CMB-2 Flashcards
1
Q
What is the functionality of proteins dependent on?
A
the 3D arrangement of the polypeptide chain
2
Q
what is the folding of the polypeptide chain determined by?
A
- the amino acid sequence
- the molecular structure and properties of its amino acids
3
Q
what equates to ‘handedness’ in amino acids?
A
D and L isomers
4
Q
how do amino acids combine to form polypeptides?
A
via a condensation reaction releasing water
5
Q
what is a residue?
A
each repeating unit of the polypeptide chain. each residue consists of an invariable unit comprising an alpha carbon, C’=O and an NH group
6
Q
how is the variable side chain usually arranged?
A
in a trans conformation (99.9%)
7
Q
how is the peptide bone arranged?
A
it is a planar structure with rotational freedom around an alpha carbon
8
Q
what does rotational freedom of peptide bonds allow?
A
allows huge variation in the conformation of the peptide chain. this freedom favours the formation of structural arrangements such as alpha helices and beta sheets.
9
Q
what is ∆G?
A
the change in free energy upon folding
10
Q
what determines the most favourable conformation of a molecular structure?
A
the minimisation of its specific energetic state
11
Q
what can affect the free molecular energy of a conformation…
A
the molecular environment, e.g.: aqueous or lipid membrane, other proteins or molecules. Changes in this environment can induce a further conformational change
12
Q
what forces determine folding (non-covalent bonds)?
A
- charge or electrostatic attractions
- hydrogen bonds
- van der waals attraction, dipole
- hydrophobic interactions
13
Q
what are van der waals forces?
A
van der waals forces occur between two atoms in non-covalent interactions. they are determined by their fluctuating charge. attraction at a close distance is balanced by repulsion due to proximity that is determined by the van der waals radius of an atom. they are induced by proximity of molecules.
14
Q
what are hydrophobic interactions driven by?
A
the minimisation of the disruption of the water lattice
15
Q
how do disulphide bonds form between cysteine residues?
A
very strong covalent bonds form between the side chains of two cysteine residues in an oxidative reaction forming. the SH groups from each cysteine cross link. this usually occurs in distant parts of the amino acid sequence but adjacent in the 3D structure
16
Q
what is the primary structure of proteins?
A
covalent bonds forming polypeptide chain - order of amino acid residues joined by peptide bonds
17
Q
what is the secondary structure of proteins?
A
regular folded form, often stabilised by hydrogen bonds - e.g. alpha helices, beta sheets and beta turns
18
Q
what is the tertiary structure of proteins?
A
overall 3D structure, stabilised by hydrogen bonds, hydrophobic, ionic and van der waals forces, and sometimes by intra-chain covalent bonds
19
Q
what is the quaternary structure of proteins?
A
organisation of polypeptides into assemblies, stabilised by non-covalent bonds and sometimes by interchain covalent bonds
20
Q
what happens as a result of protein misfolding?
A
function of misfolded protein is almost always lost and they often have a tendency to self-associate and form aggregates. others can result in cellular processing that leads to their degradation (cystic fibrosis)
21
Q
what are diseases of protein misfolding?
A
Huntingdons, Alzheimers, Prion Protein, Parkinsons, AA amyloidosis, Type 2 Diabetes
22
Q
what reasons can protein misfolding occur for?
A
- somatic mutations
- errors in transcription or translation
- failure of the folding
- mistakes of the post-transcriptional modifications or in trafficking of proteins
- structural modification
- induction of other protein misfolding
23
Q
how does Alzheimers occur?
A
- proteolytic cleavage of amyloid precursor protein
- APP has multiple functions but is involved in G-protein signalling
- cleavage results in ~40 residue peptide
- in the intact molecule this anchors the protein in the membrane
- in alzheimers the β-Amyloid peptide accumulates
- misfolding of this results in a planar arrangement and polymerisation
- this can form fibrils of misfolded protein
- the β-Amyloid fibres are formed from stacked beta sheets in which the side chains interdigitate
- aggregation interferes with working of the synapse particularly in hippocampus
- higher order insoluble aggregates form (containing much crossed beta structures) which deposit in plaques causing damage to neuronal cells of brain
24
Q
what are prions?
A
misfolded proteins that interact with other normal proteins. this interaction induces misfolding of the normal protein and polymerises
25
what do oligomers form and what is this process reliant upon?
form fibrils of misfiled protein. it is reliant upon the concept of energy minimisation
26
what forces influence protein structure and folding?
charging interactions, hydrogen bonds, hydrophobic, VdW, covalent
27
what is a structural motif?
a minimum arrangement of independently forming secondary structure combining to produce recognisable folds across many different proteins
28
what are examples of DNA binding motifs?
helix loop helix, helix turn helix, leucine zipper, zinc finger
29
what is the globin domain?
each chain of haemoglobin has a tertiary structure very similar to that of the single myoglobin chain, strongly suggesting evolution from a common ancestral O2-binding polypeptide. The globin domain is maintained in haemoglobin and haemoglobin-s. Glu is substituted with Val in the beta chains leading to polymerisation of deoxygenated haemoglobin
30
why does the Glu to Val substitution cause polymerisation of deoxygenated haemoglobin?
