Organismal genetics Flashcards

Question

What does it indicate if a trait disappears in F1 and reappears in 25% of F2?

Answer 1

The trait is recessive to the dominant trait ## Footnote This follows Mendel's laws of inheritance.

Answer 2

9:3:3:1 ## Footnote This ratio represents the expected phenotypic outcomes of a dihybrid cross.

Answer 3

He studied seed size in pea plants and introduced statistical concepts like regression and correlation ## Footnote Galton's work laid the groundwork for the biometric approach in genetics.

Answer 4

Mendelian focuses on discrete traits, while biometricians emphasize continuous variation in traits ## Footnote This conflict delayed the integration of genetics with natural selection theory.

Answer 5

Some phenotypes are controlled by one gene, while others are influenced by multiple genes and environmental interactions ## Footnote This complexity reflects the multifactorial nature of many traits.

Answer 6

False ## Footnote Many phenotypes are determined by multiple genes and their interactions.

Answer 7

He was a founder of the movement that led to racist policies and forced sterilization programs ## Footnote Galton's ideas, while influential in statistics, contributed to harmful social policies.

Answer 8

Mendelians believe variation in discrete characteristics drives evolution (new mutants have large effects) Biometricians believe that evolution is natural selection acting on continuously distributed characters

Answer 9

Phenotypes that vary along a range of values, controlled by interactions between genes and environment.

Answer 10

The inheritance pattern of phenotypes that can be measured on a continuous scale.

Answer 11

Quantitative trait loci are genes that cause quantitative traits, while Mendelian traits follow discrete inheritance patterns.

Answer 12

Genes that produce a detectable effect on phenotype.

Answer 13

Genes can be additive or dominant in their effects.

Answer 14

Each gene contributes equally to the variation in phenotype.

Answer 15

One gene has a more significant effect on phenotype than other genes.

Answer 16

To determine if variation in a trait is controlled by genetics, assessing if relatives are more similar than non-relatives.

Answer 17

Because the effects of many genes are too small to measure individually.

Answer 18

Cellular compartments that store melanin ## Footnote Melanosomes play a crucial role in determining pigmentation in humans.

Answer 19

* Eumelanin - black/brown * Pheomelanin - red/yellow ## Footnote These two types of melanin contribute to the variety of human skin and hair colors.

Answer 20

* Differences in number of melanosomes * Type of melanin * Size and shape of melanosomes ## Footnote These factors are influenced by genetic variants that affect pigmentation.

Answer 21

* Protection against UV radiation * Synthesis of vitamin D ## Footnote Geographic variation in pigmentation serves these important biological functions.

Answer 22

It generates strains that are homozygous at all loci and have different genotypes at many loci.

Answer 23

It involves identifying genomic variations between strains that affect phenotype. | QTL - Quantitative trait locus

Answer 24

F1 is produced by crossing two inbred strains and is heterozygous for all loci.

Answer 25

To study alcohol preference behaviour by offering one bottle with water and another with ethanol. -> D2 consumes less than 1g/kg/day while B6 consumes 10g/kg/day.

Answer 26

Approximately 5%.

Answer 27

Genetic factors.

Answer 28

F2 lines, each mouse having different combinations of homozygous DNA.

Answer 29

There has been recombination or crossing over.

Answer 30

Markers which are polymorphic between strains + have a known chromosomal location used: SSR used, gel electrophoresis to detect SSLP of PCR product -> animals w/ 2 bands are heterozygous. For SNPs, can detect by sequencing PCR product that amplifies SNP ## Footnote SSR - simple sequece repeat (microsatellite) SSLP - simple sequence length polymorphisms

Answer 31

Can narrow down scope of investigation depending on study. e.g. for alcoholism in mice - expressed in brain, synaptic function, gene w/ sequence change that causes functional change to protein (Stxb1)

Answer 32

Transgenic rescue - add wt allele to mutant & see phenotype disappear Can use knock-in to generate same sequence change in wt mice + induce same phenotype

Answer 33

Study of heritable changes that occur without a modification in DNA sequences of genes - histone modification - DNA methylation - nucleosome remodelling (FACT facilitates removal + reincorporation of H2A-H2B dimers) - non-coding RNA mediation ## Footnote extra readin**

Answer 34

Addition of methyl group to cytosine (C) - blocks transcription due to tightly packed chromatin. - methylated genes generally not expressed - occurs in mammals + plants Form of 'imprinting' , can change depending on parental chromosome ## Footnote 5' position of cytosine residues in a CpG dinucleotide

Answer 35

Micro-deletions on Chromosome 15. - PWS , short stature, impaired cog development, respiratory distress, obesity Paternal allele mutated + maternal imprinted (silenced) -> loss of SNRPN expression - AS, developmental delay, hyperactivity, impaired cog function, seizures Maternal allele mutated + paternal imprinted (UBE3A gene)

Answer 36

- imprint establishment in gametes - matured and then maintained in fertilisation - imprint read into differentiated cells - erased in primordial germ cells Differentiated gametes undergo widespread epigenetic reprogramming Zygotic genome activated at 2 (mice) or 8 (humans) cell stage. In mammals - 2 cell lineages of trophectoderm + ICM. ICM -> forms epiblast & primitive endoderm ICM + TE have different degree of methylation. ICM forms embryo + TE forms placenta.

Answer 37

Cloning e.g. Dolly the Sheep 1996 Nucleus removed from adult cell, oocyte transferred w/ no nucleus. Implant it into host mother -> nucleus/DNA supplied from adult w/ imprint in place - electric current applied so nucleus fuses w/ empty egg 4% results in birth of live young

Answer 38

Often unhealthy live young from cloning. - respiratory distress, 2-fold increase in birth weight - skeletal, immunological + placental defects Caused by improper placental development due to imprinting defect. - abnormal X chrom inactivation in females + imprinting (far less methylation) defects both genders

Answer 39

Cloned animals used in natural mating -> offspring do not show defects (LOS) They have same genotype but not same epigenetic imprinting SO yes. Gene expression changed -> determines survival & phenotype

Answer 40

Chromatin organisation formulates gene expression pattern. Epigenetic signatures vary by cell type & in disease. Chromatin & miRNA environment dictates cellular gene expression

