F-Box Containing Tubby Transcription Factor Family Daisy Robinton Goldberg Lab Spring 2006

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

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

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

What Gene am I Working With? 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 AT1G ’ ’ UTRIntronExon startstop

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? Inflor +RT Inflor -RT Sil +RT Sil -RT Positive Control Negative Control

What do my RT-PCR Results Mean? Inflor +RT Inflor -RT Sil +RT Sil -RT Positive Control Negative Control Gene-Specific Expected size: 160 bp Control Bands 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

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 Melissa’s primers and my primers are present. Plant 7 Plant 8 Plant 9 Plant 10* Plant 12* Plant 11 Plant 13 Plant 14 Plant 15 Plant 16 Plant 17 WT Plant Positive Control Negative Control Melissa’s DNA w/ Insert Homozygous Mutants Plants 10 and 12 Predicted Size: 291 base pairs

Where is the Insert? 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) Actual Insertion SiteVia SequencingNucleotide 642 LBb1 SALK prediction = insertion site at nucleotide 659 (difference of 17 nucleotides) 5’ ’ UTRIntronExon startstop Rv

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 StageTorpedo Stage Cotyledon Stage Mutant Globular NONE FOUND There were no other apparent phenotypic differences upon observing the actual plant.

Wild Type v.s. Mutant Torpedo Stage Wild Type Mutant

When and Where does the “Promoter” Regulate the Transcription of my Gene? Predicted size of the cloned region: 3,166 base pairs 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 Colony 5Colony 6Colony 7Colony 8 Negative ControlPositive ControlPCR Product The T7 and SP6 sequencing seem to be reliable Compare to Sequencing

What is my Second Gene? AT1G76900 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 UTRIntronExon startstop 5’ ’

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? Inf. +RTInf. -RTSil. -RTSil. +RT Positive ControlNegative Control

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. Inf. +RTInf. -RTSil. -RTSil. +RT Positive ControlNegative Control Control Bands Gene-Specific Expected Size: 498 base pairs

Are there any knockouts? 28 Plants genotyped 0 Heterozygous Mutants 0 Homozygous Mutants The separating primers genotyping shows that the LB primer was working (Thi’s 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 Multiplex Separating Primers Plant 4Plant 3Plant 2 Plant 1 Plant 5Plant 6 Plant 11Plant 12 Plant 13 Plant 14Plant 15Plant 16Plant 17Plant 18Plant 19Plant 20Plant 21 WT Plant Positive ControlNegative Control Fw/RvFw/LBRv/LB LB Test

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)

Where was the Insert Predicted to Be? 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 LB UTRIntronExon startstop FW 5’ ’

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 PCR Product Positive Control Negative Control

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. 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 What further research should be carried out? AT1G25280AT1G76900 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)

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. :) =