Part 3 Flashcards
1
Q
How do you identify a gene?
A
- Sequencing DNA
- Assembling and annotating the human genome
- Methods of gene production
- Identifying genes by micro array experiements
2
Q
Analysing gene function
A
- Reverse genetics
- Forward genetics- Genetic screens, complementation analysis assigns mutations to individual genes and Linkage analysis - genes positionally cloned
3
Q
Cost per genome
A
£7.800 for your genome
*used to be $100m
4
Q
“Data miming” genomic sequence- how do we find genes in the nucleotide sequence
A
- Use gene prediction software- scanning sequence for promoter, start and stop sequences and intron splicing sites
- Use computer to translate the DNA in all 6 reading frames- search for known similar proteins (BLAST)
5
Q
BLAST protein alignment
A
-input the amino acid sequence of the proposed protein the blast program searched databases for proteins with similar sequences
Shows alignment of uncharacteristic protein (query) to a protein called Zen (subject)
6
Q
What is the PA translation from gene Zen?
A
CG1046 Length= 353 score 150 Identities 31/57, Positives 39/57 query= 57, subject=147 similarity found between protein sequences suggests that the protein evolved from the same common ancestor and that the protein has similar functions
7
Q
Looking at the genome online
A
EST- short sequences from the ends of cDNA expressed sequence tags
- predict homolgy
- predicted genes
- genomic assembly
- reverse strands
8
Q
Micro arrays
A
Allow us to compare the transciptomes of different tissues to each other eg. normal liver tissue to cancerous liver tissue
High throughout= small scale, fast and automated
9
Q
How do micro arrays work?
A
1.A very precise robot manufactures the array
each position on the grid contains one cDNA (as single strand)
One spot for every gene in the genome
2. Purify mRNA from liver tissue and tag it with fluorescent dye
3. Put the mRNA onto the array (hybridise) then rinse off the exons
10
Q
House keeping genes and what genes are we looking for?
A
Most of the genes in the normal and tumour samples are the same
look for:
- genes that are lost in tumour tissue (potential tumour supressor genes)
- genes that are activated in tumour (potential activators)
Using grid coordinates we can look up the identity of these genes
11
Q
3 different ways to identify genes?
A
- by making a library of cDNA clones from mRNA
- by making a library of genomic clones then make predictions based upon genomic sequences
- Identify sets of interesting genes using microarrays
All of these use a gene cloned into a plasmid or sequence
12
Q
Genetic engineering in mice
A
powerful but time consuming and spenny
from endogenous genes you can either:
A) gene replacement- (maybe using found mutant gene) and test whether the mutation causes the disease symptoms - mutant active gene present ONLY
B) Gene knockout- completely remove the genes to determine its function (must have genomic clone of gene)- no active gene present
13
Q
How can we knockout a single gene in mice?
A
- acquire a genomic clone of the gene and insert NEO into an exon (destroying activity of gene)- TK placed one side of it
sequence from taerget= Homologous arms- homology of mouse - Introduce construct into mouse ES using cell culture technique
- Homologous recombination occurs sometimes (KO)when it does occur TK gene is lost
Double selection identifies KO
- selective markers (neo and tk) used to identify colonies that are the result of homolgous remcombination
- cell integrating NEO can now grow on neomycin containing media
- Cells containing TK gene along with neo will die when grown on CANC media
14
Q
How can we target a single gene in mice?