Answer 41

Mapped areas of infant mortality + low birthweight in UK 1910s, analysed death rates due to Cardiovascular + type II diabetes 1970s -> correlation w/ deprivation Hypothesised nutrient conditions during pregnancy affect health in adult life -> permanent metabolic changes (thrifty phenotype) Programming - caused persistent physiological + metabolic changes which cannot be compensated by adequate nutrition after birth

Answer 42

Maternal starvation during pregnancy limits intrauterine growth of offspring -> higher CV + diabetes rates -> placental abnormalities + maternal stress Offspring had children w/ low birthweights -> not found w/ siblings not born in famine

Answer 43

Individuals exposed to famine in gestation -> reduced methylation of IGF2 gene at age 60. Vs siblings not conceived in famine had higher levels of IGF2 methylation SO uterine environment encodes epigenetic state during development - maternal obesity increases risk type II diabetes in child monozygotic twins discordant for type II diabetes

Answer 44

Fed normal and low protein (LP) diet during gestation + lactation. - Hnf4a polymorphisms confer type II diabetes risk - Hnf4a expression reduced in LP diet rats - persists throughout life - Hnf4a levels normally decrease in old age LP diet induces DNA methylation + histone acetylation on Hnf4a enhancer region -> changes expression throughout life

Answer 45

Refers to altered phenotypes in offspring through beyond F2 generation to F3. - need to eliminate effect on gametes to confirm - offspring exist as germ cells in grandmothers pregnancy - can study w/ animal models Multigenerational exposure can be seen in grandchildren (F2) but no further.

Answer 46

Folate deficiency - folate is carrier of methyl groups needed for cellular metabolism. Mttr gene needed for normal metabolism of folate + methionine Mttr mutation (gt) - insertion leads to folate metabolism deficiency Analysed gt heterozygote intercrosses: - phenotypes did not correlate w/ maternal or offspring phenotypes - 45% zygotes showed abnormal phenotype in development - abnormal phenotypes present in all offspring genotypes equally (F2) SO hypothesised maternal + grandparental genotype might correlate w/ offspring

Answer 47

Analysed pedigree charts - pregnancies w/ abnormal litters always had mutant grandparents - global DNA methylation levels disrupted in mutant adult livers - Wt littermates show disrupted methylation levels in adult liver Wt pre-implantation embryos w/ mutant maternal grandparent transferred to wt female for development Wt embryos w/o mutant maternal grandparents used as controls -> found congenital defects only in offspring w/ mutant maternal grandparent ## Footnote Defects persisted for at least 5 generations -> transgenerational

Answer 48

Have mutliple functions. Tend to generate lethal phenotypes early on SO mosaics, temp sensitive mutants + conditional systems used

Answer 49

Flies have 1 notch Notch 1 + 2 highly conserved (similar to flies) Notch 3 has fewer EGF repeats + no TAD domain Notch 4 has lost NLS, no TAD + fewer EGF repeats ## Footnote Notch is signalling mol -> involved w/ proliferation

Answer 50

Phenotype -> genotype - Use existing mutants/ create random ones - Cross mutants w/ each other to detrmine is on same gene (complementation test) - Map where gene is (linkage analysis + positional mapping) + obtain its sequence Can then find out expression patterns, isoforms, compare to database, infer functionfrom phenotype or dtermine molecular nature of mutants ## Footnote Working hypoethesis can then be tested: biochemica/cell based assay, find partners or targets, engineer new mutants (KD, KO)

Answer 51

Developed after genome projects (human 2003) + RNAi (CRISPR-Cas9) Use gene/protein sequence to: - search database for info on protein domains/homologs - find out expression patterns - look for isoforms - engineer KD or KO, look at phenotype Working hypoethesis can then be tested: biochemica/cell based assay, find partners or targets, engineer new mutants (KD, KO) ## Footnote Finds phenotype encoded by a gene

Answer 52

LoF: Amorph (none), hypomorph (reduced), antimorph (antagonistic to wt) GoF: Hypermorph (increased), neomorph (novel) ## Footnote Many mutations often change enhancer elements (EEs) so transcription altered

Answer 53

- Often recessive (unless on X), as normal allele can compensate for loss - can show dominance if in haploinsufficient genes -> only if protein works in protein complex OR quantity of protein is critical for function

Answer 54

Interfere w/ wt function + more severe than 1 amorph Common in proteins w/ binding partner (receptor/dimer), mutation sequesters partner protein -> non-functional

Answer 55

- high transcription or failure to degrade transcript/protein -> constitutively active protein (e.g. receptor always bound regardless of ligand)

Answer 56

Novel function - commonly due tp chnage in expression pattern (mutation in reg sequences) - also due to translocations -> produce chimeric fusion protein

Answer 57

EGF-LR repeats: ligand binding altered -> amorph (cant bind), hypomorph(less efficient), possible neomorph (new ligand) LNR repeats: cleavage altered -> amorph (no cleavage), hypermorph (cleavage w/o ligand) one NLS: not directed to nucleus -> hypermorph ANK repeats: prevents regulatory binding -> hypomorph (no +ve partner in nucleus), hypermorph (cant bind -ve reg in cytoplasm) PEST: less Notch degradation -> hyermorph

Answer 58

Mobile DNA elements that can jump ~50% human genome - use cut & paste mechanism Has 2 inverted repeats (IR) at start + end (complementary to each other, 15bp) Also has gene encoding tranposase (inc promoter) -> cut + paste -> has both restriction endonuclease + integrase function | tranposase ofetn imprecise ## Footnote semi parasitic/viral origin

Answer 59

TEs that have lost genes for transposition. Use transposase from another related TE. Useful for engineering - has space to insert a COI - can have nromal/mutant gene, reporter gene, promoter + marker for integration - transposase from vector 2 binds IRs on vector 1 -> structure folds + trnaposase cuts host DNA - COI inserted randomly in progeny genome | COI is construct of interest

Answer 60

Exogenous gene added into experimental system that can be expressed under its own OR different promoter.

