Genetics Module 4

directional selection

additive effects

all become 1of the homozygous extremes

disruptive selection

underdominance

advantage for both extremes

leads to bimodal population

Stabilizing selection

Overdominance

All become heterozygous

Both alleles remain in population

effects of small population size

genetic drift

founder population

inbreeding

genetic drift

founder population

founders will affect next generations

genetic drift

random fixation or loss of alleles over time from small population

positive assortative mating

mating similar individuals results in more homozygotes but only for loci under selection

negative assortative mating

mating opposite individuals results in more heterozygotes for the loci under selection

inbreeding

changes frequency of genotypes but not allele frequency

more homozygotes

affects all loci

cancer genetics

genetic but rarely heritable

cancer multi hit

Sporadic and influenced by environment

-siblings rarely have same cancer

-populations that migrate to new regions get cancer rates typical of that region

Cancer develops over time

-changes in cancer rates from new environment take decades

types of genes involved w/ cancer

tumor suppressor genes (prevent bad cells from dividing)

proto-onco genes (allow good cells to divide)

tumor suppressor gene mutation

recessive acting- must disrupt both copies to lose cell regulation

tumor suppressor genes

BRCA1

p53

RB

RB gene mutation

Tumor suppressor gene

40% cases inherited- inherit 1 bad type then another somatic mutation happens to cause cancer

RB gene function

G1 to S transition

normally prevents E2F from activating replication

BRCA1 and 2

tumor suppressor

used to repair double strand breaks

woman vulnerable to breast and ovarian

men vulnreable to prostate and breast

p53

Tumor suppressor

Colon, lung, breast, brain and is found in altered form in half of all tumors

if DNA damaged, p53 delays cell division until DNA repaired or dies

haploinsufficiency exception

loss of function of one copy and the other copy doesnt produce enough gene product to exhibit wild type phenotype

some cases of cancer w/ mutations in only 1 copy of tumor suppressor gene: bloom syndrome

bloom syndrome

defective DNA helicase enzyme that repairs double strand breaks

homozygous for mutated BLM gene have very high rates of cancer

hetero ppl have elevated colorectal cancer risk

oncogene mutation

only need 1 copy for cancer

burkitts lymphoma

reciprocal translocation between chromosomes 8 and 14 places c-myc ongogene next to enhancer

chronic myelogenous leukemia

reciprocal translocation involving chromosomes 9 and 22 places 2 oncogenes near each other

retroviruses causing cancer

mutate and rearrange proto-oncogenes

insert a strong promoter near proto-oncogenes

clonal evolution

over time tumor cells acquire more mutations that allow them to be progressively more aggressive in proliferation

mutations affect

cell cycle regulation

signal transduction

telomere length

chromosome segregation

vascularization

DNA repair

defective nucleotide excision repair= xeroderma pigmentosum

defective mismatch repair = colorectal, endometrial, stomach cancers

defective double strand break repair = BRCA1, 2

telomere length

telomerase works in germline cells but not in somatic cells, allows them to die and be replaced

tumor cells have telomerase, thought to contribute to the immortality

vascularization

angiogenesis- growth of new blood vessels important to tumor growth

angiogenesis inhibitors may be under expressed or inactivated

growth factors for angiogenesis overexpressed

classic cancer modle

cancer is a proliferative disease

cancer prevention

HPV vaccine

avoid environmental factors

get cancer screenings

height

quantitative trait with normal distribution

multi factor hypothesis

Expression depends on the additive effects of a number of genes.

The effect of each gene is small.

Environment plays an important role in expression of trait.

-smooths curve

phenotype formula

P = Genetics + Environment

Genotype formula

G = Additive + Dominance effects

A variable

A = Average effect of substituting A for a in genotype

D variable

D = dominance effects due to specific combinations of alleles at a locus

when does D = 0

if the value of Aa is exactly between the values of AA and aa

how to determine the values for G, E, A, D

We must look at a population and determine the values for G, E, A, D. and partition the variation we see in the phenotypes of the individuals in the population into G, E, A, D.

Heritability

proportion of the phenotypic variance due to genetic effects

broad sense and narrow sense

Broad sense heritability formula

H² = VG/VP proportion of phenotypic variance due to genetics

narrow sense heritability formula

h² = VA/VP proportion of phenotypic variance due to additive genetic effects

The higher the heritability…

the more progress we can expect from selection/breeding

heritability ranges

0 = all VP from environmental variation

1 = all VP due to genetic variation

who developed PCR

Kary Mullis

temp to heat up to PCR

95 c to allow strands to separate

then cool to 50-65 to identify target region for amplification

PCR steps

denature DNA by heating and allows strands to separate

cool, primers anneal to identify target region

Taq polymerase adds nucleotides to 3’ end of primer (72c)

PCR limitations

must know about sequence surrounding gene of interest

taq polymerase does not proofread and correct errors

fragments amplified are small

amplification of DNA

VNTR

allele based on length of DNA segment

must be polymorphic

recomb DNA restriction enzyme

endonuclease that recognizes a specific DNA sequence and cleaves dsNDA at that sequence

recomb DNA palindrome

reads the same 5’ to 3’ on either strand for a segment of DNA

cloning vectors have

origin of replication

selectable/insertional markers

multiple cloning site

cloning vectors selectable/insertional markers

allow cells containing vector and recomb molecule to be identified

cloning vectors multiple cloning site

has many restriction enzyme cut sites that can be used in producing a recomb DNA molecule

ligation experiment for recomb

join foreign DNA to vector

foreign DNA and vector cut w/ same restriction enzymes

DNA mixed and DNA ligase is added

insertional inactivation for recomb

inserted DNA inactivates a gene in the vector by inserting into that gene

allows cells which contain recomb DNA molecule to be identified

transformation experiment for recomb

allow cells to take up products from a ligation experiment

identification of different cell types for recomb

cells w/ no uptake, cells that took up the original vector, and cells that took up the recomb plasmid

Why Does Variation Exist in a Population?

Neutralist Theory and Selection Theory

Reproductive isolation can occur because

They don’t choose to mate with each other or cannot mate with each other (Prezygotic).

Or their progeny are sterile or inviable (Postzygotic)

prezygotic reproductive isolation

They don’t choose to mate with each other or cannot mate with each other

Postzygotic reproductive isolation

Their progeny are sterile or inviable

prezygotic types of reproductive isolating mechanisms

Ecological- habitat differences

Behavioral- different mating behavior

Temporal- reproduction at different times

Mechanical- anatomical differences

Gametic- not compatible

postzygotic hybrid breakdown

F1 hybrids are viable and sterile but F2 are not

Allopatric Speciation

Geographic barrier initiates speciation by blocking gene flow

Sympatric Speciation

Arises within a single interbreeding population without geographical barriers to gene flow.

Hybridization that leads to allopolyploidy is another mechanism for sympatric speciation.

Races of the Apple Maggot Fly sympatric speciation example

Resource use is linked to mating preference.

Original fly fed on hawthorn tree fruit.

Mutation allowed feeding on apples.

Those with the mutation mated together more on apple trees (reproductive isolation)

Speciation not complete yet.

Gene flow only 2% between hawthorn and apple flies

Anagenesis

evolution within a lineage over time

Cladogenesis

splitting of one lineage into two

Paralogs

homologous sequences found in the same species and arrive through gene duplication.

Orthologs

homologous sequences found in different species

Molecular Clock

differences in sequence between present day organisms can be used to date past evolutionary events.

G1/S checkpoint

monitors for proper cell size and undamaged DNA.

G2/M checkpoint

holds up cycle until replication and DNA repair are complete.

M checkpoint

proper spindle formation and attachment

High MZ low DZ

indicates significant role of genetic effects

Low MZ but still much higher than DZ

indicates genetic predisposition, but environmental factors are important

how to make cDNA from mRNA

use reverse transcriptase (RNA dependent DNA polymerase)

-uses mRNA as a template to synthesize the first strand of cDNA

transformation of rice w/ daffodil PSY gene

need lots of DNA

need a promoter

need a way to get it into the rice genome

Rhizobium radiobacter

transfers DNA to plants

Modified Ti plasmid inserts cloned gene of interest into plant chromosome

Expression vector

allows inserted gene product to be produced

must contain sequences required for transcription and translation of the gene

what is needed for insertion into rice genome

PSY gene

crtl gene

promoters- endosperm specific

Poly A signals

Transgenic marker

LB and RB insertion into plant genome

blotting

process of transferring molecules that were previously separated to a membrane better able to support additional testing

Southern blot measures what

DNA fragments separated based on length

how does southern blot work

single stranded DNA fragments from a gel are transferred to a nylon membrane

Nylon membrane incubated w/ labeled single stranded probe DNA of interest

Probe binds to complimentary DNA fragments on the nylon

Northern blot

RNA fragments separated based on length

Western blot

Proteins separated based on molecular weight, isoelectric point, electric charge, etc

electrophoresis

DNA loaded at negative pole and migrates to positive pole

small migrates faster

Dideoxy Sequencing Reaction

3’-OH required by DNA polymerase to form a phosphodiester bond.

• DNA replication reaction proceeds until a dideoxy nucleotide is incorporated.

• No further extension occurs.

• Detect which position the dideoxy nucleotide was incorporated bc that’s where the sequence terminates

golden rice results

rice with maize PSY genes makes higher b carotene than rice with daffodil gene

need 72g of that a day to get enough vitamin A

Forward Genetics

Start with a mutant phenotype and seek out the gene that causes that phenotype.

• Use chromosome mapping to identify the gene

Reverse Genetics

Start with a DNA sequence (a genotype), alter its function or prevent its expression and observe the effects on the phenotype

Reverse Genetics Transgenic Mice

Inject gene of interest into fertilized egg.

Implant in female.

Test progeny for presence of gene.

Mate to obtain mice homozygous for gene.

Study gene function

Reverse genetics knockout mice

Phenotype of knockout mice reveals function of gene

Reverse Genetics Knockdown Expression Using RNAi

Excess ApoB protein leads to high levels of cholesterol.

Inject lipid coated ApoB synthetic siRNA into Cynomolgus monkeys to ‘knockdown’ expression.

Monkeys that got more siRNA had lower cholesterol

Microarray analysis of RNA from cancer and noncancer cells

shows that some genes are more expressed in cancer cells (green) and some more expressed in noncancer cells (red)

RNA sequencing to determine expression of genes

RNA of interest isolated

enzyme reverse transcriptase used to make cDNA from mRNA

cDNA broken into overlapping fragments

adapters w/ sequences for amplification and sequencing are added to ends of fragments

fragments are amplified w/ PCR