03 - Genome Maintenance and Change

do closely related eukaryotes have simmilar genome sizes

some closely related eukaryotes have large differences in genome size

What does this cross-species comparison of a gene sequence tell us about the accumulation of genetic change?

even within a gene, some sequences are more highly conserved

exons are conserved

Do all sequences accumulate change at the same rate?

no

in very important genes = mutations very harmful = dont help us and are removed = so they wouldnt persist = limmited change

most imp genes = highly conserved

what does highly conserved genes mean

highly important

Would all sequences encoding amino acids in a protein be equally important/conserved?

they are not conserved equally, some sequences are more important than others

eg active sites if you chnag eit will destroy

Multispecies sequence comparison show:

Protein-coding sequences of genes are more highly conserved than genome size and organization (including # of basepairs, # of chromosomes, # of genes, position of genes along chromosomes, amount of repetitive DNA)

Exons are more highly conserved than introns

Exons

(coding for the amino acid sequence of proteins)

introns

(spliced out during RNA processing)

Mutation

change in the DNA sequence

what can cause mutation

errors in DNA replication or repair

point mutation

switching one nucleotide for another

which error rate is higher bacteria or human

bacteria

Large-scale rearrangements in change in dna sequence

Insertions Deletions Duplications Inversions Translocations

what mechanism of change is likely to result in loss/mutation of genes?

Deletions

intrachromosomal rearrangement

within 1 chromosome

interchromosomal rearrangement

between 2 chromosomes

reciprocal translocation

swapped without any net loss or gain to the genome

reciprocal exchange

non reciprocal translocation

one-way translocations in which a chromosomal segment is transferred to a nonhomologous chromosome

no reciprocal exchange

synteny

These blocks that carry genes in conserved order (but now located in different positions)

what do you think is more highly conserved: sequences within exons or the positions of genes on particular chromosomes?

exons sequences are more conserved

positions move around and scrambled

what percent of human genome shows conservation across species.

5%

Gene sequences only make up about a third of that; There are many conserved DNA sequences of unknown function

Highly Repetitive DNA

At least 105 copies of the sequence and make up 1-10% of a genome

Tandem repeats

repeat over and over without interruption

Satellite DNA

(generally found at centromeres and telomeres)

- 5-500 bp in tandem repeats of up to 100 kb that can form very large clusters.

Base composition is distinct from bulk DNA

Minisatellite DNA

10-100 bp with up to 3000 repeats → highly variable (polymorphic), therefore # repeats differs between individuals– basis for DNA fingerprinting in criminal and paternity tests

Microsatellite DNA

1-5 bp in clusters of 10-40 bp scattered quite evenly throughout the genome → highly variable (# of repeats changes fast!), can be used to compare closely related populations

Moderately Repetitive DNA

• 20-80% of the genome depending on the organism

• Repeated a few times to tens of thousands of times

• Can include genes or non-coding DNA

Moderately Repetitive - Genes

many copies of ribosomal RNAs and histones genes

Moderately Repetitive - Non-coding

Sequences scattered throughout the genome (interspersed) caused by transposons

SINEs

short interspersed elements

LINEs

long interspersed elements

slippage

In both prokaryotes and eukaryotes, repetitive sequences can be expanded or destroyed by “slippage”

events causing misalignment during replication

“Microsatellite instability”

related to progression of some diseases

Mobile DNA

DNA that moves from one place to another in the genome

genetic rearrangement is called transposition

moving mobile genetic elements were called transposable elements or transposons

DNA transposons move by

“cut-and-paste”

“Copy-and-paste”

what is “cut-and-paste” catalyzed by

catalyzed by the transposase enzyme

what is “cut-and-paste” mechanism

1. Inverted repeats on the ends of the transposon are required for recognition by transposase, which catalyzes excision from the donor DNA site and insertion elsewhere.

2. A direct repeat is generated in the recipient DNA site

what is “Copy-and-paste” catalyzed by

RNA intermediate

what is “Copy-and-paste” mechanism

Retrotransposons use and can encode a reverse transcriptase enzyme to catalyze production of DNA from RNA. Example: LINEs and SINEs

Why do you think plants and animals can survive a higher frequency of transposition events?

survive high frequency bc of large genome with non-coding regions

transposons insert without disruption and minimize harmful effect

transposition in viruses

Some viruses use transposition mechanisms to move into host genomes and can also be a source of genetic change

Over time/generations, can different types of DNA sequences accumulate change at different rates

Can be because some sequences (ex. protein-coding) do not tolerate change without impacting health of the organism