glu is hydrophilic whiles Val is hydrophobic
31
what is the modularity of motifs in transcription factors?
proteins that bind to DNA and regulate transcription. Many different TFs but they all contain a small number of conserved motifs. these motifs are conserved across all phyla i.e. huge varieties of eukaryotes ranging from fungi to plants and animals. these motifs form DNA binding domains that allow regulatory function of their respective proteins. these proteins are formed from homo and heterodimers
32
what is the role of alpha helices in binding?
can fit within the major groove of DNA. different DNA binding domains and motifs present the binding helix using different arrangements of the structural motifs
33
what is the helix-loop-helix motif?
- binds dna in dimeric form
- exists as homo and heterodimers
- the central portion formed overlapping helices form a structure that enables dimerisation
- the terminal part of the lower opposing helices contain basic amino acids that interact with the major groove of DNA - giving rise to the b/HLH
34
what is the leucine zipper motif?
- formed from 2 contiguous alpha helices
- dimeric protein formed from 2 polypeptide chains
- dimers zip together in the top stalk to form a short 'coiled coil'
- coil is held together by hydrophobic interactions down opposing sides of the helix
- as in the b/HLH domain, basic amino acids dominate the lower part of the helix and interact with the DNA major groove
- heterodimerism expands the regulatory potential of leucine zippers
35
what is the helix-turn-helix motif?
- consists of 2 short helices orientated at right angles to each other and connected by a turn
- the motif is found in both prokaryotic and eukaryotic DNA binding proteins
- CRO protein = homodimer, recognises pellindromic sequence and by binding DNA represses transcription
- only the recognition helix interacts with the nucleotide sequence itself - other contacts are made with the phosphate backbone and like other DNA binding motifs it locates within the major groove
36
what is the zinc finger motif?
- alpha helix and beta sheet held together by non-covalent interactions with Zn
- alpha helix of each motif interacts with major groove of DNA and recognises a specific DNA sequence
37
what is the isoelectric point?
the pH of buffer at which a protein has no charge
38
if the pH
it is an acidic solution with a net positive charge
39
if the pH > pI.....
it is a basic solution with a net negative charge
40
what is the basic principle of electrophoresis?
it is the migration of charged particles (macromolecules) in an electric field
41
what is migration in electrophoresis based on?
size, shape and charge. Electrophoresis uses current and resistance
42
what are the two types of electrophoresis?
hoizontal (usually for agarose gel) and vertical (usually for polyacrylamide gel)
43
what is the apparatus needed for electrophoresis?
gel casting trays, sample combs, tank, power supply
44
what should electrophoresis apparatus ensure?
- uniform electric field across gel
- cooling to prevent thermal artefact
- access to gel loading and monitoring
45
what is the buffer there for in gel electrophoresis?
provides ions to carry current and to maintain relatively constant pH
46
what is agarose gel used for?
to separate large proteins >200kDa. has a relatively low resolving power
47
what does the buffer do in vertical slab electrophoresis?
- supplies current carrying ions in electrophoretic cell
- maintains desired pH
- provides medium for heat dissipation
48
what can buffer systems in vertical slab electrophoresis be classified as?
- continuous
| - discontinuous
49
what is a continuous buffer?
uses same buffer in gel, sample and tank
50
what is a discontinuous buffer?
non-restrictive large-pore gel, resolving gel for greater resolution, have different buffers for stacking gel, resolving gel and tank buffer
51
what does the migration of a protein in an electric field depend on?
pH and pI
52
what is SDS-PAGE?
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis: most commonly used electrophoretic technique for separation
53
what is SDS?
a strong anionic detergent to solubilise, disassociate and denature most proteins to single polypeptide chains - disrupts H bonds and blocks hydrophobic interactions. includes disulphide bond cleaving agents
54
what is SDS-PAGE not suitable for?
small polypeptides and peptides of MW
55
what are detection methods in electrophoresis?
- protein staining
- fluorescent staining
- silver staining
- radioactive methods
56
when is native (non-denaturing) gel electrophoresis used?
mainly when native conformations are to be analysed. runs without SDS.
57
what is separation based on in native gel electrophoresis?
charge to size ratio and conformation/shape
58
what are the advantages of native gel electrophoresis?
- potential of separating proteins of identical MW
59
what is Serum Protein Electrophoresis (SPEP)?
SPEP measures specific binding proteins in blood. Uses electrical field to separate proteins into groups of similar size, shape and charge. Helps to identify diseases. Blood serum contains 2 major protein groups; albumin and globulins
60
what most commonly happens in haemoglobin electrophoresis?
pH range of 8-9 is the most commonly used buffer system. the majority of proteins will be negatively charged.
61
what are examples of chromatography separation techniques?
- salting in or out
- column exchange
- electrophoresis
- isoelectric focussing
62
what are types of column exchange chromatography?
