Aging 2

Front 1

aka Baker's Yeast
(sugar mold)

Back 1

Saccharomyces cerevisiae
Many aspects of the cell cycle were first worked out with yeast

Front 2

Sirtuins

Back 2

conserved protein that regulates gene silencing in yeast and other organisms

Front 3

Sir2 and Aging

Back 3

Up-regulated: cells live longer
Down-regulated: shorter reproductive life
Essential for response to caloric restriction
NAD co-factor: couples gene restriction and metabolism

Front 4

Resveratrol

Back 4

Anti-microbial compound produced by plants
Present in red wine (French Paradox)

Front 5

Claims about Resveratrol

Back 5

Activates SIRT1 (homolog of Sir2)
Can postpone diseases of aging

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Sirtris

Back 6

Company founded to develop Resveratrol supplements for human consumption

Front 7

How many species of Nematodes?

(Phylum Nematoda)

Back 7

about 80,000

Front 8

C. elegans

Back 8

Transparent
959 cells, 300 neurons, 81 muscle cells (complete fate map!!!)
17,800 genes in the genome
Sense of taste, smell, temp, touch
Can be frozen and revived
3-week-lifespan
Primarily self-fertilizing

Front 9

Dauer Stage of C. elegans

Back 9

No feeding, growing, or reproducing
Arrested development leads to longer life (7 times longer)
Stress resistance
Remodeled tissues

Front 10

Necrotic cell death

Back 10

cells die because of trauma

Front 11

Programmed cell death
(apoptosis)

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Normal development (tadpole tail)
Purging of damaged cells (viral infection)

Front 12

Too little apoptosis...

Back 12

some cancers, autoimmune diseases, and viral infections

Front 13

Too much apoptosis...

Back 13

neurodegenerative diseases, allergies, asthma

Front 14

RNAi

Back 14

RNA interference
(2006 Nobel Prize, Fire and Mello)

Front 15

RISC

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RNA-Induced Silencing Complex
(finds specific sequences of RNA)

Front 16

Genetic Screens
(definition)

Back 16

an experimental procedure intended to isolate genetic mutations with a desired phenotype

Front 17

Genetic Screens
(3 types)

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Simple
Selective
Reverse genetics

Front 18

How does a simple screen work?
(ex. with C. elegans)

Back 18

1) mutagenize worms
2) collect F1 progeny, place in cultures
3) examine F2 progeny for phenotype of interest
4) select worms with desired phenotype
5) test F3 to see if trait breeds true

Front 19

age-1
(found in selective screen for lifespan)

Back 19

The first "aging gene"
Extends lifespan 40-60%
May reduce fertility
No effect of feeding, movement, or reproduction
Normal metabolic rate

Front 20

RNAi-Based Screen
(reverse genetics)

Back 20

Many genes that extend lifespan involve mitochondrial function
Worms genes are turned off selectively from eating particular bacteria

Front 21

Reverse Genetics

Back 21

start at the level of the gene and look at effect on organism

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Classical Genetics

Back 22

start at the level of the organism and explain the effects with genetics

Front 23

What are some factors which may increase longevity?

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Decreased DAF2 (insulin signaling)
Dietary restriction
Decreased TOR signaling
Increased JNK signaling
Decreased germline signaling
Increased Sir2 activity
Decreased mitochondrial respiration

Front 24

Insulin Signaling in C. elegans

Back 24

Decreased signaling means decreased sensory perception which leads to longer life

Front 25

Dietary Restriction in C. elegans

Back 25

Reduction of caloric intake by 30-40% extends lifespan

Front 26

TOR Signaling in C. elegans
(Target of Rapamycin)

Back 26

TOR protein affects cell growth, proliferation, motility, transcription, and protein synthesis
Rapamycin is an immunosuppressant targeted by TOR so the decreased TOR activity reduces rapamycin and extends lifespan

Front 27

JNK Signaling in C. elegans

Back 27

Plays a role in stress response and apoptosis
Increased JNK signaling induces resistance to oxidative stress and reduces insulin signaling

Front 28

Germline Signaling in C. elegans

Back 28

Signals from the gonads affect lifespan by modulating insulin signaling
Less germline signaling leads to longer lifespan

Front 29

Sir-2 Activity in C. elegans

Back 29

Over-expression of Sir-2 increases lifespan
Deletion of Sir-2 reduces lifespan

Front 30

Mitochondrial Respiration in C. elegans

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Decrease in mitochondrial respiration slows down development, feeding, movement, and reproduction

Front 31

Raymond Pearl

Back 31

Investigated:
Smoking and lifespan
Human population growth
Mechanisms of aging
Aging in Drosophila
Rate of Living Theory

Front 32

Key Points from C. elegans

Back 32

Life Cycle (Dauer)
Apoptosis
RNAi
Mutant Screens

Front 33

Rate of Living Theory

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High metabolic rate leads to a shorter lifespan
Slow metabolic rate leads to a longer lifespan
Best evidence: lower temperature leads to longer life in ectotherms

Front 34

Modern portion of the Rate of Living Theory being studied....

