BIOL 3010 Exam 3 Flashcards

Double Strand Break

both homologs are broken at a particular point

• can be caused by…

  • error in mitosis/meiosis

  • ionizing radiation

  • transposable element insertion/mobilization

Chromosomal Rearrangment

illegitimate recombination of chromosomes following a double-strand break

• leads to mutation if not repaired

• especially common in places with repeated sequences

• can occur in somatic or germline cells

Deletion

genes in the middle of a chromosome are lost

genes on the end end up too close together

Inversion

after DSB, the middle of the gene is inserted backwards

Deletion & Duplication between sister chromatids

both chromosomes are broken and joined together incorrectly

• results in 1 too short of a strand (deletion)

• and 1 too long of a strand (duplication

Translocation between non-homologous chromosomes

fusing the ends of incorrect chromosomes

• 2 completely novel genes

Acute Radiation Syndrome

-an issue in Chornobyl from the penetrating gamma rays

• death by loss of proliferative stem cell population

131I exposure lead to increased risk of papillary thyroid carcinoma - why?

• thyroid readily uptakes iodine - caused DSB and mutation

Outline outcome of RET/ELE1 Rearrangement

~35% of papillary thyroid cancer cases

inversion in chromosome 10 caused RET (receptor tyrosine kinase) and ELE1 (a transcription factor) to be too close together

• RET no longer needed a ligand for signaling and could proliferate

What are the two ways translocations can underlie cancer?

1. ones affecting regulatory elements and gene expression

   1. enhance becomes closer to a coding sequence and changes the expression

2. ones affecting open reading frames and protein function

   1. different, novel combos of the genes - fusion

Homologous recombination

ideal way of DSB repair- much like crossing over process

• exonuclease chews away strand around the break and the sister chromatid strand invades

  • replication of DNA with sister as template

  • almost perfect

Single Strand annealing (SSA)

less ideal way of DNA repair - at long homologies

• uses a similar-ish sequence of the same strand as a template to repair the DNA after exonuclease comes in

• can result in large deletions

• DNA ligase fuses the backbone back together

Alternative end-joining (altEJ)

ess ideal way of DNA repair - at short homologies

• allows reattachment of chromosome ends

• mutagenesis rearrangement that can cause insertions and deletions

Non-homologous end-joining

pretty ideal?

• joins the blunt ends of broken DNA back together

• however, if there are multiple DSB there is a potential for translocation

What is special about tardigrades?

extremely resistant to ionizing radiation due to presence of damage suppressor protein

Dsup

expressed in all tardigrade cells - a protein that binds to nucleosomes to protect against damage from ionizing radiation

• include single/double strand breaks and free radicals

• in study testing function: cells treated with Dsup had less “oozing comet tails” than those that were not - less damage

Deficiency allele

a large deletion that results in removal of entire gene

ETV6-NTRK-3

inversion of these genes accounted for ~14% cases of papillary thyroid carcinoma

• due to inversion of TF and kinase

Regions of synteny

shared gene order between or within species

• can be uncovered by comparative genomics

• big blocks of genes remaining together across evolutionary time

gar/chicken>car/human>human/mouse

Significance of Gar?

share a lot of gene order with tetrapods, especially the chicken

• it is because it has also underwent 2 WGD like tetrapods

• genome is very comparable

Muntjak example takeaway

reevesi and muntjak share a common ancestor with many small chromosomes, but a fusion event in both resulted in different numbers of chromosomes in each

• still very genetically similar

• Muntjak- 4 large chromosomes

• reevesi - lots of small ones

Paracentric v pericentric inversion

paracentric-centromere is not in the inverted portion of chromosome

pericentric- centromere is in the inverted portion of the chromosome

*we talked about pericentric

acentric & dicentric strand

acentric- after crossing over, strand without centromere

• lost

dicentric- after crossing over, a strand with two centromeres

• pulled in 2 opposite direction and breaks into 2- inviable

Immediate meiotic consequence of inversion:

heterozygote for inversion has reduced fertility

Long-term meiotic consequence of inversion:

there are no crossings over at sites of inversion

• genes not broken up so they can acquire new mutations together

Super gene

alleles always stay together and do not participate in crossing over- result of inversion

ex. Ruff bird

Ruff bird example significance

males have 3 phenotypes: wildtype, satellite, and Faeder

• used PCR priming to discover that satellite and faeder are heterozygous for an inversion

  • has supergene that has acquired multiple mutations such as ones in melanocortin 1 receptor required for pigment

Inversion loop

when a non-sister chromatid has an inverted segment, it must take on an inversion loop during crossing over to line up the base sequence

Homologous gene

genes that have shared ancestry

orthologues

homologous genes of different species

ex. between gar and chicken

paralogues

homologous genes in same species due to a duplication event

ex. Hox genes

Copy number variants

aka CNVs

major class of genome structural variation

• we all have different amount of copies of these regions

• deletions and insertions associated with disease

Rhodesian ridgeback dog example

“ridgeback” trait is dominant and predisposes dog to dermoid sinus

• there is a duplicated region that has 3 Fibroblast Growth Factor genes which play role in hair development

