Organsimal Genetics Flashcards
1
Q
Mendelian phenotypes
- what causes a Mendelian phenotype?
- what is a monogenic disease?
- give an example of a monogenic disease and describe it
A
- variations in a single gene cause a phenotypic change
- variations in a single gene cause a disease
- Phenylketonuria. Mutation causes a defective phenylalanine hydroxylase causing phenylalanine build up which disrupts brain development.
2
Q
Penetrance
- what is penetrance?
- what is the penetrance of an allele which display 40% of affected individuals?
A
- the probability that a disease will appear in an individual when the disease-causing allele is present
- 40%
3
Q
Expressivity
- what is expressivity?
- give an example of a disease that displays expressivity?
A
- the range of symptoms possible for a given disease
- Marfan’s syndrome - has mild to severe symptoms
4
Q
Pleiotropy
- what is pleiotropy?
- give an example of a disease caused by a mutation in a pleiotropic gene and describe it
A
- where one gene has many functions
- Nail-Patella Syndrome. LMX1B mutation causes nail abnormalities, causes patella to be absent, kidney disease
5
Q
Maternal effect
- what type of organisms does this occur in?
- what does this mean?
A
- organisms that undergo delayed zygotes transmission e.g. insects, frogs
- the offsprings phenotype is determined by its mothers phenotype and not its own.
6
Q
Allelic series
- what is an allelic series?
- give an example of an allelic series
A
- where different mutations in the same gene causes different phenotypes
- Firoblast Growth Factor Receptors. Mutations in different regions of the receptor cause different diseases
7
Q
Genetic heterogeneity
- what is genetic heterogeneity?
- what are the two forms of genetic heterogenity?
- give an example of a disease caused by genetic heterogenity and describe it
A
- where different mutations result in the same phenotype
- allelic heterogeneity - different mutations in the same gene cause the same phenotype
Locus heterogeneity - different mutations in different genes cause the same phenotype - Multiple Epiphesyal Dysplasia. Causes short stature and osteoarthritis.
8
Q
Multiple Epiphyseal Dysplasia
- how does this disease display an allelic series?
- how does this disease display genetic heterogenity?
A
- mutations in COMP protein can lead to several disorders such as MED and pseudoachondroplasia
- multiple mutations in different genes such as COMP and COL9A1 all result in MED
9
Q
Gene identification
- what model organism is commonly used for gene identification?
- how is this done?
A
- Drosophilia
- breeding an inheritance pattern analysis
10
Q
Genetic maps
- what are the two types and what do they show?
A
- Linkage mapping = based on recombination during meiosis (average)
Physical map = based on DNA length (physical distances)
11
Q
Recombination
- what does recombination mean?
- are markers that are further apart more or less likely to recombine during meiosis?
- are markers that are closer to each other more or less likely to recombine during meiosis?
A
- genes that are inherited together during meiosis
- more
- less
12
Q
Human Genome Project
- when was it finalised?
- what did it identify?
A
- 2004
- coding regions
13
Q
Exome sequencing
- what is an exome?
- what genetic disease was made clear by exome sequencing and how?
A
- all of an individual’s exons (coding regions)
- Celiac disease. Sequence variations between wild type and affected individuals were collected. 55 of 56 were already known. The unknown gene HATR2 was found to be the disease causing mutation which changed a conserved Leu to a Pro creating a defective enzyme.
14
Q
Exome sequencing benefits and limitations
- what are the benefits of exome sequencing?
- what are the limitations of exome sequencing?
A
- allows for the sequencing for a whole pool of samples at a time, no longer need gene specific primers
- only samples known coding regions, population specific differences (e.g. ethnic background) not necessarily known
15
Q
Linkage analysis
- what does SNP stand for and what does it mean?
- what is a haplotype?
- what does haplotype analysis show?
A
- Single Nucleotide Polymorphism. A single base alteration between individuals
- combinations of alleles possible on the same chromosome homolog
- shared haplotype indicate possible similarities in phenotypes and large scale analysis shows regions on the genome that differ between populations
16
Q
Polygenetic phenotype analysis
- what analysis method is used for polygenic diseases?
- how does this method work?
A
- genome wide association studies (GWAS)
- use SNP markers to generate haplotype for all genomic regions and asses genes in regions in affected individuals for regions linked to phenotype
17
Q
Genome Wide Association studies
- what are the advantages of GWAS?
- what are the disadvantages of GWAS?
A
- useful for polygenic diseases, identify genomic regions with small amounts of phenotypic contribution, used on large groups
- associated regions often contain no genes, association to causation difficult to demonstrate
18
Q
Gregor Mendel
- what was his initial experiment and what was its conclusion?
