Module 2 Flashcards
Genomic equivalence
Genomic equivalence
The theory that every cell of an organism carries the same genome.
Polytene chromosomes
Drosophila result from continuous rounds of DNA replication without intervening mitosis- they represent a high resolution physical genetic map ~ 5000 discernable bands
The banding pattern,
represents the organization of the genome, is the same in different tissues – and this was considered early evidence in support of genomic equivalence.
the endo cycle.
Drosophila larvae experience rapid growth involving increased nuclear ploidy and cell size without cellular division. This is a cell variation known as the endo cycle.
how can we observe gene expression in embryos/cells/tissues?
immunostaining embryos/cells/tissues using antibodies to detect proteins
in situ hybridization using labelled nucleic acid probes to detect mRNA molecules
reporter gene constructs can be used to detect transcription associated with specific enhancer or promoter elements
Antibodies (as molecular biology tools) are useful for:
visualizing proteins in fixed cells
visualizing proteins separated by electrophoresis (western blot)
biochemically isolating proteins* from cells (immunoprecipitation)
blocking protein activity in living cells.
In situ hybridization
In situ hybridization is one of the techniques that form the bedrock of molecular developmental biology; it is used to the visualize mRNA molecules, and hence we can observe patterns of gene transcription.
when is in situ useful
In performing whole mount in situ hybridization, embryos are fixed in a way that preserves the mRNA molecules (no RNAases anywhere!) and allows the tissues to be relatively permeable (treatment with proteinases).
reporter genes :
encode products that can be easily detected and are not normally expressed in the cells being studied (b-galactosidase = lacZ , green fluorescent protein = GFP, also - luciferase, b-lactamase, chloramphenicol acteyl transferase )
lacZ -
detected using a special colourless b-galactosidase substrate that produces a coloured precipitate product (X-gal) when cleaved by b-galactosidase
GFP –
and other fluorescent protein detected by fluorescence microscopy (blue light/ green filters for GFP) and can be done on living cells/ organisms.
reporter contructs:
DNA from regions flanking a gene of interest (containing enhancer sequences – more on enhancers later) are cloned into a construct containing a promoter sequence upstream of a reporter gene.
transcription:
activity of transcription factors results in differential gene transcription by RNA polymerase (RNApol II). The availability of a given promoter to transcription factors or RNA polymerase is influenced by the state of chromatin, which is regulated through processes such as histone methylation or histone acetylation as well as DNA methylation.
Why do we wish to make reporter gene constructs?
reporter constructs represent and easier method for visualizing gene expression without the need to perform immunostaining or in situ hybridization
in some cases the promoter and enhancer of a gene of interest is placed (molecular biology and DNA cloning) upstream of the reporter gene
in other cases a DNA sequence can be cloned upstream of a minimal synthetic promoter* to test if that particular sequence contains an enhancer element from the gene of interest
RNA processing:
nascent transcripts must be processed (spliced, capped & tailed) to become mRNA that can be translated in cytoplasm.
) transport and localization of mRNA:
in some cases transport of mRNA out of the nucleus can be selective, while the localization of mRNA molecules within the cytoplasm can determine whether or not it will be translated
translation:
not all mRNA’s are translated at the same rate; they can also be degraded at different rates (mRNA stability). Stability of mRNA is influenced by polyA tail length as well as activity of miRNA (microRNAs)
post-translational modification:
protein activity and subcellular localization can be influenced by protein phosphorylation, glycosylation, etc.
silencers
are DNA regulatory elements that actively repress transcription
protein that binds to the NRSE is neural restrictive silencer factor (NRSF or REST) is expressed in all cells except?
neurons
How do transcription factors work?
bind to enhancer sequences
interact with other proteins – often with result of recruiting histone modifying enzymes (histones can be acetylated or methylated – also phosphorylated & histone modification can change chromatin compaction)
transcription factors also create a stable transcription pre-initiation complex – kind of a landing pad for RNApol in front of the promoter
transcription factors all have a DNA binding domain
recognizes and binds particular DNA sequences within the enhancer
transcription factors all have a trans-activating domain
activates or suppresses transcription of the associated gene by interacting with proteins that in turn bind RNA polymerase
protein-protein interaction domain
allows the transcription factor to be modulated by transcription associated factors (TAFs) or other transcription factor
