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Organismal genetics > L 9-13 > Flashcards

L 9-13 Flashcards

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Biology 101 flashcards
1
Q

what is an example of protein isoforms

A

notch in mammals
- proteins that are similar to each other and perform similar roles within cells

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2
Q

what are the main issues found with forward genetics

A
  • production and mapping of mutants is a long process, using tools exclusive to yeast and flies
  • pleiotropic genes have multiple function, can generate lethal phnotypes (need to use mosaic or temperature sensitive conditional systems)
    -redundancy amongst genes in the same family or paralogs
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3
Q

what is loss of function

A
  • production of no or less proteins or a protein with reduced or no activity
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4
Q

what is gain of function

A
  • production of more proteins or an extended expression pattern (new time and/or new place), or a protein with an increased activity or new function
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5
Q

what are the three types of loss of function

A
  • amorph
  • hypomorph
  • antimorph
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6
Q

what are the three types of gain of function

A
  • hypermorph
  • neomorph
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7
Q

what is the LOF and GOF effect on transcription

A
  • amorph
    none
  • hypomorph
    reduced
  • antimorph
    normal
  • hypermorph
    increased
  • neomorph
    extopic
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8
Q

what is the effect of the LOF GOF

A
  • amorph
    none
  • hypomorph
    reduced
  • antimorph
    antagonistic
  • hypermorph
    increased
  • neomorph
    novel
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9
Q

what is the LOF GOF of translation

A
  • amorph
    none
  • hypomorph
    reduced
  • antimorph
    normal
  • hypermorph
    increased
  • neomorph
    normal
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10
Q

what is the LOF GOF for function

A
  • amorph
    none
  • hypomorph
    reduced
  • antimorph
    reduced and antagonises wild type
  • hypermorph
    increased
  • neomorph
    new
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11
Q

what is the LOF GOF often

A
  • amorph
    recessive
  • hypomorph
    recessive
  • antimorph
    dominant
  • hypermorph
    dominant
  • neomorph
    dominant
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12
Q

describe an amorph

A
  • often recessive (unless on the X chromosome) due to the normal allele compensating for the loss
  • when they are dominant, they occur in haplo insufficient genes
  • haploinsufficiency usually occurs when the protein is in a protein complec or if the quantity is crucial
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13
Q

describe a hypomorph

A
  • often recessive (unless on the X chromosome) due to the normal allele compensating for the loss
  • when they are dominant, they occur in haplo insufficient genes
  • haploinsufficiency usually occurs when the protein is in a protein complec or if the quantity is crucial
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14
Q

describe an antimorph

A
  • dominant negative
  • interfere with normal gene function - more severe than one copy of the amorph (null)
    common with proteins who have binding partner (dimer, receptor), inhibits the partners function
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15
Q

describe a hypermorph

A
  • high transcription or failure to degrade transcript or protein
  • sometimes constitutively active (a receptor is normal activated apon ligand binding. if mutation is on binding site, then independent of ligand and alsways active)
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16
Q

describbe neomorphs

A
  • novel function for protein arising from mutation (new catalytic activity/binding partner)
  • commonly due to change in expression pattern (in regulatory regions controlling expression)
  • can also arise from translocation from chimeric fusion proteins
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17
Q

what types of chemical mutations can be generated for forward genetics

A

chemical
- ethylmethane sulfonate (point mutation) flies. worms, plants
- nitrosourea (point mutation) mice

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18
Q

what type pf radiation mutations can be generated for forward genetics

A

radiation
- uv light (small mutations) many
- x rays or gamma rays (chromosomal rearagnements and deletions) many

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19
Q

what type of insertion mutagenesis using transposable elements can be used for forward genetics

A

insertion mutations
- transposable elements (disrupt gene/promoter or small deletions) flies, worms, plants
- retrovirus (disrupt gene/promoter or small deletion) mice

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20
Q

what is the downside of mapping mutants through chemical and radioation mutagenesis

A
  • takes a long time, can be combined with NGS for faster results
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21
Q

what is a transposon

A
  • mobile DNA element, jumping gene (make ~50% of human genome
    contains
  • 2 inverted repeats, at start and end position
  • gene coding for transposase including promoter, used for cutting and pasting transposon
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22
Q

what are the steps of a transposon

A
  • The transposase acts as a restriction endonuclease and an integrase.
  • Step 1: Once expressed, the transposase binds the inverted repeats (IR) and cuts out the transposon (EXCISION), removing some nearby sequences in the process.
  • Step 2: The transposase (bound to the transposon) cuts the host DNA like a restriction endonuclease.
  • Most transposases bind very small specific target sequences which are found frequently, so creates seemingly random mutation
  • Step 3: The transposase integrates the transposon within the cut made in the host genome.
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23
Q

what happens if a transposon is inserted into an exon

A
  • might distupt protein function
  • if it is not in a number of three then the open reading frame will be messed up
  • effect depends on exon size
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24
Q

if a trasposon is inserted into an intron

A
  • may modify bingin site of the splicing factor, may prevent the spcing from taking place, intron in the reading frame
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25
Q

if a trasposon is inserted infront of a gene

A

may effect reggulation of a gene due to trascription genes before gene

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26
Q

what should be in a COI (construct of interest)

