Week 2 - Genes, Proteins, Pharmacogenetics Flashcards
1
Q
What is an intergenic region in a gene?
A
Non coding DNA region, junk DNA used for plasticity and control of gene expression
2
Q
Describe Upstream regulatory regions
A
5 prime end, binding area of RNA polymerase to control RNA synthesis
Includes: enhancers, silencers, insulators and locus control
3
Q
Describe the Promoter region
A
controlled binding area containing the upstream regulatory regions and protein binding sites
4
Q
What is gene expression driven by?
A
RNA polymerase 2 - transcription factors bind around promoter region in specific place, TATA box
5
Q
Describe activators
A
Bind to enhancer sequence and increase expression than without them ( basal/low expression)
6
Q
What are the three RNA polymerases
A
RNA polymerase 1 - larger ribosomal RNA
RNA polymerase 2 - mRNA production
RNA polymerase 3 - tRNA + small ribosomal RNA
7
Q
Describe Transcriptional repressors + example
A
- interacts with activator to block function
- overlap the binding site - stops activator binding
e.g. Wilm’s tumour protein
EGR-1 gene switching off expression, if mutation occurs tumours in kidney form in early life
WT1 gene considered tumour suppressor
8
Q
Describe 5 prime end capping of mRNA
A
Capped by:
- methylated guanine nucleotide by removal of a phosphate via phosphatase enzyme
Addition of GMP via guanayl transferase
- Methyl group via methyl transferase
9
Q
Describe 3 prime end cleaving in mRNA
A
poly A tail of up to 200 nucleotides added by PolyA polymerase
10
Q
What occurs if mRNA isn’t capped?
A
Degraded from 5 prime to 3 prime
11
Q
Describe splicing
A
Removal of introns from coding sequences, responsible for diversity by creating iso- forms due to alternative splicing
12
Q
What are the 5 types of alternative splicing
A
- Exon shipping
- 3 prime splice sites
- 5 prime splice sites
- Mutually exclusive exons
- Intron Retention
13
Q
Describe the role of small ribosomal unit
A
Initiator tRNA carrying methionine associates with small ribosomal unit along side eukaryotic initiation factor 2 (eIF2)
Small ribosomal unit recognises 5 prime end of mRNA capped with two additional initiation factors eIF4G and eIF4E
14
Q
Describe translation termination
A
- Ribosome encounters stop codon
- Cytoplasmic release factors bind to stop codon and free carboxyl end of peptide chain
15
Q
Describe errors in gene expression
A
- cause uncommon genetic disorders
- influence predisposition of common diseases
16
Q
Describe 4 common gene mutations cause
A
- DMD gene - duchess muscular dystrophy
- SMN gene - spinal muscular atrophy
- CFTR gene - cystic fibrosis
- BMPR2 gene - PAH
17
Q
Describe transcriptional errors
A
- over expression of transcription factor causing cancers
- one copy of transcription factor gene mutated leading to:
Haploinsufficiency - one gene copy isn’t enough
Dominant mutation - exert dominance over wild type
18
Q
Describe electrophoresis
A
- 1% agarose gel + 100ml buffer TAE/TBE
- DNA sample inserted into gel wells created
- DNA will move from anode to cathode - separated via size
- DNA can be analysed
19
Q
Describe the western blot technique
A
- Gel electrophoresis
- Blotting - transfer proteins to membrane layers - filter paper, membrane, gel, filter paper
- Blocking - no specific sites in membrane - prevent antibody binding to unspecific regions - incubate membrane
- Antibody Probing -
Primary - 4 degrees overnight, wash away unbound antibody (TBST)
Secondary - Horseradish peroxidase, produces detectable signal, leave for 1 hour on shaker and wash with TBST - Detection/ Visualisation
20
Q
What is gene expression profiling?
A
- Detects how many copies of genes
- Idea of gene regulation at a specific time
21
Q
Describe PCR stages
A
- Initial denaturation
- Second denaturation
- Annealing
- DNA extension
Repeat 25-30 times
22
Q
What are pro proteins?
A
Inactive peptides or proteins that need post translational modifications to become their active form
23
Q
Describe insulin production to its active form
A
- cleavage and removal of signal peptide by signal peptidases in ER
- Oxidation of SH groups to -s-s- ion ER to crosslink the chain
- Cleavage and removal of C chain in ER - the link between A and B insulin chain
24
Q
Describe post translational modifications and their significance
A
- Processing ( proteolytic cleavage to active form)
- Covalent modification - chemical modification
Significance
- extend structural repertoire of proteins
- chemical and spatial structures
- some are reversible allowing rapid dynamic regulation of protein activity
25
What is proteolytic cleavage?