- ion exchange
- gel filtration
- affinity
- reverse-phase HPLC
63
what is the definition of chromatography?
a separation method in which components partition between a moving phase and a stationary phase
64
what properties of proteins does chromatography utilise?
- charge distribution
- molecular size
- solubility
- binding properties
65
what is Ion Exchange Chromatograhy?
stationary phase is polymer (matrix) substituted with charged groups. acidic groups of resin interact with positively charged proteins and are called cation exchangers. if groups are basic in nature they interact with negatively charges molecules and are called anion exchangers.
66
what happens in IEX?
proteins will bind to an ion exchanger with different affinities. as the column is washed with buffer, proteins with relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column. The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column. proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH
67
what is gel filtration chromatography (GFC) also known as?
size elusion chromatography, molecular sieve chromatography
68
what is GFC widely used for?
to purify biologically complex samples such as blood
69
what happens in GFC?
column consists of porous bead of dextran or agarose. separation is based on size and shape, with larger molecules eluting first, and smaller last.
70
what is elution volume (Ve)?
the volume of a solvent required to elute a given solute form the column after it has first contacted the gel
71
what is affinity chromatography (AC) used for?
AC is used to isolate antibodies, antigens, hormones and other proteins. Separation is baed on reversible interaction between target protein and specific ligand
72
what does AC require?
- beads matrix
- ligand (a molecule that specifically binds to the protein of interest)
- solution containing substance to be isolated
- a wash to elute non-bound substances
- final wash containing competitive ligand
73
how do you remove the protein of interest from the column in AC?
elute with a solution of a compound with higher affinity than the ligand (competitive). you can change the pH, ionic strength and/or temperature so that the protein-ligand complex is no longer stable
74
what is high performance liquid chromatography?
- highly improved form of column chromatography
- solvent is forced under pressure of 400 at the minute through column
- allows the use of smaller particle size for the column material, thus increasing surface area
- this allows much better separation of components
75
what are the components of HPLC?
reservoir of mobile phase, pump, an ejector, separation column and detector
76
what is normal phase chromatography?
has a polar stationary phase and a less polar mobile phase. the more polar the analyte, the greater the retention. increasing polarity of mobile phase, decreasing retention.
77
what is reverse phase chromatography?
the most widely used move of HPLC. separates molecules on the basis of hydrophobicity (non-polar stationary phase and polar mobile phase). in practice the non-polar functional group is 'bonded' to silica - "bonded phase chromatography"
78
what does the Y-axis on an HPLC graph represent?
the signal generated by molecules within the detector and may be proportional to the molecules concentration
79
what does the X-axis on a HPLC graph represent?
the time after injection of the molecules onto the column or the time after the gradient of buffers A and B was started
80
what are the advantages go HPLC?
- speed
- high resolution
- sensitivity
- reproducibility
- accuracy
- automation
81
what are the disadvantages of HPLC?
- cost
- complexity
- coelution
82
what is a gene?
a unit of heredity, containing instructions for an organism's phenotype. DNA segment containing instructions for making a particular product including the regulatory elements
83
what are transcription factors?
proteins required to initiate or regulate transcription in eukaryotes. assemble on promoter to position RNA polymerase II. They pull apart the DNA helix and expose the template strand.
84
what is needed for gene-specific regulation of transcription?
additional upstream sequences
85
what are untranslated regions?
UTRs are transcribed but not translated.
5' UTR - regulation of translation
3' UTR - mRNA stability and miRNA binding
86
how is the primary transcript of RNA processed?
- capping
- polyadenylation
- splicing
87
what is capping?
- 5' end modification
- guanine nucleotide - 5'-5' triphosphate bridge, methylated at position 7
- capping enzyme complex
- co-transcriptional modification
88
what is polyadenylation?
- cleavage by specific endonuclease
- addition of tail by poly(A) polymerase
- polyadenylated mRNA precursor
89
what is splicing?
the removal of introns, joining exons
90
what are introns?
non-coding sequences within genes
91
what is a form of alternative splicing?
exon skipping in ~95% of genes
92
in what ways is differential gene expression regulated?
```
time (development and in response to hormones, infection and other signals)
and space (different tissues or cell types express different gens)
```
93
what is the 'big goal' in the control of gene expression?
- the ability to isolate adult stem cells form all developmental compartments
- the ability to drive these cells to develop either in vivo or in vitro in a completely controlled manner so that we can regenerate tissues
- this well require a detailed and fundamental understanding of the control of gene expression
94
what is beta-thalassaemia?
a group of genetic diseases caused by insufficient expression of beta-globin. in most types of beta-thalassaemia then beta-globin protein is structurally normal (unlike sickle cell disease). there are multiple independently-arising forms of the disease. mutations cause beta-thalassaemia
95
what is the sequence of post-transcriptinal gene regulation?
- polyadenylation
- capping
- splicing
- translation
- RNA stability
96
what are examples of translational control?
