1. Masaki Ito Presented By Nina Carini and Erika Thiele
Conservation and Diversification of Three-Repeat Myb Transcription Factors in Plants
Masaki Ito
Presented By Nina Carini and Erika Thiele

2. Objective
The purpose of this study is to show that Myb transcription factors containing three repeat sequences in their DNA-binding domains have a significant role in regulating transcription of cyclin B in the G2/M phases of tobacco.

3. What is Myb ?
Myb is derived from “myeloblastosis”, which is a name for a specific type of leukemia.
This gene was first recognized as the v-Myb oncogene of the avian myeloblastosis virus.
Family of transcription factors containing 2 or 3 repeat sequences in the DNA-binding domain (Myb domain).
Each repeat sequence is composed of approximately 50 amino acids.
These proteins bind to MSA elements in the promoter regions of specific genes such as cyclin B and activate transcription late in the G2 phase.
The domains that contain three repeat sequences have been shown to be evolutionarily conserved among plants. This study will focus on proteins that contain three repeat Myb domains.

4. Myb Structure
The structure of Myb domains are represented as a helix-turn helix formation.
This structure contains a domain with three repeat sequences (in blue).

5. The Cell Cycle S phase: DNA replication
M phase: Mitosis and Cytokinesis. Mitosis has 4 stages: prophase, prometaphase, metaphase, and anaphase. Cytoplasm division occurs telophase.

6. Arabidopsis Genome
5% of the Arabidopsis genome encodes 1,500 transcription factors.
One of the largest families is the Myb family.
Arabidopsis contains 130 Myb genes and 125 of these genes encode proteins with two repeat sequences. The proteins of interest contain Myb domains with three repeat sequences.
Arabidopsis contains 9 proteins that encode cyclin B.

7. Cyclin B genes in Arabidopsis

8. Methods Five 3R Myb proteins were isolated from tobacco.
NtmybA1, NtmybA2, and NtmybB were isolated by yeast one-hybrid screening.
NtmybC1 and NtmybC2 were isolated by PCR.

9. Yeast One-Hybrid Screening
Two yeast strains carrying the HIS3 reporter gene with a six repeat MSA element and three repeat MSA element were constructed.
The yeast cells with the six repeat MSA element were transformed with cDNA fragments of mRNA that were prepared from actively dividing tobacco cells.
The yeast cells were cultured to obtain colonies with the HIS3 reporter gene.

10. Polymerase Chain Reaction
PCR amplified the specific sequences of NtmybC1 and NtmybC2.
In plants, myb factors comprise one of the largest families of DNA-binding domains.
DNA sequences for NtmybC1 and NtmybC2 have not been determine.
Amino acid sequences are similar.

11. Electrophoretic mobility shift assay
To test if NtmybA1, NtmybA2, and NtmybB bind specifically to MSA elements.
Mutations in MSA element prevent binding of proteins.

12. Binding of proteins to MSA element

13. RNA Gel Blot
To determine accumulation of NtmybA1,NtmybA2, and cyclin B late in G2 phase.
NtmybA1, NymybA2, NtmybB, and cyclin B were treated with aphidicolin, a substance that inhibits the cell cycle. Upon removal of the aphidicolin, the proteins were run on a gel.

14. Gene expression late in G2 phase
Autoregulation: NtmybA1 and NtmybA2 have their own promoters. They can bind to their own promoters and enhance their activity.
Cyclin B binds to CDK to form a complex and phosphorylate NtmybA2.

15. Future Studies
Comparison of plant 3R Myb proteins and animal 3R Myb proteins.
To determine which gene sequences NtmybC1 and C2 interact with.
The mechanism for the positive feedback loop of NtmybA1 and NtmybA2 proteins with cyclin B.
The mechanism that causes accumulation of Ntmyb proteins and cyclin B late in the G2 phase.