Molecular biology and Genetics Midterm 1

How did Mendel jumpstart field of modern genetics?

• Conducted systematic experiments and analysis using replicates and controls including applying statistics 

How did we determine dates of domestication and the birth of agriculture?

When plants and animals are domesticated, they are being selectively bred. This can cause marked genetic changes in these organisms

Examples

Figs

• discovery of seedless figs in Jericho 11,400 yrs ago

  • Called parthenocarpic figs

• Since seedless plants should be at a disadvantage, this indicates that they had been manually vegetatively propagated.

Horses

• Images of spotted horses → did exist prior to domestication 

  • Previously believed to not exist before domestication because the mutation causing spottedness also causes blindness in horses 

• Extracted bone marrow from spotted horses 

• Indicated that they were not selectively bred 

• Spotted horses did not have any other characteristics of domestication

Common Phenotypic changes associated with domestication 

• No seeds in plants 

• Piebald spotting 

  • Lack of coloring in an area on the head → indicates changes to neuro

• Floppy ears 

  • Indicates change in connective tissue 

Dogs and Siberian Fox Experiment 

• aken over by Cornell after fall of Soviet Union 

• Siberian Fox experiment aimed to try and recreate dog from foxes 

  • Bred aggressive foxes with aggressive foxes and softer foxes with softer foxes → resulted in genetic changes over time 

  • Selected for juvenile traits, many traits in domesticated animals are juvenile traits 

• Same process by which animals were domesticated for agricultural purposes 

Mendel Sheep Experimentation

• Mendel compared domesticated sheep and goats with non domesticated versions of these animals 

• Mendel’s question → what is inherited and how?

• Interest for Mendel 

  • Creation of hybrids in both plants and animals 

    • Could be interspecies hybrids or hybrids between different strains 

    • Hybrids over multiple generations were found to be unstable 

    • Instability of hybrids was a big question for Mendel and other researchers 

      • Unstable hybrids do not maintain the desired phenotype over several generations 

  • Under advising of Napp and Nestler, two sheep breeders in his monastery, Mendel conducted experiments

    • Were breeding their sheep for wool softness and mutton production

Alternate theories about life

some people believed that life could come from inorganic matter, which was disproved

discontinuous traits

can only be either or

Three hypotheses for genetics

• Uniparental (only one parent gives factors to offspring) 

• Blending (bi parental, but mixes traits) 

• Particulate (bi parental but no mixing of traits)

uniparental (preformationist)

• Spermists → Everything needed for an organism is contained within sperm. Female just provides environment that is suitable for the organism to develop 

• Ovists → Everything needed for an organism is in the egg. Sperm just provides incentive for the egg to develop

blending genetic theory

• Sperm and egg come together and blend in the hybrid 

• Stresses fusion and idea of stable hybrids 

• Focuses on the idea that by blending, a new thing is being formed 

  • Ex. mixing paint blue and yellow = green and then should stay green forever 

• The problem was that there were red + white flowers being bred to make pink flowers, but the next generation may be red, pink or white 

• Gave rise to particulate hypothesis 

particulate theory (mendel’s, right)

• Strains must be pure/true breeding (homozygous) for specific discontinuous phenotypes that are easily identified and clearly different between different striains 

• Must be an easy way to ensure paternity 

  • Good that he picked pea plants because they need to be fertilized before they bloom 

  • Tend to self fertilize 

  • Mendel cut stamen in some pea plants while they were still buds to ensure paternity 

• All genotypes should be equally fertile 

“proposes that traits are passed from parents to offspring through discrete, unchanging units called genes. Unlike the earlier "blending" theory, this model suggests that these genes do not merge but are sorted and passed down in their separate identities, and recessive traits can be masked but are not lost forever”

Law of dominance

• Phenotype transmitted entire or almost unchanged → dominant 

• Disappear in hybrids → recessive 

“when an organism has two different alleles for a trait, one allele (the dominant allele) will mask the expression of the other allele (the recessive allele), resulting in the expression of the dominant phenotype”