Answer 61

DNA transposons in flies. COI w/ weak promoter + marker for integration (w+) can rescue mutant phenotype (w-/w- or w-/Y) Can insert into germline cells - coinject 2 vectors into w- blastoderm. All females selected + crossed to w-/Y males -> orange eyed flies contain TE , partial rescue in transformed cells

Answer 62

- digest DNA w/ restriction enzyme - re-ligate to form circular DNA - digest again w/ P-element specific r. endonuclease - PCR w/ primers that bind to inverted repeats (ulta specific) - can then sequence PCR product + determine insertion by comparison w/ genomic database

Answer 63

- DNA transposons e.g. P-elements - RNA intermediate transposons/ retrotransposons ^ autonomous - Non-autonomous TEs for engineering ## Footnote retroviruses used in zebrafish + mice, more technical limitations than RNA transposons +ve used to identifiy cancer genes -ve used in lab to infect blood + mammary cells (leukemia)

Answer 64

modified SB transposon - tool of choice for insertional mutagenesis in vertebrates for cancer gene discovery - infects all tissue type - more controllable (infects subset of cells in whole tissue) - can create several mutations in one cell, tumuour results from ~6 mutations in cancer genes

Answer 65

Reverse genetics approach. - wt copy introduced as COI in a TE or retrovirus COI has wt version w/ integration marker behind a ubuquitous promoter (e.g. HSP binds sigma32 in RNA pol to initiate transcription, CAG or PGK) -> see if phenotype rescued

Answer 66

Phenotypic - alleles w/bvious phenotypes Molecular - geneome maps established using polymorphic molecular landmarks SSLP: simple sequence length repeat polymorphism (microsatellite) SNP: short nucleotide polymoprhism

Answer 67

- Mutant hom organism selected (m/m), DNA + SNPs extracted at desired locations. - Choose mapping stock + cross to mutant - Backcross F1 to mutant stock - Select m/m organisms in F2 + sequence SNPs ## Footnote m linked to nearby SNP, will segregate w/ it in meiosis -> homozygous m/m also homozygous for nearby SNP

Answer 68

- Select M/+ instead of m/m & cross to mapping stock - In F1, select M/+ not m/m, the backcross to mapping stock - M/+ selcted in F2, look for 100% progeny Always backcross to mapping stock ## Footnote M linked to nearby SNP so will segregate w/ it in meiosis. Mapping is iterative process

Answer 69

Investigating expression patterns gives insight into gene function. Enhancer trap screens - started in flies - TE random insertion allows detection of expression controlled by nearby EEs ## Footnote - EEs work independently or in combination - EEs can be located far from gene sequence or in introns

Answer 70

w-/w- embryos injected w/ 2 TE vectors (transposase + COI), weak promoter, integration marker (w+) & reporter gene not present in organism (lacZ, GFP) female w-/w- crossed to male w-/Y -> transposition cuases orange eyed flie w/ TE Can check proximity of insertion to EE by checking expression partter of reporter gene. e.g. lacZ compared in wt vs m-/m- -> TE insertion revealed vestigial expression controlled by 2 separate EEs in wing

Answer 71

Knock out - delete gene/promoter or critical portion of it, marker replaces all or great part of endogeneous gene/promoter Knock down - delete small portion of gene/promote, marker replaces small part gene/promoter Knock in - add construct to genome or replace a gene, DNA(mutation/reporter gene) often fused to marker

Answer 72

Gene of interest deleted. 2 integration markers - 1 to replace gene or promoter (M1) - 1 to ensure integration via HR (M2) ## Footnote Typically M1 and M2 code for proteins that are sensitive to drug treatment: e.g. M1 procures resistance to drug (e.g. antibiotic resistance gene), while M2 makes the cell die when exposed to drug (e.g. HSV-TK makes cells sensitive to Ganciclovir). NB: M1 can also be a reporter gene, e.g. GFP.

Answer 73

Double replacement method w/ +ve & -ve selection -> introduces subtle mutation w/ residual exogenous sequence 1) replace chosen exon w/ reporter gene (GFP) using KO -select all cells w/ GFP, apply drug -> all cells w/ no M2 will survive 2) Replace reporter gene w. mutant DNA, select cells w/ no GFP, apply drug -> cells w/ no M2 will survive

Answer 74

Cas9 - helicase + endonuclease w/ 2 'scissors' Guide RNA - matching sequence ~20nt, includes PAM sequence (NGG for Cas9) which is very common, secondary loop structure -> only specific segment binds. NHEJ -> INDELS resulting in premature stop codon (KO) HDR (homology directed recombination) requires shorter homologous sequences than HR -> can do 2 cuts for larger insert (KI) ## Footnote HR less effective - relies on mitosis for effective function, needs more flanking DNA

Answer 75

Short - Cpf1 (uses sticky ends), most efficient but only for short fragments (GFP) 10bp-2kb, requires short single strand donor DNA Long - Cas9 (uses blunt ends), same as HR mechanism but more efficient as cut stimulates repair to occur, less homology sequences needed to work insert up 6kb (0.5-15kb homology needed)

Answer 76

Edit single cell embryo + implant morula. Does not need ES cell culture or chimera - Cas9 infects single cell embryos, guide RNA + donor RNA/DNA -> KO/KD/KI - one generation process (quicker) 85-95% success, $250 min, only 4 weeks, can be done in house, several mutations possible per mouse ## Footnote vs HR, 50-55% success, min$25,000, 6 months for 1 mutation

Answer 77

CRISPR cuts DNA. Elevated p53 can induce apoptosis, surviving cells can mutate p53 in response to selection pressure & turn into cancer (dangerous in vivo) Off target Crispr effect can be toxic to cells, any edits are permanent ## Footnote p53 is guardian of genome (oncogene when mutated)

Answer 78

Catalytically inactive but can bind specifically + irreversibly to DNA. Can inhibit transcription if sitting on promoter - KO effect but no gene deletion. dCas9 can be fused to another protein which it delivers via guideRNAs- can regulate transcription by gene regulation (activator/repressor domains) OR epigenetic modification (DNA methylase/demethylase) ## Footnote repressor domains like KRAB -> zinc finger-like domain that induces chromatin compaction (HDAC recuritment)

Answer 79

for Cas9: CRISPR KO screening - looks at phenotypic effect of tretament where gene is KO (amorph) dCas9 & fusion: CRISPRi - interefence screening (KD -hypo), looks at phenotypic effect on essential genes CRISPRa - activation screening (hyper), effect when gene overexpressed ## Footnote requires library of sgRNA - can be transduced into cells whihc are then 'treated'

