Abcam.com. hutton.ac.uk. Ipmdss.dk. Bo Gong and Eva Chou

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1 Abcam.com Bo Gong and Eva Chou Ipmdss.dk hutton.ac.uk

2 What is a homeotic gene? A gene which regulates the developmental fate of anatomical structures in an organism Why study them? Understand the underlying mechanisms controlling cellular differentiation and growth

3 We like to eat! cdn.foodbeast.com.s3.amazonaws.com

4

5 Wildtype Arabidopsis Flower

6 ag-1 mutant ag-1 mutant (EMS) No stamens/carpels 6 petals in place of the 6 stamens In place of carpels there is a repeating sepal, petal, petal pattern

7 agamous mutants ag-1 EMS WT T-DNA insertion

8 They have a mutant they believe is an allele of ag-1, how would they test that? Complementation test Why is this hard to do in agamous? Because agamous mutants are sterile

9 Complementation test T-DNA ag-1 mutant -/- X -/- insertion mutant sterile

10 T-DNA ag-1 mutant +/- X +/- insertion mutant Scenario 1: allelic Scenario 2: not allelic ag-1 mutant +/- X +/- T-DNA insertion mutant ag-1 mutant +/- +/+ X +/+ +/- T-DNA?? insertion mutant +/+ +/- +/- -/- All wild-type phenotype

11 Complementation results ag-2 ¼ progeny displayed mutant phenotype Complementation failed T-DNA mutation dubbed ag-2 T-DNA insertion

12 The big picture EMS ag-1 mutant allelic ag-2 mutant T-DNA

13 Kanamycin resistance co-segregates with the mutant flower phenotype ag-1 + ag-2 are allelic T-DNA can be used as a probe to isolate the AG gene EMS ag-1 mutant allelic ag-2 mutant T-DNA

14 Plasmid Rescue

15 Figure 2a: pcit505 How could you determine if the plasmid pcit505 contains sequence of the AG gene? Determine the linkage between pcit505 sequence and AG gene Probe for a longer sequence and do transgenic rescue

16 Restriction Fragment Length Polymorphism (RFLP) Ecotype A A B A B Ecotype B Adapted from

17 Restriction Fragment Length Polymorphism (RFLP)

18 Ler Nd-0 ag-1 AG X ag-1 AG ag-1 ag-1

19 What would you see if pcit505 is linked with ag-1? ag-1 -/- Plants in F2 No crossovers observed. Therefore pcit505 is tightly linked to the AG gene

20 Making a cosmid library Cut + Ligate Infect E. Coli In vitro packaging into phage biosiva.50webs.org

21 Screening the cosmid library pcit540 Gobot.ca Incubate with a radioactive probe based on pcit505 Autoradiograph to See successful hybridization

22 Found pcit540

23 Transgene complementation ag-2 pcit540 T-DNA insertion

24 Phenotype Recovery T-DNA insertion Wild-type Rescued with pcit540

25 Phenotype rescued by pcit540 T-DNA insertion is stable What does this tell you? Rescued with pcit540 The pcit540 sequence likely contains the AG gene

26 Generate a cdna library from the WT Arabidopsis flower Reverse transcription Degrade RNA + DNA Pol I Digest and ligate in to a plasmid and transform in to E. coli mun.ca

27 Probing the cdna library Regions flanking the T-DNA were cut using restriction enzymes and used as a probe against the cdna library A cdna clone was found to span the region of the T-DNA

28 Sequence the cdna Sequence the genomic DNA How could the cdna help us determine the intronic and exonic regions of the gene? Fig 2b:

29 How can you confirm if the sequence is AG? Sequence the AG region in ag-1 mutants Difference between ag-1 and WT found to be a single nucleotide substitution from G to A in the fourth intron T-DNA insertion found to be in the second intron

30 Protein sequence Amino acid sequence obtained from the cdna sequence Longest cdna = 1043bp RNA = 1.1kb Fig. 2c:

31 AG may be a transcription factor Fig. 3: AG (Arabidopsis) ARG80 and MCM1 (Yeast) SRF (Human) DEF A (Antirrhinum) hortipedia.com devbio.biology.gatech.edu

32 AG gene expression pattern Northern (RNA) blot In situ hybridization Reverse transcription PCR RNA-seq database Promoter driven marker

33 Northern blot with radiolabelled Fig. 4: probes utoronto.ca/efp phenomics.cn F = floral bud (stage 9) S = floral stem V = vegetative tissue jamesandthegiantcorn.com

34 In situ hybridization Prepare sample Fix sample Hybridize Prepare probe Generate radiolabeled RNA utoronto.ca/efp isogen-lifescience.com

35 In situ hybridization Fig.5:

36 In situ hybridization Negative control not shown Expression in stamens and carpels but not petals or sepals Hypothesized to be a transcription factor which regulates genes that are involved in the development of stamens and carpels Bio 433 Lecture 5 Slide 13

37 Arabidopsis efp browser Stage 12: Highest expression in stamens No/low expression in petals and sepals bar.utoronto.ca/efp

38 Additional genes expressed in flowers Identified other Arabidopsis genes differentially expression in flower These genes share DNA binding motifs similar to SRF (human) and MCM1 (yeast) Possible family of genes regulating different steps of Arabidopsis floral organ development

39 Summary Identifying a T-DNA insertion mutation that is allelic to ag-1 mutant Isolating the AG gene sequence Analyzing the nucleotide and peptide sequence of the AG gene Hypothesizing the potential role of the AG gene product as a transcription factor based on peptide sequence homology Characterizing the spatial expression pattern of the AG gene

40 Future Directions Further investigate the spatial and temporal expression pattern of the AG gene. Examine the correlation between the expression pattern and function of the AG gene product. Confirming the transcription factor role of the AG gene product. How the AG gene product regulates flower development as a transcription factor. Regulatory interactions between AG gene product and other members of the later known MADS-box gene family.

41 MADS Box Genes MADS-box genes are named after 4 of the founding members: MCM1 (Yeast) AGAMOUS (Arabidopsis) DEFICIENS (Antirrhinum) SRF (Human)

42 MADS box AGAMOUS is a type II MADS-box gene. It contains MADS (M), intervening (I), keratin-like (K) and C-terminal (C) domains. In the ABC flower development model, it is a member of the class C gene. Folter and Angenent, 2006 (Trends in Plant Sci)

43 MADS box CArG box Folter and Angenent, 2006 (Trends in Plant Sci)

44 Thank You!