Organsimal Genetics Flashcards by Bailey Francis

Mendelian phenotypes

what causes a Mendelian phenotype?
what is a monogenic disease?
give an example of a monogenic disease and describe it

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.

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Penetrance

what is penetrance?
what is the penetrance of an allele which display 40% of affected individuals?

the probability that a disease will appear in an individual when the disease-causing allele is present
40%

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Expressivity

what is expressivity?
give an example of a disease that displays expressivity?

the range of symptoms possible for a given disease

- Marfan’s syndrome - has mild to severe symptoms

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Pleiotropy

what is pleiotropy?
give an example of a disease caused by a mutation in a pleiotropic gene and describe it

where one gene has many functions

- Nail-Patella Syndrome. LMX1B mutation causes nail abnormalities, causes patella to be absent, kidney disease

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Maternal effect

what type of organisms does this occur in?
what does this mean?

organisms that undergo delayed zygotes transmission e.g. insects, frogs
the offsprings phenotype is determined by its mothers phenotype and not its own.

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Allelic series

what is an allelic series?
give an example of an allelic series

where different mutations in the same gene causes different phenotypes
Firoblast Growth Factor Receptors. Mutations in different regions of the receptor cause different diseases

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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

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.

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Multiple Epiphyseal Dysplasia

how does this disease display an allelic series?
how does this disease display genetic heterogenity?

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

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Gene identification

what model organism is commonly used for gene identification?
how is this done?

Drosophilia

- breeding an inheritance pattern analysis

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Genetic maps

- what are the two types and what do they show?

Linkage mapping = based on recombination during meiosis (average)
Physical map = based on DNA length (physical distances)

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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?

genes that are inherited together during meiosis
more
less

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Human Genome Project

when was it finalised?
what did it identify?

2004

- coding regions

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Exome sequencing

what is an exome?
what genetic disease was made clear by exome sequencing and how?

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.

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Exome sequencing benefits and limitations

what are the benefits of exome sequencing?
what are the limitations of exome sequencing?

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

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Linkage analysis

what does SNP stand for and what does it mean?
what is a haplotype?
what does haplotype analysis show?

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

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Polygenetic phenotype analysis

what analysis method is used for polygenic diseases?
how does this method work?

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

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Genome Wide Association studies

what are the advantages of GWAS?
what are the disadvantages of GWAS?

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

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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?

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

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Contradictions to Mendelian genetics

who did this?
what was his experiment and what were its results?
what were these later coined?

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

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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 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

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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?

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.

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Alcoholic preference in mice

- what are the basic steps?

- identify phenotype of strains 
Identify two contrasting strains 
Inter crossing 
Linkage analysis 
Regression analysis 
Identify gene candidates 
Comparison with other strains

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Alcohol preference in mice - strain selection

how did they identify phenotypes of strains?
what two strains did they identify to be contrasting?

two bottle choice - one water, one 10% ethanol

- D2 less than 1g/kg/day of ethanol, B6 10g/kg/day of ethanol

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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?

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.

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?

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

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?

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

Alcoholic preference in mice - Final conclusions

what is done when you have chosen a gene candidate?
what were the conclusions of the experiment?

compared to different strains and tested in comparisons to phenotype
Snytaxin binding protein 1 Polymorphism related to ethanol preference

Notch

to which domain do ligands bind in Notch?
two which domain do regulators and partners bind?

EGF-like repeats domains 11-12

- ankyrin repeats

Forward and reverse genetics

what is forward genetics?
what is reverse genetics?

studying from phenotype to gene

- studying from gene to phenotype

Mullers morphs

what morphs fall into loss of function?
what morphs fall into gain of function?

amorph, hypomorphs and antimorphs

- hypermorph and neomorphs

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?

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

Antimorphs

what is an antimorph?
what are they also known as?
in what proteins do these usually occur? Why?

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

Hypermorphs

what is a hypermorph?
what can this be caused by?

where a mutation causes increased function

- failures to degrade a protein or by a constitutively active protein

Neomorphs

what is a neomorph?
how do these commonly arise?

where a protein gains a novel function after mutation

- due to changes in expression pattern

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

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

Basic transposons

- what is the mechanism for insertion with a transposon?

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.

Non autonomous transposable elements

how do they differ from autonomous transposases?
how can you use these to insert a COI?

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

P element mutagenesis in flies

- explain the process of P element mutagenesis using the white eye mutation

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

Insertion repeats as molecular markers

- explain how insertion repeats can be used as molecular markers to find candidate genes

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

Different types of transposable elements

- what are the three different types of transposable elements and what are their functions?

DNA transposons (p elements) - cut and paste
RNA transpsons - copy and paste
Non autonomous transposons - genetic engineering

Insertional mutagenesis in vertebrates

what genetic tool is used?
what are the advantages of this tool?
what are the disadvantages of this tool?

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

Sleeping Beauty transposon

what tissue types does it affect?
how is it more controllable than retroviruses?
what is another advantage?

all
targets only a subset of cells and not whole tissue
can create several mutations in one cell

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

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)

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?

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

Homologous recombination - embryonic stem cells

- explain the process

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

Making knockouts using homologous recombination

what are the three outcomes
how do you ensure you select the correct outcome?

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

Making knock-ins using homologous recombination

- explain the process

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

CRISPR-Cas9

what does this require?
what does Cas9 do?
what two outcomes can happen after this step?

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)

CRISPR-Cas9 - creating transgenic mice

- explain the process

CRISPR-Cas9 is incorporated into mice embryos and inserted into a surrogate mouse. All offspring will have their genome modified with your desired gene

CRISP-Cas9 vs. Embryonic stem cells

- what are the benefits of CRISPR-Cas9

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

Advantages of using Drosophilia as a model organism

- give four advantages

rapid life cycle - large numbers bred and screened
Infrequent genetic redundancy
Easy to obtain mutants
Polythene chromosomes allow mapping

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?

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

Drosophilia - hypomorphs and amorphic mutations

give an example of a hypomorphic mutation and an amorphic mutation?
what is the wild type phenotype?

hypo - apricot (wa). Amorph - white (w)

- red eyes

Drosophilia - haploinsufficiency

give an example mutation and what it causes
what is haploinsufficiency?

Notch, wing boundaries to be unproperly developed

- where two copies of a gene are required in order to maintain normal function

Drosophilia - Antimorphic mutations

give an example and what the mutation causes
how would you prove this mutation is antimorphic
how can this can occur?

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

Drosophilia - hypermorphic mutations

give an example and what does it cause
how would you prove that this mutation is hypermorphic?

eclipse and causes EFG-receptor signalling in the eye

- Elp/Df results in wild type phenotype

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?

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

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

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

Drosophilia - epistasis

what is epistasis
give an example

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

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