How did Mendel disprove ovists and spermists

• results did not show that the female/male trait was dominant (trying to cross specifically with female with the trait or male with the trait)  and it was found that the trait itself was dominant and not the ‘female trait’ or ‘male trait’ 

How did Mendel disprove blending believers

F2 generation shows reappearance of the recessive trait

How did mendel disprove blending theory

• plants still ended up white or purple, none were lilac or pale purple etc 

• All white flowers were true breeding and homozygous 

Mendel’s Law of Inheritance 

Genes tend to be named for which allele?

Recessive

Different opinions on what hereditary material is

• Darwin → circled around stoma and coalesces in gonads during mating 

• Galton → blood, transfused blood between rabbits thinking they would change traits 

• Weissman → germline passed on genetic information 

• Boveri and Sutton →found chromosome reduction in meiosis → oogenesis 

  • Used light microscopy observing cell nuclei in grasshopper testes 

  • Found evidence of meiosis reducing chromosome number in gonads → spermatogenesis 

spermatogenesis

Cis and trans orientation

During meiosis

dominant and dominant allele together, recessive alleles together 

Law of Segregation

alleles for a trait separate during gamete formation

during gamete formation (sperm and egg), the two alleles (gene versions) for each trait separate, so each sex cell receives only one allele, ensuring offspring get one allele from each parent

Mendel → Chromosomal Theories

Morgan and Bridges → fly studies

first to provide data of hereditary traits

chromosomal basis of sex in flies, found X linked white eyes trait in flies

• Looked at the few flies in his group that had white eyes → flies with white eyes turned out to be male 

  • Crosses white male with red female 

  • Got 100% red eyed flies 

    • Understand red to be dominant to white 

  • In the next generation males have white eyes in a 1:1 ratio to males with red eyes 

    • Slightly off because white eyed flies are less viable bc the gene responsible for making flies have red eyes is also involved in neural development 

  • He thinks this means the trait is linked to the x chromosome

• Once he got a white eyed female, he crossed them with males with red eyes 

  • Found that all the males had white eyes and all the females had red eyes 

    • This helped to show that the gene mutation was x linked and therefore was more prevalent in males 

  • After genotyping finds some rare classes 

    • Male with red eyes that are sterile (Only has one sex chromosome X and no Y, and got the X from the father)

    • Females with white eyes that are sterile (Has three sex chromosomes XXY, and got two X chromosomes from the mother and no X chromosomes from the mother)

    • These were results of nondisjunction 

  • Rare examples further proved that the rare eye color gene is contained on the x chromosome 

Observation of linked traits, flower example

• Bateson, Punnett and Saunders 

  • Studying red and purple flowers 

    • After breeding for stamen height and color between two different ones found that the ratio resulting from crossing the two did not match independent assortment

• Found 

  • Heterochromatic knob and translocation in corn

  • “Barbara McClintock's landmark 1931 study with Harriet Creighton used the conspicuous heterochromatic knob on maize chromosome 9 to provide the first direct physical proof of genetic recombination (crossing over) and chromosome translocation, linking visible traits (like kernel color/starchiness) to specific physical exchanges between chromosomes, demonstrating that genes aren't just linked but can physically swap locations” 

  • Found that these two phenotypes correlated with kernel phenotypes 

• Found that certain combinations of traits were not possible 

  • Colorless waxy and colored starchy were not found 

• Found that despite crossing over 

• Saw a few examples physically of crossing over between chromosomes 

translocation

the genetic process where a piece of one chromosome breaks off and attaches to a different chromosome

“Translocation is the movement of a piece of one chromosome to a different, non-homologous chromosome, often causing a genetic abnormality”

Pedigree female

CIRCLE

Pedigree male

square

Double line

incest

Dot in center of person

heterozygosity

Proband/propositus

• individual presenting with a specific phenotype that the consultand wants to have their genes analyzed