Back 34

oxidative damage

Front 35

Oxidative Damage
(as part of Rate of Living Theory)

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high metabolic rate leads to high mitochondrial activity leads to lots of free radicals

Front 36

What are two methods for dissecting quantitative traits?

Back 36

QTL Mapping
DNA Microarrays

Front 37

QTL Mapping

Back 37

Technique for finding the chromosomal locations of genes that influence quantitative characters
For a phenotype of interest, compare two stocks that differ in the trait with a recombinant population that carries parts of chromosomes from each of the two stocks

Front 38

DNA Microarrays

Back 38

Device for assaying the level of gene expression of every gene in the genome
Fluorescence intensity reflects amount of RNA

Front 39

Typical Microarray Results

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Up-regulated in long-long lines:
-many innate immunity genes
-a few antioxidant genes
-DNA repair genes
-nutrient reserve genes
-miscelaneous

Front 40

What are 3 single-gene approaches?

Back 40

Genetic Transformation
Mutant Screen
Homologous Genes

Front 41

Genetic Transformation

Back 41

insert a foreign gene into the germ line using P factors (a Drosophila transposable element, TE, or "transposon")

Front 42

Mutant Screen

Back 42

look for single-gene mutations induced by P-factors that extend life

Front 43

Homologous Genes

Back 43

many mutations extend lifespans in C. elegans. What about similar mutations in flies?

Front 44

P-factors in Drosophila

Back 44

Naturally occurring transposon
Horizontal transfer from another species in 1950
Cloned in 1980
Widely used for genetic transformation

Front 45

How do P-factors work?

Back 45

modified P-factor is injected into eggs to produce genetic transformants

Front 46

Orr and Sohl Hypothesis

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Hypothesis is that oxygen free radicals/reactive oxygen species (ROS) cause of senescence because ROS cause molecular damage that accumulates with age

Front 47

What are the major defenses against ROS?

Back 47

SOD and catalase
SOD converts superoxide radical to H2O2 and catalase breaks down H2O2 into water and oxygen

Front 48

What is seen in flies that over-express SOD and catalase?

Back 48

Lived about 30% longer than controls (median and maximum lifespan)
Lower levels of damage due to ROS
Delayed loss of motor ability
BUT transgenic effects on longevity only obvious in short-lived strains

Front 49

Who is C.C. Little?

Back 49

Founded the Jackson Labs in 1929

Front 50

Mouse Life Cycle

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4-12 pups per liter
Sexually mature at 50 days
20 day gestation period
2-3 year lifespan

Front 51

Who is George Snell

Back 51

discoverer of H2 locus in mice which is homologous to human HLA

Front 52

Chimeras

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mice with more than 2 parents (collect early embryos and disassociate cells, mix cells from multiple embryos, implant into foster mother)

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Gene Targeting

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insertion of foreign DNA into specific sites in the genome

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Knockout

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an organism with a chosen gene inactivated; the condition is inherited

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Rapamycin

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immunosuppresant derived from bacteria

Front 56

Sydney Brenner

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first C. elegans genetics laboratory

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T.H. Morgan

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first Drosophila genetics laboratory

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Raymond Pearl

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promoted Rate of Living Theory

Front 59

H.J. Muller

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discovered X-rays cause mutations

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C.C. Little

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first mouse model system for cancer research

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George Snell

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nobel prize for immunogenetics in mice

Front 62

Capecchi, Smithies, and Evans

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Nobel prize for knockout mice

Front 63

T or F:
daf mutation play a role in the the formation of dauer larvae

Back 63

True

Front 64

T or F:
A mutatgen is a chemical that protects DNA from mutations

Back 64

False

Front 65

T or F:
Reverse genetics means genetic manipulations to produce earlier forms of life

Back 65

False

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T or F:
Senescent yeast cells are sterile and wrinkly

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True

Front 67

Example of reverse genetics...

Back 67

RNAi allows geneticists to turn off expression of a particular gene one by one in order to see phenotypic effects

Front 68

What are insulin-like signaling pathways in nematodes and flies?

Back 68

pathways that tell the organism about environmental conditions and help determine whether resources will be used for reproduction and maintenance

Front 69

What evidence supports the Rate of Living Theory?

Back 69

ectotherms live longer at colder temps and shorter at warmer temps

Front 70

What evidence supports idea that CR will extend life in humans?

Back 70

1) CR works in almost every animal tested
2) close relatives, Rhesus monkeys, indicate health benefits

Front 71

Dicer

Back 71

enzyme active in RNAi

Front 72

Chromosome large enough to be seen under ordinary light microscope?
Drosophila transposon?