A

splice site
marker for integration
reporter gene

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27
Q

who discovered transposons

A

barbara mcclintock 1940s, nobel prize 1983

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28
Q

what diseases are trasposable elements ivolved in

A
  • Neurodevelopmental disorder
  • Myocardial infarction
  • Modulation of immune system
  • Neurodegenerative diseases
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29
Q

what are the two steps of mapping

A
  • use markers to find out on which chromosome your mutationis: association mapping
  • use markers to find out where on the chromosome your mutation is: linkage mapping
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30
Q

what are the two types of markers

A
  • phenotypic markers
  • molecular markers
31
Q

what are phenotypic markers

A
  • Alleles with obvious phenotypes - for years, with geneticists only relied on these marker and flies were the model organism of choice
32
Q

what are molecular markers

A
  • SSLP, simple sequence length repeat polymorphism, microsatallite
  • SNP
33
Q

what are the limitations of traditional mutagenesis screens

A
  • Traditional screens focus on genes that affect specific phenotypes
  • fail to identify genes whose functions overlap or are limited to specific phases of development, or genes that are expressed in a limited number of tissues.
  • Genome projects have revealed that traditional screens have assigned functions to only a minority of a given organism’s genes.
  • Can investigate gene function through an investigation of expression patterns.
34
Q

what is an enhancer trap

A
  • TE insertion allows the detection fo the expression controlled by the nearby enhancer elements
  • is called an enhancer trap as the COI is a reporter gene and a mutant insertion (we only care about the insertion near enhancer elements)
35
Q

how do we identify the gene present

A
  • sequence the transposable element flanking DNA
  • if the enhancer element is close to the gene, you can identify a gene of interest
  • 98% of the fly ET, the TE insertion doesnt modify endogenous gene expression
36
Q

what is required in the COI for reverse genetics

A
  • Promotor
  • Marker of integration
  • Normal gene
  • Mutant gene inserted randomly and then see what happens
  • Sequence we know is required for reverse genetics
  • Still inserted randomly in the genome
  • Adding a construct into the genome
37
Q

principles of targeted insertion through homologous recombination (HR)

A
  • COI surrounded by sequences homologous to targeted insertion site
  • COI delivered as a linear fragment or a vector
  • HR occurs in meiosis and before each mitosis
  • one part of the endogenous gene is replaced by one part of the vector (target insertion)
38
Q

what sequences must be known for the insertion to work

A
  • we need to know BC, DE for the crossing over to work
  • we need to know exactly where we want it
  • 2.5-6 kb of flanking homologous sequences required
39
Q

What is the benefit of targeted insertion

A

We do not have to wait for the mutant, we know exactly where it is

40
Q

what is a knock out

A
  • delete a gene/promoter or a critical portion of it
    mutant
  • marker replaces all or a great part of the endogenous gene/ promoter
41
Q

what it knock down

A
  • delete a small portion of a gene/promoter
    mutant
  • marker replaces a small part of a gene/promoter
42
Q

what is a knock in

A
  • add contruct to genome
    mutant
  • DNA (specific mutation, reporter gene, extra gene from same or other species) often fused to marker
43
Q

what is needed for a knock out

A

2 integration markers
- one to replace your gene or gene portion or promoter
- one to ensure the integration happened via HR

44
Q

how to do a knock in

A
  • replace exon X with a reporter gene using the KO method, select cells with reporter gene and apply drug , cells without M2 survive as M1 is anibiotic
  • replace the reporter gene with your mutant DNA and apply drug, cells without M2 survive
45
Q

what amorphs result from Kock out mutations

A

targeted deletion in gene/promoter

46
Q

what hypomorph result from knock down/ knock in mutation

A

targeted small deletion in gene/P or introduction of key mutation

47
Q

what anitmorphs result from knock in mutations

A

targeted small deletion or introduction of key mutations

48
Q

what hypermorph (overexpression) result from knock in mutations

A

bypass or boost promoter replace gene by mutant with increased translation or a consitutevely atived allele

49
Q

what neomorphs (misexpression) result from knock in mutations

A

place gene under the control of a new promoter or introduce a mutant that changes function