- Early embryogenesis; during the first 4-8 cell division there is virtually no gene expression. at the end of blastocyst formation, the first genes to be expressed are due to up-regulation of translation form maternally derived pre-formed mRNAs
- environmental stress; exposure to heat shock or pathogens can cause global changes in translation
- there are many specific examples - e.g. ferritin
97
what does the 5' UTR do?
doesn't determine whether a ribosome binds but it does play a major role in determining how efficiently the ribosome initiates translocation (glob in: efficiently, ferritin: inefficiently)
98
what does the 3' UTR play a role in?
determining the stability of the mRNA. it confers very different stabilities on mRNAs: glob in 3' UTRs confer stability, immune stress hormones = very unstable mRNAs
99
how are intracellular iron levels controlled?
traslationally controlled, ferritin binds iron and retains it in the cytoplasm as a store for excess. we only need ferritin at times of iron excess.
100
what are miRNAs?
they are small non-coding RNAs which are transcribed by RNa polymerase II. they act to control the post-transcriptional regulation of as many as 1/3 of all human genes. any given miRNA can regulate several target genes. they are derived by processing from a larger precursor.
101
what is the transcriptome?
the segment of the eukaryotic genome that is transcribed (most of it is never transcribed). only a fraction of the transcriptome is transcribed at any one time.
102
what is a housekeeping gene?
one that is in every cell
103
what is a promoter?
a sequence that is immediately 5' to the region to be transcribed. they recruit RNA polymerase to a DNA template - RNA polymerase can only move one way
104
what are enhancers?
sequences of DNA that are not immediately adjacent to where transcription starts that act to enhance the recruitment of RNA polymerase to a promoter. enhancers can reside 5' or 3' to a transcription unit and can even be located within an intron. they contain DNA sequences that are very strong binding sites for specificity factors of 'transcription factors'
105
what is recruitment of promotors mediated by?
initiation factors: predominantly 'sigma factor' for prokaryotes and the TFII transcriptional machinery for prokaryotes. once stably recruited to DNA, RNA polymerase is able to convert from a closed to an open complex
106
what does it mean that sigma factor and TFII's ability to recruit RNA polymerase to promotors is generic?
they happen at every promoter. this does not account for the ability to vary the level of transcription form a promoter.
107
what are regulatory transcription factors?
mediate regulatory changes. in both prokaryotes and eukaryotes they function to dramatically alter the level or recruitment of RNA polymerase and/or its ability to initiate transcription. additionally, in eukaryotes they can influence chromatin structure.
108
what is a transcriptional switch in prokaryotes?
the lac operon
109
what are transcriptional switches in eukaryotes?
- oestrogen-responsive transcription
- tissue-specific transcription (beta-globin)
- a complex regulatory circuit (cell cycle)
110
what is the lac operon?
a prokaryotic paradigm for the regulation of transcription
111
how do regulatory factors recognise their target sequence?
by interacting with DNA. they do not have to unwind the DNA double helix to see their target, therefore an intact DNA molecule can present information to the cell.
112
what is a way of targeting therapeutic drugs?
interfering with the interactions between regulatory and general transcription factors
113
what are transitions through the cell cycle regulated by?
cyclin-dependent kinases
114
what do CDks do?
target transcription factors
115
what is a key event in G1/S transition?
the transcriptional activation of genes that encode proteins involved in DNA replication
116
when is E2F activity repressed?
in G0 and early G1 by the product of the retinoblastoma gene (pRB). Rb is a common target in cancer.
117
what is chromatin?
chromosomes and associated proteins
118
what are histones?
proteins responsible for the firs level of packaging
119
what are nucleosomes?
DNA + histones
120
what is the 30nm fibre?
nucleosomes pack themselves into these fibres, constituting the second level of DNA packaging which increases packaging 6 fold
121
what is the 80-100nm fibre?
the 30nm fibres pack themselves into 80-100 nm fibres constituting the third level of DNA packaging which increases DNA packaging 3 fold
122
what is the mitotic chromosome?
the fourth level of DNA packaging which increases packaging 10,000 fold
123
what do chromosomes consist of?
predominantly DNA, histone proteins, non-histone proteins and non-coding RNA
124
what happens to DNA in the interphase chromosome?
almsot all DNA is packages at the first level (nucleosomes). much DNA is tightly packed into higher order structures.
125
how do histones assemble?
they assemble to form an octamer core with 2 molecules each of histones H2A, H2B, H3 and H4. the N-terminal tails outside the octamer core. DNA is wrapped around the histone core and electron micrographs show a 'bead on a string' appearance
126
what does the compaction of nucleosomes to form higher order structures involve?
- linker histones (eg H1)
- interaction of histone tails with adjacent nucleosomes
- binding of packing proteins to histone tails
127
what does it mean that chromatin structure is not static?
during transcription, or DNA replication, nucleosomes must be removed from the DNA in front of the polymerase, and replaced behind the polymerase
128
what are histone remodelling factors?
enzymes that remove and replace nucleosomes
129
what is euchromatin?
- lightly staining areas of chromatin
- rich in genes
- made up of nucleosomes, but not dense higher order packaging
130
what is heterochromatin?