A
Selected cells line is re-introduced in mice embryos
- first generation= mosaic (mixture of sc and mother and goads)
- mosaic are bred to generate non-mosaic carries of the transgene (2nd generation)
- carriers then interbred to create homologous mutant analysis (3rd generation)
15
Q
Transgene
A
with one copy of target gene replaced by altered gene in germ line
Genetic material or gene transfered naturally or by any of the genetic engineering methods from one organism to another
16
Q
Injecting it into mice
A
Wait 3 days
inject into ES cells- early embryo partly formed from ES cells
Introduce into pseuopregnant
Birth
Somatic cells of offspring tested for presence of altered gene
selected mice bred down germline
17
Q
Forward genetics
A
- randomly mutate the genome
- look for interesting phenotypes in the offspring
- Identify the gene that causes the defect
Bc random mutagenesis affects whole genome- one has to analysis many mutanergised animals to find defect- easier to use zebrafish, c.elegans and drosophila
18
Q
Problems for genetic screens
A
Loss of certain cells or tissues
Disease like phenotype
Biochemical abnormalities
loss of hearing, vision, behaviour and drug addiction
19
Q
Forward vs reverse
A
Forward= we find a mutant- start with only phenotype, dont know what the gene encodes (function - genes) Reverse= Know the gene and want to find function (KO)(gene to phenotype)
20
Q
Forward genetic clone of flies
A
Mutangenize male= each sperm has a different set of mutations EMS is a chemical mutagen
+/+ x +/+ = PO- heterozygous for mutations
+/+ x +/m = F1
+/+ x +/m = F2 incross to see homozygous embryos (what they look like)
=m/m =F3
-3 generations to make homozygous
21
Q
If different mutations have the same phenotype, are they different alleles of the same genes?
A
m1/+ x m2/+ = m1/m2
Mutants fail to complement they are alleles of the same gene
m1/+ x m3/+ = m1/+ ; m3/+
Mutants complement- no offspring with phenotype= mutations of different genes
Complementation analysis allows mutations to be sorted into distinct groups
22
Q
When we find a mutant, we start with only a phenotype, we dont know what the genes encode- linkage analysis is used to identify genes
A
By analysising recombination between our alleles a) and b) on the same chromosome, we determine whether the gene and marker are linked
Greater distance between genes= more frequent crossing over occurs in meiosis
23
Q
Meiosis and genetic recombination
A
Maternal (AB) and paternal (ab)– meiosis recombination– Ab and aB haploids (egg and sperm)
24
Q
Calculating recombination frequency
A
R/T x 100 = centimorgan (cm)
cm= measurement of genetic distance
R= number of recombinant gametes (counting)
T= calculatee recombination frequency
25
Calculating the genetic distance between a human disease gene and a marker
SNP- single nucleotide polymorphisms
markers that are easy tpo analyse and vary from individual
Over 1 mill SNPs have been placed in the human genome
DNA samples taken and snp tested- if a snp is present in diseased children not normal then know its gene is linked
26
Once you have found closely linked SNPs you can look for candidate genes
- All of the SNPs have been placed on human genome
| - 2 SNPs are the closest to your gene, then gene is good candidate to sequence for mutations
27
How do mutations affect gene function?
1. Changes in regulatory sequence- In DNA that affects transcription
2. Changes in non-coding sequence
3. Changes in coding sequence- May alter an important aa fold of protein- premature stop codon created= trauncated protein
- missense= amino acid sub situation
- nonsense= early stop codon
28
Examples where mutation in the coding sequence often affect primary function
1. amorphic/ non-functioning
2. hypomorphic/ weakened
3. anti-morphic/ dominant
4. hypermorphic/ overactive
29
Amorphic/ non- functioning
Missense mutation that completely inactivates the DNA binding domain
+/- = normally there is enough gene product from one wt, copy halosufficient
-/- = stong phenotype due to no transcriptional activation- recessive
30
Hypomorphic/ weakened
Missense mutation that weakens DNA binding domain
+/- = Normally enough wt , mutant may dimerize
-/- = Mild phenotype due to poor transcriptional activation, complex from on DNA
31
Antimorphic/ dominant
missence mutation that destroys dimerisation domain
+/+ = mutant binds DNA but doesnt dimerize with wt
-/- = completely inactive
32
Hypermorphic/ overactive
Missense mutation - overactivation that is independent of dimerization
+/+ = mutant binds DNA all the time
-/- = the same
33
Types of phenotypes produced by mutations
1. Loss of function
- amorphic- Complete loss of function, typically protein nulls or deletion of entire gene (early nonsense)
- hypomorphic- reduction of wt formation (enhancer and missense)
- antimorphic- competitive inhibitors, mutations that affect one domain of protein, heterozygous form still partially active, mutant interact and poison protein (dominant negative)
2. Gain of function
- Hypermophic- over expression of transcription unit, dominant
34
Control of gene expression- RNA
1. Isoforms
2. Subcellular localisation can be used to target translation to the part of the cell where its needed
3. Translation can be directly regulated by sequences in UTRs (untranslated region) or globally by regulation of eIFs (Eukaryotic initiation factor)
