Genetics Exam 4

What is the primary method of controlling gene expression in prokaryotes?

regulation of transcription

How many layers of regulation are in Eukaryotes?

5

Layers of Regulation - chromatin remodeling

2 classes, ATP dependent and histone modifying

ATP dependent chromatin remodeling

protein complexes loosen nucleosomes using ATP

Histone/DNA modifying chromatin remodeling

acetylation = expression

methylation = silencing

Why is transcriptional regulation most common?

cell doesn’t want to use energy

Layers of Regulation - transcriptional

interfering with the signaling cascade (game of telephone)

___ can act as the ligand and start a cascade

hormones

transcription factor

help/hinder RNA polymerase binding to DNA

cis activating factors example

TATA + enhancer

trans activating factors example

PIC + proteins themselves

transcription complexes

specific binding domains for specific sequences

Why are transcription complexes a great opportunity for drug targeting?

there’s specificity of the transcription complex to DNA sequence

enhancer proteins

activator proteins bind to bring transcription factors and trans-activating factors together

promoter region

transcription factors bind

silencer region

repressor proteins bind

insulator region

block transcription on non-target genes

What has the basal level of expression?

core promoter

What causes up/down regulation of transcription?

promoter-proximal elements

Layers of Regulation - RNA processing

hnRNA is converted to mature RNA

RNA transport regulation

mRNPs through large multi-protein pore complexes

miRNA - mature microRNA

stem loop, down-regulate gene expression

miRNA usefullness

seen in a lot of tumors, biomarkers

miRNA process

formation

exported from nucleus by exportin-5

attached to protein complex

passenger rna strand discarded

dicer removes hairpin loop

blocks ribosome + speeds up breakdown of poly-a tail

Where is most translational regulation?

initiation

What if no 5’ cap?

no ribosomal binding

elongation factors

bouncers of aa

ribosomal pausing

stacking ribosomes causing traffic and cleavage

Layers of Regulation - post-translational regulation

modification of aa in a protein

disulfide bonds

strengthen protein between cysteine AA

proteolytic cleavage

gets protein to right place in handcuffs

myristoylation

attachment of lipid chain confining protein to membrane

phosphorylation

adding phosphate groups, regulates enzymatic activity

genetic material must haves

transfer, storage, mutable, high fidelity

T/F point mutations are mutually exclusive

False

types of point mutations

substitutions, indels, functional mutants

point mutations - substitutions

transition - exchange with like base

transversion - exchange outside family

Do transitions or transversions happen more often and why?

transitions bc keeps carbon structure

point mutations - indels

frameshift - changes reading frame

in-frame - does not change reading frame

T/F upstream codons are impacted by frameshift mutations

False

T/F substitutions can cause indels

True

T/F change of 1st letter of codon will not usually change aa

False

Loss vs Gain of function mutation

amorphic recessive vs neomorphic dominant

somatic vs germ cell mutations

mild + not passed vs severe + passed

wobble

non-complementary base pairs due to DNA flexibility

slipped strand mispairing

polymerase slips on repeat bases, doesn’t know where to go back to repair, causes microsatellites

depurination

release of purine

the A-rule

add adenine to apurinic site

deamination

removal of amine group changing base

What is the most common single nucleotide mutation in DNA?

5-methylcytosine → thymine

free radicals

steal electrons from nearby molecules

How do antioxidants reduce free radicals?

donate electron to free radical

UV light

causes pyrimidine dimers, stopping replication

What is the primary cause of melanoma in humans?

pyrimidine dimers

causes of chromosome duplication

ectopic recombination

transposable elements

jumping genes, originally thought to be junk DNA

Barbara McClintock

discovered transposition

Class 1 vs Class 2 transposons

require vs doesn’t require reverse transcriptase

Class 2 TE

DNA transposons, cut and paste mechanism

terminal inverted repeats

inverted compliments at end of class 2 TE recognized by transposase

flanking direct repeats

marker for excision site of class 2 TE

Who/Where is TE cut in Class 2 transposition?

by transposase between TIR and FDR

Class 1 TE

retrotransposons, RNA intermediates, copy and paste

long terminal repeats

end of class 1 TE

non-long terminal repeats

only active class of transposons in humans

class 1 TE - autonomous vs non-automomous

Long interspersed elements vs short interspersed elements

retrotransposition mechanism

retrotransposon → RNA → dsDNA → pasted into target DNA

epigenome

record of chemical changes to DNA and histones

imprinted genes

keep epigenetic tags from parents

epigenetic tags

turn genes on and off without changing underlying code

What base gets modified during methylation?

cytosine

Why are there CpG islands around promoter?

to keep it stable via methylation

Are housekeeping genes CpG usually methylated?

no

How does methylation work?

blocks activator from binding to enhancer

methyl binding proteins

can block or recruit proteins like HDAC to cause heterochromatin

lyonization

inactivation of one random x chromosome, barr body

piebalding of calico cats

white spots of no color

How can there be male calicos?

XXY

nutrients that impact methylation

folic acid and vitamin b

active agouti gene = …

obese yellow mouse

What happens if you give a pregnant, obese yellow mouse methyl rich foods?

mostly healthy offspring

what experiment proved that epigenetic memory can be passed across generations?

healthy brown mice not given methyl food

cancer cells have ___ level of methylation than healthy cells

lower

suicide victims have ___ levels of methylation than control

higher

eugenics definition

improvement of humans through selecting breeding

positive vs negative eugenics

prenatal care vs sterilization and euthanasia

problems with eugenics

polygenic traits, genetic linkage + variation, who gets to decide?

What acts as the prokaryotic immune system?

CRISPR

what does crispr stand for?

clustered regular interspaced short palindromic repeats

spacer dna

matches with viral dna

CAs genes

CRISPR associated genes, code for helicases and endonucleases

infected cell without CRISPR

viral DNA replicates and lyses

infected cell with CRISPR

CAs genes → proteins → crRNA → viral DNA degrades

New virus and CRISPR

cas1 makes copy of DNA for immunological memory

CRISPR - Cas 9

method of genome editing

What does CRISPR/Cas 9 plasmid need

promoter, gRNA, nuclear signal, Cas9

crRNA

complementary to viral and spacer made by cas proteins

Protospacer adjacent motif (PAM)

cas9 binds at nGG

How cas9 works

binds at PAM, open DNA, gRNA complementary pairs, cut DNA

NHEJ vs HR

knock out vs knock in genes