Genomic Structures and Mutations in Eukaryotes and Bacteria

Describe Bacteria Genome

Circular genome in nucleoid region

Mostly coding region

One origin

Some repetitive DNAs

Compacted by loop domains and supercoiling (DNA Binding proteins)

Eukaryotes description of genome

Many millions of nucleotides, centromeres, origins evry 100 kb, telomeres, genes with introns

Repetitive DNA in Eukaryotes

rRNA repeat regions

telomeres

transposable elements

other repetitive DNA

What are transposable elements

small DNAs that move from place to place in genome

Two mechanisms for transposable elements movement

Simple: Cut and Paste Mechanism directly from DNA

retrotransposition: Copy and paste using RNA intermediate

Common Features of Transposable elements

1. Ability to move within a genome (transposition) "Jumping'

2. Transposase: DNA transposons use to cut and paste

3. Reverse Transcriptase: Create DNA copy from RNA intermediate

Are tranposable elements muatagenic?

Yes

(Chromosome breakage, rearrangements, mutation, gene inactivation, etc)

Describe nucleosomes in Eukaryotes

DNA wrapped around octamer

2x each of 4 histones, H1 is stabilizer

Supercoiled nucleosomes make short, thick 30 nm fiber

What are MARs

DNA matrix attachment regions

- DNA regions that anchor radial loops to nuclear matrix

Do Eukaryotes have even higher condensation

Yes, they have higher condensation than eukaryotes

Euchromatin vs Heterochromatin

Euchromatin: DNA not packaged as tightly, has genes (transcriptionally active)

heterochromatin: Tightly packed, no genes (transcriptionally silent)

Name the Types of DNA Rearrangements

Deletions, duplications, inversion, translocation

Describe Deletions, how it occurs, detrimental or not

Occur from one or two double stranded breaks

Off End= Terminal Deletion

In Middle= Interstitial Deletion

Is Detrimental: Lethal nearly always

Normally Heterozygous Deletion= 1 chromosome

Describe Duplications, how it occurs, detrimental or not

Occur one or two double stranded breaks/ unequal crossover

Can be detrimental OR neutral

Neutral= Cause new gene functions

Describe Inversions, how it occurs, detrimental or not

Occur from 2 double stranded breaks

pericentric= Across two arms (No offspring)

paracentric= Across one arm

Usually Not detrimental BUT

Effects fertility- Problems with segregation after crossing over

Solution= Inversion Loops (Twist so homolgous chromes.

Translocation, how it occurs, detrimental or not

Occurs from two double stranded breaks in two different chromosomes

Usually Not detrimental BUT

Effects fertility- Problems with segregation after crossing over

Diploid vs triploid

Diploid= 2 sets chromosomes

Triploid= Three sets of chromosomes

Trisomy vs Monosomy

Trisomy= (2n+1)

Monosomy= (2n-1)

Non-Disjunction

Error in meiosis in which homologous chromosomes fail to separate.

if meiosis 1- All gametes screwed

If 2- 1/2 screwed

Mitosis= Nondijunction of complete chromosome loss

why are polyploids an issue

Even numbers are fine, odd numbers segregation= no homologous chromosomes to line up with

Mutation:

A change to the DNA sequence of a gene leads to a new "allele"•

A mutation in a gene can lead to a new "phenotype"• If there is no associated phenotype, they are considered polymorphisms•

Mutations are either neutral, detrimental, or beneficial•

The wild-type (normal) allele of a gene is the one that is found inat least 1% of all alleles in a population

Forward vs Reverse Mutation

Forward= Wild-type--> Mutant

Reverse= Mutant--> wild-type

Germline vs Somatic Mutation

Germline= Mutation in reproductive cells (sperm/egg)

- Can be passed to offspring

Somatic= Mutations in non reproductive cell, not inherited.

Base Substitution

Transition= Pyridine (T,C,U)--> Purine (A+G)

Transversion--> Purine--> Pyridine

Addition/Deletion

Causes frameshift

Protein messed up, stop codon altered

silent mutation

Unaffected due to wobble position

Conservative vs non conservative missense

Conservative: Minor Change to similar amino acid

Non-Conservative: drastic change to amino acid

Missense: Base Substitution

Nonsense Mutation

Base substitution changes codon to stop codon

- Uses one change

Frameshift

mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide

Can one mutation suppress mutant phenotype of another mutation?

Yes

Name kinds of mutations outside of coding region that can cause mutant phenotype

Changes in promoters/ operator, splice sites, untranslated regions

Intragenic vs Intergenic Supression

Intragenic: second mutation reverts original to wt (same gene)

Intergeneic: second mutation reverts original- wt (In different gene)

Position effect mutations

Genes location on chromosome changes alters the experssion

Null Mutation

Mutation= No function, no protein made

Recessive

Hypomorphic Mutation

Less Function, Less protein made

Recessive

neomorphic mutation

Leads to new protein function, gain of function

Dominant

Haploinsufficiency Mutation

Less function, less made

Dominant

Hypermorphic Mutation

More protein function, more made, gain function

Dominant

Dominant Negative Mutation

Reduced protein function, screws up other proteins in complex, gain of function by the others

