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What cellular functions are needed to carry out development?
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What cellular functions are needed to carry out development?
DivideGrowDifferentiateDieMoveAdhereSecreteSignal
Cell biological properties need to be coordinated in space and time
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Where is the “program” for development encoded?
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Where is the “program” for development encoded?
In the genes
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Why do different cells behave differently?
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Why do different cells behave differently?
Have different genes?Have different histories?Experience different environments?Chance?Have different gene expression states?
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Why do different cells behave differently?
Have different genes?Have different histories?Experience different environments?Chance?Have different gene expression states?
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05_02_DNA.jpg
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05_10_Genes_info.jpgGenes
Carry out functions
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07_37_Protein.produc.jpg
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08_03_control.steps.jpg
mRNA localization control
mRNA turnover control
Protein turnover control
Protein localization control
Regulation of gene expression
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08_03_control.steps.jpg
mRNA localization control
mRNA turnover control
Protein turnover control
Protein localization control
Regulation of gene expression
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coding strand
Parts of a gene
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08_13_gene.activation.jpgRegulation of transcription
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Transcription factors
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05_24_Chromatin pack.jpgNucleosomes (histones) package DNA
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05_30_histone tails.jpgHistone modifications affect gene expression
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08_14_chromatin.struc.jpgSome transcription factors affect histones
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08_15_Reg. proteins.jpgMultiple transcription factors regulate most genes
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08_18_reporter.gene.jpgModularity of the Drosophila even-skipped promoter
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Coordinated regulation of multiple genes
Developmental functions
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Signals can regulate activity of transcription factors
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08_23_cell.memory.jpgMaintaining gene expression states
(a positive feedback loop)
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08_24_chromatin.state.jpgMaintaining gene expression states
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08_14_chromatin.struc.jpgSome transcription factors affect histones
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Maintaining gene expression states – DNA methylation
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How does one monitor which genes a particular cell expresses?
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10_14_1_Southrn.blotting.jpgSouthern blot
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10_14_2_Southrn.blotting.jpgSouthern blot – DNA on blotNorthern blot – RNA on blot
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Northern blot hybridization
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In situ hybridization of developing flowers with ARF6 probe
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From Wildwater et al., Cell 123: 1337-1349 (2005)
Rb-Related expression in Arabidopsis embryosby in situ hybridization
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08_18_reporter.gene.jpgPromoter:reporter fusion gene in a transgenic fly embryo
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PARF6::ARF6::GUS fusion expression in flowers and ovules
Promoter:protein:reporter fusion gene – reveals protein location
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Kosman et al., Science 254: 118-122 (1991)
anti-Snailanti-Twist
Immunolocalization of Snail and Twist proteins in Drosophila embryos
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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
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Gene chips (microarrays) for assaying global gene expression patterns
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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.
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The Fordham Hall DNA Microarrayer
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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
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TumorPool of Cell Lines
A Typical DNA Microarray Experiment
Reference or Control
ExperimentalSample
Lower in Tumor
Higher in Tumor
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10_15_DNA.microarrays .jpg
Microarray technology
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increased expression
decreased expression
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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.
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Chu et al., Science 282: 699-705 (1998)
Transcription response during yeast sporulation (1116 out of ~5000 genes changed)
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Iyer et al., Science 283: 83-87 (1999)
Transcriptional response of human fibroblasts to serum
(~6% of genes on microarray changed)
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10_02_cell_sorter.jpgUsing a fluorescence-activated cell sorter (FACS) to separate cells
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Birnbaum et al., Nature Methods 2, 615 - 619 (2005)
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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
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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