Tutorial Quiz 2 Flashcards
What is each abdominal segment on the ventral side of the embryo characterized by?
- denticles on the anterior portion of each abdominal segment, pointing towards the posterior
- naked region on the posterior of each abdominal segment
What is forward genetics?
- 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
What are amino acids that should be avoided in degenerate PCR?
Amino acids with high degeneracy. Leucine, Serine, Arginine (These each have 6 possible codons that code for them)
Which amino acids have low degeneracy?
Methionine, tryptophan (these each have just one codon)
Cysteine, tyrosine, aspartate, glutamine, histidine (2)
When should inosine be used during degenerate PCR?
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.
Which amino acids have intermediate degeneracy?
isoleucine
Where is sonic hedgehog expressed?
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.
Which digit forms at the posterior part of the limb bud?
Closest to the ZPA - highest concentration of SHH morphogen - get pinky forming
Which digit forms at the anterior part of the limb bud?
Furthest from the ZPA (source of SHH)
thumb or big toe forms here (where concentration of SHH is lowest/not existent)
What are orthologs?
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
What are paralogs?
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
What was the first hedgehog gene to be found in mice?
Desert hedgehog in mice
What is a genomic library?
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)
What is cross hybridization?
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
What are low stringency conditions?
low temperature and high salt
- use during cross hybridization
- allows hybridization to imperfect DNA ie. DNA sequences can be slightly different.
What does alkaline solution do when screening a genomic phage library?
Denatures the released DNA
What is degenerate PCR?
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
What happens in preaxial polydactyly?
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
What happens when the ZRS sequence is deleted?
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
What does ZRS stand for?
ZPA regulatory sequence
Why don’t snakes have limbs?
- 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
What happens when a mouse is serpentized?
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.
What are the post-translational modifications that SHH protein must undergo to become functional?
- 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
What are mutations to the gene coding for Shh that lead to ADHPE?
- 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
