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.

–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. There are five members of the SIR2 gene family in Saccharomyces cerevisiae (SIR2, HST1-HST4)

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) MAT  his3-  1 leu2-  0 met15-  0 ura3-  0 hst2::kanMX4 hst3::kanMX4 hst4::kanMX4 Strain WPD2.ii) MAT  his3-  1 leu2-  0 lys2-  0 ura3-  0 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-  1 leu2-  0 met15-  0 ura3-  0 hst4::kanMX4 X Research Genetics strain #11801: MAT  his3-  1 leu2-  0 lys2-  0 ura3-  0 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 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 Research (3) Fall 2001: Genetics (18) Summer 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.