Answer 80

Induce recombination or expression in a given space + time 2 transgenic constructs introduced using TEs, HR, CRISPR-Cas9 depedning on organism: 1) driver line - has tissue/cell specifier (drives TF/recombinase expression) 2) responder line - has inducible system (responds to recombinase/TF) - require crossing 2 established transgenic lines. Transgenic constructs are in all cells of the animal, but only produce desired effect when animals crossed. ## Footnote can add extra time specifier

Answer 81

Tool from P1 bacteriophage, used in yeast + mice -> deletion mutations (+ inversion & translocation) - Cre recombinase catalyses recombination between 2 loxP sites - Orientation of loxP sites dictates combination result -> circular DNA Cre is under control of specific promoter (driver) - where Cre is expressed, construct is floxed/deleted ## Footnote useful for pleiotropic or lethal genes

Answer 82

Cre fused to modified oestrogen receptor that only reacts when tamoxifen present (WHEN), not to endogenous estradiol. Tamoxifen binds receptor + moves it to nucleus w/ fused Cre. Floxing happens in cells: - where Cre is expressed - when Tamoxifen provided

Answer 83

GAL4 binds UAS sequences in yeast - activates transcription. No GAL4 or UAS in flies - so introduced into 2 separate trasngenic line using P-elements that contain : & Not targeted insertion so copy of transgene added

Answer 84

TE insertions just behind EEs are selected - ubiquitous in all cells. GAL4 can be behind an HSP (if P inserted randomly) -so promoter only activated when temeprature elevated to 29C temporarily Can combine GAL4 + GAL80(TS) driver: GAL4 inserted behind specific enhancer (WHERE) GAL80 inserted behind ubiquitous promoter (e.g. Tubulin) change in temp determines WHEN (see footnote) ## Footnote At 18C, GAL80TS is expressed in all cells and where GAL4 is expressed, it binds to GAL4 and prevents it to activate transcription at UAS site. When temperature rises at 29C (WHEN), GAL80TS is inactivated (change of conformation due to temperature) and stops biding to GAL4, letting GAL4 activate transcription at UAS site.

Answer 85

Similar to UAS-GAL4, tTA expression driven by specific promoter & binding to TRE (WHERE) tTa inactivated when drug added (tetracycline or doxycycline), ON -> OFF vs rtTA which only binds TRE if Dox added (OFF ->ON) TRE - tet response element | tTA - tet controlled transactivator

Answer 86

Effects comparable to mutant phenotype but notdue to mutations Caused by RNAi (KDs & KOs)

Answer 87

RNAi blocks/inhibits expression of chosen gene. Delivered via synthetic mol or inducible plasmid (short lived action) through virus thatcan insert genome. e.g. - worms given dsRNA in bateria they feed on - plants, dsRNA applied to cell culture or via virus (TMV) - flies, dsRNA injceted in developing embryo + made inducible by UAS-GAL4 - Mice, viruses used to introduce dsRNA to embryonic stem cells, inducible via Cre-loxP or Tet systems ## Footnote - Useful in organisms not amenable to other genetic approaches (worms) - Effect seen on whole organism in worms + plants

Answer 88

Pros: - KD all normal copies of a gene -> homozygous effect (good for polyploid plants) - Worms demonstrate RNAi effect easily in all cells Cons: - effects variable for different organisms/genes, can only show effect in subset of cells - can show cross reactivity + an off target effect (KD other RNAs w/ similar sequences) - does not work in yeast, xenopus or zebrafish

Answer 89

Mutations cause murine small eye + human anirida phenotypes. - it has a paired box domain - expressed in brain, spinal chord + all stages of eye development eyeless homolog in flies discovered, a.acids highly conserved (especially DNA binding domains) - ey2 and eyR (2 existing spontaneous mutations) ## Footnote Part of pax gene family Cre-loxP used to study Pax6 function in mice

Answer 90

In situ hybridisation experiments w/ eyeless RNA probe , stage 15 embryos stained for eyeless - ey2 is hypomorph Sequenced ey2/ey2 flies -> spontaneous transposon insertion in intron between exon 2 & 3 - intron has an EE so insertion prevents regulatory binding Residual transcription must be due to another EE ## Footnote Same for eyR - but has different TE

Answer 91

1. Te insertion , randomly integrate into genome 2. crossed to *need 2 UAS-ey constructs for full rescue of ey2/ey2 Can also be misexpressed in wrong location -> ectopic eyes on wing, antenna, leg + body. - eyeless master gene that controls eye development ## Footnote Mice + Human Pax6 can subsittute for eyeless in rescue/misexpression experiments.

Answer 92

In flies: [eyeless-GAL4 driver] stock mutated using EMS screen -> obtain mutations eyeless regulatory regions Cahnges intranscription monitored by crossing flies to UAS-lacZ/GFP lines, specific point mutations identified In mice: - used HR KI to replace parts of promoter region + fused to lacZ - tested which modification altered lacZ expression | EMS - ethyl methanesulfonate

Answer 93

- rapid life cycle (10 days) -> large numbers & easy/inexpensive to maintain stock - easy to obtain mutants -> chemical, x-rays, TEs in males, infrequent genetic redundancy - well studied organism -> detailed physical map from polytene chromosomes, sequenced genome, linkage analysis given detailed genetic maps

Answer 94

Has XY determination + 3 autosomal chromsomes. - chrom4 very small w/ few genes + no meiotic recombination - no meiotic recombination in males Dominance/recessive depends on encoded phenotype e.g. Curly is dominant by Cy/Cy is recessive lethal in embryos

Answer 95

Df - loss of region of chromosome that removes copy of gene of interest Dp - reintroduction of wt gene, Dp(vg+) rescues -/- back to wt phenotype (defines mutant as LoF) ## Footnote Used to identify LoF mutations

Answer 96

Hypo - wa/wa leads to orange eyes (partial LoF), weaker phenotype (more red) than wa/Df - wa has more function than Df A - w1/w1 or w1/df leads to white eyes (complee LoF)

Answer 97

Notch N/+: N is amoprhic allele - dominant function as 1 copy of wt insufficient to maintain normal function N/Dp - N phenotype completely rescued

Answer 98

Negative activity - antagonise wt gene copies e.g. ebony (e) - dominant negatives by competing w/ (+) reducing wt gene product activity Can interrupt tetrameric complexes OR prevent domain interactions w/ susbtrates

Answer 99

GoF e.g. Elipse in EGF receptor -> increased signalling Disrupts eye formation.

