Genetics Exam 3

What is a “lost” gene?

a pseudogene (inactive)

Recent example of a species with a genome duplication and how to track these duplications

Teleost fishes (like zebrafish) had a recent duplication compared to vertebrates

Hox genes: very common among species, help with body plan specification, have shared synteny

• invertebrates have 1 set, vertebrates have ~4, teleosts have ~7

• ohnologs: homologous genes from WGD

Why would genes be lost or retained

lost: extra gene is redundant or a lethal dosage

retained: extra gene is beneficial or develops a new usage; more common for developmental genes

fates of duplicated genes and how gene function may be affected

• mutation in coding: possible new protein

• mutation in noncoding: possible altered expression

  • nonfunctionalization: pseudogene forms

  • neofunctionalization: mutation changes specificity of the gene

  • subfunctionalization: both copies of gene are needed for full function

    • divide duties

what is a karyotype, how to make one, and what it can do

• whole chromosome analysis that puts them in order of largest to smallest

  • AT rich (gene poor) is darker than GC rich (gene rich)

• done by arresting cell in metaphase

  • FISH: fluorescence for easier viewing

  • spectral karyotype: various colors to show specific regions of each chromosome

• can show translocations, inversions, and aneuploidy

what is an aneuploidy, how can it happen, and how to screen for it

aneuploidies are abnormal numbers of chromosomes

• caused by nondisjunctions of chromosomes/chromatids and results in gametes that are n+-1

• use various karyotypes to screen

  • FISh

  • Spectral karyotyping (24 colors)

trends for aneuploidy occurences

• occurs in 5-25% of fertilized eggs, very few are born

  • 1-2% of sperm and ~20% oocytes

• higher maternal age leads to breakdown of cohesin and structural proteins that help with regular disjunction

  • leads to higher chances of aneuploidies

regions of human sex chromosomes

PAR1 and PAR2 are on the ends of both X and Y chromosomes, are ~30 genes; PAR genes are both active even when one X is inactive

MSY region has 3 male fertility genes and 8 other genes found on X; contains SRY which is sex determining gene (if had, are male)

sex chromosome aneuploidies

• Sex reversal by translocation

  • XX but with the SRY gene = male

  • XY but without SRY gene = female

  • when ends of X or Y are translocated

• Turner Syndrome: only 1 X chromosome

  • results in half PAR dosage

    • PAR contains SHOX (helps with growth) leading to a short stature

    • germ line is infertile because not enough gene copies

• Klienfelder syndrome: males with an extra X chromosome

  • Extra active SHOX genes (in PAR region on extra X) leads to longer limbs

  • increased breast cancer risk

double strand breaks, how they happen, types of alterations they can lead to

• phosphodiester backbone of both DNA strands is cleaved

• more common in areas with repeated sequences (microsatellites)

• Occur due to: erros in meiosis/mitosis and/or ionizing radiation

• can result in insertions, deletions, or inversions

  • Deficient alleles: large deletions that remove entire genes

ways to repair double bond breaks and their pros/cons

• Nonhomologous end joining (NHEJ):

  • results in 1-4 nt deletion

  • fastest so most used

  • ends are directly ligated back together with no template

• Homologous recombination:

  • most accurate because homolog chromosome is template

  • slower so less used

  • end of each strand is resected then strand invasion (like in crossing over) occurs

• Single strand annealing (SSA):

  • used if long repeating sequences flank the break

  • ends are resected until the repetitive sequence can allign with each other

  • large deletion that was originally between the repeats

• Alternative end joining:

  • like SSA but when the repeating sequence is shorter

  • large deletion that was originally between the repeats

• SSA and AEJ can lead to deficient alleles if deletion in the right place

immediate and long term consequences of 1986 Chernobyl disaster

immediate: acute radiation syndrome lead to death becasue of loss of proliferating cells (because they are fastest dividing and have little time for DNA repair)

long term: increased thyroid carcinoma risk in children

• ~35% of cases: ETV6 (a TF) and NTRK3 (a receptor tyrosine kinase) on two different chromosomes are accidentally fused together after the break

  • leads the growth receptor to be constantly “on”

  • a translocation

• ~14% of cases: chunk of chromosome inverted fuses two genes together that leads to a cell growth signal staying in the “on” position

evidence for roles of chromosomal inversions/translocations in human cancer and types of cells involved

• translocations can impact ORFs or regulatory elements and change the protein or the frequency of expression

• translocations/inversions that fuse genes can cause hyperactive tyrosine kinases and frequent division

