developmental genetics Flashcards
vertebrate gene nomenclature
-Species Gene symbol Protein
Homo sapiens SHH SHH
Mus musculus Shh SHH
Gallus gallus SHH SHH
Xenopus laevis shh Shh
Danio rerio shh Shh
how do Random mutations occur
-Radiation (eg X-rays)- high energy wavelengths, can modify DNA
-Chemical (eg base modifiers)- deletion, insertion snd translocation
-These changes can be point mutations (single base-pair), deletion, insertion, translocation.
-Naturally occurring mutations are important for evolution, but also are a major cause of disease.
-Germline mutations are inherited (sperm or oocyte)
-Somatic mutations are just in our body- not passed on
whats forward genetics
-phenotype —-> gene
-Researchers can cause high levels of mutations in model organisms (called mutagenesis). They raise the mutagenized animals and look for interesting phenotypes. For example animals which cannot see the colour green may result from a mutation in a gene that encodes the green photoreceptor.
whats reverse genetics
-phenotype —-> gene
-Targeted mutations: CRISPR and Gene Knock-out
-Genetic engineering in mice to target a gene:
A=Gene knock-out. This completely removes the gene to determine its function.
B=Gene replacement (knock-in) usually makes a small changes to the endogenous gene
-For example, if you find a point mutation present in human patients, you could test whether that mutation is causing the disease symptoms by making the same change in the corresponding mouse gene.
-CRISPR is a different technology – but it can also be used to knock-out or knock-in. The advantage to CRISPR is that it works in any organism.
How do mutations affect genes?
-Changes in regulatory sequences in the DNA that affects transcription. (Regulatory sequences such as enhancers)
-Changes in non-coding sequence of the transcript that may affect RNA splicing, stability or translation.
-Changes in the coding sequence:
may alter an important amino acid affecting folding of the protein or may create a premature stop codon -> truncated protein.
Missense = single amino acid substituted. Nonsense = stop codon
-need sequence in introns so they can be spliced out
How do mutations affect protein function?
-transcriptional factor e.g.:
=DNA binding-> dimerisation -> conformational change-> transcriptional activation
-A domain is a functional unit in a protein
-A dimer is when two of the same protein bind together
-A conformational change is a change in protein structure - caused by dimerisation therefore becoming activated
whats a missense mutation
-affects a single AA needed to recognise the binding site therefore domain not active anymore
Example 1: amorphic/non-functioning
-missense mutation that completely inactivates the DNA binding domain
- +/- : normally there is enough gene product from the one wild-type copy. Haplosufficient- necessary lethal mutation which are haplosufficient
- -/- Strong phenotype due to no transcriptional activation. This is recessive.
-The wild-type copy is the non-mutated version
Example 2: hypomorphic/weakened
-missense mutation that weakens the DNA binding domain.
-reduced function but not completely absent
- +/- : normally there is enough gene product from the one wild-type copy. The mutant form may also dimerize with wild type and still activate transcription.
- -/- mild phenotype due to poor transcriptional activation. The dimer forms on DNA but is often falling off. This is recessive.
Example 3: antimorphic/dominant negative
-missense mutation that destroys the dimerisation domain
- +/- : The mutant form binds DNA but does not dimerise with the WT and thus does not go through a conformational change to become active.
Transcription is compromised and it is only activated when two WT proteins land together. This is dominant.
- -/- : completely inactive
Example 4: hypermorphic/overactive
-missense mutation that results in activation that is independent of dimerization
- +/- : The mutant form binds DNA is active all the time. We call this constitutively active. This increases the overall activation of transcription. This is dominant.
- -/-: is the same
what are the Types of phenotypes produced by mutations (Muller’s Morphs)
-Loss-of-function mutations
-Gain-of-function mutations
whats loss-of-funcion mutations
-amorphic - complete loss of gene function: typically early nonsense mutations or a deletion of the entire gene. Most genes are haplosufficient in diploid organisms so these are usually recessive.
- hypomorphic - reduction of wild type function: typically missense mutations. Usually recessive.
-antimorphic - competitive inhibitors, typically mutations that affect one domain of a protein. In heterozygotes the mutant form is still partially active allowing the mutant protein to interact with and poison the wild type protein.
Also called dominant negative.
whats gain-of-function mutations
-hypermorphic - over expression of the transcription unit or over activity of the gene product: for example mutation in a binding site for a repressor. Dominant.
whats the genetic pathway
-S1 - > S2 -> S3 -> S4 -> melanin
-A, B, C and D are enzymes that are required for the biosynthesis of melanin
-Amorphic mutations in any of the genes that code for A, B, C and D may lead to the albino phenotype.
-Mutations that result is the same phenotype that are not in the same gene suggest that the genes function in the same pathway
-Mutations that result is the same phenotype may be different mutations in the same gene. Mutations in the same gene are called alleles.
-note: s= substrate and letters are enzymes
what does Generating a GFP transgenic line entail of
1) genomic DNA with all of the regulatory elements
2) genetically engineer GFP onto the end of the last exon (gene fusion) or replace the gene (reporter construct)
3) Re-introduce this into the animal
what are the Uses for GFP transgenic lines
-The movie follows a GFP tagged protein in the early embryo of C.elegans (1-4 cell).
-The protein becomes localized to one cell after each division.
-Then it becomes nuclear localized.
-(GFP fusion construct)
-Uses for GFP transgenic lines:
=To follow expression of a gene or to follow the behavior of cells in vivo.
-To follow subcellular localisation of a protein.
