Lecture 17: HOW DO GENETIC DIFFERENCES LEAD TO DISEASE? Flashcards
What do the codons within the coding region of the mRNA do?
Specify the amino acid sequence of the polypeptide chain
What does each tRNA have?
A different base sequence but about the same overall shape
What does each tRNA carry?
An amino acid to be added to the polypeptide chain
What is the first step of initiation?
Small ribosomal subunit binds to mRNA
What happens after the small ribosomal subunit binds to mRNA?
Large ribosomal subunit completes the initiation complex
What is the first step of elongation?
Codon recognition
What happens after codon recognition?
Peptide bond formation
What happens after peptide bond formation?
Translocation
What is the first step of termination?
Ribosome reaches a stop codon on mRNA
What are the stop codons?
UAG, UAA and UGA
What happens after the ribosome reaches a stop codon on mRNA?
Release factor promotes hydrolysis and release of the polypeptide
What happens after the release factor promotes hydrolysis and release of the polypeptide?
Ribosomal subunits and the components dissociate
What is the mutated enzyme which causes PKU?
Phenylalanine Hydroxylase
What is the mutation causing PKU?
In the coding strand, an A changes to a T meaning an A changes to a U in the mRNA and amino acids change from arginine to tryptophan
What is the PKU mutation (short form)?
R408W mutation in PAH gene
What does R408W mean?
Codon 408 for arginine (R) is mutated into a codon for tryptophan (W)
What does the R408W mutation in PAH protein result in?
Incorrect folding
What does the incorrectly folded PAH protein do?
Forms an aggregate that enzymes in the cell degrade
What does normal PAH do?
Folds correctly and functions to break down phenylalanine
Why will some mutations have no effect?
Because some base changes will result in the same amino acid being produced
What is a substitution mutation?
A point mutation where there is a change in one letter
What will a substitution mutation do?
May affect the protein slightly if the substitution results in a change of the amino acid
What is a deletion mutation?
Frameshift mutation where there is a loss of one letter
What will a deletion mutation do?
The amino acids following the deletion will be different and so will be consequences
What is an insertion mutation?
Frameshift mutation where there is gain of one letter
What will an insertion mutation do?
The amino acids following the insertion will be different and so will be consequences
What codon being introduced will have a large effect?
Stop codon
What do pancreatic beta cells do?
Sense how much glucose is in the blood and release insulin when glucose is high (fed state)
What is a key enzyme in glucose sensing?
Glucokinase
What does glucokinase do?
It determines how much glucose is broken down and therefore how much insulin is produced
What does a mutation in one glucokinase gene (heterozygous) result in?
Persistent mild hyperglycaemia, a type of diabetes called MODY2 (maturity onset diabetes of the young, type 2)
What is the MODY2 mutation?
Dominant so presence of the wild type can’t mask the effect of the mutant
What do homozygous MODY2 individuals have?
Sever diabetes and very high blood glucose levels
What is the mutation that causes MODY2?
Base changes from G to A so amino acid changes from glutamate to lysine
What is the first part needed for genetic testing?
Obtain some cells (such as form the mouth)
What happens after some cells are obtained?
Isolate DNA
What happens after isolate DNA?
PCR amplify a specific gene sequence
What happens after PCR amplify a specific gene sequence?
Detect genetic difference of interest
What goes in the test tube for PCR?
DNA from cells, DNA nucleotides, primers with sequences that can base pair with the region of DNA that you want to amplify and Taq DNA polymerase
What is the first step of PCR?
Heat DNA to 95 degrees to break the hydrogen bonds and separate DNA
What is the second step of PCR?
Cool to approximately 60 degrees to anneal (base pair) a DNA primer (a short sequence approximately 20 nucleotides that is chemically synthesised)
What is the third step of PCR?
Heat DNA to 72 degrees to allow Taq DNA polymerase to copy the DNA (add complementary nucleotides)
What happens when the PCR cycle is repeated?
It doubles the amount of DNA present each time
What is used to detect sequence differences?
Enzymes
What is HIndlll?
A restriction enzyme that specifically cuts the sequence AAGCTT to recognise the mutation
What does Hindlll cut?
The mutated version to give two fragments
What does Hindlll not cut?
The wild type
What does the wild type show up as on gel electrophoresis?
One band
What does the MODY2 show up as on gel electrophoresis?
Fragments
