Final Exam Flashcards
(180 cards)
1
Q
What are some characteristics of the human genome?
A
- wild and recessive alleles
- polymorphic
- lots of inser. an dels.
- SNPs
- SSRs (microsatellites)
- CNV/CNP
2
Q
What are the three main categories of genetic variants?
A
- SNPs
- SSRs
- CNV/CNP
3
Q
What are SNPs?
A
Single Nucleotide Polymorphisms.
- Particular place in the genome where alternate DNA letters distinguish people from each other.
4
Q
How do SNPs arise?
A
Mistakes in replication or mutagens.
5
Q
What are SSRs?
A
Simple Sequence Repeats (includes microsatellites).
Can also expand and mutate to multiple alleles.
6
Q
How are SSRs caused?
A
- Replication errors
2. Spontaneously from random events.
7
Q
Why are SSRs so highly polymorphic?
A
Faulty replication system. Pol pauses causing a loop (14 CA to 17 CA repeat allele)
8
Q
What are CNP/CNV’s?
A
CNP - copy number polymorphism (greater than 1% freq).
CNV - copy number variation
They are large blocks of deletions or duplications.
9
Q
Why are repeat sequences more mutagenic?
A
Non-homologous recombination occurs (hard to match up with so many repeats), can end up with larger deletions or duplications)
10
Q
How do you detect CNV/CNP’s through Array CGH?
A
- Label patient and control DNA with fluorescent dye
- Patient and control compete to hybridize on microarray.
- Microarray scans for the fluorescent signals.
- Computer analyzes and scans plot.
- Can see DNA dosage (gain or loss)
11
Q
How do you detect SNPs?
A
- Restriction fragment the sequences
- PCR amplify
- Gel electrophoresis—> Southern hybridize
- Measure the different lengths (do the SNP interfering with restriction site)
OR
SNP array
NGS
12
Q
How can expression arrays be used?
A
Label cDNA with two colours and hybridize to see which colour is prevalent. Can see which DNA is expressed and which is not.
13
Q
What are the benefits of NGS?
A
Sequenced simultaneously, can do entire genome in one reaction.
14
Q
What are the steps fo NGS?
A
- Library prep (Material DNA or RNA? How much needs to be sequenced? Whole genome? Exomes?)
- Sequencing (Real tie seauening, track addition of nucleotides)
- Data analysis (translate into sequence, map to reference, compare deletions, SNPS, CNPs etc.)
15
Q
What can rearrangements result in?
A
Altered map distances, altered pairing during prophase 1 in heterozygotes, reduces recombinant gametes/crossing over.
16
Q
How do rearrangements arise?
A
Double stranded breaks and repair, recombinantion at related sequences.
17
Q
How can you detect dupications and deletions?
A
FISH, CGH (comparative genome hybridization), Next Gen, banding pattern on chromosome.
18
Q
What is FISH?
A
fluorescent probe that complementary bind to a region (DNA denatured first)
DNA will fluoresce at the location of binding (more than 1, duplication, none deletion)
19
Q
What are the phenotypic effects of large deletions and duplications?
A
- Homozygous lethal
- Heterozygous detrimental
- Duplications more tolerated than deletions.
20
Q
What are the two types of inversions and what are they caused by?
A
Pericentric and Paracentric.
Caused by doubl stranded breaks, 180 chromosomal rotation, rare unequal crossing over beteen repeated sequences in opposite orientations.
21
Q
When are gametes balanced in Pericentric and Paracentric inversions?
A
When no recombination occurs
22
Q
What are the effects of unbalanced gametes?
A
Inviable or aneuploidy
23
Q
What is reciprocal translocation?
A
Two different parts of chromosomes switch places.
24
Q
What type of translocation segregation pattern results in balanced gametes and what are the segregation alleles?
A
Alternate.
N1 & N2) (T1 & T2
25
What are the segregation patterns of Adjacient I? Are the gametes balanced?
T1 & N2 , T2 & N1
| Unbalanced
26
What are the segregation patterns of Adjacient II? Are the gametes balanced?
T1 & N1, T2 & N2
| Unbalanced.
27
What is semisterility and what is it caused by?
When you are partly sterile. Caused by translocation of heterozygotes (<50% of the viable gametes produced through alternate segregtaion patern)
28
What is Robertsonian Translocation?
Recriprocal exchange between the ACROCENTRIC part of the chromosomes to generate large METACENTRIC chromosomes and one small chromosome that is usually lost.
29
Why do Robertsonian translocations generally have no real phenotypic effect?
the acrocentric chromosome lost does not contain a lot of information.
30
What are the characteristics of polypoloidy?
Larger in size and vigor
31
How does euploidy differ from aneuploidy?
Euploidy consists of multiples of complete sets of chromosomes.
Aneuploidy consists of multiples of a chromosome,
32
How do triploids arise and why do they give rise to unbalanced gametes?
Through the union of monoploid and triploid gametes.
| Meiosis splits up the genetic information unevenly.
33
Polyploidy is good in plants, but how does it fare in humans?
Triploidy associated with developmental defects.
| Tetraploidy results in miscarriage.
34
How does aneuploidy arise?
1. Nondisjunction (mainly in Meiosis I, but can occur in Meiosis II)
2. Familial robertsonian translocation (can end up with three copies one normal and a abnormal metacentric chromosome that can contain three copies of genetic info)
3. Mitotic nondisjunction in the zygote that can result in mosiac aneuploidy.
35
How can you test for aneuploidy?
1. Amniocentesis
| 2. Fetal karyotyping
36
Why are some aneuploidies more tolerated than others (ie. sex chormosome aneuploidy)?
Chromosomes have different threshold levels due to a the number of protein coding genes. The less amount of genes, the more tolerated. Sex chormosomes have mechanisms to turn off copies of X (Barr bodies) therefore it is not as harmful.
37
Why do trisomies increase with maternal age?
Dysfunction of cohesions increase with age.
38
Outline sister chromatid cohesion in mitosis.
1. Mitotic cohesions bind sister chromatids together
| 2. Separase degrades mitotic cohesions allowign sister chromatids to separate properly in anaphase.
39
Outline Sister chromatid cohesion in meiosis.
1. Meiotic cohesions bind sister chromatids togeher.
2. Separase degrades meiotic cohesions in the chromosome arms.
3. Shugoshin protects the meiotic cohesions in the centromere during Anaphase I
4. Separase degrades the centromere cohensions during Anaphase II.
40
What is the effect of dysfunctioning cohesions?
Leads to Anyploidy (produces trisomies).
41
What are the characteristics of bacterial genomes?
1. Circular chromosome
2. DNA condensed throughs supercoiling and packed in nucleoid body.
3. Replicates inside cell and divides by binary fission.
4. Genes can exist autonomously, replicating extra-chromosomal DNA called PLASMIDS
5. conaint IS ELEMENTS and TRANSPOSONS.
42
What are IS elements and what is their function?
They are transposable elements that do not carry selectable markers (antibiotics)
They disrupt gene function (prevents transcitiption in coding regions and cause spontatneous mutations).
43
What are transposons and what is their function?
Jumping genes.
They are useful for the identification of disrupted genes.`
Can make random mutations by incoporating transposons (antibiotics) into a gene.
44
What is the universal problem of gene regulation?
- Genes must be expressed at the correct level and the correct time and space.
- Presence and absence of gene products must be regulated according tot he needs of the organism.
45
What are principles in gene regulation in bacteria that hold true for eukaryotes?
1. Gene transcription repressed or expressed through trans-acting factors encoded elsewhere in the genome.
2. Cis-acting elements adjacent to the genes are essentional docking sites for trans-acting factors.
3. Activation by substrates (induction)
- Repression by end products of pathways (feedback)
46
What is an aspect of gene regulation that is specific to prokaryotes?
Gene attenuation!
| Translation machinery can influence transciption in prokaryotes only!
47
What are the three mechanisms of gene transfer in bacteria?
1. Transformation
2. Transduction
3. Conjugation.
48
What is the process of transformation?
DNA from donor added to bacterial growht medium --> taken up by bacterial cells.
49
What is the process of transduction?
A phage released from donor cells transfers DNA to the recippient cell.
50
What is the process of conjugation?
1. Requires cell to cell contact.
2. One cell has to contain an F plasmid which contains genes for synthesizing connections between donor and recipient cells.
3. F pilus binds to recipient cell walls
4. Pilus retracts and two cells come in contact
5. Nick in DNA, DNA passes to recipient cell.
6. Both cells now contain F plasmid and can conjugate other cells.
51
What is Hfr bacteria?
Cells that contain an integrated F plasmid in the chromosome.
52
Outline the mating between Hfr donor and F- recipient.
1. F pilus formed
2. Single strand of integrated F plasmid is cut
3. F plasmid followed by DNA transfer
4. Hfr chromosomes replicates as transfer proceeds and i
5. Recipient cell now has part of F plasmid and Hfr genome now is partly diploid (merozygote).
6. DNA is incorporated in recipient enome through crossovers.
7. leftover DNA broken down by nuclease.
8. Cell remains F-, but now contains DNA from Hfr cell.
53
Function of lacZ, lacY and lacZ in lac operon?
Encode enzymes that split lactose.
54
Function of Operator in lac operon?
Cis-acting, contains binding site for repressor.
55
What happens to the lac operon in the ABSENCE of lactose?
REPRESSION.
| Repressor binds to operator, prevents transcription.
56
What happens to the lac operon in the PRESENCE of lactose?
INDUCTION.
| Allolactose binds to repressors, repressor changes shape and cannot bind to operator, transcription occurs.
57
What are the three repressors in the lac operon and what is their effect?
1. lacI+ : normal repressor, prevents expression of lacZ (no transcription)
2. lacI- : mutant repressor, cannot bind to Operator. lacZ always expressed (constitutive expression)
3. lacI^s : Superregressor, inducers cannot bind to it. lacZ is always repressed.
58
What was the PaJaMo experiment?
- supported the existence of a repressor and an inducer.
- B-galactosidase induced in absense of lactose, therefore proves that initial lac of repressor allows synthesis of lacZ. As lacI accumulates, synthesis stops because repressor blocks lacZ transcription.
59
What are the normal and mutant operator and what function do they serve?
O+ : normal operator. Can be repressed.
| Oc : mutant operator, repressor cannot bind, lacZ expressed constitutively.
60
Do lacZ-, lacY- and lacZ- code for functional proteins?
No, they have missense mutations.
61
ex. F' lacI+, lacO+, lacZ- x lacI+, lacOc, lacZ+ F-
Lactose present: F' inducible, but no protein produce. F- consitutive expression of lacZ+
Lactose absent: F' repressed. F- constitutive expression of lacZ
62
ex. F' lacI+, Oc Z+ Y+ / lacI- O+ Z- Y+
lactose present: lacZ+ and Y+ expressed.
lactose absent: lact+ Oc Z+ Y+ expressed, but the other is repressed because the lacI+ encoded by the other plasmid transfers over and bind to lacI- O+ Z- Y+ operator.
63
How is the lac operon regulated by positive controls?
cAMP binds to CRP which binds to regulatroy region which increases transcription.
cAMP levels are high when glucose levels are low.
Even when there is lactose, high glucose means little induction which results in repression of transcription.
64
How can you see the binding sites on a lac operon?
With DNA footprinting.
When a protein binds to a site, DNAase cannot digest it therefore in gel electrophoresis there will be a gap in fragmentation. (Gap occurs whenever there is a repressor or a CRP bound to a region)
65
How is the trp operon controlled?
Through feedback inhibition.
When tryptophan is present, it represses the operon.
Trp acts as a co-repressor of transcription and incluences the rate of transcription through attenuation.
66
What are the uses of the lac operon?
1. Reporter gene (lacZ can be a reporter gene, fused to regulatory region, and expression of B-galactosidase dependant on signals received in regulatory region)
2. Protein expression system (lac regulatory region fused to gene X to control expression of genes, or grow hormones---> overproduce a product through induction)
67
How does Trp act as a co-repressor?
no Trp ---> transcription
| Trp presen ---> binds to repressor (TrpR) ---> binds to operon ---> no transcription.
68
What is the attenuation of transcription?
High amounts of Trp ---> ribosome moves quickly ---> formation of 3:4 stem loop ---> transcription terminated.
Low amounts of Trp ---> ribosome moves lsowly ---> formation of 2:3 stem loop ---> transcription continues.
69
What are the assumption of HWE?
1. Infinite population
2. Random mating
3. No new mutations apprea in gene pool.
4. No migration
5. Different genotypes have no impact on survival.
70
What is positive assortative mating?
Individuals with similarity (of a given trait) reproduce with each other.
71
What is negative assortative mating?
Individuals wtih dissimilarty (of a trait) reproduce together.
72
What is the impact of postive, negative assortative mating and cosanguinity on zygosity?
Positive: increase in homozyosity
Negative: increase in heterozygosity
Cosanguinity: Increase in homozygosity
73
What leads to population stratification?
Mating with respect to ethnicity.
74
What causes changes in allele frequency?
1. Genetic Drift
2. Founder/Bottleneck
3. Migration
75
In what situations is HWE good fxor?
```
Recessive disorders
(contribution of new allels small, ballanced by selection against homozygotes)
```
76
When do you use positional cloning and what does it do?
When you have no info known about the protein.
Enables researchers to obtain the clone of a gene without any prior knowledge of it's protein product or function. Uses genetic and physical maps to locate mutations repsonsible for phenotype.
77
What is the process of positional cloning?
1. Identify the DNA marker that shows linkage to disease locus
(though analyzing pedigrees of multiple families, 2 point crosses---> map disease locus to small chromosomal region)
2. Look for candidate genes
(Analysis between the region between recombination sites that define the smallest areas within which the disease locus can lie should reveal the presence of candidate genes
3. Idenfitication of candidate genes responsible for disease.
78
What is a DNA marker?
An identifiable physical location on a chromosome whose inheritance can be monitered.
79
What does it mean when you know the markers phase?
Whether or not it came from the mother or the father.
80
How are markers used?
In pedigree analysis to determine disease marker or phase.
81
What are the two patterns of segregation of markers?
1. Unlinked
(Marker 1 equally associated with both marker 2 alleles: 50% Parental)
2. Linked
(Marker 1 associated more than 50% with marker 2 alleles: more than 50% parental)
82
Hint for markers!
A B
A B
visualize AA on one chromosome and BB on the other.
83
If you have a child how has a speculated disease marker, yet does not express the disease, how is that explained?
Through recombination of the disease locus. So the child has the marker, but not the disease locus.
84
What are the limitations of positional cloning?
Gene identification is laborious and expensive.
85
What are the limitatons of homozygostiy mapping?
Only viable in a small, closed population
86
What is homozygosity mapping?
- Using affected individuals to identify the region of homozygosity to find disease allele.
87
How is homozygosity mapping performed?
1. Genotype homozygous affected
2. Exclude homozygous loci that look like affected, but are not. (comparison with family)
3. Determine a candidate region of homozygosity
4. Saturate region with markers
5. Determine the extent of homozygosity and recomibination break points.
88
How do you identify the candidate gene responsible for the disease?
Compare affected individuals with unaffected controls.
| Com[pare DNA, expression pattern at RNA level, protein level through Northern hybridization etc.
89
Explain the function of SNP microarray + Homozygosity mapper.
SNP microarray you genotype thousands or millions of SNPs. Homozygosity mappers looks for blocks of contiguous SNPs that show a homozygous phenotype. See candidate --> sanger sequence
No candidate--> next gen.
90
What are the ways that transcription is regulated?
1. Chromatin structure
2. Methylation of Lysines
3. Transcription
3. Protein-Protein Interactions
91
What are the cis-acting elements in organisms?
Promoter and enhancer
92
How does Chromatin structure influence transcription?
1. heterochromatin (compact, no transcriptional activity)
2. euchromatin (loosely compact, transcriptoinal activity)
3. Position of euchromatin and heterochromatin influences gene expression---> heterochromatin can spread. (invesion can bring them closer together
4. Chromatin remodelling: unpacking DNA precedes Transcription. Dislodging nucleosomes allow initation of transcription---> exposes the promoter.
5. Histone modification
93
How does histone modification influence chromatin structure and gene activity?
1. Acetylation of lysine on Histones 3&4
(increases chromatin remodelling, increases transcription. HATs promote transcription, HDACs inhibit transcription)
2. Methylation of lysines
(methylation of H3K4 activates genes, methylation of H3K9 and H3K27 inactivates gens)
94
What transcriptional regulation affects transcription?
1. Initiation
(basal factors bind to promoters---> recruit RNA pol---> basal transcription)
2. Efficiency
( - Activators bind to enhancers, increase basal level
- Activating transcription factors can recruit chromatin remodelling complexes ---> displace nucleosomes
- Repressors bind to promoter, block activators ---> no basal transcription
- Repressors can indirectly impede activator function: compete with activators for binding site, bind to an activator (no activator protein interaction), exclude activator from nucleus, Heterodimerization to change activator to a repressor)
95
How do protein-protein interactions affect transcription?
- change DNA-binding specificity (ex. Jn: Fos)
- Change transcriptional activity (Max:Max repressor, Max:Myc activators that bind to activators)
- Allow one protein to regulate another or create a functional multimeric unit
- transcription factors function as a dimer
96
How does RNA splicing affect gene expression post transcription?
1. Can generate protein diversity
(intron/exon splicing can affect phenotype)
2. Regulates mRNA function
(splicing can result in productive or unproductive splice)
97
How does RNA stability affect gene expression post transcription?
- 5' and 3' UTR contain short sequences or complex structures that bine proteins that regulate translation, cellular location and stability.
ex. Poly A tail.
98
How does Nonsense-mediated decay affect gene expression post transcription?
mRNA with a premature stop codon with have Exon junctions or EJC (gap between two exons that are normally removed) remaining on mRNA. Ribosome release factors interact with EJC, signal decaying enzymes and degradation occurs.
99
How does mRNA editing affect gene expression post transcription?
Changes sequence of RNA after transcriptions (base subsitutions, insertions or deletions).
Consequently alters amino acid sequence of the protein, alters function of protein.
100
How does microRNA mediated RNA interference affect gene expression post transcription?
- Small RNA moecules prevent translation of mRNA
- miRNA within RISC complementary base parit with sequence on mRNA --> perfect homology ---> degradation.
partial homolgy ---> translation repression
101
What are the two main types of translational regulations?
1. Protein modification
| 2. Portein degradation.
102
How does protein modification affect gene expression?
Hydroxylation, Acetylation, Carboxylation, Phosphorylation, Glycosylation, Addition of fatty acids and carbohydrates.
Result in conformation or biochemical property change that affect protein function, localization and stability!
103
How does protein degradation affect gene expression?
Ubiquitination ---> targets for degradation.
| Removal of an unwanted protein.
104
What is imprinting and through what mechanism is it caused by?
Parental copy of a gene is transcriptionally inactive.
Due to the methylation of cytosines in C & G's (CpG dinucleotides within imprinted region)
Imprinting reduces transcription --> no expresion--> "gene silenced"
105
What are the characteristics of imprinting?
- stable enough to last one generation
- erased and reapplied during meiosis ON BOTH GAMETES. (gametogenesis)
- epigenetic: does not alter DNA sequences.
- Can be carried through the generations depending on the nature of the imprint and the sex of the individual.
106
What is the effect if you have a paternal deletion and a maternal imprint?
Most likely disease-causing.
107
How does X-inactivation work?
1. Begins at X inactivation center
2. XIST gene transcried only the inactive X
3. produces transcript that remains in the nucleus and coats inactive X chromosome.
4. Modifications to histones by methyl groups + other proteins that bind to XIST- coated DNA --> inactive heterochromatin state.
108
What genes escape inactivation?
pseudoautosomal regions, tips of x regions, tips of short and long arms.
109
How does X inactivation lead to mosaicism in females?
Because X inactivation is random. Since females have two, one of the X's will be randomly shut down in the cells. Can cause different phenotypes in each cell.
110
What is skewed X inactivation and what problems can it cause?
X inactvation is normally 50:50 for each X in females. But skewed X can is whn the inactivation is skewed so one x is more activated than the other. This can cause the manifestation of x-linked diseases if the wild type x is mostly inactivated.
111
Under what conditions can there be forced expression of one x?
- extra embryonic tissue, paternlly derived x is inactivated
- Turner syndrome
- Structurally abnormal X
- X:autosome translocations---> if balanced, normal X off.
- mutation of the x-inactivation center---> unmutated one turned off.
112
What is uniparental disomy (UPD)?
Inheritance of 2 copies of a chromosome from one parent only.
113
How does UPD occur?
Aneuploidy ---> trisomy rescue of monosomy rescue.
114
What is trisomy rescue?
In trisomies, a zygote can try to repair it by throwing out one copy randomly. If you throw out the the parental copy that you have ONE from, you will end up with UPD
115
What is monosomy rescue?
When you have a monosomic cell, the zygote may try to rescue it by duplicating the one copy that you have.
116
What are the characteristics of the mitochondrial genome?
1. Mitochondrial DNA ---> organized in condensed structures ---> nucleoids.
2. Genome is circular / double stranded (contains its own transcriptional and translational apparatus)
3. Extremely compact no introns
4. mitochondrial genomes have higher variation
5. mtDNA have higher rate of mutation. (more replication errors, less efficient repair mechanisms)
117
Why is mtDNA only inherited through the mother?
MtDNA is exclusively in the ovum, mtNA in the sperm ar destroyed following fertilization.
118
Why does mitochodrial DNA have non-mendelian inheritance patterns?
Genes are carried in organelles rather than in the genome.
119
What are the two types of mtDNA cells?
1. Homoplasmic
( one type of organelle, one type of mtDNA)
2. Heteroplasmic
( more than one type of mtDNA, mutant & normal)
120
Can mitotic progeny of heteroplasmy cells change to homoplasmy?
YES.
There are also different degrees of heteroplasmy and disease and severity level is due to the degree of mutant over wild.
121
Why do different tissues tolerate different levels of heteroplasmy?
Depends on the energy requirement of the tissue. The higher the energy requirement, the less tolerant.
122
How do mitochondrial mutations contribute to the aging process?
Mitochondrial mutations increase with age.
123
Will identical twins have identical mitochondrial genomes?
NO!
| They may have different levels of heteroplasmy
124
What is fragile X?
Defect on an x chromsome--> can be disease causing
| Is a triple nucleotide repeat (CGG) that has a tendency to expand on the tip of the long arm on the x chromosome.
125
How does fragile X cause developmental problems?
Fragile X repeats CGG is near the promoter of the FMR1 gene which is vital for development. Full expansion of fragile X can attract methylation which will prevent the transcription of FMR1.
126
How does the size of fragile x repeats impact disease?
Full expansion = disease
Small repeats = no disease
Premutation carriers: the larger the number of repeats, the higher the likelihood of full expansion
127
What is the inheritance pattern of fragile x?
Male premutation carriers will have children who are premutation carriers.
Female premutation carriers will have children who are affected.
**fragile x expands in oogensis.
128
How do you determine the length of premutations?
PCR amplify ---> Capillary electrophoresis. From the size you can determine the length of mutation.
129
What is genetic anticipation?
Earlier age of onset through successive generations.
130
What is biochemical genetics?
The study of genetic disease as it relates to metabolism
| Focusses on the pathophysiology, diagnosis and treatment of IEMs
131
What is metabolism?
The sum of all chemical reactions constituting the breakdowns and renewal of our bodies.
132
What are inborn errors of metabolism (IEMs)?
An inherited disorder resulting from mutations in a gene encoding a protein involved in metabolism.
Typically autosomal recessive, x-linked or mitochondrial.
133
Genes causin IEMs encode what?
1. Enzymes
(Catalyze rates of reactions)
2. Transporters
(transport substances or class of substances across the membrane)
134
What is Intoxication and what does it cause?
The build up of substrates.
| Can cause abnormal symptoms and health conditions. ie. SIDS, brain swelling, coma etc.
135
What are the three categories of IEMs?
1. Intoxication
2. Energy metabolism
3. Complex molecules
136
What causes IEMs?
Typically a block in the metbolic pathwa that results in a bild up of substrates, deficiency in products, or increased conversions to an alternate metabolite.
137
What are the techniques used in a biochemical genetics lab?
1. Complementation analysis
2. Enzyme Analysis
3. Chromatography
4. Mass spectrometry
138
What are treatment strategies for IEMs?
REVIEW LECTURE NOTES
139
What is the basis of medelian inheritance?
- Dominace/recessiveness
- autosomal or x-linked
- predictable ratios / risk assessment
140
What is the basis of non-mendelian genetics?
- exceptions to biallelic expression (mitochondrial, imprints, UPD)
- dynamic mutations
141
What is the basis of polygenic inheritance?
- Additive effect of a large about of alleles
- Increasing the number of genetic risk factors (multiple alles at one or multiple genes), increases the number of possible phenotypic classes
142
What are multifactorial/complex traits?
Combination of small variations in genes that together in combination with environmental factors prouce or predispose a disease (multifactorial disease)
143
Factors that influence risk of multifactorial disease
1. Higher, the more related you are to affected
2. Higher, if your liability factor is high (burden of disease on family)
3. Higher, if the severity of the disease/condition in relatives is more sever.
4. Sex, depending on the affected proband.
144
How do you identify multifactorial genes?
1. Genome Wide Association Study (GWAS)
| 2. Case Control.
145
What is the process of GWAS
1. Genome microarray ---> obtain whole genomic profiles
2. Tag SNP
3. Disase association ---> find differences in SNPs or traits
4. Manhattan plot ---> lowest P value = highest dots (best place to look for disease associated alleles)
146
What is the process of case control studies?
1. Compare frequency of risk factor (at risk alleles) in a patient population to a control popultion
2. Disease association: an allele present in an affected population at a higher frequency than in control
** Increase populationto detect small genetic effects.
147
What are the hallmarks of cancer?
1. Uncontrolled growth
(Cancer cells grow independantly of externally provided growth factors. Do not respond to inhibitory signals)
2. Evasion of apoptosis
(Lack p53 DNA damage checkpoint, evade cell death)
3. Immortality
(Divide indefinitely, Telomerase is activated)
4. Bypass barriers to growth
(Can invade surrounding tissue, secrete grwoth factors for blood vessels for nutrients [angiogenesis], evade immune system detection)
5. Genomic and karyotipic instability
(decreased DNA repair---> increased mutation rate. Increase mitotic defects & chromosomal aberrations, increased tolerance of aneuploidy and translocations)
6. Clonal
(Cancer cells are dervied from a SINGLE cell)
148
What role do cell-cycle regulators play in the control of the cell cycle?
- Stimulate cells to enter the cell cycle, or divide more rapidly
- control progress of the cell through the cell cycle
- arrest the cell when DNA or cellular machinery is damaged
149
What role do Cyclin-dependant kinases (CDKs) play in the control of the cell cycle?
Central role in the progression of cells through the cell cycle. Regulate cell cycle transitions G1--->S and G2--->M
Phosphorylate proteins that can activate or inactive protein function.
150
What are the cell cycle transitions and checkpoints controlled by?
CDKs
151
How are the G1-->S and G2-->M transitions controlled?
By inhibition and activation of CDKs (CDKs cycle betwen inactive and active states)
152
What are the purpose of cell cycle checkpoints?
To minimize effects of damaged DNA defects in cell cycle apparatus.
153
What is the relationship between CDKs and cyclins?
Stage specific cyclins activate CDKs. Cyclins specifiy the set of target proteins to be phosphorylated.
There is a specific cyclin that are synthesized in each different stage of the cell cylcel that specifies which proteins CDK should phosphorylate
154
Describe the G1--->S transition
1. E2F (transcription factor) repressed by Rb
2. Transitions initiated by CDK-cyclin
3. CDK-cylin phosphorylate Rb
4. E2F active, induces genes for DNA synthesis
5. S phase cyclins induced, promote degradation of G1 cyclins
155
Descibe the G2--->M transition
1. CDK complexed with cyclin throughout G2, but in an inactive state (phosphorylated)
2. Dephosphorylation of CDK-cyclin activates, leading to M phase.
156
When is it disadvantageous for the cell to complete the cell cycle?
- DNA damage
(environmenta or random errors, must pause cell cycle to repair DNA and avoid replicating errors)
- Errors in cellular machinery
(failure to replicate DNA, nondisjuction, failure to divide all lead to aneuploidy!)
157
What is the purpose of cell cycle checkpoints?
1. Check integrity of genome
(look for damage by random errors or environmental factors)
2. Check integrity of cell cycle machinery
3. If damage is detected, cell does not progress through cell cycle
158
When do the major checkpoints occur?
Before S phase
Before M phase
In M phase
159
Describe the checkpoint before S phase (G1--->S)
This the p53 checkpoint!!!!
1. p53 induces expression of CDK inhibitor (prevents cell from moving to S phase.
2. Induces expression of DNA repair genes
3. If DNA repair does not ocur, undergoes Apoptosis.
160
What are the consequences of a failure of the p53 checkpoint (G1--->S checkpoint)?
- Aneuploidy
- Double stranded breaks
- Chromosomal rearragnements
161
Describe the checkpoint before M phase (G2--->M)
1. Detects DNA damage that occurs during G2 (usually double stranded breaks)
2. Mitotis delayed (CDK-cyclin inhibited) ---> repairs break
162
What are the consequences of the failure of the G2--->M checkpoint?
- Chromosome breaks
- Rearrangments
- Aneuploidy
163
Describe the spindle checkpoint at the M phase
1. Detects failure of the formation of spindle and chromosomal attachment
2. CDK-cyclin inhibited--> pause between metaphase and anaphase
3. Reattach chromosomes together
164
What are the consequences of the failure of the spindle checkpoint during the M phase?
Aneuploidy in daughter cells.
165
How is the cell cycle regulated?
- Growth factors, hormones and ligands can bind to cell surface receptors or stimulate or inhibit cell proliferation.
- Ligand binding can activate or repress receptors
- Receptors relay signals into the cell/nucleus
166
What are stimulating signals?
- Activate gene expression that promotes cell growth
OR
- Inhibit gene expression that inhibits cell growth.
1. Stimulating growth factor binds to receptor
2. Signal transducers---> nucleus---> transcription factor---> protein that stimulates cell division
167
What are inhibitory signals?
- Activate gene expression that blocks cell growth
OR
- Inhibit gene expression that stimulates cell rowth
1. Inhibiting growth factor binds to receptor
2. Signal transducers---> nucleus---> transcription factor---> protein that blocks cell from dividing.
168
How does cancer form?
Accumulation of mutations over time in the cells of a clone ---> cancer.
Arise in two types of genes:
1. Oncogenes
2. Tumour suppressors
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What are Oncogenes?
Dominant mutatiosn that inappropriately activate genes for cell proliferation.
ACTIVATION ---> CANCER
170
What are tumour suppressors?
Recessive mutations that inactivate genes that are normally reponsible for preventing excessive cell proliferation
LOSS OF FUNCTION ---> CANCER
171
What are proto-oncogenes?
normal, wildtype version of the gene.
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How are Oncogenes activated?
1. Rearrangement/Insertion
(insertion of retroviral DNA---> activation
rearrangements bring promoter closer to oncogee---> activation)
2. Point mutation
(Can cause proliferation without growth factor)
3. Amplification
(trisomies increase oncogene dosage, extra chromosomal copies of oncogenes can cause over expression)
4. Gene fusion
(Philadelphia chromosome rearrangement---> increase kinase activity---> inhibits apoptosis, contains dominant oncogene)
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How are tumour suppressorgenes inactivated?
Recessive mutations in BOTH alleles--> loss of function
174
How is all cancer genetic, yet only a small percentage is actually hereditary?
- Most cancers are sporadic, as a result of random mutations
- Environmental factors and lifestyle influence cancer rates (exposure to mutagens)
- Cancer also increases with age (mutations accumulate over time)
175
What is the key difference between hereditary cancer and sporadic?
Hereditary most often caused by inactivation fo tumour suppressor genes.
In Hereditary cancer, a single germline mutation is present in all somatic cells, predispositng invidiual to cancer. Individual just needs ONE more mutation---> loss of function of Tumour suppressor---> cancer
In sporadic, you need TWO mutations events---> loss of function of Tumour suppressor---> cancer
176
What is transgenesis?
Adding genes to the genome.
| Take a needle of DNA---> inject into early embryo
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What are the applications of Transgenesis?
Insertion of: wild type genes, dominant mutations, GFP markers etc.
178
What is forward genetics?
Induce mutations and scren for phenotype of interest ---> Identify mutated gene responsible for phenotype (positional cloning, transposon tagging, etc.)
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What is reverse genetics?
Choose gene of interest with known sequence---> Generate mutation in gene of nterest (knockout by homologous recomibination, RNA intereference, targeted deletions and insertions with specific nucleases)
180
Model Organisms
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