Consultand

• person who approaches genetic counselor 

Alternate sex/chromosomal approaches

• sex is determined by female and are the heterogametic sex 

  • Males are ZZ and make sperm 

  • Females are ZW and make egg 

• In these organisms, females would be more affected by disorders that are Z linked 

How do biologists determine sex?

who makes sperm? = male

who makes egg? = female

Hardy Weinberg Equation

p² + 2pq + q² =1

Penetrance

percentage of individuals with a specific genotype that will develop or express the mutant phenotype

Expressivity

• Degree to which an individual with a specific genotype displays the mutant phenotype

Microsatelite Region

• Microsatelite region -> Repeat regions are prone to expansion or contraction 

  • The repeat region can cause the polymerase to stutter 

    • Creates polymorphism 

    • If it’s all the time → poor marker 

    • If it’s occasional -> good marker 

Pseudoautosomal Region

• XY → on y chromosome but there is a corresponding region on the X chromosome 

• Homologous between X and Y → pseudo autosomal region 

  • This region can be protected from inactivation 

• “a small, homologous segment on the tips of the human X and Y chromosomes where they pair and recombine during meiosis, behaving like autosomes rather than sex chromosomes, allowing genes within these regions to be inherited non-sex-linked, escaping X-inactivation and present in two copies in both sexes”

my understanding - since theyre both activated on x and y chromosomes, these regions can also both be activated on both regions for females (in females, there tends to be x chromosome silencing to match expression of males) this is what it means by escaping inactivation

why are repeat genetic regions used as markers?

• Repeat regions in the genome are less stable than other regions so they can be used as markers 

  • They are also more prone to mutation → polymorphic

Capillary electrophoresis

• gives a decimal number of the number of base pairs in the DNA region/sample 

Allele binning

• label the smallest result in a pedigree as 1, and then the largest as 4 and then number upwards and label 

Allele binning in genetics involves grouping similar genetic variants (alleles) by size or function, especially for markers like microsatellites, to simplify analysis, while pedigrees visually map family inheritance patterns to track traits or diseases across generations

FA on polar body testing results

• failed PCR amplification 

Whatever allele the second polar body gets, the egg will get the __________.

opposite

Polar body testing and oocyte

• Whatever the second polar body gets, the egg must get the opposite 

  • Ex. if 2nd polar body gets the WT allele, then the egg will get mutant 

• Homozygous for the allele, no recombination = write down only once 

  • Increased peak size or band thickness → indicates this

• Nondisjunction in the polar body for 10 shown 

  • The oocyte would get no alleles and the 2nd polar body got both 

  • Then oocyte is not viable 

How does recombination affect relationship between second polar body genotype and the ootid genotype

Without recombination → ootid genotype is the same as the genotype of the second polar body 

With recombination → ootid genotype is opposite genotype of the second polar body 

Allelic series

multiple different kinds of alleles for a trait

Writing one gene on one chromosome

Writing two genes on one chromosome

Writing two genes on two chromosomes

An allele is dominant if ____________ + example

• An allele is dominant if it has the same phenotypic effect in heterozygotes and homozygous 

  • Example with flies → red eyes are the same in both +/+ and w/+ 

  • w/w is white 

plus means

WT

haplosufficient

• Those loci are haplosufficient 

• Only need one copy of the gene in order to achieve the wild type phenotype

Dominant allele (+)

• an allele that expresses its phenotypic effect even when heterozygous with a recessive allele 

Recessive allele (w)

• an allele whose phenotypic effect is not expressed in heterozygote 

• Graph depicts amount of pigment made by each genotype of fly → can see that there is a threshold to depict the trait 

Wrinkled vs Round Peas

• Because round ones have lower osmolarity to start with, they have less water to lose meaning they maintain a more round shape 

• Because wrinkled ones have higher osmolarity to start with and they have more water to lose, the loss of water causes a more noticeable physical effect. 

