What cellular functions are needed to carry out development?

Divide Grow Differentiate Die Move Adhere Secrete Signal Cell biological properties need to be coordinated in space and time

Where is the “program” for development encoded?

In the genes

Why do different cells behave differently?

Have different genes? Have different histories? Experience different environments? Chance? Have different gene expression states?

Why do different cells behave differently? Have different genes? Have different histories? Experience different environments? Chance? Have different gene expression states?

05_02_DNA.jpg

05_10_Genes_info.jpg Genes Carry out functions

07_37_Protein.produc.jpg

08_03_control.steps.jpg mRNA localization control mRNA turnover control Protein turnover control Protein localization control Regulation of gene expression

08_03_control.steps.jpg mRNA localization control mRNA turnover control Protein turnover control Protein localization control Regulation of gene expression

coding strand Parts of a gene

08_13_gene.activation.jpg Regulation of transcription

Transcription factors

05_24_Chromatin pack.jpg Nucleosomes (histones) package DNA

05_30_histone tails.jpg Histone modifications affect gene expression

08_14_chromatin.struc.jpg Some transcription factors affect histones

08_15_Reg. proteins.jpg Multiple transcription factors regulate most genes

08_18_reporter.gene.jpg Modularity of the Drosophila even-skipped promoter

Coordinated regulation of multiple genes Developmental functions

Signals can regulate activity of transcription factors

08_23_cell.memory.jpg Maintaining gene expression states (a positive feedback loop)

08_24_chromatin.state.jpg Maintaining gene expression states

08_14_chromatin.struc.jpg Some transcription factors affect histones

Maintaining gene expression states – DNA methylation

How does one monitor which genes a particular cell expresses?

10_14_1_Southrn.blotting.jpg Southern blot

10_14_2_Southrn.blotting.jpg Southern blot – DNA on blot Northern blot – RNA on blot

Northern blot hybridization

In situ hybridization of developing flowers with ARF6 probe

From Wildwater et al., Cell 123: (2005) Rb-Related expression in Arabidopsis embryos by in situ hybridization

08_18_reporter.gene.jpg Promoter:reporter fusion gene in a transgenic fly embryo

P ARF6 ::ARF6::GUS fusion expression in flowers and ovules Promoter:protein:reporter fusion gene – reveals protein location

Kosman et al., Science 254: (1991) anti-Snail anti-Twist Immunolocalization of Snail and Twist proteins in Drosophila embryos

Utility of looking at expression of single genes at a time: Markers of cell type, differentiation Visualization of regulatory events Utility of looking at expression of many genes at once: Global view of tissue identity Comparing different tissues or states Global view of regulatory events

Gene chips (microarrays) for assaying global gene expression patterns

Spotted Microarrays PCR products (direct from genome, or from cDNA clones), or oligonucleotides are spotted by capillary action onto a glass microscope slide. Up to ~44,000 features per slide Typically hybridized with 2 differentially labeled samples simultaneously.

The Fordham Hall DNA Microarrayer

Spotted DNA Microarray 18,000 PCR-amplified cDNA clone inserts Printed on poly-lysine coated 1 X 3 inch glass slide Each spot corresponds to a different gene circa 1998

TumorPool of Cell Lines A Typical DNA Microarray Experiment Reference or Control Experimental Sample Lower in Tumor Higher in Tumor

10_15_DNA.microarrays.jpg Microarray technology

increased expression decreased expression

these genes have higher expression in normal tissue than breast cancers these genes have lower expression in basal-like breast cancers than in normal tissue or luminal breast cancers Expression profiling: overall patterns of gene expression can be used in diagnosis Each column is from a different tumor. Each row represents one gene. Rows are clustered by similar expression pattern.

Chu et al., Science 282: (1998) Transcription response during yeast sporulation (1116 out of ~5000 genes changed)

Iyer et al., Science 283: (1999) Transcriptional response of human fibroblasts to serum (~6% of genes on microarray changed)

10_02_cell_sorter.jpg Using a fluorescence- activated cell sorter (FACS) to separate cells

Birnbaum et al., Nature Methods 2, (2005)

K. Birnbaum et al., Science 302: (2003) Global expression map depicting major patterns of gene activity in the Arabidopsis root ~4,000 genes have cell-type- specific expression patterns

Are you interested in doing UNDERGRADUATE RESEARCH at top UNC labs this summer? Now taking applications for CURE (Carolina Undergraduate Research Enrichment) Summer 2009 The CURE program is run by graduate students in the Cell and Molecular Biology (CMB) program at UNC. The CURE program pairs undergraduates with graduate student mentors to participate in research. CURE students also meet weekly to participate in journal clubs and career development workshops. Requires hours per week in the lab between May 20th and July 31st Stipend Provided: $4,200 Application Deadline: March 27 th, 5pm More details and applications available for download at: