Functional genetics Flashcards
1
Q
what general shows that ageing is a genetically determined trait?
A
different species have different maximal lifespans
2
Q
if ageing is genetically determined, what does this mean you can do?
A
- take a classical genetic approach
- isolate mutants with altered rates of ageing
- ap, clone and sequqence genes concerned
- identify lifespan determining proteins and biochemistry
3
Q
what are the four species used for lifespan genetics?
A
- S. cerevisiae
- c.elegans
- drosophila melanigaster
- mus muscus
4
Q
what does inbreeding do to lifespan ?
A
it decreases again in flies
5
Q
what are the signs of ageing in c.elegans?
A
- reduced fertility
- increased molecular damage(protein carbonyl)
- organ deterioration ( intestine, gonad)
6
Q
what is better, long lived or short lived mutants and why?
A
- long lived, if you have a short lived mutant then it could be dying from something other than decreased ageing
7
Q
who was the first to screen and isolate a long lived mutant and in what animal?
A
Klass- worm
8
Q
who isolated the age-1 mutant?
A
Johnson
9
Q
what phenotype does the age-1 mutant express? (2)
A
65% mean lifespan increase
110% maximum lifespan- remains youthful for longer.
10
Q
who isolated the daf-2 mutant?
A
kenton
11
Q
what is the phenotype of the daf-2 mutant?
A
greatly increases lifespan
12
Q
what was found in an age-1 mutant in 2007?
A
10 fold increase in maximal lifespan
13
Q
what is the dater larvae?
A
Developmentally arrested alternative third stage larva
• Forms in response to high population density (dauer pheromone), high temperature, low food• Non-ageing: post dauer adults have normal lifespans
• Stress resistant (heat, ROS) • Reduced levels of movement (but can move fast)
14
Q
what does daf mean?
A
daher abnormal formation
15
Q
why were people interested in looking for mutants which affect dater formation?
A
- the dauer phenotype seems to resemble deferred ageing- they are non ageing- so whatever is involved in forming the dater phase may increase lifespan
16
Q
what two genes arose from looking for mutations which affect dater formation?
A
daf-c (dauer constituitive)
daf-d (daf defective)
17
Q
how were the dat-2, dat-16 mutants ordered?
A
daf-2 mutants are long lived. daf-16; dat-2 mutants are not long lived. So daf-16 mutant suppresses the increased longevity of dat-2. So daf-16 wild type extends longevity and daf-2 inhibits dat-16 normally
18
Q
in what way are daf-2 mutants temperature sensitive?
A
at non-permissive temperatures, daf-2 mutants are dauer constituitive but at permissive temperature the mutant is long lived
19
Q
how do we know that the increased lifespan we see in age-1 and daf-2 mutants is not due to dormancy or arrest but instead due to decelerated ageing?
A
only non-permissive temperatures results in constituitive dauer state but at permissive temperatures they are just long lived. Furthermore, the age-1 mutants are only daf-c in extreme alleles. So we know that the longevity is due to the isexpression of longevity genes rather than constituitive dauer expression state
20
Q
once the daf-2, age-1 and the daf-16 (daf-d) had been found, who and how was this shown to be relevant to other species that didn’t have a dauer state?
A
Ruvkun found that these genes do indeed have a human homologue!
-age-1 :Catalytic subunit of phosphatidyl inositol 3-kinase
daf-2: Insulin or IGF-1 receptor
daf-16.Daf-c=FoxO-class forkhead transcription factor
- all of these genes have homologues in humans
21
Q
what is daf-2 pathway and what are the components of each of the genes that were discovered in the worm?
A
the daf-2 gene: encodes an insulin/IGF-1 age-1: catalytic subunit of PI3K. the activation of PI3K converts PIP2 to PIP3. PIP3 then recruits AKT to the membrane where it can be phosphorylated by PDK-1. AKT then phosphorylates DAF-16 which is the FOXO- class forkhead transcription factor, this prevents it from transcribing genes associated which dauer formation and increased longevity . the phosphorylation of daf16 causes it to bind to proteins in the cytoplasm and leave the nucleus.
22
Q
describe an experiment which was used to identify the tissues in which daf-16 was required to increase longevity? what did they find?
A
because they knew that the daf-2 mutant required daf-16 expression to increase longevity, they investigated, by using tissues specific promoters to drive daf-16 expression, where daf-16 expression needed to be resorted in a daf-2;daf-16 mutant . they found that daf-16 as required in the intestine.
23
Q
what does phosphorylation of daf-16 stimulate?
A
it to bind to proteins in the cytoplasm and leave the nucleus
24
Q
since they found that daf-16 expression is required in the worm intestine for longevity, what does this tell us about mammalian tissue?
A
the worm intestine combines the role of intestine, liver and adipose tissue. so maybe these tissues control longevity?
25
how did people seek to find out whether the insulin pathway that was found in c.elegans, is conserved in other animals?
- they looked at the insulin/IGF-1 signalling pathway in flies. They found that chico mutants (insulin receptor substrate) and dINR (homologues to the daf-2 insulin/IGF receptor) were both long lived mutants
26
what were the two fly homologues of the fly insulin signalling pathway which were looked at in flies?
```
chico= the insulin receptor substrate
dINR= the homologue to the insulin receptor
```
27
by how much is the lifespan of female flies increased in chico mutants?
85%
28
because flies and worms have one insulin/IGF-1 receptor, and mammals have an insulin receptor, , an IGF-1 receptor and a inulin-like receptor, what did people look at to investigate conserved mechanisms- what did they fine
they tried to look at the consequences of reducing the functions of the insulin receptors- in human slight reduction causes type-2 diabetes and severe reduction causes leprechaunism. KO in mice is lethal. this showed that it was unlikely that insulin signalling was involved in ageing in humans. but they did find that fat-specific KOs of insulin receptor in mice increased lifespan by 18%- they then turned to IGF-1 signalling. this was more successful- ames mouse.
29
what is the somatotropin axis?
the anterior pituitary release growth hormone, this stipulates the liver to release IGF-1. IGF-21 progress cell survival, growth, puberty, godly function and reduces adiposity..
30
in what organism and what mutant revealed the connection between the IGF-1/insulin signalling pathway in worms and flies, with mammals?
The ames dwarf mouse- has a mutation for prop-1 which is a transcription factor involved in reduced anterior pituitary development. This results in detectable levels of IGF-1 in the circulation and low circulating insulin and glucose levels and mean lifespan increased by 70-80
31
in addition to the ame dwarf mouse, what other mutants revealed the connection between the somatotropin axis and ageing? (3)
snell, little and baron all have reduced IGF-1 signalling and GH
32
what is important about the fact that GH reduces lifespan and is linked to ageing in mice
it mainly acts through IGF-1 signalling
33
describe an experiment which looked at IGF-1 signalling and ageing in mice (2)
- heterozgous mice for IGF-1 recetor deletion were resistant to oxidative stress and females had an increased lifespan
- insulin receptor substrate mutants also showed increase in lifespan
34
describe the studies which looked at the soamtotropic GH/IGF-1 axis in humans in relation to ageing .
- body size and longevity: mice, rats and dogs- clear negative correlation between body size and lifespan
- study on ecuadorian dwarfs wit hGH receptor deficiency showed that the they were resisted to cancer and diabetes but died of other things
35
describe three man studies that looked at allele frequency in genes related to the somatotropin axis.
- they basically looked at the alleles that were most frequent in different genes between the oldest surviving people:
- Allele A of IGF-1R is ore frequent among long lived people
- SNP variant of GH1- carriers were 2cm shorter
- FOXO allele- less cancer and CVD
36
why was it first postulated that longevity of the IIS mutants was a resistant to stress?
in Johnsons ab, the flies were exposed to hurricane cartoon, the incubators ovrheated and all of the worms died apart from the age-1 worms! this means that they were thermotolerant. They then found that exposure of wild type worms to short burs of heat made them thermotolerant too and also extended lifespan. This led to a closer look at the role of molecular chaperones and their role in protection against cold stress, oxen deprivation and other stress responses.
37
which heat shock proteins were shown to be able to increase life span when over expressed? in flies?
-70,- 22
38
describe the experiments on heat shock.
the thermotolerance of IIS mutants suggests that daf-16(daf-2 mutant also thermotolerant) switches on the expression of heat shock proteins- molecular chaperones. They then found that the over expression of hsp-16 can increase c.elegan lifespan. this implies that protein misfloding contributes to ageing.
- HSF-1 is a transcription factor which activates the expression of hsps. RNAi of HSF-1 causes progeria!! accelerated ageing.
- over expression of HSF-1 increases lifespan
-Life extension dependent on • DAF-16
• (in part?) on small HSPs proteins
- RNAi of HSF-1 suppresses daf-2 mutant longevity
39
what are molcular chaperones?
- ensure proteins are in the right place and correctly folded. They also protect against cold stress, oxygen deprivation and other stress responses
40
once it was fond that age-1 and daf-2 worms were resistant to heat, what other things were these mutants found to be resist to? in worms..
- UV radiation, hydrogen peroxide, paraquat (superoxide generator)
- hypoxia
- pathogens
41
is the resistance of age-1 and daf-2 mutant worms conserved in flies?
- yes!
- chico mutants are resistant to starvation, heat stress but not paraquat
- insulin deficient flies are resistant to paraquat, starvation but not cold
42
what are chico mutants not resistant to?
paraquat
43
is the resistance of age-1 and daf-2 mutant worms conserved in mice?
yes! ames and GH receptor mutants have fibroblast resistance to heat stress, paraquta and UV
44
generally, what is the correlation between long lived mutants and stress resistance?
they have increased resistance
45
after all of the data showing that these mutants had resistance, what was the problem?
lifeextension and DDR (insecitices) resistance are dependent on dfoxo in flies (??)
46
at is functional genomics?
use of high throughput techniques to exploit the vast wealth of data produced by genome sequencing projects and unbiased direct approach to understand biological processes associated with ageing and longevity
47
what are the 4 main types of functional genetic techniques?
- RNA-mediated intereference
- gene knock out or mutation
- micoarrays` RNA seq (transcriptomics)
- proteomics
- metabolomics
48
how has RNAi been used in ageing studies?
knock down of mitochondrial transport chain genes in c.elegans extend lifespan independentl of daf-16 - this is unrelated to IIS
49
how has gene knock down been used to study ageing genes in yeast?
here is a "library" of complete deletions of each non-essential S. cerevisiae gene. Kenedy and Kaeberlein screened this library for chronological lifespan (ability to survive long-term culture in absence of cell division): deletion of components of the protein synthesis machinery extends lifespan (Reduced protein load? Altered protein profile?). This is linked to TOR and is also observed in C. elegans.
50
how can microarray be used to study ageing and IIS signalling?
can use microarray to look at the difference in genes expression in daf-2/daf-16 vs wild type or daf-2 vs daf-2;daf-16- this will identify the different in expression and link genes to longevity
51
what is the protocol for carrying out a micro array?
a microarray is a set of DNA spots of different genes attached to a solid surface.
In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs. The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA or cRNA "targets" derived from experimental or clinical samples.
- you isolated the RNA from the two samples beng comapred- you create cDNA for each and then label the different sample with its own colour. You then wash on and look at whether one colour is stronger or weaker in each spot- this will tell you whether thee expression has increased or decreased.
52
what type of array is more sensitive than spotted away?
gene chip arrays- affemetrix arrays
53
what are the downsides to a spotted microarray?
0. 5kb is a large chunk of DNA- this means that you can't distinguish between prologues and you may miss out genes that have similar sequences and think that they are the same.
- the comprartive sequences do not tell you much about the expression levels really
54
due to the downfalls of the spotted DNA microarray, what other type of array can be used?
a DNA ne chip can be used- these contain 25bp long oligonucleotides that can be used to distinguish between between paralogues and you can have many more probes in the chip
55
what is the downside of DNA chip analysis?
you have to perform the analysis for each sample independantly and then compare them
56
what was uncovered using microarray analysis? what animal were they performed in
?
- by comparing the transcriptome of daf-2 mutants vs daf-16:daf-2 mutants, Gems found that daf-16 upregulated 1348 and down regulated 926: around 10% of all genes showed altered expression. c.elegans
57
what percentage of the c.elegans genome is moderated by daf-16
around 10%
58
what is the problem with using the 10% of genes whose expression is altered by daf-16 to find evidence for the pathway that you're interested in? what is this approach called and how can it be circumvented?
- with such a long list of genes you could find evidence in support of any of your theories really, this is called fishing. In order to prevent this bias you need to take a statistical approach
59
what is the statistical approach that can be taken to find genes that are particularly implicated in daf-16 mediated longevity increase?
```
Compare list of regulated genes with given gene class: Are more members of that
gene class present than expected by change (use a statistical test)? check that the difference is significant- normally a p values of less that 0.05 or 0.1- use observed vs expected
```
60
once a set of genes were identified which were upregulated or down regulated by daf-16, how could you test that these were involved in longevity?
you can perform RNAI of those that are uprgeulated in daf-2 mutants and see if these shorten their lifespan (if the increase of longevity in daf-2 mutants acts via these genes) or the other way around for those that are downrgulated by daf-16 and see if RNAi against these genes in normal worms increases lifespan
61
once you have identified sets of genes that are regulated or downs regulated by daf-16, how can you go about identifing process and what is wrong with this approach?
you can start to look for groups of genes with similar function- find lots of hsps or superoxide disumustase genes- but becase the list is so long you could do this for many processes
62
when they carried out significance tests on the similarity between the genes regulated in daf-2 mutants and in dauer regulated genes, what did they find?what conclusion was drawn from this?
there as a strong significant overlap- daf-2 mutants switches no the dauer longevity programme in the adult and that therefore daf-16 regulates processes in the dauer that it also regulates in the long lived daf-2 mutant.
63
e you have determined that daf-16 regulates similar processes in due formation as it does in daf-2 mutants, how can you begin to identify what these processes are? what was found by doing this?
- use gene ontology classes (functional groups or other defining features such as proteins domains) which are represented in both significantly. hey fond that cytochrome p450, short chain dehdrogenases/reductases, UGTs, glutathione s-transferases . These are enzymes involves in phase 1 and 2 of drug detoxifications- upreg of enzymes involved in drug resistance- xenobiotic (dont have to be foreign compounds) that need to be detoxified .
64
once they found that insulin down regulation increases drug detoxification enzymes in c.elegans, how can the conservation of this mechanism be investigated and what was the outcome?
- you can measure if increased detoxification in the fly or the mouse, increases lifespan
- they found that the chico and the ames dwarf mouse are resistant to xenobiotics and that deotixificaiton is up-regulated in these long lived mutants
65
what are the4 enzymes found to be unregulated by daf-16 and are in common with the dauer process in c.elegans that were later fund to be involved in stage 1 and 2 of xenobiotic detoxification?
cytochrome p450, short chain dehdrogenases/reductases, UGTs, glutathione s-transferases .
66
what is a key piece of evidence that demonstrates that IIS signalling and xenobiotic detoxification are conserved between c.elegans and flies?
droosphila IIS mutants re resistant to paraquat, starvation and inseciticides but only the insecticides resistance is dependent on dfoxo (daf-16 homologue)
67
why is the premise that ageing is mediated by genes reasonably true?
the difference in lifespan between different species.
68
why are the transcriptional targets of daf-16 important?
because the longevity of daf2 mutants is dependent on daf-16 expression and daf-16 is a FOXO class transcription factor
69
what does fishing mean in terms of looking for the functional ageing targets of daf-16
this means that when faced with a list of genes that are controlled by daf16, people are likely to go for the sites of genes that they know are related to a certain process or function and it is therefore bias
70
what is the general premise of a statistical test looking to identify groups of genes involved in ageing following a microarray?
you look to find groups of gene types or functional classes that are present to an extent that is greater than would be expected by random gene assortment. Gems used a p vale of 0.001
71
what are the two phases of the detoxification system?
phase 1: addition of chemically reactive functional groups, which allow further metabolism of otherwise unreactive compounds
phase 2: the addition of side groups which increase solubility and increase excretion
72
which are the 4 enzymes that were identified to be involved in xenobiotic detoxifcation, are involved in each phase of detoxification?
- cytochrome P450 and short chain dehydrogenase/reductases are in involved in phase 1 but UGTs and GSts are involved in phase 2
73
what is the green theory of ageing ?
Among IIS-regulated gene groups where increased expression is correlated with longevity, we identified gene groups linked to detoxification, and chaperonins. Based on this, we suggest that ageing entails accumulation of damage to macromolecules due to a range of toxic by-products of metabolism. We propose that longevity assurance mechan- isms involve either removal of diverse molecular species that cause damage (e.g. by CYPs, SDRs, UGTs, GSTs), or repair of damaged proteins (e.g. by smHSPs). According to this view,view, the cell is under constant threat from metabolic waste products and xenobiotics. We suggest that the smooth ER works as a cellular filter, deploying phase 1 and phase 2 metabolism to mobilise and excrete these mainly lipophilic toxins. This clears the cell of molecular rubbish, thereby preventing molecular damage, and ageing.
74
why is the green theory named as such?
Gems argues that the process of detoxification and safe removal of waste by the cell parallels that campaigned for by green supporters
75
How does Gems incorporate the green theory with the oxidative stress theory of ageing?
We suggest that the oxidative damage theory is only partially true. It is likely that molecular damage caused by ROS contributes to ageing, and antioxidant defences to longevity. The exquisitely ordered molecular machines that living systems are must also somehow deal with an enormous diversity of other unwanted molecular species, some of them highly toxic. This is presumably a particular problem with organic (typically lipophilic) compounds, where molecular diversity is almost infinite. This would explain why treatment with antioxidants does does increase longevity- have to target all of the sources of molecular damage
76
What is David Gems argument for why treatmentt with antioxidants or increase ROS clearance does not affect longevity?
- molecular damage can derive from many different sources within the cell- toxins that are enodbiotic and lipophilic can induce stress and damage just as ROS can too. This makes getting rid of just ROS redundant
77
where are the toxins thought to come from in the green theory of molecular damage?
they are endobiotic and lipophilic
78
how did gems microarray analysis incorporate the oxidative stress theory?
our microarray analysis showed significant up-regulation of a number of antioxidant defense genes in daf-2() mutants, including sod-1 and ZK430.3 (Cu/Zn SOD), sod-3 (Mn SOD) and ctl-1 (catalase) (McElwee et al., 2004). Consistent with earlier studies, this suggests an association between longevity assurance and increased defences against a broad range of sources of molecular damage
79
How does the green theory fit into the disposable soma theory?