4. Some mRNAs have a 2nd ORF (Open reading frame) that can be regulated independently
4. RNA degradation
35
Isoform gene
A variety of different proteins made from a single gene
36
Where does regulation of gene expression occur
At almost every level
| Transcription - splicing (final mRNA) - translation (RNA)
37
Alternative splicing creates isoform
40% of drosophila and 75% human genes alternatively spliced
Splice donor and acceptor are only 2 bases so very frequent
Other sequences and secondary structure in RNA affect choice of splice
38
Choices of splice site
- Optional exon
- Optional intron
- Mutually exclusive exons
- Introns splice site
39
Regulation of alternative splicing
Sex determinationn in drosophila
| 3 genes= male and female differentiation- sxl, tra and dsx (sex lethal, transformer, double sex)
40
Difference in splicing in males and females
Males= ( 1 x chromosome)- sxl and tra spliced out to give rise to inactive proteins, dsx transcripts give rise to male specific repressor proteins which repress transcripts from genes for female development
Female= (2 x chromosomes)- small amount of sxl protein made (alternative promoter) which represses splicing by blocking binding of U2AF, feeds back to itself and makes more sxl, binds to tra= female specific dsx produced
41
The site of polyadenylation on an mRNA can be regulated
B lympocytes produce 2 antibody isoforms
| -This antibody has 2 positive positions for cleavage
42
Transcription with different size transcripts
Long RNA transcript
- first stop codon is spliced out
- transmembrane domain translation
- membrane bound antibody
- Terminal hydrophobic peptide
Short RNA transcript
- Splice acceptor lost and the first stop codon is not lost (Intron not spliced out)
- Antibody is secreted
- Hydrophilic peptide
*Alternative endings allows different isoforms
43
Alternative start sites can be regulated
Sequences around the start help to initiate translation
Optimal sequence= Kozak sequence- ACCAUGG
Sequence is less than perfect, the small ribosome can scan past the first AUG and stop a a second or third AUG
All in the same reading frame- Different isoforms of the protein differ only by the sequence of the N terminus
44
Kosak sequence
ACCAUGG
45
What is it called when there are different N terminus's
Leaky scanning
| many isoforms from 1 gene
46
The first AUG is favoured by
High levels of eIF-4F
47
Regulated nuclear transport (HIV)
HIV has a small genome that is integrated into the host genome:
- after integration the ENTIRE genome is transcribed into one piece- alternative splicing allows for many different protein products to be made
- Full length RNA is needed to make new virions and unspliced RNA cant leave the nucleus
48
Signals in the untranslated region (UTR) of an mRNA can target it to be part of the cell
Intermolecular base pairing in the 3'UTR forms stem loops that are recognised by cellular proteins
mRNA binds to the RNA binding protein
49
mRNA translational control elements in the 5' and 3' of UTR
- Ferritin= protein, stores iron in the cell thus reducing the available fe
- Transferrin receptors import iron into the cell= reducing availability
50
Controlling the reduction of Iron
1. Aconitase bind to stem loop in the 5'UTR of feritin mRNA and block translation
2. Aconitase binds to stem loop on 3'UTR of transferrin mRNA and blocks its degradation
51
What happens when there is high FE in the cytoplasm
mRNA is translated
mRNA is degraded
Aconitase binds to iron in the cytoplasm and goes through a confirmational change. (ferritin)
Aconitase then releases the mRNAs. This provides rapid and strong regulation. (transferrin)
52
Global regulation of translation by EIFs and EIF-2B
EIF-2/ GTP binds to met tRNA to start ribosome scanning
EIF-2b required for dissociation of GDP from EIF-2
Cell entering G0 (resting) or infected with virus- turn down global translation by phosphoryating EIF-2
53
IREs allow more than one gene to be present on an mRNA
Internal ribosome entry sites
= stem loop in the rna that can initiate formation of the ribosome independent of the cap/polyA initiation complex
EIF-4G required for IRES based initiation and binds to the IRES Stem loop
54
Where are IRES often found
Viral transcripts- fabour translation of their transcripts by cleaving EIF-4E but still binds IREs
55
During apoptosis what happens to EIF-4G
Similarly cleaved to viral transcripts
| Certain genes required during cell death ultilize IREs and they continue to be translated
56
RNA stability is a common method for regulating transformation
Half life of different mRNA varies from hours to minutes
PolyA tails start about 200 in length- endonucleases chew down to 30nt at which point they are decapped and degraded
Some mRNA are deadenylated in the cytoplasm to activate them or extend half life
57
Factors that promote translation block degradation
DAN competes with EIF-4e for binding to the cap
58
Non genetic anylsis of F box
Where is a protein in the cell/ organism?- Antibodies
Where is a gene transcribed in a tissue or organism?- RNA in situ - quick way to look at gene expression
Visualizing gene expression and protein localisation in living cells- GFP
59
To make a protein specific antibdody what do we need to do?