- what was his seed shape experiments and what were its conclusions?
- what was his two trait inheritance experiment and what were its conclusions?
A
- planted white flower seeds in a yellow flower bed and monitored offsprings flower colour. Flowers were white. Concluded traits were inherited by offspring from their parents
- crossed smooth peas with rough peas and observed all smooth in F1 gen but 25% rough in F2 gen. Concluded each trait must be controlled by two factors - dominant and recessive
- crossed smooth yellow peas with rough green peas. Observed a 9:3:3:1 ratio. Concluded that traits are inherited independent from each other
19
Q
Contradictions to Mendelian genetics
- who did this?
- what was his experiment and what were its results?
- what were these later coined?
A
- Francis Galton
- conducted studies on seed size in plants. Concluded that some phenotypes do not have only two states but a wide range of results.
- quantitative traits
20
Q
Trait inheritance
- for a quantitative trait with two extreme parents where will the offspring lie?
- for a Mendelian trait with two extreme parents where will the offspring lie?
A
- a range of values in the middle of the two extremes
- all F1 offspring will have the dominant trait but there will be 25% recessive in F2
21
Q
Inbred Mouse Strains
- how many times are mice selectively bred before experimentation?
- how do different strains differ from each other?
- why are inbred mouse strains useful?
A
- 20
- different genotype at many loci, different in non-coding regions of DNA
- wide range of phenotypes allow for segregation into groups. Differences between two strains can be used to identify major genes contributing to QTL.
22
Q
Alcoholic preference in mice
- what are the basic steps?
A
- identify phenotype of strains Identify two contrasting strains Inter crossing Linkage analysis Regression analysis Identify gene candidates Comparison with other strains
23
Q
Alcohol preference in mice - strain selection
- how did they identify phenotypes of strains?
- what two strains did they identify to be contrasting?
A
- two bottle choice - one water, one 10% ethanol
- D2 less than 1g/kg/day of ethanol, B6 10g/kg/day of ethanol
24
Q
Alcoholic preference in mice - intercrossing
- what is the genotype of the F1 generation?
- why do we cross the F1 generation?
- what is done with the F2 generation?
A
- heterozygous at all loci
- creates different combinations of homozygous DNA
- F2 generation phenotype tested to identify those that match chosen parental trait. DNA is then compared.
25
Q
Alcoholic preference in mice - linkage analysis
- where are markers chosen in the two strains?
- what are the two forms of markers? How are they detected?
A
- regions that are polymorphic between the two strains
- Simple Sequence Length Polymorphisms - different lengths of inserted repeats detected with gel electrophoresis
Single Nucleotide Polymorphisms - single base changes between two groups - detected by sequencing of PCR products
26
Q
Alcoholic preference in mice - regression analysis
- why do we use regression analysis?
- how are gene candidates chosen?
- what were the candidates chosen in the experiment?
- what is then done with these regions?
- which was the best candidate?
A
- to identify areas where QTL might be located.
- Based on logic and previous studies
- syntaxin binding protein 1 , glutamate decarboxylate 1 and 2
- analysed for differences between the two strains
- syntaxin binding protein 1
27
Q
Alcoholic preference in mice - Final conclusions
- what is done when you have chosen a gene candidate?
- what were the conclusions of the experiment?
A
- compared to different strains and tested in comparisons to phenotype
- Snytaxin binding protein 1 Polymorphism related to ethanol preference
28
Q
Notch
- to which domain do ligands bind in Notch?
- two which domain do regulators and partners bind?
A
- EGF-like repeats domains 11-12
- ankyrin repeats
29
Q
Forward and reverse genetics
- what is forward genetics?
- what is reverse genetics?
A
- studying from phenotype to gene
- studying from gene to phenotype
30
Q
Mullers morphs
- what morphs fall into loss of function?
- what morphs fall into gain of function?
A
- amorph, hypomorphs and antimorphs
- hypermorph and neomorphs
31
Q
Amorphs and hypomorphs
- what is an amorph?
- are these morphs often recessive or dominant? Why is this?
- in what cases do these genes show dominance?
- in what systems do hypomorphs usually occur?
A
- where a protein has no function at all after mutation
- where a protein has a reduced function after mutation
- recessive because the normal allele can compensate for their loss
- if they occur in haplo-insufficient genes
- when the protein is a part of a protein complex or the amount of protein produced is essential for its function
32
Q
Antimorphs
- what is an antimorph?
- what are they also known as?
- in what proteins do these usually occur? Why?
