Lecture 17 - How do genetic differences lead to a disease?

Question 1

Translation

Answer 1

Process by which mRNA is converted into a protein

The codons within the coding region of the mRNA specifics the amino acid sequence of the polypeptide chain.
Each tRNA has a different base sequence but also about the same overall shape.
Each tRNA carries an amino acid to be added to the polypeptide chain

Question 2

Translation initiation

Answer 2

Small ribosomal subunit binds to mRNA and finds the start codon. Large ribosomal subunit completes the initiation complex

Question 3

Elongation

Answer 3

Codon recognition, peptide bond formation, translocation

Question 4

Termination

Answer 4

Ribosome reaches a stop codon on mRNA, release factor promotes hydrolysis, ribosomal subunits and other components dissociate.

Question 5

Template strand and coding strand

Answer 5

Template strand is used to make a replica of the coding strand

Question 6

Redundancy of the genetic code

Answer 6

Different sequences encode for the same amino acids, exceptions like ATG is the only sequence that codes for Met (M)

Means that some mutations do not have an affect (silent mutation)

Question 7

Mutation causing PKU

Answer 7

Codon 408 for arginine (R) is mutated into a codon for tryptophan (W), this results in incorrect folding. This incorrectly folded protein forms an aggregate that the cell degrades. Hence no active PAH protein is made which normally functions to break down phenylalanine

Question 8

Types of mutations

Answer 8

Substitution, deletion, insertion

Question 9

Substitution mutation

Answer 9

Change of one letter for another , it is known as a point mutation

Question 10

Deletion mutation

Answer 10

Loss of one letter, known as a frameshift mutation

Question 11

Insertion mutation

Answer 11

Gain of one letter, known as a frameshift mutation

Question 12

Frameshift mutation

Answer 12

a genetic mutation caused by indels of a number of nucleotides in a DNA sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame, resulting in a completely different translation from the original.

Question 13

Glucokinase and pancreatic beta cells

Answer 13

Pancreatic b cells ‘sense’ how much glucose is in the blood and release insulin when glucose is high (feed state).
Glucokinase is a key enzyme in glucose
sensing as it determines how much glucose is broken down – therefore how much insulin is produced.
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).
Homozygous individuals – severe diabetes and very high blood glucose levels

Question 14

MODY2

Answer 14

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).
Homozygous individuals – severe diabetes and very high blood glucose levels

MODY2 is said to be dominant because you can see the phenotype of this mutation even though there is a wild type copy present (incomplete dominant). Having one functioning enzyme present is sufficient to catalyse a reaction that it doesn’t matter whether or not you have another copy because there is still plenty but because the amount of enzyme being produced is part of the signalling pathway then they have a small amount of enzyme which will only break down a significant amount od glucose when there is a high amount of glucose present in the cell. If you knock out 1/2 of the copy then you are getting 1/2 of the amount of enzyme being produced.

Question 15

How do you detect MODY2?

Answer 15

Wild type = GAG —> Glutamate
Mutant = AAG —> Lysine
Sequence that is mutated is recognised by the restriction enzyme Hind III
Wild type cannot be cut
Mutant can be cut
Use gel electrophoresis to detect DNA of different lengths

Question 16

Genetic testing

Answer 16

Obtain some cells, isolate the DNA, PCR amplify a specific gene sequence, detect genetic difference of interest ( PCR doubles the amount of DNA each time)

Question 17

PCR

Answer 17

A technique for amplifying DNA in vitro by incubating with special primers with sequences that can base pair with the region of DNA that you want to amplify, DNA polymerase molecules, and DNA nucleotides.

Steps
1- Heat DNA to 95 degrees Celsius to separate the DNA strands
2- Cool to approximately 60 degrees to anneal (base pair) a DNA primer (primer is a short sequence of approximately 20 nucleotides that is chemically synthesised)
3 - Heat DNA to 72 degrees Celsius to allow DNA polymerase to copy the DNA
4- Copying complete, repeat cycle (three steps 95, 60 and 72 degrees Celsius)

Question 18

Temperatures associated with PCR

Answer 18

three steps 95, 60 and 72 degrees Celsius

Question 19

Gel electrophoresis

Answer 19

Gel electrophoresis is a technique used to separate DNA fragments according to their size. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode.