT-PCR of the
  mutant should be carried out

• Get new seeds
• More plants need to be grown and genotyped
• Once/if an insert is found, the appropriate
  experiments should be conducted (as with the
  first gene)

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Thanks!
Thanks to SALK, who made our plants (and mixed
things up so as to provide valuable learning
experiences. Thanks to Ahnthu, Ria, John and
Mike for testing, retesting, explaining,
teaching, reteaching, etc etc etc thanks a
million times. Thanks to Tomo, Javier, Brandon
and Xingjun for behind the scenes work and
Nomarski. Thanks to Jessica for food and
pictures haha What do you mean in the wrong
place? And finally, thanks to Dr. Goldberg for
making it all possible. And the pictures I
borrowed for my presentation. )