genetics unit 3

DNA, chromatin, and nuclear architcture

how DNA packaging helps to regulate gene expression

epigenetic changes

“above” DNA sequence

“persistent”

reversible

make changes to the structure around DNA and changes how genes are used

chromosome structure

nucleosomes

nucleosomes

dna rapped around histone proteins

can be wrapped together to different extents

heterochromatin

closed; usually not expressed

DNA wrapped tightly

can wrap info away that isnt needed

euchromatin

open; often expressed

DNA wrapped loosely

usually is expressed and DNA is used

what is organization affected by

DNA and histone modificaitons

chemical modifications of histones

methyl, acetyl,, phosphate groups can affect structure and interactions w/regulatory proteins

me (methylation)

one methyl group

me2 (diimethylation)

two methyl groups

how does adding an acetyl group affect a histone

it loosens it up

chromatin remodeling

DNA methylation

primarily cytosine

most common at CpG islands

often linked w/gene silencing

methylated DNA can recruit enxymes that modify histones

heterochromatin formation and blocks transcription

what happens when you add bisulfite to cytosine

it turns into uraccil

what happens when you add bisulfite to methylated cytosine

it doesnt turn into uracil

what does methylation do

it turns off genes

the cell will remember which genes are methylated

we can mark parts of DNA that will be and will not be used

epigenetics

changes in expression due to environmental cues can be inherited

growing evidence that can happen across organismal generations (parent to child)

nuclear organizaiton

DNA is HIGHLY organized in the nucleus

how can 3-D organization change

in response to the environment

by cell type

the same DNA can be used in different ways

Pitx-1 gene

regulates expression of other genes

promotes hind limb development

expression controlled by the PEN enhancer region

PEN enhancer region active in both forelimb and hindlimb

kragesteen et al (2018)

DNA organized differently in forelimb and hind limb cells

PiTX1 expressed in forelimb if DNA folding is changed

causes forelimb to have hindlimb traits

liebenburg syndrome in humans

how do enhancers regulate specific genes

gene location can influence expression

many genes can be “near” an enhancer

insulater sequences influence promoter-enhancer interactions

how do insulators work

insulators binding proteins organizes chromatin into loops

enhancers can interact w/promoters but cannot interact w/other promoters

insulators act as boundaries between chromatin domains

Epigenetics/Health

psychiatric/neurological disorders influenced by the environment

effects can persist after environmental factor is gone (eg PTSD)

can sometimes be treated w/neurological drugs

how these drugs work isnt always clear

what can cause changes in chromatin structure

environment

neurological disorders incresingly associated with changes in chromatin structure in brain tisseu

Perisic et. al 2010

do antidepressant drugs alter epigenetic markers

exposed brain cells isolated from mice to different drugs

do antidepressent drugs alter epigenetic markers

glt-1 (glutamate neurotransmitter membrane transporter)

known to be associated w/psychological disorders

reporter construct= glt-1 promoter and luciferase gene

dna mutations and repair

dna is inert —> doesnt really react

this makes it better for storing info since theyre so stable

mutations

heritable changes in genetic info

they’re the ultimate source of alleles

diffferences of the same gene

sources of mutations

spontaneous error: inappropriate base paring

atypical base pairing

• not as stable as typical base pairing

dna poly cn back up and takeout hte incorrect base pairs→ they self correct

what is atypical base pairing immediately considered

its referred to as incorporation/replication error

it eventually becomes a permanent change (mutation) after the 2nd round of replication

mutations

permanent changes in DNA sequences

transition point mutation

purine ←→ purine

pyrimidine ←→ pyrimidine

transversion point mutation

purine ←→ pyrimidine

A to G or C to T

what would flipping the template strand affect

transcription

duplicaitons, deletions, and slippage

base pairing can allow dna strands to align incorrectly during replication

daughter strand slips

daughter strand gains nucleotides

template strands slips

daughter strand loses nucleotides

size of gain/loss depends on waht

length of repeated sequence

if the repeat is 2 nucleotides…

the insertion/deletion would be multiples of 2 in length

spontaneous error:

nucleotide lesions

depurination

loss of a purine base (A/G)

happens about 5,000/cell/day

DNA poly will guess that hte original nucleotide that was lost was A

deamination

loss of a nitrogen gropu from A, G, or C

up to 100 times/cell/day for cytosine

mutagens

increase teh rate of replication erros

base analogs

mimic bases but can base pair w/multiple partners

5- bromodeoxyuridine can pair w/A or G

modifying agents

chemically alter bases

external chemicals and metabolic byproducts

cause DNA lesions (e.g. depurination, deaminations)

add chemical groups —> alter/block base pairings

intercalating agents

insert into and distort DNA helix

promote slippage

radiation

alter/break chemical bonds

UV light

cross-links adjacent pyrimidines

dna poly stalls and skips

group 1 carcinogen

what does UV cause on the molecular level

double deletions that can’t be repaired

what leads to apoptosis

too many mutations

what break covalent bonds in the dna backbone

x rays, oxygen free radicals

creates nicks in the backbone

sometimes parts of chromosomes are lost

mutation rates

how often a nucleotide changes per genome

mammal has about 1/10^8-9 change a given nucleotide will change

are larger or smaller genes more likely to expreeince mutaitons

larger genes

is changing an amino acid from nonpolar to polar going to change anything

yes it can change a lot

are mutations completely random

no

CpG sites are more suceptible to mutations due to deamination

cytosine at CpG is often methylated

histones protect sections of DNA

is a methylated cytosine a noticeable mutation?

