Genetics Lab Midterm

Model organism definition

simple, easily bred, short generation time, characterized genome, similar enough to the organism of interest to be useful for studying a trait/process/disease

Core criteria

simple, accessible, easily bred, identifiable/heritable phenotypes, rapid development, small size, affordable, strong genetic toolkit

Model organism examples

Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio, Mus musculus, Arabidopsis thaliana

schmidtea mediterranea

Escherichia coli

bacteria

30 minute generation, one chromosome, prokaryote

Saccharomyces cervisiae

budding yeast

doubles every ~100 minutes, first eukaryotic genome sequenced

Drosophila malanogaster

fruit fly

2 week life cycle, high homology to humans

Caenorhabditis elegans

nematode

48 hr generation, 959 somatic cells fully fate-mapped, RNAi by feeding, first multicellular genome sequenced

Danio rerio

zebrafish

90 day cycle, transparent embryos, morpholino knockdown

Mus musculus

house mouse

9-11 week cycle, first mammalian genome sequenced, knockouts available

Arabidopsis thaliana

flowering plant

2 month cycle, 5 chromosomes, easily transformed/mapped

Chromosome structure

centromere (spindle attachment)

telomeres (protect ends)

kinetochore (microtubule attachment site)

Interphase

G1, S (DNA synthesis), G2

M phase

mitosis + cytokinesis

What enforces checkpoints in the cell cycle?

CDK/cyclin complexes

(G1/S = cell size, G2/M = DNA damage, vis p53)

mitosis

1 division, somatic cells, 2 identical diploid daughters, no crossing over

meiosis

2 divisions, germ cells, 4 unique haploid daughters, crossing over increases genetic variation

Hartwell, Hunt, and Nurse

2001 Nobel Prize for discovering cyclins/CDKs as cell-cycle regulators (sea urchin embryo model)

Conditional mutation

normal phenotype under permissive conditions, mutant phenotype under restrictive conditions (ie temperature-sensitive alleles)

Planaria / schmidtea mediterranea

flatworm

somatic stem cells are self-renewing and multipotent

whole-organism regeneration from a cut fragments models human stem cell biology

planaria feeding

thaw a liver sliver, add to worm stock, agitate water to draw worms out, feed ~30 minutes, remove remaining liver

Gene

segment of DNA that codes for a specific trait

allele

a variant form of a gene

dominant

an allele that masks the effect of a recessive allele when both are present

recessive

an allele whose effect is masked by a dominant allele

genotype

genetic makeup of an organism

phenotype

the observable physical or biochemical characteristics of an organism

Mendel’s peas

P0 —> F1 (uniform) —> F2 (trail reappears, 3:1)

Law of Segregation

two alleles separate during gamete formation

Monohybrid cross (Aa x Aa)

1:2:1 genotypic ratio, 3:1 phenotypic ratio

Test cross

unknown dominant x known recessive

Dihybrid cross

9:3:3:1 ratio, Law of Independent Assortment

Complete dominance

heterozygote = dominant phenotype

Incomplete dominance

blended/intermediate phenotype

Co-dominance

both alleles expressed

X-linked traits

males are hemizygous, one recessive X allele

Drosophilia life cycle

9-15 days

instar larva (day 1-3), pupa (day 7), adult eclosion (day 11-12)

Drosophila: body plan built hierarchically by 3 gene classes

1. Maternal effect genes

2. segmentation genes

3. homeotic/HOX selector genes

Homeotic mutation

transforms one segment’s identity into another (ex. antenna —> leg)

Immunostaining

detects a specific protein in fixed tissue using a primary antibody + a dye-linked secondary antibody

Sexing flies

females = larger + striped abdomen

males = smaller, solid dark thorax, se x combs on forelegs

Chi-square test

goodness of fit test comparing observed vs expected ratios

null hypothesis (H0)

no real difference exists; any deviation is due to chance

degrees of freedom

number of categories - 1

decision rule for chi square

p > 0.005 —> accept H0

p < 0.005 —> reject H0 (deviation not due to chance alone)

Sordaria fimicola

ascomycete fungus, mostly haploid life cycle, meiosis, 8 ascospores per ascus

wild = black / mutant = tan

Ascus patterns

4:4 = no crossover

2:2:2:2 or 2:4:2 = crossover

gene to centromere map distance

(% asci showing crossover) / 2

White spotting (cat genetics)

a dominant trait analyzed via pedigrees/punnet squares to infer kitten and parent genotypes

candidate gene (KIT)

linked to white spotting in horses

cates carry a longer and a shorter KIT allele

Cat genetics: workflow

extract DNA —> PCR to amplify KIT region —> agarose gene electrophoresis

Cats as a genetic model

38 chromosomes, high homology to humans, 365+ known hereditary diseases

RNAi mechanism

Exogenous double-stranded RNA (dsRNA) is cut by Dicer into ~21-nt siRNAs —> loaded into RISC —> RISC guides cleavage of complimentary mRNA —> gene sliced

Is RNAi a mutation?

No

DNA sequence is unchanged, only the expression is knocked down

effects transmitted across generations and conserved across eukaryotes

RNAi use

silencing a gene of interest to oberve the resulting phenotype

RNAi delivery

feeding, microinjection, plasmid, viral vector

Forward genetics

phenotype —> genotype

mutagenize a population —> screen for a phenotype of interest —> analyze mutants —> map causative gene

Reverse genetics

genotype —> phenotype

start from known gene, disrupt it, and observe the resulting phenotype

Forward vs Reverse genetics. which is faster?

forward: slower but finds causes of naturally occurring phenotypes

reverse: faster once a gene is known and directly tests its function

Forward genetics: Cat example

Lykoi “werewolf” cat (HR gene)

Reverse genetics: Cat example

engineered infections clones of feline viruses (FIP) to study pathogenesis and test vaccines/antivirals