Can be because some sequences are more prone to mutation (ex. misalignment in repetitive DNA)

exon shuffling

Beyond just mutation or disruption, transposition can also result in moving/adding or removing gene coding or regulatory sequences

how can new Genes Generated from Pre-existing Genes

Small substitutions, insertions, deletions can create different variants of genes

Gene duplication may can give rise to multigene families

Translocations can also create new genes (by exon shuffling mixing together different protein-coding segments)

Horizontal gene transfer (from other organisms/viruses) is another source of new genes

what results in gene duplication

Gene duplication can result from unequal crossing over and large chromosomal rearrangements

What happens when a change occurs that is very damaging to the organism?

dies an ddamages chain

What happens to changes in DNA of somatic cells?

not inherited by offsprings

orthologs

speciation gives rises to two separate species

paralogs

gene duplication and divergence

Gene “homologs”

(meaning sharing similarity because of common ancestry/origin

include orthologs and paralogs

Evolution of Globin gene

Globin gene family includes hemoglobin, myoglobin, and plant leghemoglobin

Pathway of globin gene evolution: Creation of a multigene family from a single ancestral gene via gene duplication, mutations, deletion and transposition

Pseudogenes

have a similar sequence to the gene family but have accumulated so many mutations that they are now non-functional.

Which of the following are true about globin genes in humans?

A)They are paralogs

B)They are orthologs

C)They are homologs

D)They are alleles of each other

E) They are all pseudogenes

F) Comparing their DNA sequences will show a lot of similarities but also some divergence

a,c, f

Why do we care about small genetic changes? Do they really matter?

• Human and chimpanzee genomes differ by ~4% • Some minor genetic differences can result in major phenotypic differences!

Genetic variations within human populations

Single Nucleotide Polymorphisms (SNPs, “snips”)

Copy number polymorphisms (CNPs)

Structural Variation

Single Nucleotide Polymorphisms (SNPs, “snips”)

• Most common variability among humans

• SNPs in protein-coding regions contribute to the phenotypic differences in humans (alleles, some linked to diseases, drug interactions, etc.)

Copy number polymorphisms (CNPs)

• Differences in the number of copies of a particular sequence

• In protein coding region, extra copies = more protein → phenotypic differences

Structural Variation

Large segments of the DNA can be changed by duplication, inversion, deletion etc.

Which of the following are TRUE about comparing DNA sequence of genomes across species (select all that apply):A) All regions of DNA accumulate change at the same rate

B) Exons are more highly conserved then introns

C) All conserved sequences encode for proteins

D) Protein-coding sequences are more highly conserved than the location of genes on particular chromosomes

E) Highly repetitive DNA is more highly conserved as it is easier to replicate without errors

b, d

You want to use comparisons of genomic DNA for the purposes of identification or phylogenetic classification. Select the best strategy below:

A) Use the number of copies at a site of microsatellite DNA to trace species divergence from

the last common ancestor of bacteria and humans

B) Figure out if your poodle mix dog is more Bernedoodle, Bordoodle, or Springerdoodle by comparing the protein coding sequence of DNA polymerase

C) Use the number of repeats in minisatellite DNA to figure out who the father of an individual is (a paternity test)

D) Use the number of chromosomes in a blood sample to identify a murdered in a criminal investigation

c

Imagine 10 years from now you are working in medicine and you read a patient’s chart and tell them they will be switching to a different medication, because they have a SNP associated with a particular drug interaction. They say… “Huh?” and start asking you a LOT of questions and want to have a full lesson in genetics and molecular biology. Which of the following are TRUE things that could come up in this discussion? (select all that apply)

A) SNPs only occur in somatic cells, so don’t worry; they won’t be inherited by your children

B) SNPs can create different versions of a gene (also called alleles) that result in different traits in a person

C) You inherit one copy of each gene from each of your parents, but these might be different versions (alleles)

D) A SNP is a particular change or difference at 1 base pair in the sequence of your DNA

E) Proteins encode RNA which encode DNA, so damage to a protein results in a mutation in the associated gene in DNA

F) SNP stands for Separate Nuclear Polymorphism

b, c, d

What would happen to life on earth if DNA replication (and repair) were always perfect?

no variation

Perfect replication = no evolution = eventual extinction

What would happen to life on earth if DNA replication (and repair) were much more error-prone?

