March 2, 2011 Introduction to development Gene expression
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 Other influences on development: Maternal components Environmental signals
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 Steps in gene expression
08_03_control.steps.jpg mRNA localization control (tether mRNA to localized proteins) mRNA turnover control (plant microRNAs) Protein turnover control (ubiquitylation and proteasome digestion) Protein localization control Regulation of gene expression
08_03_control.steps.jpg mRNA localization control (tether mRNA to localized proteins) mRNA turnover control (plant microRNAs) Protein turnover control (ubiquitylation and proteasome digestion) Protein localization control Regulation of gene expression
coding strand Regulation of transcription: Parts of a gene
08_13_gene.activation.jpg Regulation of transcription
Transcription factors Dimer (Two proteins)
05_24_Chromatin pack.jpg Nucleosomes (histones) package DNA Histones affect gene expression
08_14_chromatin.struc.jpg Some transcription factors affect histones
05_30_histone tails.jpg Histone modifications affect gene expression Can affect recruitment of transcription factors to promoter H3K27m 4
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
The histone code also needs to be maintained in daughter cells
Maintaining gene expression states – DNA methylation
How does one detect 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 (tests one gene at a time)
RT-PCR: 1) Reverse transcription (tests one gene at a time)
RT-PCR: 2) Polymerase Chain Reaction (tests one gene at a time)
RT-PCR summary (tests one gene at a time) Quantitate on gel or by measuring rate of DNA product accumulation
In situ hybridization of developing flowers with ARF6 probe Reveals which cells in a complex tissue express the gene Earlier stages, longitudinal sections Later stages, transverse sections
From Wildwater et al., Cell 123: (2005) Rb-Related expression in Arabidopsis embryos by in situ hybridization Why is this pattern punctate (spotty)?
coding strand Making reporter gene fusions Examples: lacZ, GUS, GFP Insert construct into transgenic organism Promoter Reporter gene
P ARF6 ::ARF6::GUS fusion expression in ovules Promoter:protein:reporter fusion gene In situ hybridizations
08_18_reporter.gene.jpg Promoter:reporter fusion gene in a transgenic fly embryo
Kosman et al., Science 254: (1991) anti-Snail anti-Twist Immunolocalization of Snail and Twist proteins in Drosophila embryos Reveals protein location
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