Hipfner 2 (Non-mandelian Genetics)

Question 1

How is position-effect variegation non-mandelian genetics?

Answer 1

The genotype does not changed, but the phenotype does

Question 2

A women inherited an X-linked recessive mutation that caused her to have liver failure. What is the probability that she develops liver failure?

Answer 2

We don’t have enough information to predict → have to consider epigenetic silencing, X-chromosome silencing, etc.

Question 3

How does dosage compensation occur so that males and females have equal expression levels of ~ 1000 genes located on the X chromosome?

Answer 3

X-chromosome inactivation:
- In early female mammalian embryo (~8-32 cell stage), each cell randomly inactivates one of the 2 X-chromosomes → forms heterochromatic “Barr body”
*Not all cells inactivate the same, but from now one, every progenitor from that cell will have the same X-chromosome inactivated → mitotic epigenetic inheritance

*Makes all femal mosaic → can lead to non-mandelian effects

Question 4

What is the Barr body?

Answer 4

It the X chromosome that was inactivated in each cell → constitutive heterochromatic X chromosome

Question 5

What explains mosaic cat coat color? What do they all have in common?

Answer 5

They are all females!
Not the same X is inactive in all cells and X-chromosome codes for coat color (black allele on 1, orange allele on the other)

Question 6

How can females be colour-blind?

Answer 6

red-green colour blindness often dur to mutation of the OPN1-LW gene on the X chromosome

Females can be mut/mut OR mut/+:
- For many X-linked mutations, females carriers can have partial symptoms
- Variable penetrance and expressivity due to different levels of X-inativation of the chromosome with the WT allele

Ex: 50-50 X-inactivation → normal vision, 30-70 WT X-inactivation → a bit of defect, but not too noticeable, 10-90 WT X-inactivaiton → color blind even if mut/+

Question 7

Why would Rett syndrome only be seen in female?

Answer 7

It is an X-linked disease:
- Male mut/Y → not viable
- Female mut/+ → show Rett syndrome
- Female mut/mut → not viable
- Female +/+ → Heathly

Question 8

How is X inactivated in humans?

Answer 8

By repressive histone and DNA marks (DNA methylation)

Question 9

How is dosage compensation done in flies? worms? mice?

Answer 9

Flies → don’t inactivate female X, they hyperactivate male X (twice expression levels of Y or of female each X) → more heavily acetylated

Worms → Each of the 2 female (XX) X chromosome expression in decreased by half to match the X chromosome expression of male (X0)

Mice → like human, inactivate one of the X chromosomes

Question 10

Where do DNA histone methylations primarily occurs?

Answer 10

On Cytosine → on C5, addition of a methyl group

On Cytosine in the CpG dinucleutide (C-G and next pair is G-C and both C are methylated in that pair, one on each side)
*60~80% of CpG are methylated genome-wide in vertebrates

*DNA methylations passed on through mitosis

Question 11

How are CpG methylations distributed? What about CpG islands?

Answer 11

Not randomly distributed!!
- Mostly associated with intergenic regions
- Correlated with repressed chromatin state (Heterochromatin)

CpG islands → CpG rich clusters located near promotors (60% of genes)
- Mostly (>95%) not methylated and transcriptionally active
- Methylation of CpG islands is associated with repression/silencing of gene expression

Question 12

How can methylation of CpG islands lead to repression/silencing of gene expression? (2 pathways)

Answer 12

Direct effect: DNA methylation block transcription factor from binding to DNA

Indirect effect: Recruitment of HDACs and HMTs lead to repressive histone modifications

Question 13

How are DNA methylations passed on through mitosis?

Answer 13

When replication, 2 parent strands have methylations on them → DNMTs have high affinity for hemimethylated sites
Methylation pattern on the parental strand guides methylation pattern on newly synthesized strand

Question 14

What is genomic imprinting?

Answer 14

It is the process by which only one copy of a gene in an individual (either from their mother or their father) is expressed → monoallelic inheritance

Which allele gets expressed depends only on biological sex of the parent from which it came → sex-specific gene silencing

Imprinted copy is inactivated by mechanism involving DNA methylation in paternal/maternal germline → methylation imprint is maintained throughout life of the progeny (in somatic cells)

Question 15

What is the inheritance regulation/pattern of Igf2 and H19 genes?