Make a lot of protein:
- Expressions vectors use bacteriophage promoters to drive high levels of RNA synthesis. The promoters have to be inducible otherwise the bacteriophage would die rapidly
- Chemicals or temperature shifts are used to induce protein expression
- After induction the bacteria are harvested in a centrifuge and lysed to make a crude extract
- Expression plasmids often include a epitope tagging system that allows for rapid and efficient purification of the protein Tag fused in frame to cDNA during cloning
60
Making proteins from vectors
1. double stranded plasmid DNA expression vector cut with restriction enzyme
2. Insert protein coding DNA sequence
3. Introduce recombinant DNA into cells
4. Grow in culture
61
Antibody- affintiy purification
Epiptope tags are peptides for antibodies already available
Bacterial cells are lysed and resulting solution poured onto the column
Small beads about the size sand grains have been coated with antibody
- Load in PH buffer, wash, elute with PH3 buffer and collect different fractions
62
Making a protein specific antibody
Antibodies bind to small regions of protein with very high specificity= epiptope
Tagged antibodies have dyes or enzymes attached so you can determine their location
63
Detention methods
Some conjugates are dyes that are fluorescent allowing us to detect the location using specific wavelength of light
Antibodies= examine subcellular localisation of proteins
64
Commonly used enzyme conjugates
Alkaline phosphatase- substrate turn blue
| Horseradish peroxidase- substrate turns brown
65
2 antibody sandwich
1^o= Antibody made in rabbit
2^o= Antibody made in mouse, binds rabbit antibodies and carries the tag
We use a 2 antibody sandwich as it amplifies the signal - as many 2^o bind to each 1^o
66
How to use antibodies
Chemically fix (formaldehyde) animal or tissue to stabilise the structure
- Incubate with tagged antibodies
- Antibody binds to target protein
- Wash off excess antibody
67
RNA in situ analysis
Purified vector containing the cDNA of interest
- Synthesise RNA antisense probe- incorporating epitpe tagged nucleotide
- Incubate embryo with antisense probe
- Wash off excess probe
- Areas of embryo where is isnt washed off tells us where the RNA remains
* Doesn't tell us anything further
68
Bicoid in situ
Shows detection different
| Bicoid RNA remains put whilst protein diffuse away
69
GFP
Green fluorescent protein
- purified from jellyfish- excite with different wavelength eg. Excite 475 nm- emits 510 nm
-Dozens of fluorescent proteins that have been cloned and characterised
70
Generating the GFP transgenic line
1. Clone the entire gene (genomic DNA) with all the regulatory elements of the plasmid
2. Genetically engineer GFP onto the end of the last exon (gene fusion) and replace the gene (reporter construct)- generating a GFP transgenic line
3. Integrate the GFP fusion gene back into the organisms genome- usually involves micro injected a solution of DNA into a one cell zygote (randomly integrates)
71
Uses for GFP trangenic line
GFP tagged protein in the early embryo of c.elegans, the protein becomes localised to one cell after each division then becomes nuclear localised
1. Follow expression of gene in the WHOLE animal
2. Follow subcellular localisation of a protein- some TF become nuclear after activation
3. Follow of behaviour of cells in vivo - grp marks so can be distingushed
72
The phylogenetic tree of animals
Most genes same in all animals
BLAST aligning protein sequence shows significant sequence homology
Differences= changes in expression of common genes
73
Molecular clock
Apes and man change in nucleotide sequence is 1% every 10 million years
Compare fossil records to genomic data- estimate rate of clock
74
What is the simplest model to assemble the tree?