- darkly staining areas of chromatin
- few genes
- dense higher order packaging of nucleosomes
131
what is facultative heterochromatin?
contains genes not expressed in that cell type. DNA tightly packages as heterochromatin, but may be packaged as euchromatin in other cell types.
132
how are histones and non histone proteins extracted form chromosomes?
the chromosomes are treated so that they don't completely fall apart. they appear as long DNA loops attached to a scaffold of tightly bound proteins
133
what does evidence suggest about loops and chromatin domains?
- each loop may have a different degree of chromatin compaction
- the scaffold isolates the chromatin in one loop from the next loop
- so one loop may have open chromatin and active genes
- the neighbouring loop could be tightly packed as heterochromatin
134
what does DNAse I do?
cutes double stranded DNA and histone binding proteins, protecting DNA from DNAse digestion
135
what are DNAse I sensitive sites?
sequences of DNA without histones
- may be naked DNA or binding transcription factors
- cut by very brief digestion with DNAse I
- found in promoters and enhancers
136
what is chromatin immunoprecipitation?
- break the chromatin into small fragments
- add antibody to a target protein e.g. GATA-1 or acetylated histones
- the precipitate contains the target protein, associated proteins and DNA
137
what are regulatory transcription factors?
TFs bind promoter and enhancers, they recruit general transcription factors, assemble initiation complex which recruits RNA polymerase.
138
what are histone acetyl transferases (HATs)?
acetylate lysine residues on histones, lead to the unpacking of chromatin
139
what are histone deacetylases (HDACs)?
de-acetylate histones, lead to the compaction of chromatin
140
what happens in methylation?
histone tails are methylated by his ton methyl transferase (HMTs), demethylated by histone demethylases (HDMs). a lysine residue can be mono-, di- or tri- methylated
141
what are histone marks?
histone marks are read by binding proteins. related domains are found in multiple code reading proteins. histone marks are not read in isolation - multiple lysine residues on each histone, multiple modifications, other histone modifications.
142
what is evidence for 'code readers'?
protein complexes that read combinations of marks
143
what is the general mechanism for memory in cell differentiation?
cell remodelling (states of DNA methylation and histone modification can be copied to daughter cells), positive feedback ('feed-forward')
144
what two examples illustrate positive feedback in master gene regulators in differentiation?
- melanocytes, MITF
| - skeletal muscle, MYOD1 family
145
what is a master gene regulator?
a transcription factor that co-ordinately regulates many/all of the specialised genes expressed by a particular cell type
146
what is MITF?
'micropthalmia-associated transcription factor'
- master gene regulator for melanocytes
- severe mutation in MITF gene, when homozygous, cause loss of all melanocytes in the body (skin, hair, eyes)
- eyes become small (loss of pigmented retina) 'micropthalmia'
- so MITF is needed for all normal melanocyte development
147
what is Waardenburd syndrome 2?
- mutation of one copy of the MITF gene in humans
- deafness and congenital patchy loss of pigment - in skin and can include iris
- so MITF also needed for human pigmentation
- human eye with no melanin is light blue
148
what are myogenic factors?
master gene regulators in skeletal muscle. there are 4 knows - MYOD1, MYF5, myogenin and MRF4
149
what are E proteins?
widely expressed transcription factors. myogenic factors normally work as dimers with E proteins
150
what is ID1/the ID family?
inhibitor of differentiation.1. a protein in myoblasts, that can strongly bind E proteins but no DNA.
151
what is cell senescence?
one of the basic cellular processes. a major defence against cancer. it is strongly implicated in the symptoms of ageing. our understanding is recent and still emerging. there is permanent cell growth arrest, following extending cell proliferation (replicative senescence)
152
what is the Hayflick Limit?
the number of times a normal human cell population will divide until cell division stops. Empirical evidence shows that the telomeres associated with each cell's DNA will get slightly shorter with each new cell division until they shorten to a critical length.
153
what are examples of molecular markers in senescent cells?
- lysosomal beta-galactosidase
| - p16 a cell cycle
154
what are telomeres?
1000s of repeats of a hexamer sequence (TTAGGG) at chromosome ends
155
what is telomerase needed for?
3' ends of linear DNA can't be replicated normally, because an RNA primer has to bind beyond the part to be replicated so the enzyme telomerase is needed to maintain telomere length
156
what does telomerase do?
can replicate the telomeric DNA by reverse transcribing DNA hexamers (TTAGGG) from its own RNA sequence and joining them to the chromosome end. telomerase activity is highest in germ cells which therefore have the longest telomeres
157
what is TERT?
telomerse reverse transcriptase
158
what is TERC?
telomerase RNA component
159
why is there no telomerase activity in more human somatic cells?
because they express no TERT so there is no telomerase activity and therefore telomeres shorten as cells divide
160
when is replicative senescence triggered in normal cells?
when telomeres get to a specific short length
161
why do normal germ line cells maintain full length telomeres?
because they do express TERT. hence the germ line is immortal - cells can divide forever
162
what evidence is there fore cell senescence being behind many of the symptoms of normal ageing?