  • Wrinkled peas are homo for an insertion in the SBE1 coding sequence that disrupts the function of the gene that converts the sugar to starches 

  • Means that they have no functional starch branching enzyme, because the sugars can’t get converted into starches they just have sugars 

All loci that Mendel studied were ___________

• All of the loci that Mendel studied were haplosufficient, and all of the mutant alleles are loss of function 

Haploinsufficient Gene + Examplez

• In a diploid cell, a gene that can promote wild type function with only one functional copy (common) 

  • Example in people 

    • PAH human 

      • Loss of one copy -> normal 

      • Loss of both gene copies → end up with PKU disease 

    • Disease (phenotype is inherited as a Mendelian recessive disorder 

• Haploinsufficient 

  • In diploid cell, gene that cannot promote wild-type function with only one functional copy (rare) 

    • Example in people 

      • Connective tissue disorder making people tall, long-limbed, predisposed to aortic tearing 

      • Loss of one gene copy → Marfan syndrome 

      • Loss of both gene copies → very rare and lethal

List all of Mullers Morphs

amorph, hypomorph, hypermorph, neomorph, antimorph

Loss of function mutations

amorph, hypomorph

Gain of function mutations

hypermorph, neomorph, antimorph

Amorph

• complete loss of a functional product (null) 

Hypomorph

•  incomplete loss of function (leaky)

hypermorph

• increase in normal function 

Neomorphs

 new function, often with loss of normal function

Antimorph

opposes the wild type function (dominant negatives)

“genetic mechanism where a mutated gene product interferes with the normal function of the wild-type gene product, leading to a more severe loss of function than if the gene were simply non-functional”

Dominant negative

antimorph, “genetic mechanism where a mutated gene product interferes with the normal function of the wild-type gene product, leading to a more severe loss of function than if the gene were simply non-functional”

What did Muller do/find?

• Exposed flies to x-rays

• Collected flies that had different colored eyes due to different mutations in the white gene (on scale from white, apricot etc… red) 

• White ones were complete homozygous → zero functional protein made

• Found that the wild type was dominant to all of the alleles and it was a haplosufficient locus 

• Used deletions and rearrangements as tools for his experiment 

• Found some chromosomes with complete deletion of the white locus 

  • Something about banding that I didn’t catch 

  • Referred to as a deficiency chromosome in flies 

• Found some mini X chromosomes 

  • Only contained centromere and the white coding region 

More 

• White apricot was incompletely dominant to white 

  • Transheterozygote → two different alleles to the same gene 

• Those with one white apricot allele had less function than both apricot but more than white 

  • Indicates that white apricot has some more function 

• He found a hierarchy of the white eye alleles in these flies 

• All these flies were females though 

• He next did experiments with males 

What did Muller do to males?

• Instead of silencing the X in the females, they amplify the X in males (hyperactivation) 

• White apricot in male looked just like the white apricot in females that were homozygous 

  • This because the males are upregulating the expression on that chromosome 

• Added mini X chromosome to some females and gave them three copies of apricot 

  • Allowed them to look more close to the wild type 

• In males 

  • Added mini chromosome to make XxY flies 

  • The mini x chromosome was not enough to make the males female 

    • He was finding these flies with these genotypes naturally ?? 

• Transheterozygote

carrying two different mutant alleles for the same gene

Flies and eyes, distinguishing amorphic and hypomorphic alleles

• Looking at the presence or absence of an eye in flies 

• Typical for alleles to be of different strengths 

  • Weak allele (weak hypomorph) 

  • Strong alleles (strong hypomorphs) 

  • Null alleles (amorphs) 

• Hypomorph gene/hypomorph was wild type 

• Enhancer screen 

  • e^+ / e^ null → wildtype 

  • Mutagenize these 

• Everything else ends up +/+, +/e^h, e^h/e^n 

  • Ended up with some with an intermediate phenotype 

  • Way to isolate hypomorphic alleles 

• Hypermorph → what would you expect the phenotype to be of a fly that is B/def?