Too many harmful mutations → short-term survival risk

proposed models of dna replication

semi conservative replication

conservative replication

So which is it? Conservative, semi-conservative or dispersive? 34

DNA Replication is Semi - Conservative

DNA strand is synthesized in what direction

5’ to 3’

Incoming nucleoside triphosphate base pairs with the _____ strand and is then covalently attached to the ___

template

3’ OH

what provides the energy to create this new bond

hydrolysis of the bond between phosphates

Could NOT work if trying to extend the 5’ phosphate)

Replicating DNA with a low error rate - how

DNA polymerases add new nucleotides to a growing DNA strand in the 5’ to 3’ direction

1 existing DNA strand serves as a template, and if an incorrect nucleotide addition disrupts complimentary base-pairing, then it is usually detected and corrected

Proofreading function of DNA polymerase:

• DNA polymerase has a separate catalytic sites for polymerization and for editing

• DNA is pushed into the editing site when a mismatch is detected

three Proofreading mechanisms during replication

5’ → 3’ polymerization

3’ → 5’ exonucleolytic proofreading

strand- directed mismatch repair

Strand directed mismatch repair (MMR)

1. Recognition of mismatch by MutS

2. Identification of newly synthesized strand

3. Removal of incorrect nucleotides from new strand

4. Resynthesis of excised segment (using other strand as template)

5. Ligation to seal DNA backbone

what is used for recognition of mismatched base-pairing

Distortions of the double-helix

Why edit the newly synthesized strand instead of the template strand?

Ability to reversibly make single-stranded DNA (and use it as a template) is critical for transcription and DNA replication AND allows us to probe and synthesize DNA in the lab

How does the structure of DNA make replication possible?

- Hydrogen bonds between bases allow DNA strands to be separated.

- Opening up DNA (making it single stranded) allows each parent strand to serve as template for synthesis of a new complimentary strand.

- Complimentary base-pairing allows the selection of the correct nucleotides to properly replicate the sequence.

Renaturation also referred to as

Reannealing or Hybridization

process of DNA Denaturation and Renaturation

dna double helices → heated → denaturaturation to single strands (hydrogen bonds between nucelotide pairs broken) → slowly cool → renaturation restores dna double helices (nucleotide pairs reformed)

DNA Denaturation

Experimentally when DNA is slowly warmed to a certain temperature, DNA strand separation begins

Different DNA sequences denature at different temperatures

Thermal denaturation of DNA

DNA Melting

why is uv radiation useful for determining dna concentration

The nitrogenous bases of a nucleic acid absorb UV radiation (maximum at 260 nm), which can be used to determine DNA concentration

Single stranded (ss) DNA absorbs ___ as much UV light compared to double stranded (ds) DNA

twice

Melting Curve - y axis and x axis

UV absorbance on Y-axis, temperature on X-axis

Tm

the temperature at which the shift in absorbance is half completed

do all dna sequence denature at same temp

no

The higher the GC content of DNA, the ____ the Tm

higher

DNA Renaturation

Occurs when denatured DNA is allowed to cool and complementary sequences ‘find’ each other and base pairing begins

After renaturation, DNA is once again a double-stranded helical molecule

Renaturation Rates in diff size genome

Small genome = faster renaturation

Large genome = slower renaturation

how to make and measure a C0t plot

1. Shear DNA into equal-sized fragments – to avoid size effects on renaturation speed.

2. Heat the DNA to denature it (separate strands).

3. Cool slowly, allowing complementary strands to renature (hybridize).

4. Measure how much DNA has renatured over time.

C0 t value:

Combination of two values: 1. DNA concentration 2. Incubation time

• X-axis: C0 t plot

Initial DNA conc. (C0 ) X time of reaction (t)

• Y axis: C0 t plot

Fraction (percentage) of original DNA conc. that is renatured (dsDNA)

C0 t plot Curve shape:

* Single symmetrical curves because all sequences are present at the same concentration

Will repetitive sequences anneal faster or more slowly?

more faster

Experiment involves recording data on denaturation of DNA

“DNA Melting Curve” or “C0 t plot”

neither

Experiment involves recording data on DNA polymerization

“DNA Melting Curve” or “C0 t plot”

C0 t plot

Experiment involves recording data on DNA hybridization

“DNA Melting Curve” or “C0 t plot”

DNA Melting Curve

Experiment involves treating sample with various temperatures

“DNA Melting Curve” or “C0 t plot”

DNA Melting Curve

Experiment involves quantifying changes in the sample based on absorbance of UV light

“DNA Melting Curve” or “C0 t plot”

DNA Melting Curve

Results reflect the %GC base pairs in the DNA

“DNA Melting Curve” or “C0 t plot”

DNA Melting Curve

The shape of the results graph depends on whether or not the DNA contains a mix of both unique & repetitive sequences

“DNA Melting Curve” or “C0 t plot”

C0 t plot