- telomere length is variable but on average falls with age, typically very short in people >100
- p16 and other senescence associated proteins are expressed increasingly in ageing tissues
- telomere length at birth varies between people; genetically linked to age at death
- defective genes for telomerase subunits give syndromes with premature ageing and early death
- p16 locus also genetically associated with human senile defects - cardiovascular disease, frailty, type II diabetes, neurodegeneration, cancer
163
what is a stem cell?
a cell that is capable of both self-replication and differentiation into one or more kinds of specialised functional cells
164
what types of stem cell are there?
unipotent - can form only one functional cell type
puripotent - can form several functional cell types
totipotent - can form ALL functional cell types
165
why is the zygote not normally considered to be a stem cell, despite being totipotent?
it doesn't divivd to form more zygotes - however the inner cell mass of the early mammalian embryo can act as totipotent stem cells, called embryonic stem cells
166
what are somatic stem cells?
those that remain as a proliferate reservoir after birth
167
do some somatic stem cells have telomerase?
some do but in general too little to make the cells immortal, telomeres shorten less per division in somatic stem cells than in other somatic cells, but they do shorten. so somatic stem cells do senesce gradually
168
what are examples of connections between stem cell ageing and our ageing?
- bone marrow - older people show decreased immunity, increased bone marrow failure, decreased success rate as bone marrow donors
- hair greying - linked to decreased melanocyte stem cell maintenance win hair follicles (data from mice)
- reduced healing ability of skin with age, increased risk of skin ulcers. senescence in dermal fibroblast - although epidermal stem cells have very little telomere shortening and remain able to divide.
169
how can cancer cells be similar to stem cells?
tend to be poorly differentiated and tend to have telomerase. sometimes stem cell markers can be found in some of the cancer cells
170
what is the cancer stem cell idea?
this is largely hypothetical for many types of cancer. cancer stem cells are proposed to be:
- a small subpopulation in the cancer
- rarely dividing
- the only cells in the tumour that are immortal
- thus the only cells that can form large clones or regenerate a tumour after depletion
- affects design of therapies. if rarely dividing, such cells may evade standard therapies, because these tend to target proliferating cells
171
what is the definition of genetics?
inheritance of genes that are activated or inactivated by mutation. change in DNA sequence
172
what is the definition of epigenetics?
inheritance of a state of gene activation or inactivation without any change in DNA sequence
173
what is mechanism I of DNA differentiation?
- nucleosomes are removed in front of the replication fork and replaced behind it
- new nucleosomes are added, since there is now twice as much DNA
- old histones carrying histone marks e.g. methylation, acetylation
- recruit HATs, HDACs etc
- add similar marks to new histones
174
what is mechanism II of DNA differentiation?
- DNA methylation
- maintenance methylase binds to hemimethylated DNA and methylates unmethylated CG
- histone modifications and DNA methylation interact
- this is a form of positive feedback
175
what does carcinogenesis require mutations in?
- dominant oncogenes
| - tumour supressor genes
176
what do tumour suppressor genes require in order to allow cancer to develop?
both copies of the gene in a diploid cell to be inactivated
177
what may typical TSGs be?
- cell checkpoint genes e.g. Rb, p53
- DNA repair genes
- genes regulating cell death
178
why is P21WAF an important regulator of the cell cycle?
- inhibits cell cycle progression
- acts as a TSG
- inactivated in many cancers
179
what is the G1 checkpoint for DNA damage?
- normally p53 is degraded quickly, unstable and very low levels
- phosphorylated (active) p53 is not degraded
- active p53 promotes transcription of genes that induce cell cycle arrest, in particular it binds to promoter region of p21 gene and stimulates p21 expression
- p21 binds and inactivates G1/S-Cdk and S-Cdk complexes
- cell arrests in G1 (allowing time to repair the damaged DNA)
180
what is the presentation of Sickle Cell Disease?
- anaemia
- pain crisis
- infection
- organ crises
- chronic organ failure
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what is SCD due to?
a point mutation in the beta globin gene
- HbA abnormal (mutant beta gene)
- HbF normal (no mutation in the gamma gene)
- but gamma gene is inactive in adults
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what investigational drugs are being used on SCD?
- HDAC inhibitors (butyrates)
| - DMT inhibitors (deciabine)
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what happens in the human genome project?
whole genome sequenced
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what are gene chips?
a large number of DNA probes for individual genes, gridded on a solid substrate
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what happens in DNA microarrays?
- prepare single stranded DNA sample
- label DNA molecules with a fluorescent
- hybridise to array
- each DNA will only bind to its complementary sequence
use fluorescence to see which spots 'light up'
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what is NextGen sequencing?