Back 72

P-factor

Front 73

Experimental modification of germline?

Back 73

genetic transformation

Front 74

Over-expression of this gene in yeast causes tighter packaging of DNA and longer life span.

Back 74

Sir2

Front 75

Investigations that begin by asking what happens if a particular gene is altered.

Back 75

reverse genetics

Front 76

Chromosome large enough to be seen under ordinary light microscope?
Drosophila transposon?

Back 76

P-factor

Front 77

Experimental modification of germline?

Back 77

genetic transformation

Front 78

Suppresses immune system and extends life in mice.

Back 78

Rapamycin

Front 79

Over-expression of this gene in yeast causes tighter packaging of DNA and longer life span.

Back 79

Sir2

Front 80

Investigations that begin by asking what happens if a particular gene is altered.

Back 80

reverse genetics

Front 81

Suppresses immune system and extends life in mice.

Back 81

Rapamycin

Front 82

Chromosome large enough to be seen under ordinary light microscope?
Drosophila transposon?

Back 82

P-factor

Front 83

Experimental modification of germline?

Back 83

genetic transformation

Front 84

Over-expression of this gene in yeast causes tighter packaging of DNA and longer life span.

Back 84

Sir2

Front 85

Investigations that begin by asking what happens if a particular gene is altered.

Back 85

reverse genetics

Front 86

Suppresses immune system and extends life in mice.

Back 86

Rapamycin

Front 87

Chromosomal segment containing genes that influence a multi-gene trait.

Back 87

QTL

Front 88

Stocks are routinely frozen and revived
Developmental origin of every cell is known

Back 88

C. elegans

Front 89

P-factors in the genome

Back 89

D. melanogaster

Front 90

Suitable for mutant screens
Lays eggs

Back 90

C. elegans and D. melanogaster

Front 91

Possesses insulin or insulin-like pathways
Dietary restriction known to extend lifespan

Back 91

C. elegans, D. melanogaster, and mice

Front 92

Brenner, Little, and, and Morgan
(mice, worms, or flies?)

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Brenner - worms
Little - mice
Morgan - flies

Front 93

Advantages of using mice?

Back 93

mammals (more similar to humans), relatively short lifespan, inbred stocks available, knockouts, can be used to study cancer

Front 94

Advantages of using worms?

Back 94

small size, can be reared and analyzed like bacteria, rapid generation, transparent, multicellular differentiation

Front 95

Advantages of using flies?

Back 95

small, rapid generation, sexual reproduction, polytene chromosomes, can do genetic transformation

Front 96

T or F?
siRNA's gives RISC their specificity

Back 96

True

Front 97

T or F?
Transgenetic flies are sterile

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False

Front 98

T or F?
Linkage and recombination, and X-ray induction of mutations were first discovered in Drosophila

Back 98

True

Front 99

T or F?
Polytene chromosomes have many tenits

Back 99

False

Front 100

T or F?
Sir2 and similar genes are found in yeast, nematodes, and humans

Back 100

True

Front 101

T or F?
Resveratrol induces sirtuins

Back 101

True

Front 102

T or F?
Pleiotropy means genes interact to produce the phenotype

Back 102

False

Front 103

How do you obtain knockout mice?

Back 103

Embryonic stem cells are isolated from donor mice and then infected with a viral vector that carries foreign DNA into the cells. Insertion of the foreign DNA inhibits normal gene function. Cells are screened for foreign DNA; if they carry it, they are implanted into foster mother. Progeny will have gene knocked out in either somatic tissue or germ tissue; in the latter case the knockout will be transmitted to the next generation.

Front 104

“There are genes for lifespan”

Back 104

Disagree. There are certainly genetic influences on life span, but the genes have other primary functions and their effect on aging is pleiotropic.

Front 105

“Caloric restriction (CR) is likely to extend lifespan in humans”

Back 105

Agree. CR extends life in all other species that have been tested, including other mammals
(mice and rats). Further, there are CR experiments underway with Rhesus monkeys that are incomplete but do demonstrate positive health effects of CR.

Front 106

Researchers are likely to find a master switch in the genome that regulates lifespan”

Back 106

Disagree. The evidence from mutant screens in C. elegans is that there are a half dozen different mechanisms by which lifespan can be modified, including insulin-signaling, JNK signaling, TOR signaling, modification of metabolic rate, and modification of germline signaling. It seems unlikely that a single switch will regulate all those different functions.

Front 107

“Based on recent research in mice, it would be a good idea for humans to take rapamycin to increase the chances of a long, healthy life.”

Back 107

Strongly disagree. Rapamycin suppresses immune function, so anyone who takes it would be unusually susceptible to infectious disease. It extends life in mice that live in semi-sterile conditions, not in the wild.