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					    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   • Silencing can be
  to a mechanism     modified by
  by which gene      changes in
  expression in      chromatin
  regions of the
  genome is          structure
  repressed
• In eukaryotes,     • SIR2p is a
  altering             component of
  chromatin states     chromatin
  around a gene      • SIR2p sets up
  allows cells to      silent
  achieve complex      chromatin
  patterns of          states around
  regulation.          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.

				
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