Download Using Escherichia coli and Saccharomyces

Using Escherichia coli and
Saccharomyces cerevisiae as Model
Systems for Teaching Genomics to
Undergraduates
Breaking the silence: researching
the SIR2 gene family.
Dr. Myra K. Derbyshire and undergraduate students
Mount Saint Mary's College,
Emmitsburg, Maryland.
Collaborations and affiliations
Dr. Jeffery Strathern and Dr. Donald Court
NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland.
GCAT
Genome Consortium for Active Teaching
Dr. Patrick Brown
Stanford Microarray Database
Dr. Frederick Blattner
University of Wisconsin-Madison E.coli Genome Project.
Yeast SIR2 is a member of a
family of genes conserved
from E.coli to humans.
Yeast SIR2 is important for
• The structure of the nucleolus
• For silencing near chromosome
telomeres
• For silencing of special
sequences involved in cell-type
differentiation
Silencing :
• The term given
to a mechanism
by which gene
expression in
regions of the
genome is
repressed
• Silencing can be
modified by
changes in
chromatin
structure
• In eukaryotes,
altering
chromatin states
around a gene
allows cells to
achieve complex
patterns of
regulation.
• SIR2p is a
component of
chromatin
• SIR2p sets up
silent
chromatin
states around
the genes it
regulates.
Yeast SIR2p and SIR2-like proteins have an
NAD+-dependent deacetylase activity which is
responsible for underacetylated histones
within silent chromatin.
This deacetylation is coupled to NAD cleavage.
NAD is cleaved into : an ADP-ribose moiety
and nicotinamide
A novel cellular metabolite:
O-acetyl-ADP-ribose is generated.
3 primary domains of cellular life:
Bacteria, Archae, Eucarya
•
Eukarya: Regulation at the level of
chromatin.
•
Archae: Regulation at the level of
chromatin?
• -Archaeal Sir2p deacetylates the major
•
archeal chromatin protein Alba (Bell et
al., 2002)
•
Bacteria : Studying the E. coli SIR2
homolog cobB may tell us something
about DNA compaction in E.coli.
There are five members of the SIR2 gene
family in Saccharomyces cerevisiae
(SIR2, HST1-HST4)
– Little is known of the roles of the
HST1-HST4 genes.
– HST1-HST4 gene products may
modify chromatin structure at
different chromosomal locations
than those regulated by SIR2p.
Using standard yeast genetic manipulations,
we generated differently marked
multiple null mutants
as a resource for
Functional genomics
Among these we have identified
two potential triple null mutants by PCR .
Strain WPD1. i) MATa his3-D1 leu2-D0 met15-D0
ura3-D0 hst2::kanMX4 hst3::kanMX4 hst4::kanMX4
Strain WPD2.ii) MAT a his3-D1 leu2-D0 lys2-D0
ura3-D0 hst2::URA3 hst3::kanMX4 hst4::kanMX4
Students are currently focusing on
microarray analysis of a
hst3::kanMX4 hst4::kanMX4
double mutant generated as follows
Cross:
Research Genetics strain #3550:
MATa his3-D1 leu2-D0 met15-D0 ura3-D0
hst4::kanMX4
X
Research Genetics strain #11801:
MATa his3-D1 leu2-D0 lys2-D0 ura3-D0
hst3::kanMX4
The resulting diploid was sporulated,
tetrads were dissected and spore clones
were screened for the hst3::kanMX4
hst4::kanMX4 double mutant by Southern
analysis.
• Summer 2000. MSM undergraduates
generated an E.coli cobB null mutant
• Using a defective  prophage mediated
recombination system described by (Yu
et al 2000)
• U.W. Madison E.coli genome project
E.coli mutant strains are available for
functional analysis.
Milieu
• Liberal Arts College situated close to
Frederick Maryland
• College missions include: teaching,
scholarship and community service.
• Research for 20 years post PhD. Full
time teaching last 4 years.
• Support from NCI-FCRDC and GCAT.
Number of students in
MSM Genomics initiative
Fall 2000:
Genetics (11)
Spring 2001:
Research (1)
Summer 2001.
Research (3)
Fall 2001:
Genetics (18)
Summer 2002.
Research (2)
Fall 2002:
Genetics (25 ?)
Research (4)
MSM students through genomic
research
Strengthen
- conceptual understanding
- problem solving skills
Have access to the virtual learning
/teaching/research/service community of
GCAT.
Immerse themselves through Genomics in
different model genetic systems.
Global Goals of Genomics:
– To assess an organisms genetic
complement.
– To determine which genes are
expressed and under what
conditions.
– To determine the function of
protein products.
Students in their study of
the SIR2 family: predict
gene function
Identify SIR2 family homologs
– Interact with GenBank (database of all
publicly available DNA and derived protein
sequences)
– Carry out data base searches to identify
family members using: PSI-Blast and
know motifs.
Students in their study of
the SIR2 family appreciate :
– multiple sequence alignments.
– Structural similarity does not imply
common function.
– To predict function one needs
experimental proof of function
MSM students studying the
SIR2 family
Compared gene expression patterns of two
meiotic segregants (HST3 HST4 and hst3 hst4)
using microarray technology (GCAT)
This data is viewable on a public search of the
Stanford Microarray database.
Undergraduate access to Yeast
Microarray technology was made
possible through affiliation with
GCAT/Stanford Microarray database.
MSM Undergraduates are:
– Repeating this microarray
comparison using three independent
RNA isolations
– They will analyze data from multiple
experiments and correlate patterns of
gene expression
Planned student microarray experiment:
made possible through affiliation with
GCAT/ UW-Madison E.coli genome
project
Gene expression patterns of an E.coli
mutant having a null mutation in the
cobB gene and an isogenic wild-type
strain
Feedback from some MSM students who
participated in the collaboration
•
–
–
–
I am working on protein structure
predictions algorithms…very neat stuff!
(Pursuing a Ph.D. in Bioinformatics).
Thank you again for the wonderful
opportunity and experience you afforded me in
genetics class (Pursuing a graduate program in
Cellular and Molecular medicine)
Thank you for everything you have given
and taught me. I carry it with me all the
time. (Pursuing a Masters in Bioscience
and technology).
Collaborations and affiliations
Dr. Jeffery Strathern and Dr. Donald Court
NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland.
GCAT
Genome Consortium for Active Teaching
Dr. Patrick Brown
Stanford Microarray Database
Dr. Frederick Blattner
University of Wisconsin-Madison E.coli Genome Project.