Dominant

Ectopic Mutation

Gene is expressed in wrong part of body

Very low

yes, they are random

Is rate of spontaneous mutation high or low? Are they random or not/

Describe general process of damage to DNA

1. Lesion

2. Repair

3. If no repair, permanent mutation through replication

Deamination definition, fix

Deaminated cytosine acts as a U (T)

- Replicates, pairs with A (C-A) = transition

Fix: Base excision repair

Depurination

Drops a purine, makes apurinic site

FIX: BER

Can get transition or transversion

Tautomeric Shift

More likely to shift to uncommon form of a base

Ex: G pairs with A, T pairs with C

FIX: Mismatch Repair

ROS DAMAGE

Reactive Oxygen Species

- Wants to stick to more electrons

ROS attacks g--> pairs as an A

FIX: BER

What is Induced Mutation

Caused by UV Light, radiaion, X ray, chemicals

Deamination

Makes base pair like the opposite

(C, A)

FIX: BER

Alkyl Agents

1. Lesion

2. Alkyl group added

3. Base pair like other purine

FIX: BER

Intercalating Agents

1. proflavin/mutagen bullys into DNA

2. Take up space

3. Polymerase puts in any base= Frameshift

FIX: Nucleotide Excision Repair

Base Analogs

Chemicals that look like a base

1. bromouracil= Base pair like a T

NOTE: tautomerize more often

Physical Agent

DSB are one

X-Rays, Gamma rays

UV Light Damage

causes bond formed between two Ts on same strand instead of H bonding to opposite

FIX: Nucleotide Excision Repair

AMES TEST

1. Treat Salmonella with mutagen

2. Let grow

3. A lot of colonies= Mutagen cause mutation

4. If few colonies, not the mutagen

5. Add liver to test humans

Direct Repair

1. Photolyase breaks same side bond (UV LIGHT)

2. Alkyltransferase fixes lesion--> base

Base Excision Repair

1. Enzymes find lesion

2. Cleave Uracil

3. Chews region out

4. DNA polymerase works, ligase

Replaces oxidized bases, damaged, innapropr. base

Nucleotide Excision Repair

1. UvrA/UvrB scan for DNA

2. Damage Signaled brings in UvrC

3. UvrC expands, cuts DNA around Thymine Dimer

4. UvrD chews out DNA

5. DNA polym, ligase

Xeroderma Pigmentosum

Child with mutation in one of many NER genes

Leads to more mutations

Mismatch Repair

- Fix innapropriate base pairs

- DNA polymerase made mistake

1. Identify new strand (Doesnt have methyl group)

2. chew, replicate, ligate

homologous Recombination Repair

fixes DSB when sister chromatid present (G2)

1. Break Occurs

2. Chews DNA on both strands

3. Single Strand tail displaces DNA on other sister, H bonds

4. Fill in gaps with DNA polym+ligase

5. CROSSOVER occurs, but then gets cut/ligated

Non-Homologous End Joining

- Fix Double stranded Breaks any point in cell cycle

1. find Lesion, bind protein to them

2. proteins recruited to bridge gap

3. DNA polym, ligase

ISSUE: cant recognize chromosome #s

- Lead to inversions, translocations

Repressor vs Activator

repressor - Proteins bind to DNA to shut off transcription (-)

Activator;- bind to DNA to initiate transcription (+ control)

Inducers

Turn gene on by binding to an activator

co-repressors

Turn gene off

Bind to inactive repressor and activate it (change shape) so it can bind

Inhibitor

locks enzyme activity or gene expression

Causes change of shape of activator and it detaches from DNA

Inducible Genes vs repressible

Inducible: Normally Off

Get turned on when inducer present

catabolic pathways (ex lac operon)

Repressible= On, turned off, corepressor, anabolic (trp operon)

What is the inducible system of E. Coli

lac operon

What is lac repressor protein

Produces tetramer, binds as 4 proteins

What happens when no lactose is present

Binds to operator, which inhibits transcription- promoter can't bind

What happens when lactose is present

Inducer makes lac repressor change shape so it cant bind to the operator

Transcription occurs

Negative vs positive control

negative= protein binds DNA to shut off transcription

Positive= protein binds to DNA to turn on transcription

Lactose permease

move lactose into cell across membrane

Beta galactosidease

Produces allolactose

Allolactose

Causes change of shape of repressor, binds to lac repressor

Types of Mutants

P- = RNA polym cant bind to promoter

Oc (O-) = LAc repressor cant bind (Always transcription)

I-= Loss of repressor (cant bind to Operator)

Is= Gain of fxn for repressor (Cant bind allolactose)

What does E. Coli consume primarily

Loves glucose, some lactose

Describe relationship between glucose and cAMP

Increase Glucose= Low cAMP and vise versa

Describe cap protein

Binds at cap site

Only binds when it has cAMP

Binding of RNA polym is advamced

Glucose, no lactose

Glucose= low cAMP

Operator bound by repressor, no txn

Lactose, glucose

Glucose= Low cAMP

Lactose= Txn occurs

txn occurs at slow rate

No glucose or lactose

Operator bound to repressor

Transcription occurs super slow because high cAMP

Lactose, no glucose

High cAMP, txn occuring super fast

What is the repressible system

trp operon

Goal of trp operon

Stop transcribing where lots of tryptophan