A
- where a mutation causes the protein to antagonise the wild type protein
- dominant negative
- proteins that require a binding partner, the antimorph will sequester binding partners meanings that they are not available for wild type proteins
33
Q
Hypermorphs
- what is a hypermorph?
- what can this be caused by?
A
- where a mutation causes increased function
- failures to degrade a protein or by a constitutively active protein
34
Q
Neomorphs
- what is a neomorph?
- how do these commonly arise?
A
- where a protein gains a novel function after mutation
- due to changes in expression pattern
35
Q
Mullers morphs in Notch
- give an example of an amorph in notch
- give an example of a hypomorphs in notch
- give an example of an antimorph in notch
- give an example of a hypermorph in notch
A
- if ankyrin repeats are deleted, the protein has no function because it can no longer bind to proteins in the nucleus
- if one of the NLS is deleted, the protein still has function but there is less efficient trafficking to the nucleus
- if the intracellular domain is deleted, the ligand still binds but is sequestered from other Notch receptors
- if a mutation occurs in EGR like repeat domains 11-12 then it can cause a constitutively active protein
36
Q
Basic transposons
- what is the mechanism for insertion with a transposon?
A
- transposase binds to inverted repeats on either side of the transposon creating a loop, the transposase will also bind to another sequence somewhere in the genome. The transposase will then cut into the gene in a palindromic fashion (like a restriction endonuclease) and will fill in any missing nucleotide bases inserting the transposon into the gene.
37
Q
Non autonomous transposable elements
- how do they differ from autonomous transposases?
- how can you use these to insert a COI?
A
- the transposon function has been lost meaning a construct of interest can be inserted instead
- inject two vectors, one containing a nonautonomous transposon and one containing a transposase. The transposase will bind at inverted repeats and insert your COI into your gene of choice
38
Q
P element mutagenesis in flies
- explain the process of P element mutagenesis using the white eye mutation
A
- inject two vectors into blastoderm, one with an integration marker (e.g. white eye allele) and one with transposase. Vectors are incorporated into germ-cell precursors so vector is passed onto offspring. Offspring from the F1 generation are then crossed with a white eyed male. Any F2 offspring with the integration marker (orange eye phenotype) will have taken up the vector, those with white eyes have not
39
Q
Insertion repeats as molecular markers
- explain how insertion repeats can be used as molecular markers to find candidate genes
A
- Digest DNA with a restriction enzyme. Relegate the product to form a circular genome. Digest with a restriction enzyme that cuts inside the P element. PCR with primers that bind to inverted repeats. Sequence the PCR product and compare it to databases to find your candidate genes
40
Q
Different types of transposable elements
- what are the three different types of transposable elements and what are their functions?
A
- DNA transposons (p elements) - cut and paste
RNA transpsons - copy and paste
Non autonomous transposons - genetic engineering
41
Q
Insertional mutagenesis in vertebrates
- what genetic tool is used?
- what are the advantages of this tool?
- what are the disadvantages of this tool?
A
- retroviruses
- often target sequences close to oncogenes - useful for studying cancers
- targets blood and mammary cells, useful for some forms of cancers but not others
42
Q
Sleeping Beauty transposon
- what tissue types does it affect?
- how is it more controllable than retroviruses?
- what is another advantage?
A
- all
- targets only a subset of cells and not whole tissue
- can create several mutations in one cell
43
Q
Enhancer trap screens
- where are the transposable elements inserted and why?
- explain how you can monitor expression patterns using an enhancer screen trap in flies
- what do you do after you have an expression pattern of interest
A
- inserted downstream from enhancer elements
- inject two vectors into the blastoderm - one with transposable element containing an integration marker and a reporter gene e.g. GFP and one with a transposase. Vectors enter germ-cell precursors and are passed onto offspring. F1 generation are crossed with a white eyed male and any F2 generation with orange eyes are selected and screened for the reporter gene to check expression patterns
- use inserted repeat flanking to identify any candidate genes and create mutants with knockouts and observe what effect this has on expression pattern (the gene will have the opposite effect)
44
Q
Creating precise mutants - Homologous recombination
- when does this usually occur?
- how is this used in genetic engineering
- what is also included in the vector?
- what two forms of HR cause loss of function and how?
- what form of HR causes gain of function and how?