50
Q

how are knock out’s done with crispr

A

Non homologous end joining
KO with 1 cut (indel) or 2 cuts (large deletion)
- repairs are very rarely in multiples of three
- easy production of amorphs

51
Q

how are knock in’s done with crispr

A

homologous directed repair
easier than HR because requires much shorter homologous sequences (can do 2 cuts for large insert

52
Q

what are the two types of HDR

A

short range HDR
long range HDR

53
Q

what is short range HDR

A
  • most efficient but only inserts small fragments (GFP)
  • use Cpf1 instead of cas9
  • doesnt do blunt ends when it cuts rather sticky ends
  • different repair mechanisms
  • allows very small construct to be inserted into the genome
54
Q

what is long range HDR

A
  • same sa HR mechanism but more efficient because cut pushes repair to occur, therefore needs less homology sequence to work
  • more effective than HR
55
Q

what is the size of insert with short range HDR

A

insert 10bp-2kp, 30-40 bp of homology

56
Q

what is the size of insert with long range HDR

A

insert: up to 6kb, 500-1500bp of homology
normal HR: 2.5-6kb of homology

57
Q

why is ca9 not used in HDR

A

p53-mediated DNA damage response
- uses Cpf1
- p53 prevents cancer

58
Q

what are the cons of using CRISPR-cas9 in making mutants

A
  • off target effect
  • can be toxic to cells (p53-medaited apoptosis of p53 mutated
  • edits are perminent (not always desired
59
Q

what is the use of CRISPR-dead-Cas9

A

regulates transcription
- dead Cas9 can be fused to another protein. it binds specifically to DNA where it delivers the fused protein
- can be fused with a transcription repressor or activator domain
- can also be transcripted with epigentic modifications such as dimthylase and methylase

60
Q

what is CRISPR KO screening

A

look at a phenotypic effect of treatment when a gene is KO (amorph)
-uses cas9

61
Q

what is CRISPRi screening

A

interference screening (knock down - hypo) - look at effect on essential genes
- Dead cas9 fusion

62
Q

what it CRISPRa screening

A

activation screening (hyper) - look at effect when gene is overexpressed

63
Q

what is the driver line

A

specific complementary DNA

64
Q

what is an example of sanctio-temporal iduction

A

tet on/off

65
Q

how does tet-off work

A

addition of a drug (tetracycline or doxycycline), inhibits binding of tTa to TRE (target transgene) causing the gene expresseion not to occor

66
Q

how does tet-on work

A

reverse tetracycline transactivator (rtTA) alone does not bind to TRE meaning no activation
however addition of rtTA with a drug such as doxycycline causies transcription

67
Q

what are the two systems of TET

A

TET technology comprises two complementary circuits: the tTA-dependent circuit (TET-Off system) and the rtTA-dependent circuit (TET-On system).

68
Q

when is RNAi typically used

A

to make amorphs or hypomophs (only affecting transcription)

69
Q

how does RNAi work

A

blocking or reducing the expression of chosen gene (knockout or knockdown)

70
Q

what is the prefered organism for RNAi

A

worms (not amenable to other genetic approaches)

71
Q

what are the steps for RNAi functio

A
  • dsRNA is longer than needed
  • Recruits dicer
  • Dicer makes it a 22 nucleotide system
  • System finds complementary RNA
  • Binds to and degrades it
72
Q

how is RNAi delivered

A
  • Synthetic molecule or in inducible plasmid (by drug/nutrient) - short lived action.
  • In a virus that can insert in the genome (only way to create stable RNAi lines). Choice of virus: infect all or specific cells
  • Worms are given the dsRNA in the bacteria they feed on.
  • For plants, the dsRNA is often applied to a cell culture or through a virus that can insert in the genome.
  • For flies, the dsRNA can be injected in the developing embryo or inserted and made inducible via the UAS-Gal4 system
  • For mice, the dsRNA is introduced in embryonic stem cells using viruses and made inducible via the Cre-loxP system/ Tet On/Off system
73
Q

what are the pros of RNAi

A
  • In theory should knockdown ALL the normal copies of the gene (i.e. create a homozygous effect) particularly interesting for polyploid plants and for non model organisms whose genome has been sequenced (e.g. mosquitoes, trypanosome, etc)
  • Worms are very good at showing the RNAi effect easily in all cells
74
Q

what are the cons of RNAi

A
  • Effects are variable for different genes and different organisms. Worms are very good at showing the RNAi effect easily in all cells, but some organisms only show it a subset of cells.
  • RNAi shows sometimes cross-reactivity and an off target effect - i.e. knockdown other RNAs with similar sequences.
  • RNAi does not work in yeast

Organismal genetics (4 decks)

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