- massively parallel DNA sequence
- sequence 50,000 or more DNA molecules at the same time
- several different technologies
- one example is to trap single DNA molecules on a chip
- perform sequencing reaction with fluorescent nucleotides, then scan the chip with microarray scanner
- read the sequence for each DNA molecule
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how are eukaryotic genes dispersed along a chromosome
genes are separated by long intergenic regions of mostly unknown function. 97% of genomic DNA is intergenic
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what is an intron?
a sequence in the genes DNA that is transcribed as part of the primary RNA transcript but is removed form the mature mRNA
189
what is an exon?
a sequence in the gene and primary transcript that is retained in the mature mRNA
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what is an untranslated region (UTR)?
a sequence in the mature mRNA (therefore exon sequence) that does not code for amino acids, so also called a 'non-coding region'
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what is the ENCODE project?
"ENCyclopedia Of DNA Elements"
| international consortium to map features of interest throughout the human genome
192
what happens if you perform an Chromatin Immuno-Precipitation (ChIP) using an antibody to P300?
precipitates sequences enriched for H3K4me1
- monomethylated histone H3 lysine 4
- a chromatin mark typically associated with enhancers
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are the sequences in ChIP reactions really representative of enhancers?
- analyse DNA sequences by microarrays or NextGen sequences
- confirm presence of transcription factor binding sites
- map DNAse I hypersensitive sites
- confirm that these putative enhancer sequences are co-located with HSS in chromatin
194
what are the clinical features of Trisomy 21?
- antenatal: excess nuchal skin
- diagnosis on facial appearance; small nose and flat nasal bridge, eyes slant up and outwards
- single palmer crease
- newborns: hypotonia - weak muscles
- cardiac malformations (40%)
- delayed development
...therefore trisomy 21 can be tested prematurely or when born
195
what is meiotic non-disjunction?
the mother cell creates two gametes and they are meant to separate but they are both going to one, and the other will have none
196
what is an aneuploid?
not an exact number of the haploid number
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what are examples of aneuploidies?
- trisomy 18 ; Edward's Syndrome
- trisomy 13 ; Patau Syndrome
- 45,X ; Turner Syndrome
- 47,XXY ; Kleinfelter Syndrome
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what is translocation?
the non-homologous end joining tried to repair breakage but this doesn't always work
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what is balanced translocation?
no gain or loss of genetic material
200
what is unbalanced translocation?
net gain or loss of genetic material
201
what is reciprocal translocation?
exchange of genetic material between non-homologous chromosomes
202
what are the three centromere positions called?
metacentric, submetacentric, acrocentric
203
what are robertsonian translocation?
this occurs in the five afrocentric chromosome pairs. it is a type of non-reciprocal translocation where the chromosomes break at their centromere and the long arms fuse to form a single chromosome with a single centromere. the two short arms also fuse to form a reciprocal product which is usually lost within a few cell division
204
what other structural changes can occur?
- terminal deletion
- interstitial deletion
- inversion
- duplication
- ring chromosome: sticky ends come together to form a ring
205
what are mendelian disorders?
- monogenic
- environment has little or no impact
- rare
- familial pattern of inheritance; autosomal recessive, autosomal dominant or sex linked
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what are mutations?
heritable changes in genetic material. mutations can be caused by exposure to mutagens or errors in DNA replication
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what types of substitutions are there?
- silent/synonymous: no amino acid change
- missense/non-synonymous: change in amino acid, sickle cell
- nonsence: premature stop codon
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what are the functional effects of mutations?
mutations could cause loss of function, resulting in reduced activity, complete loss of product of haploinsufficiency.
they could have a dominant negative effect.
they could also result in again of function, causing an increased amount of product or even a new function
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what is copy number variation?
DNA segments >1kb present variable frequency compared to the reference genome
210
what is the 'equation' for common disease?
disease = genetics + environment
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what are examples of common disease?
cardiovascular disease, hypertension, stroke, diabetes, rheumatoid arthritis and many more
212
what is the gross structure of the human genome?
23 chromosome pairs
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what is the fine structure of the human genome?
coiled helix
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what is the molecular structure of the human genome?
the DNA sequence
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what is the definition of an allele?
each of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome - AN ALTERNATIVE FORM OF A GENE
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what does biallelic, triallelic and multiallelic mean?
biallelic: 2 possible alleles
triallelic: 3 possible alleles
multiallelic: more than 3 possible alleles
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do two populations of the same species need to have the same frequency at the same locus?
no
218
how does an allele arise etc?
an allele arises by mutation, spreads through migration and selection determines whether the allele survives in the population or not
219
what are the four main evolutionary processes that populations are subject to?
natural selection, genetic drift, mutation and gene flow
220
what is a mutation?
the changing of the structure of a gene, resulting in a variant form which may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion or rearrangement of larger sections of genes or chromosomes
221
what is the difference between mutation and polymorphism?
mutation: minor allele frequency 1%
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what are types of common genetic variants?
- single nucleotide polymorphisms (SNPs)
- microsatellites
- copy number variants (CNVs)
223
why does genetic variation occur?
errors in DNA replication and repair...