Answer 100

Complementation test using recessive, LoF mutants -> can see if 2 mutants on same gene or not. Complementation groups worked out using phenotype observed. Analysis cn then be used to mpa mutations - use deficiencies in chromosome compared with lethal mutants on another to see if outcome is viable or not - need to know endpoints of DFs e.g. salivary gland polyten chromosomes in Drosophila

Answer 101

when two different mutant alleles are present at the same locus in a diploid organism e.g. Hypermorhic Elipse complemented by hypomorphic allele despite being mutations on same gene -> intragenic complementation ## Footnote if assumptions not satisfied

Answer 102

Comeplex gene locus Has dpo/dpo oblique & dpv/dpv vortex pehnotypes. Intagenic complementation can arise when different parts of protein have different functions. OR different mutations lie in dfferent control regions needed for expression of gene in different tissues ## Footnote makes 1MDa protein

Answer 103

Alkylating agents - ethylmethanesulphonate (EMS) -> very efficient, induces point mutation Radiation - X-rays + gamma-rays -> chromosomal rearrangements, less efficient, easier to map P-elements - transposons -> insertions, non-random, low frequency, easy to map

Answer 104

Single cell has many nuclei - very different mechanism to vertebrates Acquired identitiy along AP axis (3 thoracic segments) ## Footnote Become primordial germ cells (polar) - forms cellular blastoderm

Answer 105

Development via genes invovled in defining AP axis Looked at stereotypical sgemented structures - bristly + naked cuticles ## Footnote AP axis defined at single cell resolution

Answer 106

Need to use F2 genetic screen. Unknown heterozygote mutant males produced in F1, then crossed to wt females. Then self cross F2 progeny -> identify those carrying embryo lethal mutations (a-/a-) BUT cannot distinguish +/+ from a-/+ so a- easily lost from stock. SO use "balancer chromosome" e.g. CyO -> homozygous cn flies mutated + F1 males cross to females w/ balancer chromosome -> orange eyes selected + wt rejected -> F2 progeny self crossed w/ cn phenotype form each line + identify embryo lethal mutants If mutated chromosome carries recessive lethal mutation then all viable progeny will have curly wings ## Footnote no recombination in male meiosis

Answer 107

Aims to generate many mutant lines so there is high probability of collecting relevant mutations - can colect dead embryos + screen for phenotype of interest (AP defects) Complementation testing then used to differentiate mutants to comp groups

Answer 108

Allows distincton between a-/+ & +/+ so a- not lost from stock - carries many inversions which suppress recombination - carries dominant mutation w/ visible phenotypic marker (Cy) - carries recessive lethal mutation - also can have recessive mutation w/ visible phenotype (cn, orange) CyO is a 2nd chromosome balancer (carries Cy, cn mutations) Want to end up with a-/Cyo, Cy, cn in our screen -> stable stock (viable w/ Cy phenotype) a-/a- is lethal CyO/CyO is lethal

Answer 109

Bicoid (TF) mutants end up w/ 2 abdomens instead of head, thorax + abdomen So early development of embryo in drosophila depends on maternal genotype Cytoplasmic transplant experiments: - drosophila germ lines have 1 oocyte + 15 nurse cells connected via sytoplasmic bridges - nurse cells secrete bicoid mRNA into oocyte - diffusion or bicoid mRNA restricted by binding cytoskeleton -> diffusion gradient SO nuclei can 'read' position in embryo + transcribe genes accordingly after nuclei division ## Footnote Not good model for vertebrates where cell dividion occurs from 1st nuclear division

Answer 110

Several segments of embryo missing. Gap genes encode TFs regulating gene expression e.g. Giant & Kruppel gap genes regulated by bicoid gradient -> needed for AP axis formation

Answer 111

Missing alternate segments - expressed in narrow stripes in affected segment + encode TFs e.g. Even skipped & Fushi terrazu Even skipped has complex regulation by +ve & -ve acting DNA binding proteins (Hunchback +, Giant -)

Answer 112

Missing parts of segment - expressed in narrow stripes down to single cell width, regulated by pair rule genes. Frequently encode signalling pathway components (more homology w/ vertebrates) e.g. Wingless & Engrailed ## Footnote Human homologues mutations (upregulation Wingless + Hedgehog) associated w/ cancer Defective Hedgehog in humans -> cyclopia

Answer 113

Alter identity of segment not number - selector genes so initiates gene expression that confers segment identity. Family of genes (evolved by gene duplication) that encode TFs + have homeobox domain (60 a.acid helix-turn-helix DNA binding domain) - fits into major groove of DNA Regulated by gap + pair rule genes -> determines combinations of homeotic genes expressed e.g. Ultrabithorax (Ubx)- haltere becomes wing (LoF - identity changed to more anterior segment)

Answer 114

Homeotic genes repress function of next most anteriorly expressed gene. Ubx LoF - region of expression reduced, so does not repress Antp (extra wing produced) Ubx GoF - region of expression progresses more anteriorly, Antp function turned off (haultere duplication + no wings) ## Footnote Similar effects seen in co-linear order of Hox gene expression in vertebrates. e.g. Human HoxD13 mutation has extra toe

Answer 115

Model for signalling pathway regulating photoreceptor differentiation. ~800 ommatidia (highly ordered), each one comprised of 20 cell types w/ specific roles + locations - identity of each cell easily recognised from position (flies lacking R7 unable to moved towards UV light - forwards behavioural screen) Both sevenless (sev) and Bride of sevenless (Boss) are LoF causing loss of R7 -> form ligand receptor pair which controls R7 fate ## Footnote Boss & sev have no other developmental function

Answer 116

identify genes or regulatory elements that increase or enhance the expression of a particular phenotype Synergistic interaction - if 2 mutations in same pathway then phenotype will be greater than expected from simple addition of phenotype

Answer 117

Mutation in 1 gene pathways suppresses phenotypic consequences of a mutation in another pathway component

Answer 118

Highly conserved GTPase protein - molecular switch, biochemically activated byt RTK in mammals. - GTP/GDP exchange factor Son of sevenless (sos) is homologue of yeast cdc25 which activaes Ras

Answer 119

Downstream of receptor kinase - homologue of mammalian grb2 Is adapator protein between RTK & sos -> allowing recuritment & activation of Ras. Binds Pro rich sequence in sos, binds phosphorylated Tyr on sev.

