Tutorial Quiz 2

Question 1

What is each abdominal segment on the ventral side of the embryo characterized by?

Answer 1

• denticles on the anterior portion of each abdominal segment, pointing towards the posterior
• naked region on the posterior of each abdominal segment

Question 2

What is forward genetics?

Answer 2

• Used to identify genes or genotypes that are responsible for particular phenotypes
• induce a mutation in the gene and then look at the resultant phenotype - make assumptions about function

Question 3

What are amino acids that should be avoided in degenerate PCR?

Answer 3

Amino acids with high degeneracy. Leucine, Serine, Arginine (These each have 6 possible codons that code for them)

Question 4

Which amino acids have low degeneracy?

Answer 4

Methionine, tryptophan (these each have just one codon)

Cysteine, tyrosine, aspartate, glutamine, histidine (2)

Question 5

When should inosine be used during degenerate PCR?

Answer 5

Sparingly! Use when the amino acid in the primer has 3-4 codons that can code for it. Do not use when the amino acid only has 1-2 codons coding for it.

Question 6

Which amino acids have intermediate degeneracy?

Answer 6

isoleucine

Question 7

Where is sonic hedgehog expressed?

Answer 7

midline mesoderm of the head, notocord, ventral neural tube (floor plate), zone of polarizing activity (ZPA) which is in the posterior part of the limb bud.

Question 8

Which digit forms at the posterior part of the limb bud?

Answer 8

Closest to the ZPA - highest concentration of SHH morphogen - get pinky forming

Question 9

Which digit forms at the anterior part of the limb bud?

Answer 9

Furthest from the ZPA (source of SHH)

thumb or big toe forms here (where concentration of SHH is lowest/not existent)

Question 10

What are orthologs?

Answer 10

Develop when an ancestral gene in an ancestral organism underwent a speciation event of a genome. Orthologs share similar function ex. Shh in mouse and chickens

Question 11

What are paralogs?

Answer 11

Related genes that diverged due to a duplication event within one genome of a species. After the duplication, there are two copies within the same genome that accumulate different mutations and thus end up having slightly different functions.

• Shh, Ihh, Dhh
• If they are within the same organism, it is a good guess that they are paralogs

Question 12

What was the first hedgehog gene to be found in mice?

Answer 12

Desert hedgehog in mice

Question 13

What is a genomic library?

Answer 13

A DNA library containing an organism’s complete genome, in the form of small DNA fragments (oligonucleotides)
- These fragments are then inserted into carrier molecules called vectors (such as a viral DNA molecule or plasmid)

Question 14

What is cross hybridization?

Answer 14

Combining phage molecules containing fragments that together make up the animal’s complete genome with gene of known sequence (trying to find its homologue in another organism)
- Combine this under LOW Stringency conditions

Question 15

What are low stringency conditions?

Answer 15

low temperature and high salt

• use during cross hybridization
• allows hybridization to imperfect DNA ie. DNA sequences can be slightly different.

Question 16

What does alkaline solution do when screening a genomic phage library?

Answer 16

Denatures the released DNA

Question 17

What is degenerate PCR?

Answer 17

A method used to amplify genes from different organisms - genes that are similar but not identical
Goal is to obtain a specific DNA sequence of one organism from a probable protein sequence

Question 18

What happens in preaxial polydactyly?

Answer 18

There a gain-of-function mutation to the ZRS enhancer (which is found in intron 5 of Lmbr1 gene) which results in SHH being expressed ectopically in the anterior side of the limb bud. Results in multiple extra digits forming

Question 19

What happens when the ZRS sequence is deleted?

Answer 19

Loss of SHH expression in the limb buds (bc ZRS is a limb-specific enhancer) and degeneration of the distal skeletal elements
Still get appearance of the anterior most digit (bc this is where there is usually no SHH signalling anyways)
- deletions in the human ortholog of the mouse Lmbr1 gene (which contains the ZRS enhancer sequence) result in a similar phenotype

Question 20

What does ZRS stand for?

Answer 20

ZPA regulatory sequence

Question 21

Why don’t snakes have limbs?

Answer 21

• basal snakes have vestigial hind limbs
• advanced snakes are limbless
• sequence degredation/accumulation of mutations led to gradual loss of the enhancer activity and thus SHH expression here

Question 22

What happens when a mouse is serpentized?

Answer 22

Replace mouse ZRS enhancer sequence with the snake ZRS enhancer sequence
- Results in the mouse losing distal portions of limbs and only maintaining the anterior most digit.

Question 23

What are the post-translational modifications that SHH protein must undergo to become functional?

Answer 23

• Signal sequence cleavage
• C-terminal domain auto-proteolysis
• Addition of cholesterol to the C-terminus (lipid modification)
• Addition of palmitic acid to the N-terminus

Question 24

What are mutations to the gene coding for Shh that lead to ADHPE?

Answer 24

• A single amino acid change adjacent to the signal sequence
• Two different mutations introducing stop codons at amino acids 100 and 105
• Two different mutations changing a highly conserved Trp to Gly or Arg

Question 25

What are examples of mutations that lead to HPE?

Answer 25

• single amino acid change adjacent to the signal sequence
• two diff mutations changing a highly conserved Trp to Gly or Arg
• two diff mutations introducing stop codons at amino acids 100 and 105
• *These mutations all occurred in the highly conserved N-terminus protein that lead to a non-functional Shh protein

Question 26

What is the eyefield?

Answer 26

The part of the embryo that becomes the eye.

Question 27

How is the eye field separated into two during normal development?

Answer 27

Pax6 is necessary to form the eyes. SHH expression along the midline represses Pax6 expression in the middle, separating the eye field into two. If SHH is not produced to repress Pax6, only one large eye will form - cyclopia.
SHH repression can occur by a chemical or a mutation

Question 28

What are examples of SHH inbibitors?

Answer 28

Jervine (plant chemical)

cyclopamine

Question 29

What did Volhard and Wieschaus do?

Answer 29

Screened thousands of embryos for patterning effects

- Previously, everyone else had been looking at adults

Question 30

Why might developmental mutations often go unnoticed?

Answer 30

They often cause embryonic lethality