  • ex: ETV6 and NTRK3 in breast cancer and thyroid carcinoma

• frequently diving cells have less chance for DNA repair making cancer more common

• can see all of this through karyotyping and flourescence

tardigrades vs humans in extreme environments and evidence about Dsup

• have strong stress resistance (freezing, chemicals, vacuum, radiation)

  • up to 6500 Grey compared to the ~1 Grey that killed Chernobyl firemen

• Dsup gene is in all of their cells and binds to nucleosomes (acts as a barrier)

• experimenters tested how tardigrades responded to stress with and without Dsup:

  • 4 groups: 2 with Dsup and 2 without; one of each type got radiation treatment

  • comet tails in gel electrophoresis indicated fragmented DNA

    • less seen in cells with Dsup

• also tested impact and used flourescence as visualization tactic

  • tested no Dsup, Dsup, and Dsup + shDsup (supressor)

  • groups A and C had more flourescence (DNA damage)

synteny definition and how it helps with inferring evolutionary chage; compare gar and tetrapod

• synteny is shared genome order seen between different species; helps track WGD

  • Hox genes have synteny that has been easy to track between species

• gar have one less WGD that other teleotsts (other fish)

• gar and chicken have many ortholog genes (homologous in different species)

  • from common ancestor

  • discovered by comparing synteny

• mouse and human have little synteny

homologous vs orthologous vs paralogous

homologous: genes with similar positions in chromosomes and with similar structure/origin even if different now

orthologous: homologous between species

paralogous: homologous genes due to genome duplicaitons

synteny and chromosome evolution across Muntjac deer

• have varying sized chromosomes and number of chromosomes due to fusions

  • M. muntjak is 2n=6

  • M. reevesi is 2n=46

• synteny shows they have the same genes just on different chromosomes

• in phylogeny: outgroups (cattle) have similar genomes to those that are 2n=46

  • ancestors had higher number of chromosomes

  • proves fusions

consequences on inversions for gamete production and why does it suppress recombinant phenotypes for genes in the inversion

• paracentric inversion: centromere is not in the inversion

  • homolog chromosomes can only pair up for possible crossing over by forming an inversion loop

  • can result in multiple or no centromeres on each chromosome

    • if have multiple: broken apart in anaphase

  • gametes are nonviable because wrong number of chromosomes

  • only offspring from nonrecombinant gametes (no crossing over) can survive because correct centromeres

    • suppresses recombinant genotypes

    • have to be heterozygous for the inversion to prevent crossing over

• paricentroc inversion: the centromere is a part of the inversion

consequence of inversion if they persist over time and evidence

• can lead to super genes: areas of large inversion where inverted genes are locked together

  • have to be heterozygous for the inversion

    • homozygous would allow for crossing over and gametes would be nonviable

  • offspring have the same combination of genotypic traits

• Ruff species: 4.5 Mb region is inverted (super gene)

  • super gene for bird’s size, color, mating behaviors, etc

  • Mc1r is a melanocortin receptor in the region that impacts pigmentation in the species

copy number variants and how common they are

when a large region of DNA is duplicated; is ~5-10% of the human genome

CNV and ridge/dermoid sinus phenotypes in dogs

• ridge coats are a result of a 33kB duplication that contains 3 Fibroblast growth factors (FGFs) which have a role in hair development

• ridge allele is dominant

  • most dogs are heterozygous

  • if homozygous: dysregulation of FGF genes can lead to dermoid sinus

• observed as a larger than normal amount of DNA on a particular chromosome/in a particular area

consequences of simple sequence repeats

~20 human diseases associated with trinucleotide expansion

• ex: making a cisregulatory element further from the promoter leading to decreased efficiency

• huntington disease (neurodegenerative disorder): doe to a PolyQ expansion in exon 1

  • glutamine repeat in HTT leads to misfolding of the protein and toxicity

how simple sequence repeats ca accumulate through DNA replication

• polymerase slipping occurs when the polymerase temporarily pauses, allowing the synthesized strand to slip/form a small loop

  • a repeat of that slipped area is added again

• this repeat will be used as a template in future replications

  • process repeats and repeats are accumulated

how increases/decreases in repeats influence genome size across organisms; give examples; explain C-value paradox

• repeat copy numbers make up majority of genomes as the genome size gets larger

  • Drosophila, humans, and australian lungfish have ~same amount of protein coding sequence but various sized genomes