Answer 120

GTPase activating protein - stimulates enzyme to convert Ras to inactive formation (dephosphorylates) Suppressor screen for negative regulators of signal used - temperature screen perofmed but with not enough sev to produce wt phenotype

Answer 121

Human Ras is an oncogene - mutation to Val locks it in GTP bound conformation Similar mutant (RasV12) engineered into Dros R7 precursor cells -> viable but extra R7 cells RasN12 locks Ras in GDP bound confromation -> dominant negative by competing w/ wt Ras to bind upstream regulatory proteins -> loss of R7 cells

Answer 122

Used to determine if gene function in same pathway + order of function - uses 2 mutations w/ opposite phenotypes e.g. hypermorphic GoF mutation of receptor (b) -> always activated SO increased response - does not matter if ligand (a) mutated or not b mutation is epistatic to mutation of a as presence of ligand is not relevant to activity of mutant receptor encoded by b Phenotype of b* = a* + b*

Answer 123

iMutant sev is GoF, extra R7 cells. Suppressor screen using EMS mutagen identified Raf as downstream effector of actuvated sev receptor. Raf is a ser-thr kinase homologous to mamallian MAPKKK - activated by RTK signalling - has oncogenc version (always active) - extra R7 when mutant expressed in Dros eye

Answer 124

Constitutively active form of Raf expressed. - mutations that suppress phenotype must be downstream Dsor - drosophila homologue of mammalian MAPKK rolled - drosophila homologue of mammalian MAPK Dsor biochemically shown to phosphorylate/activate rolled.

Answer 125

Maize mutations caused by TE insertion - altered pigment phenotypes. Insertions of Ds in pigment gene -> colourless kernals - Ac presence allowd Ds to jump out of gene, so back to Wt OR retrotransposon (LINEs & SINEs) make up large proportion human genome - could be transposition competent - linked to human disease: neurofibramatosis, MSD, haemophilia

Answer 126

+/- --> -/- can occur by partial/complete chromosome loss OR mitotic recombination (sister chromatid exchange)

Answer 127

Abnormal, caused by radiation -> chrom breaks + then recombined - sister chromtaid exchange Two daughter cells can either have same parental genotype OR different non-parental genotypes (hom mutant + hom wt)

Answer 128

Can study consequences of recessive mutations that would otherwise be lethal at early dev stage. Spontaneous MR rate too low - can be increased FLP/FRT system (geenrates MR at defined spot in chrom, high efficiency): Transgenic stocks created carrying FRT sequence + FLP recombinase - heat shock induced FLP expression allows induction of clones at defined dev stage

Answer 129

Use visible recessive markers on chromosomes - phenotypic marker must be appropriate for tissue + be distal to FRT site e.g. white- in eyes, loss of GFP (Deltex - ZFD protein - endocytoses cell surface Notch)

Answer 130

N & Dl bind each other + regulate differentiation N-/N- mutant clone differentiates extra neural cells - shows autonomy -> phenotype of N-/N- not rescued at clone boundary by present of wt N in adjacent cells. Dl-/Dl- mutant clone differentiates extra neural cells - shows non-autonomy -> extra neural cells only arise in centre of clone, those at boundary rescued by wt Dl+ in durrounding cells ## Footnote Cell autonomous: gene product required in cells where it is expressed e.g. signalling receptor, TFs Cell non-autonomous: gene product not needed in cells where it is expressed e.g. ligands for signalling receptors.

Answer 131

Inducing heat shock FLP can induce some pathces of cells homozygous for mutant - can then screen for phenotypes. - can screen for recessive phen in adult F1 screen - can create precise deletions, useful for complementation analysis - can detect late mutation effects otherwise lethal at earlier stage Can also help identify tumour suppressor genes - loss of heterozygosity induced by MR caused tumours. e.g. mammalian homologues, lats mutant in mice -> soft tissue + ovarian tumours ## Footnote Contrast to labour intensive F2 screens by Vollhard + Wieschaus

Answer 132

Lineage analysis - negative marking MR can generate negatively marked clones e.g. GSC marked by absence of lacZ staining In mammals: engineered Tamoxifen inducible recombinase used e.g. recombination removes STOP transcription sequence, switching lacZ gene on

Answer 133

Lat inhibition signal refines neural potential. - set of precursor cells acquire neural or epidermal cell potentials - neural cells inhibit other cells around them becoming neural cells. Neurogenic phenotype if lat inhibition absent - caused by Notch or Delta mutants ## Footnote dominant gene mutation which is recessive for other phenotypes.

Answer 134

Saturating large scale F2 screen to detect disruptive mutations in axon guidance pathways. Axons can either go along AP axis or across midline. 2 opposite phenotypic classes - roundabout (robo) & commisureless (comm). Robo is cell mem protein that detects repulsive signal to prevent midline crossing (axon guidnce in CNS) Comm is a protein required to keep robo levels low in cells that cross midline, axons can overcome repulsive signal + cross over ## Footnote Increased robo - axons move away from midline LoF robo - axons move closer to midline

Answer 135

Dros are good MNJ model - large size, easily accessible + ammenible to different studies. Temperature sensitive shibire mutant (dros dynamin) reveals dynamin required for synaptic transmission (vesicle recycling) 29C not recycled -> paralysis 18C dynamin able to recycle vesicles

Answer 136

Benzer - pioneer of genetic behaviour research - electrically shocked flies at tube A + used learning index to see if they remembered to go to other tube B. - screened for mutant lines w/ learning defect, identified dunce mutant. Dunce encodes drosophila cAMP phosphodiesterase

Answer 137

Tully set up massed trial + genetic analysis identified phases of memory - used CXM drug (protein synthesis inhibitor) to detrmine that LTM requires protein synthesis Disruption of cAMP pathway similarly affected STM. Yin: Used temperature inhibition of TF CREB to show that it blocked LTM but not STM - use of temp sensitive Notch mutations -> it is required for LTM formation as is regulates Klingon (cell adhesion protein expression

Answer 138

Behavioural assay screen set up. Dopamine uptake mutant found to reduce sleep. - fumin is spontaneously occurring mutant -> highly active flies at all times of day - fumin gene cloned + identified as uptake transporter of dopamine * dopamine signalling implicated in drosophila sleep regulation ## Footnote fumin mutants show no rebound period after deprivation + instantly rejoin circadian rhythm