- it is believed that ageing evolved as a secondary consequence of selection for other fitness traits which maximise reproductive success. biological processes that sure longevity are energetically costly and fitness ay be optimised by investing into somatatic maintenance processes only the reproductive period. After which point, hey are no longer activated. Accordingly, somatic maintenance processes should be energetically costly. Phase 1 and pause 2 metabolism of toxins requires a lot of energy. Moreover, disposing of toxins should require more energy that the neutralisation of ROS as ROs products do not need to be secreted, but lipophillic and organic junk must be.
80
what evidence is there to suggest that enodbiotics, rather than xenobiotics, are involved in the ageing process?
SYP and UGT are both upregulated in dauer formation and both have xenobiotic and endoiotic substrates. However, the anus and the buccal cavity of the dauer are sea;led- so these enzymes are needed for endobiotic detoxification
81
where might endobiotic and lipophilic toxins come from?
stochastic errors in a rename of metabolic pathways generate molecular intermediates which are unrecognisable to the cell and which therefore accumulate. An example of this is lipofuscins, this is a poorly characterised, fluourescent molecular waste product that accumulates in c.elegan lysosomes. lipofuscins are a consistent link between ageing and failure to detoxify molecular waste- the accumulation is retarded in daf-2 mutants.
82
summaries the link between the green theory, lipofuscins and the oxidative stress theory of ageing...
So the general theory is a combination of the oxidative stress and green theory. This is that ROS will contribute to the formation of hard to degrade endobiotic lipophillic toxins that rresult from metabolic and stochastic errors in the cell. These toxins are energetically costly to digest and require the detofixifcation pathways which involve CYP and UGTS n the endoplasmic reticulum and SDRS and GDTs in the cytoplasm. Because this process is costly and according to the disposable soma theory, longevity mechanisms after reproductivity are negatively selected for, most cells will turn these endobiotic lipophilic toxins into lipofuscins., which are stored in the lysosomes are lipofuscins. This toxic lipophilic rubbish and ROS causes molecular damage which contributes to ageing.
83
why would ageing processes, processes that are not selected for, be conserved between species?
stems from the increasing awareness that lifespan may be intimately linked to cellular processes which are under tight selection (metabolismm, DNA repair, response to oxidative damage) genes controlling these processes are highly conserved and their effect on longevity may be product of their roles which are under greater selectiv e pressure
84
describe an example where simple epistasis analysis was not reliable..
deletion of SIR2 in year shortened replicative lifespan and blocks lifespan extension by DR This latter finding was a lynchpin supporting the model that DR extendded yeast lifespan through upregulation of Sir2 activity . However, in a mutant background of SIR2 and FOB1 (another yeast longevity linked gene) DR caused a robust increase in lifespan
85
what has RNAi in c.elegans been used to uncover? (2)
- RNAi against genes on chromosome I and II uncovered genes involved in mitochondrial function h as electron transport chain -
- screening of 80% of the genes uncovered 90 RNAi clones that induced lifespan extension
86
at are the downfalls of RNAi?
false negatives
87
by using RNAi in c.elegans, how many genes have been implicated longevity in total?
around 300
88
t should be down after identifying genes controlled by daf-16 in order to find functional relevant targets?
use RNAi to screen- feed to c.elegans
89
what is systems biology?
the study of how the components of a biological system interact to give rise to functions and behaviours of that system
90
why is systems biology needed?
the behaviour of the whole system arises as a result of multiple components of the system
91
what are the general stages of systems biology?
- make a model
- test it
- make observations
- then adjust the model accordingly
92
ow can epistasis analyis be used s order longevity pathways?
you can make a mutant of daf-2, a mutant of daf-2;daf-16 and then a mutant of daf-16 and do an epistasis. You find that daf-2;daf-16 has the same phenotype as daf-16. this shows that daf-16 is epistatic. And then you can look at the interactions: if daf-2 mutants are long lived and daf-16 mutants ar short lived and daf-16 is downstream to daf-2 then daf-2 must inhibit longevity via inhibiting daf-16 and daf-16 must activate longevity
93
describe a scenario in which epistasis analysis of the affects of double mutants vs single mutants may not be reliable?