no because it turns into thymine

what sequences are subject to slippage

repeated sequences

STRs and DNA fingerprints

what are two techniques that cells use to protect themselves from mutations

DNA poly proofreading— during replication

insertion of incorrect base —> mis pairing of bases, replication stalls

when does 3-5 exonuclease become active

when DNA poly recognizes a sequence out of line and the exonuclease acts as a backspace

when does mismatch repair occur

after replicaiton

proteins recognize mis paired bases and in-del loops

reduces errror from 10^-7-9

how do repair enzymes differentiate between the daughter and template strand in prokaryotes

because of methylation in the parent strand in prokaryotes

how do repair enzymes differentiate between teh daughter strand and template strand in eukaryotes

repair enzymes only recognize daughter strands and fix DNA poly mistakes

the daughter strand is shorter and has fragments

direct repair

nucleotide repaired

base excision

single nucleotide replaced

nucleotide excision

multiple nucleotide replaced

trans-lesion synthesis

what do cells do when they recognize dna damage

replication sstalls at damage sites (e.g. UV damage)

two options: if too much damage or stall too long—> death

bypass DNA poly

pyrimidine dimers

render the DNA irreplicable

they cant be replicated

bypass dna poly

able to replicate pass damage

lacks 3’ —> 5’ exonuclease

“error prone repair”

what are the tradeoffs between the two options

death→ no errors, but no dna

error prone repair → dah, a lot of errors

double stranded breaks

50 ds breaks every cell generation

greatest risk during replication bc of okazaki fragments

non-homologous end joining

primary way to repair ds breaks during interphase

ds break

trimback damaged ends

ligated ends

this still shortens dna

error prone repair→ introduces mutations

can attach the wrong ends → chromosomal rearrangements

homology directed repair

recombination repair

second copy of chromosome used as a repair template

gaps fille dby dna poly

nicks sealed by ligase

caviot: the strands could get mixed up

HDR

would use sister chromatids for repair in mitosis

could use homologus chromosomes in meiosis (different versions)

double stranded breaks in mitosis

recombination

meiosis takes advantage repair process to generate variation

ds breaks created deliberately

what does helping to generate variation help with

organisms to survive in a changing environment

free radicals

atoms/molecules w/unpaired electrons

highly reactive- tend to attack double bonds

reactive oxygen species

naturally produced by cells

can damage cells and tissues including dna

antioxidants protect against them

antiodixants

compounds/enzymes that neutralize free radicals

can reduce damage caused by free radicals including to dna

fruits/vegetables

vitamens

p53 proteins

promotes antiodant production

dna damage increases p53 expression

tumor supression protein → works against cancer formation

turns on dna repair pathways

triggers apoptosis if dna damage is too high

how does taking a lot of antioxidants affect you

lower dna damage but also p53 production

reduces tumor supression activity

can allow cancerous cells to survive

genetic engineering types

recombinant dna

dna cloning

CRISPR

Gene therapy

diabetes and insulin

critical for maintaining healthy blood sugar levles

produce too littel to no insulin

take insulin injections to manage blood sugar levels

in 1978 they would isolate insulin from pigs and cows

difficult and expensive

impure and non-human

genetic engineering

make specific changes to genes/genomes

genes are relatively small in comparison w/chromosomes

dna cloning

copy of an isolated region of dna

much easier to do

mostly in vitro (outside of the cell/tissue)

can modify w/out altering other dna

can use as a resource

cna put back into the organism

cloning resources

DNA restriciton enzymes, cloning vectors

restriction enzymes

recognize and cut palindromic DNA sequences

both strands have same sequence and directionality

what ends does the enzyme Rsal generate

blunt ends

which enzymes generate staggered cuts

Kpnl

EcorI

kpnl

3’ overhands

EcorI

5’ overhangs

sticky ends

dna overhangs

recombinant dna

combine DNA from different sources

can be from same chromosome or even from different organisms

cloning vectors

used to carry (clone) foreign dNA fragments being examined

can be replicated independently of the chromosomes

plasmid

small circular piece of dna

posses traits we desire for cloning

contains an origin of replication

selective marker (antibiotic resistance)

multiple cloning sites (has unique Re sites)

how many molecules are you working with when cloning dna

many

transformation

bacteria takes up foreign dna

99.9% take up how much

nothing

0.1%

take up either the plasmid or dna

0.0001%

take up both

what does the antibiotic kill

everything that doesnt have a plasmid

will an inserted sequence make a plasmid funcitonal or nonfunctional

nonfunctional

directional cloning

use two different enzymes to cut plasmid and fragment

vector cant close without insert

Fragment can only insert in one orientation

what happens if you take a eukaryotic gene and put it into a prokaryote

the prokaryote wont be able to take out the introns and will produce a different protein