Download Conservation and Diversification of Three

Conservation and
Diversification of ThreeRepeat Myb Transcription
Factors in Plants
Masaki Ito
Presented By Nina Carini and Erika Thiele
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.
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.
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).
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.
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.
Cyclin B genes in Arabidopsis
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.
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.
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.
Electrophoretic mobility
shift assay
 To test if NtmybA1,
NtmybA2, and NtmybB
bind specifically to MSA
elements.
 Mutations in MSA
element prevent
binding of proteins.
Binding of proteins to MSA
element
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.
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.
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.