 Neomorph in depth with examples 

• Most neomorphs are giving a new function because it’s expressed in a time or place where is usually is not 

• Example of sickle cell anemia 

  • People who are homozygous for mutation related to making a subunit of hemoglobin

  • Make a hemoglobin susceptible to lower oxygen levels 

    • Usually fetal hemoglobin gene turns off and then you get adult hemoglobin 

  • People with sickle cell have some persistence of the fetal hemoglobin

    • Neomorphic because it’s expressed at a time when it shouldn’t be

• Example of lactase persistence 

  • Ability to breakdown lactase in milk should usually turn off around the age of 10 years old 

  • Due to single point mutation C → T caused lactase persistence and lactose tolerance 

    • New alleles for lactase persistence are emerging in subsaharan african populations where keeping cattle is relatively new 

Antimorph in depth with examples 

• Loss of normal function and opposes the wild type function 

• Very useful to knock out wild type function 

• Example in fly eyes 

  • Antimorphic dominant negative mutation in sine oculis (so) 

  • +/+ → normal eye 

  • so/so → poor eye development 

  • soD/+ –. Small eye development 

  • soD/ added so+

• In antimorph version →Transcription factor binds to promoter region but is unable to recruit the coactivator → sits on promoter region and blocks access even if heterozygous

  • Think about breaking your key off in the lock, then you can’t even use a spare key

protooncogenes

• Gene inserts randomly into the genome 

  • The mutant copy is still present in the genome 

Most dominant inherited diseases are probably..

semidominant 

Gene therapies

gene augmentation therapy, gene inhibition therapy

Gene Augmentation Therapy

• Insert a functional gene into a cell with a nonfunctioning gene 

• Treat amorphs and hypomorphs 

• Treat cystic fibrosis

Gene inhibition therapy

• Insert a blocking gene that will allow the functional gene to act normally 

• Can be used to treat hypermorphs and neomorphs and antimorphs

• Cancer caused by proto oncogene mutation 

Which are more common, completely dominant or semi/incompletely dominant genes?

Semidominant / incompletely dominant genes

Recessive lethality example in cat

• Gene in cats relating to their tails 

  • tt or mm is wild type 

  • Tt or Mm is a tail-less cat 

  • TT or MM is dead 

    • Phenotype expresses itself variably 

      • Can be a stub to a no tail to losing vertebrae to form rectum 

    • Manx cat development = shortening of the vertebrae 

  • MM → dead 

semidominance example, flowers

white pink red flowers

• Why not increase the gene dosage so that even when you lose a copy you would still be above the threshold? 

Too much and too little both will have deleterious effects

Result of incomplete dominance

leads to a continuum of different phenotypes 

• Phenotype varies in proportion to the amount of gene product 

Bistability

two genes work to modulate and stabilize each other with their products

• If you have only one functional copy (null/+ or def/+) = making 50% of gene product? 

  • Almost never true, but there are some examples 

• Bistability is common in developmental loci 

  • Threshold is around wildtype level so that having one copy doesn’t matter 

In which loci is bistability common?

developmental loci

Order of Actions for determining flies

• Order of things to do 

  • 1) Cross the mutant found back to the wildtype 

  • 2) Look at phenotype of the F1 individuals 

    • ey/ey (eyeless) or eya/eya (eyes absent) X with wildtype resulted in all wildtype offspring 

    • What can we conclude? 