Use highly conserves foxP2 protein - differences in aa sequenceat protein 80,303.325
compare mouse, chimpanzee and human
Mouse and chimpanzee= 303T (likely ancestor had it)
Human chimpanzee= 80D
75
Parasimony
Assume the simplest model
accomplish tree with 2 changes
Programmes related to BLAST consider all the possible scenarios
76
Convergent evolution
80D could have arisen twice by chance.
Less likely but does happen
Molecular phylogeny compared with morphological phylogeny and fossil records
77
FGFs in molecular phylogeny
22 vertebrates fgfs that fall into 4 clusters based upon protein sequence
Ci- single representatives in each of 4 groups
Suggests common ancestor of sea squirt and vertebrate had 4 FGFs
78
How do so many FGFs arise?
Gene duplication
Changes in ploidy and local duplications
New copy of gene= Paralogues
79
Duplication of chromosome or region of a chromosome
Ancestor -- genome duplication -- Gene loss/ fractionation
after duplication it is likely that the duplicate genes are at the first redundant
Major role in evolution- Refinement of function or new function
80
The extra copy can change in the following ways:
1. pattern of expression
2. Structural in the protein
both small, caused by point mutations or big changes in domain swapping
81
Why are changes in expression patterns of gene thought to play a major role in morphological evolution?
Because enhancers can change easily
Eg non homologous recombination
Could bring a new enhancer closer to the gene
Position of enhancer usually unimportant
82
Relatively easy to add or delete bases by?
Rearrangement, insertion, deletions or base pair substitution
Changes that affect protein structure would have to be more precise
83
Evidence that changes in the expression of single genes has played a major role in morphological evolution
Changes in expression of regulatory genes- morphological changes
Hox genes- c5,c6,d9, limb
C6 starts more posterior in chick= longer neck and less chest than mice
84
Further evidence- regulatory genes responsible for large evolutionary changes
Experimentally changing expression= Ectopic organs
Master regulatory genes- genes capable of this
Genes result in whole gene networks
Adaptability takes place in devel- Misexpress pax6- fly with eye in leg position
Crustacens have legs in abdomen
85
Ubx evolution explains why insects don't have legs in their abdomen
Fly- dxl specifies leg precursor cells, ubx is expressed in abdomen which represses dxl
Legs only form in throrax
Crustacears have legs in abdomen and thorax- Ubx not expressed BUT truth is ubx abd dxl expressed but ubx doesn't repress
ANTIREPRESSION
86
Endocytosis
Allows capture of molecules from outside
87
Exocytosis
Allows secretion of molecules from inside
88
What does the plasma membrane include
Proteins- transport function
Lipids- continuity and flexibility of cell membrane
Carbohydrates- Cell protection and tagging
89
Bilayer rich in amphipathic molecules carrying- 3 main phospholipid types
1. Phosphatidyl-ethanolamine
2. Phosphatidyl-serine
3. Phosphartidyl-choline
90
Properties of membrane
1. flexible- Fully saturated lipids= rigid, unsaturated gives disorder= flexibity (double bond)
- omega 3 (plants, fish, nuts) - Omega 6 (land plants and animals)
2. seal interior from outside- cholesterol= preserving internal molecules
3. Assymetric ( sugars on the outer leaflet of plasma membrane
91
Topography (key features ) of cell membrane
- Extracellular space sugars on outside
- cytosol neg charge on inside- molecules intrinsically repulsed by each other and plasma membrane
- Phosphatidylserine- negatively charged and found on inside 8%, ps is matter of life or death, flipsmto outer side for apoptosis
92
Endocytosis and nutrient uptake
Cholesterol is transported into cells by receptor- mediated endocytosis
Low density lipoprotein (LDL) carries cholesterol in the blood
LdL bind to LDL receptor and undergoes endocytosis, it is uncoated and fuses with the endosome as the proton pump acidifies endomes breaking LDL binding to receptor. LDL receptor is then transported back to plasma membrane by budding off a transport vesicle
93
Adaptors in receptor mediated endocytosis
Bind not only to receptors but PIP2 lipid when recruiting clathrin to form vesicles from the planar cell membrane
94
Phosphatidylinsitol
Major component of plasma membrane, important in signalling
Inner leaflet of cell membrane
Targeted by phosphatidylinositol kinase (PI kinases) which add phosphate groups to the inositol sugar in response to activation
95
What does defective endocytosis cause?