Strains lacking gene X are short-lived due to an increased contribu- tion of pathway 2 to mortality. Strains lacking gene Y are long lived and the question is whether Y inhibits activity of X (affecting pathway 2) or whether Y affects another pathway. The standard approach,described above for epistasis involving both SIR2 and daf-16, would be to generate a double mutant lacking genes X and Y. If the double mutant is long lived rel- ative to gene X mutants, then gene Y is said to be affecting lifespan independently of gene X. If double mutants lacking gene X and Y have the same lifespan as single X mutants, then X is said to be epistatic andY upstream. At best, the data are consistent with this interpretation. Consider an alternative whereby the relative contribution of pathway 2 to mortality in the absence of gene X becomes so high that nearly all individuals of the X mutant strain succumb for one reason (Fig. 1b, right panel). The causes of age- induced mortality have been dramatically altered. In this scenario, a double mutant lacking gene X and Y would be expected to have virtually the same lifespan as a single mutant lacking X even though Y may pro- mote ageing through pathway 4 (Fig. 1a, right). Loss of Y has little effect as pathway 4 is not a big con- tributor to mortality in the absence of X. This prob- lem is likely to be most severe when the X mutation shortens lifespan on its own, the case for a number of genes used for epistasis in yeast and worms.
94
in addition to daf-16, which other proteins does does daf-2 inhibit in order to inhibit increased longevity?
skn-1 and hsf-1
95
what is the interaction that skn-1, hsf-1 and daf-16 have with each other and with daf-2?
they are all required for the daf-2 mutant to be long lived, however, daf-2 on interacts with sf-1 and daf-16 directly and they know this from biochemical investigation. so some how you hsf-1 interacts with daf-16 and collectively they all act to extend longevity
96
how can a microarray be used to look at what genes are unregulated by daf-16 and which ones are down regulated?
you can compare the microarrays from a daf-2 mutant with that of the double mutant of daf-2 and daf16. You then compare the genes that are upregulated in the first and down regulated in the second and know this are involved in increasing lifespan maybe and are upregulated by daf-16 , and those that do the opposite are involved in decreasing lifespan and are down regulated by daf-16
97
ce you have used microarray to find out which genes are regulated by daf-16, how can you find out which gene is expression is directly mediated by daf-16 or indirectly by daf-16 ?
- you can find the binding preference of daf-16 for DNA binding: they find that AAACA is the preferential binding sequence. If daf-16 is directly regulating genes, this sequence will be enriched in all genes being regulated- we find this is the case for genes which are being upregulated by daf-16.
98
what RNA polymerase control the activity of?
RNA polymerase 2.
99
if daf-16 isn't directly upregulating the genes which are down regulated, how else might they be regulated?
- It may induce the expression of other transcription factors which then mediate the expression of genes
100
in order to find out which genes are directly regulated by daf-16, what can you do? (3)
- look for the binding sequence in genes
- use chromatin immunoprecipitation (CHIP)
- DAMid
101
what is the process of chromatin immunoprecipitation?
- you can chemically cross link the TFs to the DNA on which it is bound via cross-inks.
- then you shear the DNA into fragments
- and then we apply an antibody which will specifically identify that transcription factor and then yo can purify the immunocomplexes that have bound to the TF. You can then use an array and see what type of genes are being bound to by daf-16.
102
what is the process of DamID?
Another technique is DamID (DNA adenine methyltransferase identification). This uses an enzyme which can methylate specififc sequences in the DNA. so you would construct a hybrid gene which has daf-16 fused to dam-16. So whatever daf-16 binds to, the part of the DNA gets methylated at the point o binding. You can then use Dpn1 which will cut at methylated sites and produce fragments which are methylated and separate them form non methylated. You then add dpnII which digest all of the unmethlayted fragment sections so that only those intact are the methylated which have primers at each end. So after amplification of the fragments, you will get an enrichment of methylated regions of DNA so these are the fragments bound by daf-16- can then do an array to work out where they are in the genome.
103
after carrying out DamID and CHip, what did they find?
they found that it only directly regulates a few of the genes that are differentially regulated in daf-2 mutants and that the rest are regulated by kinases and other transcription factors
104
how does daf-16 regulate its own activation?
Daf-16 is also regulating a lot of its regulators- it regulates transcript levels of the trancript levels of the kinases upstream of it which are regulating its activity. such as AKT-1- it forms a negative feedback loop- it tries to stop its down activation. The AKT-1/2 and SGK-1 pathways are post-translational. This means that the proteins need to be activated once they have been translated. Therefore, it is thought that these components are being made but not activated. The idea is that the pathways which are being activated upstream of daf-16 are nutrient sensing pathways. Daf-16 is acting as though there is no nutrients and its increasing the amount of upstream components so that when nutrients does become available, it can be rapidly turned off. So links with the idea that nutrients and diet inhibits daf-16 so decreases lifespan?
105
what does the negative feedback loop that daf-16 is involved inn mean about possible daf-16 orientated treatments for ageing?
If you want to intervene into ageing, if you do something that is going to activate daf-16, its going to do something to deactivate itself. so target downstream effectors?