      • Indicates recessivity of this gene 

      • Indicates that both loci are haplosufficient 

      • Recessive alleles = easier to work with 

  • 3) Cross eyes absent flies with eyeless flies and look at the phenotype 

    • Cross and find that F1 generation all has eyes (wildtype) 

      • Indicates that they are different genes 

      • If it was the same gene then F1 would be eyeless 

    • Since they are different genes they are in two different complementation groups 

  • 4) Sibling crosses 

    • Expect 9:3:3:1 ratio 

      • 9 WT, 3 Eyes absent, 3 eyeless, 1 double eyeless/eyes absent 

      • Double mutant looks like eyes gone 

  • 5) Cross eyes gone to wild type 

    • eyg/eyg x WT → all wildtype in F1 

  • 6) Sibling cross 

    • Eyes gone and eyesless doubles are dead 

synthetic lethality and fly eyes

• Indicates that eyes gone gene would have another function that is essential for life 

• Arrest early in larvae development, they don’t make a properly functioning head and nervous system

Example in humans and drosophila of synthetic lethality

• Genetic redundancy 

• RAD52 family gene 

  • Loss of RAD52 OR RAD 59 is tolerated because the other can partially compensate 

  • However, double mutants are inviable because homologous recombination repair of double strand breaks is completely crippled 

• Drosophila tubulin isoforms (aTub84B and aTub84D) 

  • Functionally redundant 

  • Mutation in one is tolerated but double mutants are lethal because no alpha tubulin protein remains to form microtubules 

Built in redundancy is a simple explanation for synthetic lethality 

• Some are not paralogs or orthologs 

conditional expression

Epistasis

• type of interaction between the alleles of different genes, 

  • Phenotype of individuals with mutations in two different genes are analyzed 

  • Deviates from 9:3:3:1

epistasis vs dominance

• Incomplete dominance/semidominance and codominance all describes a relationship or interaction between alleles of the same gene 

  • deviation from 3: 1

Complementation testing example (forward genetics)

• Plant model 

• Small, weedy plant with a relatively small genome, 6 chromosomes

• Not a lot of redundancy compared to other model systems 

• Genetically tractable → can be mutagenized and cloned 

• Glandular trichomes produce chemical compounds for plants, ex. Cannabis, tea, mint 

• Visual screen for abnormal trichomes 

  • Mutagenize by soaking in chemical compound to result in changes in DNA 

  • Grew up seeds, put in a greenhouse, then screened for plants in which trichome structure was different from the wildtype 

  • One was called rastafari and one was called polychome (NOT comb)

    • Want to see if these are alleles of the same gene or if they are different genes at different loci 

  • If there are two different genes, then you want to know what their relationship is 

    • Need to do a double mutant 

  • Isolate two pure breeding lines 

• If the cross yields a wildtype result, we know they are both recessive 

• If the cross yields a mutant result, we have to stop and think 

• Do Rastafari and polychome lines have mutations in the same gene? 

• Complementation test 

complementation test process

• We start with recessive mutations (ideally nulls) in genes that act in the same process (have similar phenotypes) 

• If mutations complement (F1 is wildtype) the mutations are in different genes 

• If the mutations fail to complement (F1 looks like either parental) they are alleles in the same complementation group

Endoreduplication

• cell copies its genome but does not reduplicate (2C → 4C → 8 C) keeps increasing the copies of its genome without dividing 

trichome formation

• One cell receives a signal telling them that they need to develop into a trichome

  • Endoreduplication→ cell copies its genome but does not reduplicate (2C → 4C → 8 C) keeps increasing the copies of its genome without dividing 

  • Allows to make extra transcripts and proteins 

• Other mutation examples increased the number of rounds of endoreduplication → caused a greater number of branches to be present 

• Looking at polychome and rastafari, they hypothesized that this same situation was occurring in them 

• RFI inhibits excess endoreduplication → increased branch number 

• PYM inhibits excess endoreduplication → increase in branching 

neurofibromatosis type 1

dominant autosomal

huntington disease

dominant autosomal

Lethal allele

An allele that causes death when homozygous is known as a:

Epistasis

When one gene masks or modifies the effect of another gene, this interaction is called: BIG POINT THAT TWO DIFF GENES 

9:3:4 ratio is evidence of 

recessive epistasis