Atherosclerosis
- due to accumulation of LDL in blood forming plaques blocking blood arteries
- Mutations in LDL receptor account for familial cases of atherosclerosis a cardiovascular disease
coated pit cannot form properly due to mutation in the receptor and the inability of the receptor to interact with adaptor proteins
96
How does clathrin help form vesicles from the planar cell membrane
Shapes the vesicle, dynamin pinches off the cell membrane by hydrolysing GTP - GDP
Mutated dynamin cannot hydrolyse GTP and thus pinches off the endoyltic vesicle
Er and Golgi use clathrin-like coat proteins to pinch off vesicles- newly synthesised er lipids and protein are packaged into cop11 vesicles
97
Phagocytosis
Example of vesicle formation without clathrin
actin driven membrane invagination
1. Microbe adheres to phagocyte
2. phagocyte forms pseudopod that eventually engulf the particle
3. phagocytic vesicle is fused with a lysosome
4. Microbe in fused vesicle is killed and digested by lysosomal leaving a residual body
5. Indigestible and residual material is removed by exocytosis
98
Autophagy
3rd pathway towards lysosomal digestion- helps eliminate malfunction cell elements
- happens by fusion and engulfment of organelles
1. nucleation and extension- engulf cytosol and organelle
2. Closure- autophagosome
3. Fusion with lysosome despite intrinsic neg charge (merged by SNARE proteins)
4. Digestion- release of lipids, aa into cytosol
99
Exocytosis
Responsible for secretion of hormones, digestive enzymes, recycling of plasma membrane receptors and neuronal communication
100
Regulating exocytosis
Release of highly concentrated insulin from pancreatic beta cells, it happens only in response to high glucose
- Insulin is tightly packaged inside secretory granules before secretion
- Fusion of vesicle with target membrane must overcome repulsion of negatively charged membranes
101
What are the 3 snares
V SNARE- (vesicle associated membrane protein)- 1 synaptobrevin (vamp)
T SHARE - (target membrane)- 2 syntoxin and 3 SNAP25
102
How do SNARE proteins interact with the cell membrane
Tranmembrane and peripheral proteins
-Only syntoxin and synaptobrevin transvere the membrane
- Long stretch of aa form a transmembrane helix
SNARE protein form a tight 4 helical coiled coil on the initial contact
- Synatoxin= 1 Helix
- snap-25= 2helices
- vamp= 1 helix on the vesicle
works by coiling into 2 opposing membranes thereby forcing their fusion
103
What causes dissociation of SNARE coils
NSF- works by hydrolysing ATP
104
Bacterial infections that cause neuromuscular paralysis
Botulinum neurotoxin attacks SNARE proteins
Both produce toxins that block exocytosis
Botulism= happens upon consumption of contaminated food
Tetanus= Injection after skin cuts during childbirth or dirty needle injection
SNARE responsbible for release of ACh at neuromuscular junction
105
Mechanism of botulinum neurotoxin action (BOTOX)
Botulinum binds to gangliosides in neuronal membrane
Enters the luminal spaces of recycling synaptic vesicles
- Following endocytosis, 1 subunit (SNARE protease) escapes the vesicle enters the synaptic cytosol and cleaves specific SNARE proteins
- Cleaved SNARE protein cant support the fusion of synaptic vesicles= long blockage of neurotransmission (several months of paralysis)
- re synthesis of damaged proteins lead to full resumption of neuronal transmission
106
Why is botulin used in medicine?
Local muscle paralysis - long lasting inactivation( 4-6 months)
Neurotoxin type A become a successful medicine and a cosmetic when locally injected in picogram amounts- cleaves snap-25
Used to treat muscle spasms, dystonia and anti-wrinkle
