Developmental genetics Flashcards
Vertebrate gene nomenclature
Species symbol Gene symbol(Sonic) Protein
Homo Sapiens SHH SHH
Mus musculus Shh SHH
Gallus gallus SHH SHH
Xenopus laevis shh Shh
Danio Rerio shh Shh
Genes named after protein product or other molecular feature begin with an uppercase letter
Species name and gene name are italicised
When doing proteins always capitalise first letter so yk its a protein
Mutations - alternations to DNA sequence
Occur naturally but also induced in laboratory
Random (how naturally occur but sped up process in lab):
- Radiation ~ UV light, X-rays, radioactivity etc
- Chemical ~ base analogous, base modifiers, intercalating agents (insert themselves into DNA and cause problems during replication) etc
These changes can be point mutations (single bp), deletion, insertion, translocation etc
Targeted:
- CRISPR
- Knock-out
Genetic engineering in mice to target a gene
Endogenous gene - original gene
Transgene - new gene after change
Ways of change:
A. Gene knockout - completely removes gene to determine function
B. Gene replacement (Knock-in) - Usually makes small change to endogenous gene
If find point mutation in human can test whether mutation causing disease by making corresponding changes in corresponding mouse genes
CRISPR
Very new technology - only around 5 years old
Different tech but can also be used to knock-out or -in
Advantage of it is that works in any organism and not the expensive
How do mutations affect gene function?
Changes in regulatory sequences (enhancers) in DNA affect transcription
Changes in non-coding sequence of transcript may affect RNA splicing, stability or translation
Changes in coding sequence may alter important amino acid affecting folding of protein or create premature stop codon which causes truncated protein
Mutation in coding sequence often affect protein function
DNA binding -> dimerization -> conformational change -> transcriptional activation
Domain is functional unit in protein
Dimer - when two same proteins bind together
Example 1 mutation in coding sequence
Amorphic/non-functioning
E.g. missense mutation that completely inactivates DNA binding domain
+/-: Normally is enough gene product from one wild-type copy - Haplosufficient
-/-: Strong phenotype due to no transcriptional activation, recessive
Example 2
Hypomorphic/weakened
E.g. Missense mutation that weakens DNA binding domain
+/-: Normally haplosufficient, mutant form may also dimerize with wild type and site activate transcription
-/-: Mild phenotype due to poor transcriptional activity, complex forms on DNA but often falling off, recessive
Example 3
Antimorphic/dominant
E.g. Missense mutations that destroys dimerization domain
+/-: Mutant form binds DNA but doesn’t dimerize with WT, thus doesn’t go through conformational change to become active, transcription compromised and only activated when two W proteins land together, dominant
-/-: completely inactive
Example 4
Hypermorphic/overactive
E.g. Missense mutation that results in activation that is independent of dimerization
E.g. Missense mutations that destroys dimerization domain
+/-: Mutant form binds DNA is active all time and makes dimer site active all time, call this constitutively active, doesn’t need conformational change to bind and only needs one copy to bind, increases overall activation of transcription and is unregulated, Dominant
-/-: is same as +/-
Types phenotypes produced by mutations - Muller’s Morphs
Loss-of-function mutations:
- Amorphic - complete loss gene function, typically early nonsense mutations or deletion entire gene, most genes haplosufficient in diploid organisms so these usually recessive
- Hypomorphic - reduction of wild type function, typically missense mutations, usually recessive
- Antimorphic - competitive inhibitors, typically mutations that affect one domain of protein, in heterozygotes that mutant form still partially active allowing mutant protein interact with and poison WT protein, also called dominant negative
Gain-of-function mutatio:
- Hypermorphic - over expression of transcription unit or over activity of gene product, e.g. mutation in binding site for repressor, dominant
Genetic pathways
E.g. A B C D are enzymes required for biosynthesis of melanin
Lose-of-function in any genes code A B C OR D may lead to albino phenotype
Mutations that result in same phenotype may be two different mutations in same genes (alleles)
Mutations that result in smae phenotype that are not in same gene suggest proteins are in same pathway
Reporter constructs: Green Fluorescent Proteins (GFP)
Are now dozens different fluoresent proteins have been characterised
Encoded by one protein
You always absorb a wavelength that is short than the wavelength you emit In GFP it absorbs 475 nM and emits 510 nM
Generating a GFP transgenic line (transgenesis)
- Clone entire gene with all regulatory elements into a plasmid
- Genetically engineer Beta galactosidase onto end of last exon (gene fusion) or replace gene (reporter construct)
- Re-introduce this into animal, usually not into the endogenous gene
Uses for GFP transgenic lines
To follow expression of gene or to follow behaviour of cell in vivo
To follow subcellular localisation of a protein