A
- during meiosis and before each mitosis
- constructs in vectors can be incorporated into the genome via HR
- marker M
- KO - whole gene or crucial part of gene is replaced, KD - reduced expression as M usually replaces small part of gene or promoter
- KI - M is a construct that replaces gene with another piece of DNA
45
Q
Homologous recombination - embryonic stem cells
- explain the process
A
- remove the inner cell mass from the dominant allele mice blastocyst of and culture them on a disc. Insert your transgene and select the stem cells which have taken it up using drug resistance markers. Inject these cells into the blastocyst of a recessive mice and a chimeric mice is produced. These mice are selected and bred with recessive mice - all offspring with the dominant allele will all have the genetic modification
46
Q
Making knockouts using homologous recombination
- what are the three outcomes
- how do you ensure you select the correct outcome?
A
- no integration at all, targeted intergration by HR or random intergration
- vector with donor gene contains two markers M1 and M2, M1 provides drug resistance and M2 causes death when exposed to drug. Only correct intergrations will only have M1 so these will be the only ones that survive when exposed to the drug
47
Q
Making knock-ins using homologous recombination
- explain the process
A
- replace gene of interest with reporter gene using knockout method. Only intergrated cells will survive. Replace your reporter gene with desired DNA - select cells without reporter gene
48
Q
CRISPR-Cas9
- what does this require?
- what does Cas9 do?
- what two outcomes can happen after this step?
A
- guide RNA to your desired gene and Cas9 endonuclease
- creates a double stranded cut at PAM sequences
- Non homologous end joining (NHEJ) - random insertions of nucleotides are made to create knockouts by altering reading from
Homologous Directed Repair (HDR) - a construct of interest can be produced to insert desired genes into your chosen area (knock-ins)
49
Q
CRISPR-Cas9 - creating transgenic mice
- explain the process
A
- CRISPR-Cas9 is incorporated into mice embryos and inserted into a surrogate mouse. All offspring will have their genome modified with your desired gene
50
Q
CRISP-Cas9 vs. Embryonic stem cells
- what are the benefits of CRISPR-Cas9
A
- ten times cheaper, only takes 4 weeks instead of 6 months. Only requires one generation rather than many in stem cells. 85-95% success rate compared to 50-55%. Can create several mutations at once
51
Q
Advantages of using Drosophilia as a model organism
- give four advantages
A
- rapid life cycle - large numbers bred and screened
Infrequent genetic redundancy
Easy to obtain mutants
Polythene chromosomes allow mapping
52
Q
Drosophilia - loss of function mutations
- give an example of a loss of function mutation and what it causes
- explain how you would prove this is a LOF mutation?
A
- vg (vestigial), wings do not grow
- in a vg/Df fly, the wings have still not grown. But in vg/vg Dp(+), the wild type phenotype is restored meaning the normal function of the wild type gene is to grow the wings
53
Q
Drosophilia - hypomorphs and amorphic mutations
- give an example of a hypomorphic mutation and an amorphic mutation?
- what is the wild type phenotype?
A
- hypo - apricot (wa). Amorph - white (w)
- red eyes
54
Q
Drosophilia - haploinsufficiency
- give an example mutation and what it causes
- what is haploinsufficiency?
A
- Notch, wing boundaries to be unproperly developed
- where two copies of a gene are required in order to maintain normal function
55
Q
Drosophilia - Antimorphic mutations
- give an example and what the mutation causes
- how would you prove this mutation is antimorphic
- how can this can occur?
A
- ebony, darker pigmentation of fly
- e/+ dp(+) still has a darker pigmentation that the WT proving e is interfering
- mutation causes truncated protein that removes one binding site but keeps another so the protein will sequester the wild type version
56
Q
Drosophilia - hypermorphic mutations
- give an example and what does it cause
- how would you prove that this mutation is hypermorphic?
A
- eclipse and causes EFG-receptor signalling in the eye
- Elp/Df results in wild type phenotype
57
Q
Drosophilia gene interactions - complementation tests
- what is their purpose
- what type of mutations are required
- how do two mutations complement each other
- what does this mean?
- what can this be used to produce?
A
- to test whether mutations on two strains are on different genes
- recessive loss of function mutations
- if a double heterozygote produces a wild type phenotype
- two mutations occur on different genes
- physical maps
58
Q
Drosophilia gene interactions - intergenic complementation
- what are the two forms?
- give an example of each
- what is dumpy’s function
- explain how it can show intergenic complementation
A
- gain of function mutations and complex gene locus
- eclipse and dumpy
- produces tension in tissues and defines wing shape
- two different mutations, oblique and complex, can complement each other and give a wild type phenotype even though they occur in the same gene
59
Q
Drosophilia - epistasis
- what is epistasis
- give an example
A
- where one mutation will mask another mutation
- double homozygotes of white and cinnabar still have a white mutation phenotype so white is epistasis to cinnabar