Answer 15

Igf2 gene is maternally imprinted → only Igf2 allele inherited from the father is expressed, maternal copy in imprinted and inactive

H19 gene is paternally imprinted → only H19 inherited frm the mother is expressed, paternal copy is imprinted and inactive

Order of loci: Igf2 - ICR - H19 - Enhancer
*They both share the same ICR (imprinting control region) and Enhancer

Sex-specific CpG methylation of ICR only in paternal gametes (sperm) → CTCF can’t bind → H19 promotor is methylated because of spreading → Enhancer acts on Ifg2

In female, unmethylated ICR binds CTCF (enhancer-blocking insulator) → enhancer can’t reach over to get to Igf2

Question 16

When are genomic imprints established? (Specifically in Igf2 and H19)

Answer 16

Established during gematogenesis:
*Each male and female parents have a paternal (methylated) and maternal (unmethylated) chromosome

1. In premordial germ cells, all imprints are erased in female and males (methylation on paternal chromosome, X-inactivation)
2. Imprints are initiated → in paternal parent, both ICRs are methylated, in maternal no methylation of ICRs
3. Sperm has ICR methylation, Oocyte has no methylation

Question 17

What is the difference between Igf2/H19 imprinting and X-inactivation in female (imprinting)?

Answer 17

Igf2/H19 established during gematogenesis
X-inactivation established during embryogenesis

Question 18

What is one reason uniparental embryos are inviable?

Answer 18

Because of improper imprinting
Ex:
- Mutations affecting non-imprinted copy
- Epimutations → defect in histone tail modification or DNA methylation that affect gene expression
- Uniparental disomy (Robertsonian carrier, non-disjunction, somatic cell recombination

• A mutation in an epigenetically silenced gene would not affect the phenotype

Question 19

What is the effect of a mutation in the Igf2 gene in the maternal chromosome?

Answer 19

Nothing, it is an imprinted gene

Question 20

What is Silver-Russell syndrom (dwarfism)?

Answer 20

~ 50% cases, hypomethylation of paternal allele at the ICR (epimutation) → binding of CTCF → repression of the paternal Igf2 expression

Question 21

Which organelles are inherited maternally?

Answer 21

• Mitochondria
• Chloroplast

Question 22

What are the characteristics of the mitochondrial genome?

Answer 22

• 16,569 nt circular DNA molecules
• dozens - hundreds of mitochondria/cells —> each mitochondria has multiple mtDNA copies
• mtDNA ~ 15% of total DNA
• encodes 13 proteins and 24 rRNA/tRNAs
• Endosymbiotic theory ~ Gene sequences most similar to a-proteobacteria
• Not all mitochondrial proteins are encoded for by mtDNA (big part encoded for in the genome)

Question 23

What is the importance of nuclear contribution to the mitochondrial proteins?

Answer 23

• 99% of mt proteins encoded in the nuclear genome
• 13 mtDNA-encoded proteins all ivolved in electron transport chain
• Produced in the cytoplasm —> imported

Question 24

Which are the factors influencing replication of the mitochondrial genome?

Answer 24

1. Mitochondrial DNA is replicated within the nucleoids
   - Independently from cell cycle/nuclear DNA replication
2. nucleoids can divide within an organelle (mt)
3. Mitochondria themselves divide
   - Arise by division of existing ones (not made de novo)

Question 25

What are mitochondrial nucleoids?

Answer 25

protein/DNA particles in which mtDNA is package
*Several mitochondrial genomes/nucleoid

Question 26

What is needed for a cell to show a defective mitochondrial phenotype?

Answer 26

1. Need mutation in 1 nucleoid
2. The mutated nucleoid need to replicate/divide

Question 27

How are mitochondriae inhertied?

Answer 27

Both sperm oocyte and sperm contain mitochondria, but far more in oocyte (> 100,000 vs ~100) → oocyte eats paternal mitochondrion by autophagy upon fertilization

Question 28

What is heteroplasmy? vs Homoplasmy?

Answer 28

Heteroplasmy ~ Spontaneous mtDNA mutations can lead to 2 distinct mt populations within a single cell
- In some cases, mother might have mitochondrial disease because above a specific threshold, but the child might be normal because better ratio of good mitochondrials

Homoplasmy ~ Random segregation of organelles at mitosis (or meiosis) leading to all mitochondriae in a cell having the same mutation or same WT version

Question 29

True or False, Mutation in mitochondrial proteins from the mother will always lead to a mutant phenotype?

Answer 29

False, If the mutation is encoded in the nucleus, it will be rescued by the paternal copy

Question 30

How can mitochondrial diseases be prevented in the offspring if the mother are affected?