One or several amino acids removed from N-terminus or protein - peptide bond = cleaved in the internal protein
26
Describe proline isomerisation
change in proline residue spatial conformation can seriously affect protein structure adopted
27
Describe PTMs phosphorylation
Phosphate group donated via ATP transferred to acceptor protein catalysed by protein kinases - Reversible
28
Describe the cell cycle control
Controlled by cyclins and cyclin dependent kinases
- type of cyclin influences cyclin dependent kinases
- cyclin concentration changes the cell cycle
29
Describe protein acetylation + most characterised target
Acetyl group added by acetyl CoA and transferred to acceptor amino acids catalysed by protein acetyl transferases (PATs)
Deacylation catalysed by Protein deacetylase (PDAC)
Main target = histones with specific enzymes
- Histone acetyl transferase (HATs)
- Histone deacetylases (HDACs) - makes DNA less accessible to transcription factors
30
Describe protein methylation
Donated by s-adenosylmethionine catalysed by methyl transferase/ demethylase - not all are reversible
31
What is a nucleosome?
DNA wound around 8 histones
| Active chromatin activation when histones are accessible
32
Describe change in chemical nature in the immune system - protein attack
Immunesystem attacks citrullinated proteins - citrullination or deamination of arginine converting it to citrulline - cause of auto immune diseases and arthritis diseases
33
Describe glycosylation
addition of mono/oligosaccharides significant in affecting protein folding increasing protein stability, trafficking and recognition
34
Describe N- Linked glycosylation
Polysaccharide added as 14 unit to asparagine residue and polypeptide in ER
-3 glucose and mannose residues (ER) removed and others added (Golgi)
35
Describe O-linked glycosylation
sugar added one at a time in the golgi or cytosol added usually to hydroxyl of serine or threonine
- golgi for secreted proteins
- cytosol for cellular proteins
36
Describe protein polyubiquitination
Ubiquitin = small protein contains 76 amino acids
- attachment of ubiquitin to proteins changes protein structure
- attachment of polyubiquitin marks protein for degradation in the proteasome requiring ubiquitin activating enzyme, ubiquitin conjugating and ubiquitin ligase
- Deubiquitinatingenzyme removes ubiquitin
37
What are the functions of polyubiquitination?
- Removal of damaged and misfiled proteins
- control protein lifespan
- control of multi cellular processes by regulating availability of key regulating proteins
- role in neural activity - regulate synaptic transmission with protein channels and calcium receptors
38
Describe lipidation and the 4 types
Method to target proteins to membranes in organelles
1. G-C terminal glycosyl phosphatidylinostitol (GPI anchor)
2. N-terminal myristoylation
3. S- myristolyation
4. S-prenylation
Each type gives distinct membrane affinities and increases hydrophobicity
39
Why is PTM important?
- Defects in protein PTM and cell signalling are crucial in pathobiology of numerous diseases
- Used (enzymes) as therapeutic targets
- GPCR Regulation
40
Describe how mutations arise
1. strand breakage + nucleotides lost
2. Base lost - glycosidic bond is broken or enzymatically cleaved
3. Base change - guanine is oxidised to 8-oxyguanine and base pairs with adenine, cytosine loses amino group and becomes uracil. Thymidineglycol blocks replication
4. DNA cross linking - UV light cause cyclobutqane dimers and anti cancer agent cis-plating cause adjacent guanines to crosslink
5. DNA replication errors - some not corrected
41
What are some issues caused by DNA repair failure?
1. Cancer susceptibility
2. Progeria (accelerated ageing)
3. Neurological defects
4. Immune deficiency
42
Describe Missense mutations
Change in nucleotides, point mutation frame shift, gain or loss of functions
43
Describe nonsense mutations
Premature stop codon formed via frame shift usually resulting in a non functional protein
44
Describe Expanding trinucleotide repeats
same part of sequence repeated many times - further increased In replication
45
What are transposons?
sequence of DNA that can move around the genome and acts as recombination hotspot
46
What are retrosposons?