- proof reading
- mismatch repairs
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what is a single base substitution - TRANSITION?
where something is swapped with something the same (more common) purine --> purine, pyrimidine --> pyrimidine
225
what is a single base substitution - TRANSVERSION?
where something is swapped with something different (less common) purine pyrimidine
226
what is a microsatellite?
variable number of tandem repeats. the number of repeats varies between individuals. the total length of micro satellite sequence varies between individuals.
Microsatelling = length polymorphism
227
what is the genotype of SCD?
aa
228
what are the Hardy-Weinberg equations?
p + q = 1
| p2 + 2pq + q2 = 1
229
what is the hardy-weinberg principle?
if an infinitely large, random mating population is free from outside evolutionary forces then the genotype frequencies will not change over time
230
what is disease gene mapping?
this is a way of identifying which gene causes a disease when not working properly. there are over 20,000 genes in the genome - we know which ones cause disease because of mapping.
231
what is suggested if genotype frequencies in cases deviate from HWE?
suggests an association between the variant and the disease. for recessive diseases there will be an increase in homozygotes
232
what are genetic markers?
a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed
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how are cases enriched with genetic markers?
cases will be enriched for the mutation that causes that disease. if a marker is very close to that mutation then cases will also be enriched for a specific allele of that marker.
234
what happens to a marker if there is massive deviation from HWE?
the marker is co-inherited with disease variant, the marker is NOT the causal variation
235
what are the various inheritance patterns?
autosomal dominant, autosomal recessive, sex linked
236
what is homozygosity mapping?
a technique for mapping the genetic locus of an autosomal recessive trait in a consanguineous relationship
analysis of genetic markers, they may not necessarily be the gene itself but it is a DNA sequence with a known location on a chromosome that can be used to identify individuals or species
237
through which ways does the human genome become polymorphic (has many forms)?
- allelic variation
- CNV
- SNP
- microsatellites
238
how are micro satellites detected?
1 - isolate DNA from individuals to be studied
2 - design primers specific to the flanking sequences
3 - PCR amplification
4 - gel electrophoresis
5 - use fragment length to determine number of repeats
239
why do we have to look at hundred of micro satellites along chromosomes and across the genome in diagnoses?
because if we look at just one marker, the chance it is associated with the disorder is very small
240
what is the reason that patients do not pass on perfect duplicates of entire chromosomes to offspring?
recombination occurs during meiosis
241
how is homozygosity mapping done?
1 - establish trait follows autosomal recessive inheritance in a consanguineous family
2 - isolate DNA and perform micro satellite analysis
3 - analyse data to identify smallest shared regions of homozygosity
4 - evaluate which regions of homozygosity co-occur with disease status...
THE DISEASE GENE MUST LIE IN THIS REGION
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what re MTPAP and mitochondrial disorders?
- nuclear encoded gene
- mitochondrial poly(A) polymerase
- creates poly(A) tail on mtRNA transcripts, stabilises mtRNA transcripts
- mitochondrial disorders - neuropathies, freidreich's ataxia
243
what is meant by identical by descent (IBD)?
the segment is identical because its descended form a common ancestor
244
what is meant by identical by state (IBS)?
segment between individuals is identical by chance - nothing to do with your family
245
what is meant by linkage?
points close together are said to be linked, points far apart (or on different chromosomes) are unlinked
246
what is linkage disequilibrium (LD)?
when alleles at 2(+) loci are found together more frequently than would be expected by chance. this relates to the study of populations.
247
what is allelic association?
refers to the situation in which the distribution of a measured outcome (the trait) varies according to the distribution of one or more genetic variants.
248
what can knowledge of linkage disequilibrium be used for?
knowing which chromosomes are in LD is useful for association studies and linkage analysis in Mendelian disease
249
in what way should case and control populations differ?
they should differ only in terms of the trait under investigation. factors to consider include race, age and lifestyle/environment
250
where might you find an SNP?
- GENE: no amino acid change (synonymous), amino acid change (non-synonymous), introduce stop codon (nonsense)
- PROMOTER: protein level changed
- NON-CODING REGION: introns etc, doesn't code for protein but important for splice sites
WITHOUT DELETERIOUS EFFECT OF POPULATION ANNIHILATION, SNPS DO NOT DISAPPEAR
251
how do we detect SNPs?
microarrays
DNA from one person is on one microarray. they can detect up to 2.5 million SNPs simultaneously. the result is either homozygous or heterozygous for each SNP
252
what is involved in the Wellcome Trus Care Control Consortium?
- 200 scientists
- 50 UK groups
- £9 million
- 17,000 people
- 500,000 SNPs
253
what do peaks identify in manhattan plots?
the peaks do NOT identify the gene causing the disease. the peak identifies the genomic region associated with the disease.
254
what is ANRIL?
ANTISENSE NON-CODING RNA IN THE INK4 LOCUS
expressed in cells affected by atherosclerosis
the expresson level correlates with atherosclerosis
it possibly regulates expression of other genes
it is probably involved in a novel mechanism of atherosclerosis
255
what are limitations of case control studies?
- utilise lots of SNPs - at least 500,000
- need to analyse thousands of people
- labour intensive project