    • due to different amounts of repeats/transposable elements

      • introns, repetition, synteny

  • C value paradox states that the size of the genome does not correlate to the compelxity of the organism

    • Ex: humans and salamanders have ~same number of genes but salamanders have 10X bigger genome

    • genome size driven by repeats not the coding sequence amounts

DNA polymerase error rates and how are they corrected

• very common: errors every 10^24 to 10^6 nucleotides

  • totals to around 1.2×10^4 to 12×10^6 errors per cell per round of DNA synthesis

  • A to G and C to T are most common

    • transitions are more common than transversions

  • transversions are when a purine becomes a pyrimidine or vice versa

• mutations only occur if errors aren’t fixed

• DNA repair mechanisms:

  • proofreading by 3’ to 5’ exonuclease in the DNA pol complex (Polδ, Polε)

  • first line of defense

    • mice with a mutation that block proofreading abilities die younger from cancer

  • mismatch repair: follows polymerase and detects mismatch, cuts and resects region, a new polymerase tries again

    • mismatch recognized by MSH proteins

    • other proteins are recruited to cut the DNA

    • Nick sealed by DNA ligase

how DNA damage can lead to point mutations and how this compares to polymerase error

• hydrolysis, oxidation, and methylation occur everyday; ~20000 events per cell per day

  • hydrolysis: either depurination (OH at base site) or deamination (amine in base replaced with O)

  • oxidation: O added onto the base causes wrong base pairing

    • GO acts as a T

      • GO/C → T/A

  • UV induced thymine dimerization: caused by UV light, blocks replication

• DNA damage happens continuously because of environment and metabolism

  • polymerase errors are only during S phase

  • DNA damage only becomes a mutation if damage is template for new synthesized strand

what is Fanconi anemia and how does it occur

• impacts 1 in 160000 individuals, die ~16 years old

• Seen as growth retardation in 70%, hyperpigmentation in 80%, and skeletal/mental/heart defects in 10-30%

• also at 15000x risk for cancer because of progressive bone marrow failure

• arises from a mutation in a gene that is a part of the Fanconi Anemia (FA) core complex

• FA complex usually repairs DNA interstrand crosslinks

  • covalent bonds that form when linking shouldn’t occur

  • can result in lined chromosomes

men and de novo point mutations and why is it higher than women and the evidence

• comes from trio sequencing: mother, father, child

  • observe mutations/abnormalities in the child that are not in the parents

• Sperm is constantly produced by men; as they age the cells have replicated many more times, giving more chance for mutations to accumulate

  • female gametes only finish meiosis once the oocyte is fertilized

    • i.e. less chance for de novo point mutations to occur

• linear relationship by whole genome sequencing when comparing man’s age to number of mutations

aneuploidy and age

• caused more by women during cell division (nondisjunction)

  • because as a woman ages, the structural proteins and cohesins break down while arrested in meiosis

  • when meiosis finally finishes, chromosomes/chromatids are less likely to separate correctly if the mother is older

spontaneous mutants

mutations that occur naturally, often due to polymerase error or spontaneous chemical changes

ways to classify mutations and their effects

• region affected:

  • coding: deletion/insertion, nonsense, missense, synonymous substitute

  • intro/exon junctionL missplicing of RNA

  • regulatory region: altered transcriptional efficiency

• phenotypic consequences: depends on how gene function is affected

  • loss of function allele

    • null is full loss

    • hypomorphic is reduced product/activity

  • gain of function allele increases amount of product/activity

    • overexpression: higher expression

    • hyperactivity: higher activity

    • neomorphic: new expression or activity

how to increase mutation frequency in labs

• mutagenize individuals to induce mutations

  • often done to males because they produce more gametes

• Use EMS (or ENU depending on species)

  • goal is to increase copy of mutant alleles and them make homozygous species for the mutant

  • Cross EMS male with female and get F1 heterozygous male

  • Cross F1 male with regular female to get F2 of both species (hetero)

  • cross heteros and get homozygous mutant

• difficult because don’t know which is heterozygous so have to do many crosses

• can also use radiation or transposable elements

strengths and weaknesses of Drospphila as a genetic system and how does it compare to other species

• strengths: 10 day generation time, only 4 chromosomes (a polytene), exoskeleton allows for easy viewing of phenotypic changes

• weakness: have to maintain a live culture

• comparisons:

  • mouse: closer to humans and can do knockouts but longer generations and more expensive

  • Zebrafish: transparent embryos and large clutch sizes but past genome duplications complicates screens