Analogue to copy and paste system, exhibit intermediate RNA stage prior to insertion into genome
47
What are DNA transposons?
cut and paste transposable element TE
48
What are All repeats?
short interspaced elements (SINEs)
| most abundant element in genome, large number of pathogenic deletions
49
What is haploinsufficiency?
one copy of chromosomes is deleted or inactivated by a mutation - one functional gene is not enough
50
What is the dosage effect?
gene product = quantitative signalling system, amount determines how active a gene may be
gene products compete to determine metabolic or development switch
51
What is stoichiometry?
Relationship between quantities of substances involved in reactions - one can't occur without the other
52
What is penetrance?
How frequently disease is manifested
53
What is localisation proteins?
Direct pathways - proteins moved from cytosol to mitochondria, nuclei and peroxisomes
-Proteins moved from ER in secretory pathways
54
What are two types of secretion?
Constitutive secretion -all cells, continual exporting of substances
Regulated secretion - specialised secretory cells, substances stored in secretory vesicles for release in response to signal
55
Describe pancreatic cells
```
Exocrine and endocrine organ
Islets of langerhan -
B cells - insulin
A cells - glucagon
Delta cells - somatostatin
```
Pancreatic acinar cells - secretion of mainly digestive enzymes (zymogen)
56
Describe the protein secretion pathway
1. initiation of protein synthesis
2. synthesis segregated from cytosolic proteins
3. processing of proteins in the ER first then the golgi
4. packaging and condensation
5. release via exocytosis
57
Describe the golgi secretion
Cis face takes transported vesicles bringing proteins, contents released into lumen of golgi
Trans face secretes packaged proteins according to location and final destination
58
Where are zymogen vesicles stored?
The cytoplasm until signal initiates exocytosis
59
Describe DNA polymerase in DNA replication
DNA polymerase alpha binds to strand at primer site, once strand reaches 20 base pairs DNA polymerase epsilon takes over.
DNA polymerase epsilon and delta have proof reading 3 prime to 5 prime exonucleases
60
Describe DNA replication in the lagging strand
- multiple RNA primers needed
- DNA polymerase delta generates complementary DNA to the strands between RNA primers creating Okazaki fragments
- Replication is not continuous as fragments are not joined
- RNA primers are degraded and filled by action of RNAse H and DNA polymerase delta
- DNA ligase joins the breaks in leading and lagging strands to generate a continuous double stranded DNA
61
Why can replication not originate from one site?
- genome replicates in 8 hours
| - multiple origins of replication with forks leading in opposite directions to increase replication rate
62
Describe epigenetics
Heritable change in phenotype by changes other than DNA base sequence
63
What is consanguineous mating?
Insest lol
| Mating between family members - first cousins
64
What is a true homozygote?
When a child has the same genes due to consanguinity
65
What is multifactorial inheritance?
Inheritance and expression of a phenotype being determined by the cumulative action of multiple genes at multiple loci - gene and environmental factors
66
What is mendelian manner?
Order where genes are passed from parents to children
67
What are monogenic diseases?
Single strong high penetrant phenotype, rare, primary gene dominates
68
What are polygenic diseases?
Common, low penetrant phenotypes, environmental contribution reduced penetrance, involves 2 or more genes
69
Describe the threshold model
Frequency disorder among relatives compared to frequency of disorder in general population
70
What is heritability?
Proportion of disease variation due to genetic variations 1=high 0=low
71
What are monozygotic twins?
Identical twins
72
What are Dizygotic twins?
Non identical twins share 50% of DNA
73
What is concordance?
Probability that identical twins will both have the disease or how often the non identical twins will have the disease given on of the pair has the disease % shows heritability
74
What are SNPs?
Single nucleotide polymorphisms
DNA sequence variations that occur when a single nucleotide in the genome sequence is altered at the same genetic location between different chromosomes
75
What are haplotypes?
Disease linked genomic loci that are physically linked on a chromosome and segregate together
76
What is GWAS?
Genome wide association study to identify variants associated with a disease
77
What is aneuploidy?
Presence of an abnormal n.o of chromosomes in a cell
78
Describe Precision medicine
Influence of genetic variation on drug response in patients by correlating gene expression or presence of SNPs with drug efficacy and toxicity
79
What is a non synonymous mutation?
Mutation that results in a change in protein sequence
80
Describe personalised medicine
- Advanced screening
- Better drugs
- Customised drugs
- Improved dosing - genetic information bell curves
- Improved drug discovery
81
Describe warfarin treatment pharmacogenomics
Specific genes show resistance (VKORC1) and high sensitivity (CYP2C9*3), hence us SNP information to maximise dosing for each patient as warfarin has a narrow therapeutic range, also racemate with S-warfarin being more active form