106
describe how functional genomics has been used to look at transcription factors which regulate genes that have age-related changes in expression..
- the kim lab looked at genes whose expression changed in an age-related manner- they then wanted to identify the TFs that regulated these genes. To do this they identified motifs that were conserved between them
- they fond the sequence GATA
- transcription factors called GATA factors regulate these
- c.elegans have 14 gata-like TFs
- they then focussed RNAI experiments on those GATA-like factors whose expression changes over time
- this led them to ELT-3 which is down regulated over time - elt 3 is part of a regulatory circuit involving ELT-5 and ELT-6
- elt-5 and -6 inhibit elt-3. they also increase in expression over time - furthermore, IIS long lived mutants have increase ELT-3 expression and the effects of the IIS mutants appear to be ELT-3 dependent.
- RNAI of ELT-5 and -6 increase lifespan
-It is not know if elt-3 is regulated by DAF-16. However, it is not likely to be directly regulated by DAF-16.
-• However, a more recent study (Tepper et al, 2013) examined genome-wide data on daf-16-regulated gene expression and the knowledge of in vivo binding locations for a number of C. elegans TFs.
• They found that the GATA-like motif (also called DAE for DAF-16 Associated Element) was actually bound by another TF called PQM-1.
• They propose that changes in PQM-1 activity due to altered IIS underlie the regulation of DAE-regulated genes in daf-2 mutants
• Indeed pqm-1 is partially required for daf-2 longevity
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rather than daf-16, what have others suggested is the TF for GATA-like elements
- for both genes activated an repressed : TATC the copliment of GATA. rather than assuming this is actually bound by a GATA factors, they took advantage of the fact CHIP has been performed on c.elegan TFs binding sequences, they fond that PQM-1 had the highest liklihood of binding the GATA sequence- another one other than ELT-1. So they did epistasis with PqM-1 and found that when you RNAi against it, it reduces the longevity of daf-2- another TF that is required for lifespan extension of daf2.
108
describe how systems has been sed to look at mTOR
target of rapymysin is a kinase that controls metabolism and is directly linked to insulin signalling and if you inhibit it then it will extend lifespan- this is conserved across all model organisms
- mTOR can work in two complexes.
- they found that mTORC2 was regulated by Pi3K not TSC1`SC2.
109
what is growth hormone normally involved in other than ageing?
affects somatic growth, sexual maturation, body composition and metabolism
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what is the major mediator of GH's role?
IGF-1
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what happens to mice that over express GH or IGF1?
short lived
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how would the somatotropiic axis have developed
in terms of evolutionary fitness the apparent ‘pro-aging’ effects of GH might be more or less irrelevant, or instead are outweighed by the stimu- latory effects of the somatotropic axis on growth, sexual maturation, and fecundity
113
what ware the main IIS pathway mutants that confer long life in mice ? (3)
- deletions that a fact the bio-available IGF-1
- mutations that alter levels of insulin receptor substrates-1 or
- IGFR
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what is the link between mTOR and IIS?
it stimulates mTOR
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what are two pieces of evidence for the role of mTOR in longevity?
- mouse longevity was extended byy deleting S6 kinase
| - rapamycin is an inhibitor or mTOR which extends lifespan
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what is the difference in phenotype seen between mutations which alter GH biosynthesis or action and mutations interrupting IGF-1 levels or signalling?
more extreme longevity in former and can affect both sexes but this could be because complete loss of IGF-1 is lethal so can't fully KO
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describe evidence for IGF-1 and GH having not entirely the same affects on ageing? and hence explaining the differences in phenotypes seen when perturbing each
- because IGF-1 signalling is almost entirely independent from GH signalling, GH interference does not perturb this expression in the heart etc but does reduce serum levels of IGF-1 so protecting from cancer
- GH resistance or deficiency can influence ageing by mechanis unrelated to the regulation of IGF-1 expression: Gh promotes insulin resistance and IGF-1 is an insulin sensitiser
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what is a link between long lived GH mutants and stress resistance?
- GH deficient or resistant mice show increased distance of dermal fibroblasts to cytotoxic stresses such as paraquat
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what happens to the resistance of Ames dwraf derived fibroblasts when they are injected with GH?what does this suggest?
they lose their resistance to multiple cytotoxic stresses. This suggests that the Somatotropic axis inhibits detoxifying mechanisms
120
what has been shown about Ames dwarf mice hippocumps'
they are resistant to beta-amyloid plaque toxicity and oxidative stress
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give a piece of evidence relating to GH signalling in humans?
here is evidence that some hypopituitary and GH resistant individuals can reach very advanced old age
122
what is the link of autophagy with the daf-2/daf16 signalling pathway and what is thought to be its indirect contribution?
