Download Introduction to development

What cellular functions are needed to
carry out development?
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?
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
Genes
05_10_Genes_info.jpg
Carry out functions
07_37_Protein.produc.jpg
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
08_03_control.steps.jpg
mRNA localization control
mRNA turnover control
Protein turnover control
Protein localization control
Parts of a gene
coding strand
Regulation of transcription
08_13_gene.activation.jpg
Transcription factors
05_24_Chromatin pack.jpg
Nucleosomes
(histones)
package DNA
Histone modifications affect gene expression
05_30_histone tails.jpg
Some transcription factors affect histones
08_14_chromatin.struc.jpg
Multiple transcription factors regulate most genes
08_15_Reg. proteins.jpg
Modularity of the Drosophila even-skipped promoter
08_18_reporter.gene.jpg
Coordinated regulation of multiple genes
Developmental functions
Signals can regulate activity of transcription factors
Maintaining gene expression states
08_23_cell.memory.jpg
(a positive feedback loop)
Maintaining gene expression states
08_24_chromatin.state.jpg
Some transcription factors affect histones
08_14_chromatin.struc.jpg
Maintaining gene expression states – DNA methylation
How does one monitor which genes a
particular cell expresses?
Southern blot
10_14_1_Southrn.blotting.jpg
Southern blot – DNA on blot
Northern blot – RNA on blot
10_14_2_Southrn.blotting.jpg
Northern blot hybridization
In situ hybridization of developing flowers with ARF6 probe
Rb-Related expression in Arabidopsis embryos
by in situ hybridization
From Wildwater et al., Cell 123: 1337-1349 (2005)
Promoter:reporter fusion gene in a transgenic fly embryo
08_18_reporter.gene.jpg
PARF6::ARF6::GUS fusion expression in flowers and ovules
Promoter:protein:reporter fusion gene – reveals protein location
Immunolocalization of Snail and Twist proteins in Drosophila embryos
anti-Snail
anti-Twist
Kosman et al., Science 254: 118-122 (1991)
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
A Typical DNA Microarray Experiment
Reference
or Control
Pool of Cell Lines
Experimental
Sample
Tumor
Higher in
Tumor
Lower in
Tumor
10_15_DNA.microarrays .jpg
Microarray technology
increased expression
decreased expression
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.
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
Transcription response during
yeast sporulation
(1116 out of ~5000 genes changed)
Chu et al., Science 282: 699-705 (1998)
Transcriptional response
of human fibroblasts to serum
(~6% of genes on microarray
changed)
Iyer et al., Science 283: 83-87 (1999)
Using a fluorescenceactivated cell sorter
(FACS) to separate
cells
10_02_cell_sorter.jpg
Birnbaum et al., Nature Methods 2, 615 - 619 (2005)
Global expression map depicting major patterns of gene
activity in the Arabidopsis root
~4,000 genes
have cell-typespecific
expression
patterns
K. Birnbaum et al., Science 302: 1956 -1960 (2003)
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
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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:
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