Answer 30

3-person in vitro fertilization:
1. Normal parents’ fertilised egg with unhealthy mitochondria
2. Take the parents nuclei → replace by the donor’s nucleus in donor’s fertilized egg (father + other mother with healthy mitochondria)
*pronuclear transfer

Question 31

Name a type of gene that might be an E(var).

Answer 31

E(var) = enhancer of variegation → type of gene that encodes for a protein whose function involves blocking the spead → E(var) genes are likely to encode proteins that activate transcription
Ex: Histone acetyltransferase (HATs)

Question 32

What are the different stages of the 1st 3 hours of Drosophila development?

Answer 32

1. Single-cell diploid zygote (sperm and egg nuclei fuse to create a single-cell diploid zygote)
2. Multiple nuclear divisions of the nucleus → multinucleated cell with a single cytoplasm (No transcription, development controlled 100% by mRNAs povided by mother through oocyte cytoplasm) → multinucleate syncytium
   (70mins)

Here, transcription of embryo’s own gene begins (zygotic transcription)

3. The nuclei migrates to the periphery of the embryo and divides several more times (+ pole nuclei at the posterior) → syncytial blastoderm
   (120mins)
4. Cellularization → individual nuclei enclosed in plasma membrane to form cells at the periphery + pole cells at posterior end
   (180mins)

Question 33

What are the major stages of embryo determination in drosophila in the 1st day of development?

Answer 33

Main axis → 2-hour embryo: Anterior, Posterior, Dorsal, Ventral

Body segments → 10-hour embryo: Head, thoracic, Abdominal segments ~ 14 segments with each an anterior/posterior end

Each segment will go on to form specific structures of the body

Question 34

What was the Heidelberg screen?

Answer 34

Screen in drosophila for mutations disrupting early embryonic patterning
2 screens: Maternal vs zygotic genes

Screen 1 → What mutations in the mother prevent offspring from completing embryonic development (to complete early embryonic dev.)?
- Cytoplasmic inheritance of maternal-effect gene

Screen 2 → Which of the embryo’s own genes are needed for normal development?
- Zygotic genes (follows Mendelian patterns)

Question 35

What are the main stages of the cascade of transcription factor expression regulating early Drsophila development?

Answer 35

Single-celled embryo
1. Egg-polarity genes → Determination of major body axes
2. Gap genes → Regional sections of embryo defined
3. Pair-rule genes → Induvidual segments defined
4. Segment-polarity genes → Polarity of individual segments defined
5. Homeotic genes →Indentity of individual segments defined

*Each genes encode for TF required to turn on the genes from the next step

Question 36

Where are egg-polarity genes come from?

Answer 36

Maternally loaded / Maternal effect genes

Egg-polarity genes encode for TF needed to tuen on transcription of Gap genes

Question 37

What is the A-P axis determined by?

Answer 37

Determined by maternal-effect genes:

Anterior (A): bicoid
- mutants lack head, thoracic segments
- Encodes a TF forming a concentration gradient

Posterior (P): nanos
- mutants lac abdominal segments

Question 38

How is the bicoid protein gradient formed in the drosophila larva?

Answer 38

1. Bicoid mRNA (from the mother) localized to the anterior embryo
2. Bicoid protein is translated in anterior from maternal mRNA
3. Bicoid proteins diffuse towars the posterior region making a concentration gradient → information about distance from anterior
   *mRNA does not diffuse only protein

Question 39

Is bicoid sufficient to form anterior structures?

Answer 39

Yes, Injected bicoid mRNA directs formation of anterior stuctures anywhere it is injected in the drosophila larva
Ex: if injected in the middle, see thoracic segment-Head-thoraci segment

Question 40

Which 2 genes are responsible for posterior patterning in the drosophila larva?

Answer 40

Nanos + maternal Hunchback

• Maternal nanos mRNA is localized to the posterior end of the embryo, translated protein forms concentration gradient from A[low] → P[high]

→ Nanos inhibits translation of uniformly-distributed Hunchback mRNA → creates A[high] → P[low] concentration gradient of the TF Hunchback

Question 41

How are nanos and hunchback maternal mRNAs distributed in the drosophila embryo?

Answer 41

Hunchback mRNA → equally distributed every where
Nanos mRNA → Only at the posterior end
Nanos protein → gradient from P[high] → A[low]
Hunchback protein → gradient from A[high] → P[low]

Question 42

What would the offspring appearance of the cross between nanos-/- male and bicoid +/- female look like?