autophagy is required for the inhibition of daf-2 to extend lifespan. But daf-16 is not required for autophagy in c.elegans daf-2 mutants. This suggests that autophagy might act t provide raw material to be recycled into new protective proteins by the actions of daf-16 and other transcription factors
123
are hsf-1 and skn-1?
transcirption factors that are normally inhibited by daf-2
124
what is the dros homologue of daf-16?
foxo
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is there a correlation between IGF-1 levels in mice and longevity?
yes
126
give an example of an IIS competent allele that supports IIS signalling in humans
Mutations known to impair IGF-1 receptor function are overrepresented in a cohort of Ashkenazi Jewish centenarians38 and DNA variants in the insulin receptor gene are linked to longevity in a Japanese cohort
127
why is the IIS a good candidate for mediating the longevity affects of DR?in c.elegans?
Because the insulin/IGF-1 pathway senses nutrients, and because this pathway also affects the development of C. elegans in response to nutri- ent limitation
128
ow might DR restrict cancer development?
Finally, dietary restriction may inhibit cancer by downregulating insulin/IGF-1 signalling, because it fails to inhibit cancers caused by mutations that constitutively activate the insulin/IGF-1 pathway
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at is the evidence for DR acting through IIS signalling in mice?
Dietary restriction may also extend mouse lifespan by inhibiting insulin/IGF-1 signalling, as it does not extend the already long lifespans of mice with mutations in the gene for the growth hormone receptor
130
what is the interesting example of DR and smell in flies ?
In addition to eating less, smelling or tasting less (or differently) can also increase the lifespan of C. elegans16, and this effect seems also to be due to decreased insulin/IGF-1 signalling. Sensory perception also influ- ences lifespan in flies; in fact, dietary restriction does not extend lifespan as much if the flies are able to smell their food
131
what happens when you add 2% glue to c.elegans food?
In C. elegans, in fact, adding 2%glucosetothebacterialdietshortenslifespanbydownregulating DAF-16/FOXO and HSF-1 activity
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what is the evolutionary theory for DR increasing life span?
lowers IIS signalling so activates stress response which can then stimulate cellular maintenance and repair to get the animal through harsh conditions but then also increase longevity
133
what is TOR kinase?
a major amino acid and nritent sensor that stimulates growth and blocks salvage pathway suhc as autophagy when food is plentiful . inhibiting this pathway increases lifespan in many species.
134
in c.elegans, does TOR work via IIS?
no - acts independently from daf-16
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why is there a link between DR and TOR?
- TOR is a signalling pathway that is stimulated when nutrient levels are high
- TOR inhibition mimics DR
- seems to be epistatic follows tests S6 kinase mutant mice exhibit gene expression patterns similar to those triggered by DR
136
when Tor activity levels fall, what happens?
transcription levels fall
137
what is the link between TOR and autophagy?
TOR inhibition also stimulates autophagy, which, as in insulin/ IGF-1 mutants, is required for lifespan extension (at least in worms and flies)31,61,62. The stimulation of autophagy by TOR inhibition may be indirect, effected by changes in gene regulation, as in worms it has been shown to require the PHA-4/FOXA transcription factor
138
what is a hypothesis about the different triggers of IIS and TOR?
Reducing methionine intake in mammals, or protein intake in flies, extends lifespan. Perhaps different kinds of diet trigger different cell-protec- tive and longevity responses. The response to low insulin/IGF-1 signallingcould be triggered by a low-glycaemic-index diet, and the response to low TOR could be triggered by amino-acid limitation. Hopefully, one day we will know what kind of diet will best keep us youthful and healthy.
139
what transcription factor is required for TOR longevity?
skn-1
140
what ages against the reproductive trade off in c.elegans ?
removing the entire reproductive system does not extend lifespan, arguing against a simple reproductive trade-off
141
descrie evidence for the role of the reproductive system priming the age of the worm for reprod?
When the animal’s germ cells (but not its somatic reproductive tissues) are removed, its lifespan is extended by approximately 60%. Thus, an ‘empty gonad’ extends lifespan. Perhaps this phenomenon allows the animal to coordinate its rate of ageing with the timing of reproduction: if the germ line is not ‘ready’, the animal will ‘wait’ to age. This pathway, too, may have an ancient origin, as forcing germline stem cells to exit mitosis and enter meiosis during adulthood extends lifespan in both flies and worms18,19,85. Furthermore, FOXO activity, which is known to be required for the lifespan increase in worms, increases in both cases.In C. elegans, germline loss activates DAF-16/FOXO by a novel mechanism
142
Other than long life, what are the other symptoms of long lived mutants in flies, mice and c.elegans?
- the chico mutant (substrate for receptor) are smaller
- GH mice: Greatly reduced fertility
Obesity
Low circulating insulin, glucose Dwarfism
