What cellular functions are needed to carry out development?

What cellular functions are needed to carry out development?

DivideGrowDifferentiateDieMoveAdhereSecreteSignal

Cell biological properties need to be coordinated in space and time

Where is the “program” for development encoded?

Where is the “program” for development encoded?

In the genes

Why do different cells behave differently?

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.jpgGenes

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.jpgRegulation of transcription

Transcription factors

05_24_Chromatin pack.jpgNucleosomes (histones) package DNA

05_30_histone tails.jpgHistone modifications affect gene expression

08_14_chromatin.struc.jpgSome transcription factors affect histones

08_15_Reg. proteins.jpgMultiple transcription factors regulate most genes

08_18_reporter.gene.jpgModularity of the Drosophila even-skipped promoter

Coordinated regulation of multiple genes

Developmental functions

Signals can regulate activity of transcription factors

08_23_cell.memory.jpgMaintaining gene expression states

(a positive feedback loop)

08_24_chromatin.state.jpgMaintaining gene expression states

08_14_chromatin.struc.jpgSome 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.jpgSouthern blot

10_14_2_Southrn.blotting.jpgSouthern blot – DNA on blotNorthern blot – RNA on blot

Northern blot hybridization

In situ hybridization of developing flowers with ARF6 probe

From Wildwater et al., Cell 123: 1337-1349 (2005)

Rb-Related expression in Arabidopsis embryosby in situ hybridization

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

PARF6::ARF6::GUS fusion expression in flowers and ovules

Promoter:protein:reporter fusion gene – reveals protein location

Kosman et al., Science 254: 118-122 (1991)

anti-Snailanti-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 insertsPrinted 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

ExperimentalSample

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: 699-705 (1998)

Transcription response during yeast sporulation (1116 out of ~5000 genes changed)

Iyer et al., Science 283: 83-87 (1999)

Transcriptional response of human fibroblasts to serum

(~6% of genes on microarray changed)

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

Birnbaum et al., Nature Methods  2, 615 - 619 (2005)

K. Birnbaum et al., Science 302: 1956 -1960 (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 35-40 hours per week in the lab between May 20th and July 31st

Stipend Provided: $4,200

Application Deadline: March 27th, 5pm

More details and applications available for download at:

http://cmb.unc.edu/cure