  • results in reversal of hair direction

Fibroblast Growth Factor

3 extra copies in Rhodesian ridgeback mutant

• secretes growth factor that results in reversal of hair direction if you have too many copies

Dermoid Sinus

congenital disease that is associated with ridgeback phenotype of Rhodesian dog

Whole Genome Duplication

occurs through different abbreviations of the cell cycle

• polyploid cells are common and preferred in some cases

has happened throughout evolutionary history

Endomitosis

mitosis occurs but doesn’t result in cellular division

• if the nucleus divides- multiple nuclei

• if not - 1 nucleus

Endocycle

cell cycles between the G1 phase and S phase repeatedly without mitosis

Rediploidization

after a whole genome duplication, some of the duplicated genes are maintained, but most are lost

• because of redundancy in function

• if no selection to maintain both copies they are lost

ex. zebrafish underwent an additional duplication, but only has 30% more protein-coding genes than humans

WGD’s in vertebrate lineage

2 WGD in tetrapods and Gar

3 WGD in teleosts, some underwent a 4th

WGDs in Hox genes

conserved synteny with evidence of WGD in hox genes

• found as 1+ clusters of multiple genes

• remain synteny within/between species even though there were losses and duplications

ohnologues

homologous genes arising by whole genome duplication

Neofunctionalization

when cis regulatory element involved in duplication..

with additional mutation, results in complete loss of new gene

nonfunctionalization

copies of alleles diverge functionally because one of them acquires a new function domain

• made possible because the top gene is covering original function

• additional mutation is in regulatory element

subfunctionalization

loss of an additional regulatory element

• to fulfill the initial function, you now need two different genes

Why do polymerase errors lead to expansion/contraction?

especially in repeated sequences, it is easy for DNA polymerase to slip forwards or backward

-repeats aren’t paired correctly and a portion of nt sticks out

-one round of replication happens: have 1 larger and 1 shorter copy

-results in the divergence of alleles

Huntington Disease

poly Q expansion

• symptoms

  • late onset neurogenerative disorder typically appearing ~30/40

  • progressive chorea, defects in speaking and swallowing, mental decline

because of a glutamine expansion in exon 1 of the huntington protein

Point mutations

single nucleotide additions, deletion, or substitution

• can result from polymerase errors

Transversion

point mutation going prom purine←> pyrimides

ex. A to T or C, T to G or A, C to G or A, G to T or C

Transition

point mutation between purines or pyrimides

ex. A - C, G - T

Replication-coupled DNA repair

DNA polymerase has exonuclease activity that proofreads sequence 3’ to 5’

• corrects vast majority of point mutations

• takes out wrong base, allows for new, puts correct one in its place

Mismatch repair Pathways

corrects polymerase errors and DNA damage

• associates with DNA and scans for missmatches

  • exonuclease digests region→ template strand protected by RPA → recruits accessory proteins that are stimulated to cut strands → new replication by DNA polymerase → nick sealed by DNA ligase

DNA Damage: Hydorlysis

depurination - guanine released

deamination - the amino group is taken off cytosine and it turns into uracil

• after replication, turns site into TA

DNA Damage: Oxidation

oxidized guanine looks different to polymerase - pairs with A

• after replication, results in TA site

Nucleotide/base excision repair

general steps:

• proteins scan for problems

• portion chewed away by exonuclease

• polymerase lays down correct nucleotides

• backbone ligated together

Crosslinked DNA (intra v inter)

inappropriate covalent bonds within (intra) or between (inter) strands of DNA

• blocks transcription and replication

• intrastrand fixed by base excision repair

• interstrand requires a big protein complex to fix it during replication

Fancomi anemia

symptoms:

• growth retardation, hyperpigmentation, renal/skeletal abnormalities, mental impairment, hear defects, cancer risk

Why?

• due to damage resulting from interstrand crosslinked DNA

de novo mutations

mutations that arise in the germline of the parent

• observed at a 4:1 ratio in paternal gametes

• can be calculated through a trio sequencing test

trio-sequencing

whole genome sequencing of the mom, dad, and progeny to determine if a mutation arose in the germline or somatic cells of the parents

• found that mutations 4x as likely to cone from paternal allele and increased as paternal age increased

Why are de novo mutations more common in paternal gametes and aneuploidy mutations more common in female oocytes?

de novo is more common in males because there is continuous replication of DNA in the sperm cells

• more likely to have error

aneuploidy more likely in female oocytes because of delayed completion of meiosis

Loss-of-function alleles: null

mutation resulting in complete loss of gene product

Loss-of-function alleles: hypomorphic

mutation causing gene to make less product or be less active than wildtype

Gain-of-function alleles: overexpression

mutation causing higher expression of a gene than the wildtype

Gain-of-function alleles: hyperactivity

mutation causing higher activity than wild-type in the product itself

ex. a receptor no longer needs a ligand to produce product

Gain-of-function alleles: neomorphic

mutation causing a gene to take on a new expression domain/protein activity

Advantages of drosophila as model organism

• short generation time

• many progeny

• can evaluate exoskeleton under a microscope

• only 4 chromosomes - often polytene

• many similarities in genes/functions with vertebrates

Polytene chromosome

natural polyploid where multiple copies of the same gene are present in the same area

