Tutorial Quiz 2 Flashcards

1
Q

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

A
  • denticles on the anterior portion of each abdominal segment, pointing towards the posterior
  • naked region on the posterior of each abdominal segment
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2
Q

What is forward genetics?

A
  • 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
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3
Q

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

A

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

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4
Q

Which amino acids have low degeneracy?

A

Methionine, tryptophan (these each have just one codon)

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

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5
Q

When should inosine be used during degenerate PCR?

A

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.

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6
Q

Which amino acids have intermediate degeneracy?

A

isoleucine

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7
Q

Where is sonic hedgehog expressed?

A

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.

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8
Q

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

A

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

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9
Q

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

A

Furthest from the ZPA (source of SHH)

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

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10
Q

What are orthologs?

A

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

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11
Q

What are paralogs?

A

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
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12
Q

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

A

Desert hedgehog in mice

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13
Q

What is a genomic library?

A

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)

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14
Q

What is cross hybridization?

A

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

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15
Q

What are low stringency conditions?

A

low temperature and high salt

  • use during cross hybridization
  • allows hybridization to imperfect DNA ie. DNA sequences can be slightly different.
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16
Q

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

A

Denatures the released DNA

17
Q

What is degenerate PCR?

A

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

18
Q

What happens in preaxial polydactyly?

A

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

19
Q

What happens when the ZRS sequence is deleted?

A

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

20
Q

What does ZRS stand for?

A

ZPA regulatory sequence

21
Q

Why don’t snakes have limbs?

A
  • 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
22
Q

What happens when a mouse is serpentized?

A

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.

23
Q

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

A
  • 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
24
Q

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

A
  • 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
25
Q

What are examples of mutations that lead to HPE?

A
  • 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
26
Q

What is the eyefield?

A

The part of the embryo that becomes the eye.

27
Q

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

A

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

28
Q

What are examples of SHH inbibitors?

A

Jervine (plant chemical)

cyclopamine

29
Q

What did Volhard and Wieschaus do?

A

Screened thousands of embryos for patterning effects

- Previously, everyone else had been looking at adults

30
Q

Why might developmental mutations often go unnoticed?

A

They often cause embryonic lethality