Answer 139

Chem mutagenesis + TE insertion screens used. shaker - 1/3 amount of sleep + reduced lifespan, encodes VG ion channel sleepless - reduced shaker expression, encodes cell surface protein similar to snake neurotoxins *indicates 2 component regulatory mechanism

Answer 140

Conditional expression of Na+ channel. Each Gal4 expressing line crossed to a line which express Na+ channel in response to Gal4. - identify which Gal4 lines increased sleep + identify regions of overlapping expression Also used same Gal4 driver to express temp dependent ion channel - flies fell asleep at high temps 31C

Answer 141

Organisms that have multiples of normal chromosome set. e.g. diploid, triploid, tetraploid

Answer 142

(n), an organism belonging to a normally diploid species which has only one set of chroms. Zygotes fail to develop due to deleterious receessive mutations. If hey survuve to adulthood, normally sterile (no normal meiosis)

Answer 143

Intraspecific - multiple chromosome sets due to hybridisation within a species. Can arise naturally by doubling 2n complement to 4n (meiotic error), or be artifically inudced in a lab Odd numbered polyploids mostly infertile - inbalanced gametes. Tetraploids (4n) can be fertiles as balanced gamates are generated ^meiotic pariring

Answer 144

Interspecific - multiple chromosome sets due to hybridisation between different species - must be very closely related e.g Karpachenko's radish + cabbage - 2 different species so gametes not recognised, needs polyplooidisation event in Brassica for (18+18) New species as cannot reproduce w/ eiter to give fertile offspring - allopolyploidy major force of plant evol ## Footnote banana only sterile polyploid plant (3n), 33 chromosomes

Answer 145

In nature: more common in invertebrates but amphibians, reptiles + fish exhibit it, also seen in hepatocytes + 1 rodent (viscacha rat) In lab: - drosophila triploids + tetraploids - yeast can have various levels of polyploidy

Answer 146

Haploid (n) - fungi + human gametes Diploid - human cells (somatic) or yeast cells after cell fusion + mitosis

Answer 147

Changes in parts of chromosome sets. Do not generate viable offspring, but can be tolerated well in some species. In diploids: 2n + 1 (trisomic) 2n - 1 (monosomic) 2n - 2 (nullisomic) In haploids: n + 1 (disomic) Caused by non-disjunction/mis-segregation in anaphase I or II - anaphase I mistakes more profound - humans + animals, it is usually fatal

Answer 148

2n - 1 Missing one copy, usually deletrious. All autosomal monosomy die in uterus (humans), if XO instead of XX, tolerated but causes Turner's syndrome Only trisomic + monosomic autosomal aneuploids in chrom 4 of drosophila survive.

Answer 149

2n + 1 Extra copy of chromosome, deleterious or causes abnormal phenotype. In humans most ddie in uterus: - Patau (chr 13) - Edwards (chr 17) - Downs (chr 21), least deleterious XYY & XXX are fertile, XXY (Klinefelter) is tolerated due to X chromosome inactivation

Answer 150

Aneuploidy generally more dleterious than aberrant euploidy. - Euploidy ratios all 1:1 - monosomy 2:1, trisomy 2:3 mRNA level directly proportional to gene copy number - transcription related to amount of protein SO normal physiology depends on proper ratio of gene product IF reduction in gene copy number causes abnormal phenotype, then it is haploinsufficient

Answer 151

Majority of proteins function in complexes. Aneuploidy causes unlabanced stoichiometry in protein complexes. Accumulation of subunits can be toxic to the cell e.g. high levels of undimerized B-tubulin is lethal, even low levels disrupt MT assembly Aberrant euploidy conserves the gene/protein balance

Answer 152

Deletion Duplication Inversion - important source of cariation in human lineage (human chr2 from chimp 2A + 2B) Translocation Can be caused by either: - DNA breakage + repair - crossing over between repetitive DNA

Answer 153

Parts of chromosome arms swapped between non-hom chromosomes. Meiosis segregation in heterozygte translocation carriers -> chromosomes try to bend to try and pair w/ homologous counterparts -> quadrivalents Alternate segregation (50%)-> 2 viable T type gametes + 2 viable wt gametes, balanced dyads Adjacent segregation (50%) -> all dead gametes due to unblanaced dyads -> segregation leading to viable or dead gametes is random ## Footnote Human reciprocal translocations diagnosed genetically by semi sterility

Answer 154

Non-reciprocal where 2 acrocentric chromosomes fuse near centromere region w/ loss of short arms. -> forms large metacenrtic chromosome + fragment from small arms that fails to segregtae & is lost Causes Down syndrome: extra info of chrom 21 is fused to chrom 14 . Skewed meioctic pairing can cause Down syndrome, monosomy in autosomal chrom can cause lethal gamete

Answer 155

Can relocate a proto-oncogene to a new promoter region. e.g. Burkitt lymphoma - chr 14 promoter always active translocated next to MYC TF activating proliferation genes on chr 8 -> MYC always expressed Can form a hybrid gene e.g. chronic myelogenous leukemia (CML) - hybrid oncogene from translocation of chr 9 + 22 -> permanent protein kinase activity ## Footnote Cause uncontrolled cell proliferation

Answer 156

Changes gene order on chromosome e.g. CDEF becomes CEDF During segregation, dicentric bridge breaks as chromosomes try to resolve loop structure -> 1 normal product, 2 deletion products, 1 inversion products

Answer 157

Can move centromere comparative to a gene. e.g. AB-CD becomes ADC-B Chrosomes trying to anneal w/ partners causes inversion loop -> 1 normal product, 2 duplication/deletion products, 1 inversion product e.g. A or D duplicated after meiosis I ## Footnote Larger inversion w/ crossing over inside inversion loop -> problems w/ gametes

Answer 158

Inragenic - small deletions within a gene, can inactivate gene (similar to null point) Multigenic - several genes missing, severe consequences -> genetic imbalances Can exhibit pseudominance. ## Footnote pseudominance - recessive allele shows dominance due to deletion of dominant alleles on corresponding homologous chromosome

Answer 159

IN meiosis: failure of pairing between normal segment + its corresponding homolog deleted segment -> deletion loop Can be viewed in polytene chromosomes in drosophila