• advantageous for examination of model organism

Disadvantages of drosophila as model organism

stocks must be maintained as “living” cultures

N-ethyl-N-nitrosourea (ENU)

an alkylating, mutagenic agent that increases mutation rate per locus past what would be spontaneous

• to use mutation as a genetic tool

adds ethyl group to induce random base changes throughout the genome

Ethyl methane sulfonate (EMS)

an alkylating, mutagenic agent that increases mutation rate per locus past what would be spontaneous

• to use mutation as a genetic tool

adds ethyl group to induce random base changes throughout the genome

Main goal of Nusslein-Volhard & Wieschaus

sought to identify genes required for segmentation, anterior/posterior or dorsal/ventral differences of Drosophila

• conducted forward genetic screens to identify certain genes important for embryonic development and body patterning

Forward Genetic Screen

mutants identified by phenotypes of interest and then genes responsible for phenotypes are determined later

ex. drosophila experiment

Reverse Genetic Screen

targets a specific gene and see if it produces specific phenotype

Saturation of Genetic Screen

when you are finding fewer mutation at new genes

• saturated when new alleles are at loci for which mutants have already been found

• could get new alleles, but on the same gene

Gap gene mutants in Drosophila

mutants have large regions of banding missing

ex. krupel, knirps

Pair-rule gene mutants in Drosophila

mutants have alternating segments lost, the defects are in pairs

ex. even/odd-skipped, paired, runt

Segment polarity mutants in Drosophila

portion of the segment is missing or altered

ex. gooseberry, patched

General cascade of genes impacting body development in drosophila

hierarchy of genes that establish body plan

• cytoplasmic polarity → hunchback protein gradient → gap genes→ pair-rule gene → segment polarity genes

Outcrossing

crossing a heterozygote F1 with a homozygous parent

Complementation Test

use to see if two mutations are alleles of the same or of different genes

• cross two mutants and get a mutant progeny

  • the same gene!

• cross two mutants and get wild-type

  • different gene! heterozygous

• only works for recessive alleles

Meiotic mapping

uses recombination that occurs during meiosis to narrow down the location of a mutation in the genome

recombination frequency and cM

recombination frequency = # of recombinants due to crossing over/ total alleles

multiply this by 100 to calculate the genetic distance in cM

• follows idea that the closer two genes are on a chromosome, the less likely they are to be separated during crossing over

what does little crossing over between two points indicate

genes are really close together

Loci markers

Identifiable physical location on the DNA where the sequence is variable and can be detected

• provide a landmark on a gene that we can compare mutant gene to by seeing which alleles at marker loci across genome phenotype most closely associates with

What are stretches of DNA that can be used as marker loci?

1) microsatellites

• repeated stretched of CA can be variable across individuals

  • can detect size differences on gel

  • 2 bands = heterozygote 1= homozygote

2)Restriction Fragments

• at a locus, specific site is cut by particular restriction enzyme

  • homozoygous if 1 or 2 bands, heterozygote if 3 bands

3) Use of PCR and Sanger Sequencing

• shorter wave at a site indicates variable alleles

in general we want to look for nucleotides that are variable within population

Effect of gene redundancy and compensation

some mutant genes won’t show phenotype

• other genes may be picking up the slack

fusion

Smaller chromosomes join together to make larger ones

fission

Larger chromosomes split to becomes smaller ones

Gray

a measure of radiation

genotyping

process of determining differences in the genetic make-up of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence

• important in meiotic mapping

ligase activity

Joins DNA strands together by catalyzing phosphodiester backbone to form a bond

important in DNA repair!

spontaneous mutant

mutations are randomly identified by a breeder, no mutagenic agents were intentionally used to cause damage

• very rare and hard to predict

informative meiosis

During genetic mapping, when an individual is heterozygous for an allele, their gametes will undergo crossing over to create two distinct sites where genetic backgrounds are switched

You can use these to determine relative position of gene on chromosome

inbreeding heterozygote - 2

outcrossing w homozygous parent - 1

recombinant

Chromosomes that carry a mix of alleles due to crossing over of non-sister chromatids

Cans use the frequency to calculate cM, genetic distance

complementation

When mutations are in different genes - outcome is a heterozygous wildtype progeny when crossed

non-complementation

When mutations are on the same gene, the result when crossed is a mutant

allelism

The same gene having variable sites

• important for marker loci to have variability so you can compare genetic backgrounds

phenotypic rescue

An important strategy for identifying the right gene of interest

• By either replacing sequence itself or adding protein activity

• Switching the mutant phenotype to the wild-type by adding in what gene is lacking

revertant allele

A wild-type allele present that was once a mutation

• Researcher targeted the gene to alter sequence and see if the issue was resolved

transgene

cloned segment of DNA in a test tube that researchers have inserted into the genome of an organism

• Could be used to produce lacking protein activity/products

genetic background

all of the alleles of genes in an organism’s genome; the set of unknown modifier genes that influence the action of the known genes that control specific aspects of phenotype

• needs to be different between mutant and marker loci so you can compare to see where crossing over took place