Using Escherichia coli and Saccharomyces cerevisiae as Model Systems for Teaching Genomics to Underg

1
Using Escherichia coli and Saccharomyces
cerevisiae as Model Systems for Teaching Genomics
to Undergraduates

• Breaking the silence researching the SIR2 gene
  family.

2
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.
3
Yeast SIR2 is a member of a family of genes
conserved from E.coli to humans.
4
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

5
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

6

• 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.

7
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.
8
3 primary domains of cellular lifeBacteria,
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.

9

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.

10
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
hst2kanMX4 hst3kanMX4 hst4kanMX4 Strain
WPD2.ii) MAT a his3-D1 leu2-D0 lys2-D0 ura3-D0
hst2URA3 hst3kanMX4 hst4kanMX4
11
Students are currently focusing on microarray
analysis of a hst3kanMX4 hst4kanMX4 double
mutant generated as follows

• Cross
• Research Genetics strain 3550
• MATa his3-?1 leu2-?0 met15-?0 ura3-?0
  hst4kanMX4
• X
• Research Genetics strain 11801
• MAT? his3-?1 leu2-?0 lys2-?0 ura3-?0
  hst3kanMX4

12
The resulting diploid was sporulated, tetrads
were dissected and spore clones were screened for
the hst3kanMX4 hst4kanMX4 double mutant by
Southern analysis.
13

• 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.

14
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.

15
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)
16
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.

17
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.

18
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.

19
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

20
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.
21
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

22
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

23
Feedback from some MSM students who participated
in the collaboration

• I am working on protein structure predictions
  algorithmsvery 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).

24
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.
Thank you