  • Nematode: can freeze and has the exact same cells in very organism but lacks complex organs

why multiple crosses for trying to analyze recessive mutant alleles

• don’t know which are heterozygous (lots of chance)

  • only a portion are the intended/needed offspring

• only after 3 crosses of correct individuals can the mutant phenotype be apparent

  • EMS and normal get some mutant heterozygous

  • heterozygous with regular female and 50% are heterozygous

  • cross two heteros and 25% are homozygous

what is saturation in a screening and what to do to assess saturation

• saturated is when a new mutant allele is discovered in a locus that another mutant allele was previously found at

  • reach full saturation if a mutant is found for every gene in the genome; further screening leads to repeat of mutations

• assessing saturation using a complement test:

  • done if two mutant phenotypes arise and look similar at similar loci

  • cross homozygous versions of each mutant

  • if they appear a phenotypic mutation then they are the same gene

    • becasue if they are different genes the offspring will be heterozygous and not show the phenotype

Nüsslein-Volhard and Weischaus phenotypic classes and what to infer if genes in the same class

• segment polarity: either the anterior or posterior end of each segment is altered; like a mirror

• pair rule: every other segment is removed

• gap gene: large gaps in the body plan

• if genes cause the same phenotypic mutations: can infer they play roles in similar/related processes

how early pattern screening of Nüsslein-Volhard and Weischaus contribute to animal development and what was novel

• revealed 3 classes of mutant phenotypes

• performed saturated mutagenesis screen to identify all recessive mutants of genes that change visible boy plan in flies

  • discoveries and testing of developmental cascades (like hedgehog) are applicable to many species

    • hierarchial order:

      • cytoplasmic polarity, hunchback protein gradient, gap genes, pair rule genes, segment polarity/homeotic genes

what are marker loci and how are individuals genotyped

• are places with allele variation in the genome (ex: microsatellites)

  • are usually noncoding and don’t impact organism’s phenotype

• genotyping individuals: use PCR and gel electrohphoresis

  • specific enzymes cut at particular sequences, fragments will be the wring length if marker loci are mutated

how recombination of phenotypes and genotypes at marker loci determine critical interval and what is genetic distance

• crossovers happen randomly in meiosis

  • the closer together two genes or markers are on the genome, less likely for crossing over to occur

  • the marker closest to the gene causing the mutant phenotype will very rarely have a recombinant gamete

• area of continuous homozygosity and minimal marker heterozygosity (little crossing over) while still seeing mutant phenotype is critical interval

  • only see mutant phenotype if gene is homozygous

• genetic distance is in centiMorgans and is equal to 1% recombination frequency/likelyhood

  • the distance between two points on the genome

marker loci on other chromosomes

• marker locus on other chromosomes assort independantly of the mutant gene

• around 50% recombination compared to mutant locus

why is it important to sample many chromosomes for mapping

• crossing over is very rare already, have to observe crossing over very close to the mutant gene at a marker loci just once or so (much less frequent than other places)

• looking for pattern of homozygotic marker loci while observing a mutant phenotype

recombinant frequency of 200 individuals

do number of recombinants/ 200×2 because 2 chromosomes each * 100

dominant mutant recombinant genotype and phenotype

• has mutant phenotype but genome doesn’t have the dominant allele

• indicated crossing over to get allele

forward vs reverse genetic approach

• forward: mutant is identified by its phenotype, determine which gene is impacted, infer gene function

  • can randomly mutate the genome and cross individuals until a mutation is visible

• reverse: determine the gene of interest, inactivate the gene, infer gene function based on phenotypic difference

  • ways to inactivate: homologous recombination, knockdown, CRISPR

  • usually no phenotypic difference

steps of making a homologous recombinant and why embryonic stem cells and why selectable marker

• introduce neo marker into DNA segment of inactive gene (flanked with homologous arms)

• get blastocyst cells from early embryos (stem cells)

  • exploit homologous recombination mechanisms and introduce synthesized DNA

    • only a small portion will properly integrate the segment of DNA containing th marker

• treat all cells in the culture with neomycin to kill any cells that didn’t get the knockout gene

  • neo marker has a resistance to neomycin so they don’t die while all else do

• inject knockout cells into blastocyst and put into female mouse

  • produces chimera

• breed chimera to homozygosity

• embryonic cells are still stem cells and can contribut to all tissues in the offspring including germ cells

  • essential for crossing to homozygosity

• selectable marker to make eliminating the cells that didn’t properly integrate the knockout gene