Answer 160

Dri du chat syndrome: het deletion of tip of chr 5 - microencephaly, metal retadation - low fatality William syndrome: 1.5Mb deletion of chr7 homolog (17 genes) - learning disability, CV disease - frindly + social personality, good language skills *thought to be caused by unequal crossing over between repetitive sequences -> haploinsufficiency

Answer 161

Tandem - duplicated regions adjacent to each other Insertional - extra copies somehwere else on genome Segmented - large scale, most of the duplication is disperesed but some may be in tandem -> important substarte for nona-alllelic recombination ## Footnote duplication loops can be formed in meiosis - homolog duplicated segment cannot pair

Answer 162

Alter gene dosage e.g. Drosophila Bar eyes

Answer 163

We are 99.9% same but vary in our copy number variation (CNV) e.g. CNV of AMY1 - people w/ high starch diet have more copies of salivary amylase (AMY1) than those w/ low starch diet. -> positive selection on a copy number variable gene + showed functional relationship between AMY1 copy number & amount of amylase protein in saliva ## Footnote Whole genome duplication event (changes chromosome number) is usually followed by extensive genome reshaping - changes to chromosomes structure + number.

Answer 164

Identify homologs: orthologs - genes inherited from common ancestor (same locus in differenet species) paralogs - genes arose from duplication event (different genetic loci in same organism)

Answer 165

Chromosomal rearrangements can cause loss of synteny so conserved synteny can indicate functional relationships between genes Macrosynteny - mouse + human genome have large syntenic blocks in common (human chr 17 w/ mouse chr 11) Microsynteny - DAL clusters in yeast, DAL used to convert allantoin/allantoate to ammonia in low N conditions - DAL genes clustered on same chrom region in Saccharomyces + Candida (colocalised) All genes must expressed simulatensouly for degadation of allantoin. ## Footnote synteny regions - blocks of co-localised genes on chromosomes of different species

Answer 166

Can look at known & proposed polyploidy events in eukaryotes. Humans speculated to have undergone 2 WGDs. Saccharomyces had WGD event - proven. Found that duplicated regions were created simultaneously by single tetraploidisation event. - 50 of duplicated regions maintained same oreinatations w/ respect to ceontromere - no triplicate clusters - none of clusteres are overlapping c. 100mYa WGD event is root of yeast evol. New S. cerevisiae (NCYC505) is crypto tetraploid (16 chromosomes) + pre-WGD species K. waltii has 8 chromosomes

Answer 167

Responsible for genetic redundancy e.g. yeast genome ~6000 ORFs but <20% genes essential Genetic redundancy: implies that 2 or more genes carry same biological function + that a mutation in 1 of these genes has little or no effect on overall phenotype, as other would function as backup copy. So redundant gene is dispensable + can freely mutate: - sub-functionalisation - neo-functionalisation - deleted/lost Even in absence of WGD, 50% of genes in a genome would be expected to duplicate once every 35-350 million years ## Footnote C. elegans have widespread gene duplication - twice number local duplications as Drosophila 1/3 largest clusters duplicated gene are olfactory recpetors (7TM GPCRs)- 1/3 of which are peudogenes (non-fucntional) -> aenahnced survvial rate as increased genetic pool may be beneficial dependning on selection pressure

Answer 168

Redundant genes should be lost eventually due to accumulation of dleeterious mutations (not evolutionarily stable). So need to break symmetry between duplicated genes by sub or neo-functionalisation.

Answer 169

fbf-1 & fbf-2 are gene pair in germ line development of C. elegans - encode closely related RNA binding proteins that reciprocally modulate one another to regulate size of mitotic region citrate synthase in yeast: CIT1 & CIT3 in mt are active in TCA cycle. CIT2 is peroxismal form - active in glyoxylate cycle - protein functions in novel cellular context (localisation function)

Answer 170

lin-12 & glp-1 receptors in C. elegans have overlapping + seprate functions. if only lin-12 mutated, larvae doe not die (post-embrionic defects) - glp-1 has retained lin-12 functions

Answer 171

One copy of gene is not sufficicnet alone to maintain original fitness - not eough gene product Compromised phenotype compared to wt discovered by gene dosage studies.

Answer 172

Probable genetic contribution of an individual to succeeding generations. In yeast, relative fitness given by growth rate of different type of cells as they compete for a pool of resources. Competition experiments give direct measurements on growth rate + fitness in chosen environment

Answer 173

Uses an available set of oligonucleotide -tagged deletion mutants, each marked by 2 unique 20bp 'bar codes' - quantitatively identified using hybridisation array analysis or sequencing Can monitor impact of gene on fitness in different growth conditions (env effect) by measuirng its up- + down- tag. Showed haploinsufficiency can be environmentally dependent yeast - 12-20% genes show haploinsufficiency in minimal nutrient medium

Answer 174

Used in screening to discover molecular drug targets. Identify yeast losing out in competition assay - that strain carries heterozygote mutation in gene that encodes drug target. Can screen medically relevant drugs using yeast genome profiling to create library. ## Footnote Can also identify beneficial mutations in presence of drug - confer resistance

Answer 175

Homozygote cannot be distinguished from heterozygote at phenotypic level e.g. phenylketonuria (PKU) is full recessive - only 1 wt needed to carry out function (haplosufficient)

Answer 176

Phenotype of heterozygote is intermediate between the 2 homozygous. e.g. F1 between white + red -> pink (w/r)

Answer 177

Both alleles equally expressed in heterozygote e.g. IA/IB -> AB blood group

Answer 178

Mutant allele of a gene reverses effect of a mutation in another gene -> allows active protein complex formation despiyte mutations.

Answer 179

Interaction between genes at 2 or more loci, so phenotype differs from expected if the loci were expressed independently negative/antagonistic: 2-locus epistatic interaction -> smaller combined fitness than sum of their own positive/synergistic: 2-locus epistatic interaction -> enhances fitness of individual more than sum of their own ## Footnote overriding mutation is epistatic + overridden one is called hypostatic

Answer 180

2 genes have a synthetic lethal relationship if mutants in either gene are viable but double mutation is lethal - environementally dependent

Answer 181

- Test single mutants for dominance - Check if mutants at same allelic locus - Make double mutants to look for genetic interactions, if 2 genes interact, phenotype different to simple combination of 2 single mutations

Answer 182

Auxotrophic mutants cannot synthesise cellular components from inorganic nutrients.