Answer 42

100% of offspring would be WT because both only inherited maternally

Question 43

What is the importance of the Gap genes?

Answer 43

Translate maternal A-P gradients into broad subdomains

9 Gap genes identified:
- All encode TF
- Gap genes are transcriptionally regulated by maternal-effect gene products
- ex: zygotic hunchback (hb-z), kruppel, knirps, giant
- The regions of every gap genes can overlap

Question 44

What is the phenotype of gap gene mutants?

Answer 44

Large gaps in their body plans → loss of several consecutive segments corresponding to the region of the embryo where the gap gene is transcribed

Question 45

How is zygotic hunchback regulated?

Answer 45

Regulated by the presence of 3 binding sites for Bicoid (Bcd) in hb promotor

zygotic hunchback mRNA equally distributed everywhere, but transcribed in the anterior, where Bcd is present in higher concentration

Question 46

What is the importance/pattern of expression of the pair-rule genes?

Answer 46

8 pair-rule genes identified:
- All TF
- Each expressed in 7 stripes (out of 14) → different levels of expression depending on the maternal factors and gap gene levels in that specific stripe
- Position of the stripes are shifted depending on the gene
- Expression is regulated by maternal effect and gap gene

Question 47

What is the phenotype of a pair-rule gene mutant?

Answer 47

Pair-rule gene mutatns are characterized by absence of every other segment → missing/mutated 7 of the 14 segments
(Even-skipped vs Odd-skipped mutants)

Question 48

How does stripe specific regulation occur for pair-rule genes?

Answer 48

Take the example fo the Stripe-specific eve enhancer:

Even-skipped (eve) → nuclei in stripe 2 will have specific concentrations of maternally loaded and zygotic factors

eve gene has different enhancer sequences for different stripe, each enhancer has different arrangements of binding sites for maternally loaded factors and Gap genes → COMBINATORIAL CONTROL OF TRANSCRIPTION

*Each stripe = a few nuclei

Question 49

How are the segment polarity domains established?

Answer 49

Segment polarity genes:
- encode components of 2 cell-cell signaling pathways (Hedgehog, Wingless) → includes secreted proteins, membrane receptors, TF, etc.

• Activated/repressed by pair-rule genes
• Function to define A and P within each segment
  *mutants have mirroring of one hald of each segment
• Expressed in each of the 14 stripes

Question 50

What are the effects of homeotic mutations?
Give 2 examples.

Answer 50

Mutant animals lack particular structure → replaced by another structure (homeotic transformation)

Ultrabithorax (Ubx) → second thorax and set of wing in place of halteres

Antennapedia (Antp) → legs instead of antennae

*Homeotic genes tell each segment which structure it should form

Question 51

What are the characteristics of the hox genes?

Answer 51

8 genes → all encode homeodomain family TF:
- Expressed in specific segments, some overlap
- Activated/repressed by gap and pair-rule gene product
- Get different structures depending on which hox genes are expressed in a segment

Question 52

What are the 2 main complexes of Hox genes in the drosophila embryo?

Answer 52

1. Antennapedia complex (head, front legs, wings)
2. Bithorax complex (Ubx, abd-A abd-B)

Question 53

How might a CpG island function differently than an isolated CpG?

Answer 53

The clustering of CpGs in islands may serve to help recruit binding protein (i.e., proteins that bind either unmethylated CpGs or methylated CpGs) through cooperative interactions between the proteins. Without this cooperativity, binding of an individual binding protein at an isolated CpG would not be very stable.

Question 54

which gap protein regulates the posterior boundary of eve stripe 2? Describe how it does so in molecular terms.

Describe the expression pattern of the Drosophila gene eve in the early embryo, and the phenotypic effects of mutations in the eve gene.

Answer 54

The Krüppel protein regulates the posterior boundary of eve stripe 2. As the concentration of Krüppel increases, the expression of eve decreases. Thus, a high concentration of the Krüppel protein produces the posterior boundary of stripe 2. From a molecular perspective, Krüppel is a transcription factor that binds to DNA sequences in the eve stripe 2 enhancer to repress transcription of eve posterior to stripe 2.

The primary pair-rule gene eve (even-skipped) would be expressed in seven stripes along the A–P axis of the late blastoderm.

Question 55

How could you test predictions for a homeogene?

Answer 55

• Visualize mRNA expression in specific organs
• In situ hybridization
• Through genetic manipulation → put mRNA in other parts of body and see expression of different organs in other segments