TS 2 Flashcards
How many segments are in the abdomen region?
8
How many segments are in the thorax region?
3
At what cycle is the syncitial blastoderm?
10-12
At what cycle is celularization?
13+
At what cycle is segmentation?
14
What is a dentricle?
These hair-like follicles that are present in the ventral side segments and they point posteriorly
What are the naked regions?
These are the posterior segments that are in between the dentricle containing segments that lack dentricles
What is forward genetics?
This is a phenotypic driven process where a gene is mutated and the phenotype is observed then the gene responsible is determined
How was forward genetics conducted in flies?
- Adult male flies mutated using EMS
- The mutated gametes were crossed with WT female flies
- The mutated progeny (1000s of them) were observed
What was the mutant phenotype?
- Same number of segments
- Deleted naked regions
- Mirror duplicated dentricles
How did they know the dentricles were mirror duplicates?
Further posterior there were dentricles (duplicate) but they were pointing anterior (mirror)
What is a homolog?
Genes that relate because they possess an ancestral similiarity but then they differ due to a speciation
What is a paralog?
Ancestral genes with 2 duplicates in the genome that accumulate mutations
How do you determine homology?
With a DNA probe from a known gene
What is high stringency?
When the complementary DNA sequence has to be perfect for the probe to bind
What is low stringency?
When the complementary DNA sequence can be imperfect and the probe can still bind
What is the ZPA?
The zone polarizing activity is a signalling centre and is the source of a morphogen responsible for the patterning of the limb so, it is the area of the limb that secretes the morphogen
The identity of the ZPA morphogen was unknown so where did they start?
Looking for Drosophila HH as a homolog for patterning in the body segments in the chick since the HH was a secreted morphogen
Where is ZPA expressed?
It is a morphogen with the highest concentration in the posterior and lowest in the anterior
What happened when the ZPA from the posterior was grafted to the anterior?
There was a duplication of the digits
Which 2 conserved sequences did they compare?
The mouse desert HH sequence and the Drosophila HH sequence
What method did they use?
Degenerate PCR where primers that are similar not identical from the mouse DHH and the drosophila HH to probe for homologs in the chicken
What did they use to make the probe/primer the most conserved?
The amino acid sequence from the mouse DHH and the drosophila HH
Where can inosine be present?
Wherever there are 3 or 4 base pair options but you do not want too many inosines in the primer otherwise it will not be specific enough to bind to a specific sequence
How was the chicken DNA amplified?
Degenerate PCR was used to amplify this DNA
What was the probe used for?
This sequence was used to screen a chicken limb cDNA library
What was produced in the posterior of the limb bud in the ZPA?
Sonic hedgehog
How did they determine if the SHH is the ZPA morphogen?
Replication competent retroviral vector (RCAS-E)
What does replication competent retroviral vector (RCAS-E) do?
The vector contains the mRNA and is inserted into the cell and is then reverse transcribed to cDNA and is translocated to the nucleus and then expresses the protein for SHH
Where does the virus infect?
Specific cell lines to ensure that not all cells produce HH
What was used to make the full length human cDNA?
Mouse DHH
Drosophila HH
What is the most conserved region of a protein?
N-terminus
What is holoprosencephaly (HPE)?
Failure of the forebrain to separate into 2 hemispheres
What happens to homozyous HPE?
Lethal
What happens to heterozygote HPE?
Range of mild to severe
How was SHH thought to cause HPE?
Through single stranded conformation polymorphism (SSCP) the SHH is in at least one of the 4 loci
What is the difference between the mutation carrier and the wild type of ADHPE?
There is an additional band which represents the new restriction digest sites
What happens to any mutation in the N-terminus?
Produces a non-functional protein
Where is SHH expressed along the midline?
Notochord
Prechordal plate mesoderm
What is the relationship between Pax6 and SHH?
SHH is expressed between the eyes and the expression is controlled by Pax6 expression
What is hypotelorism?
Loss of midline space due to a decrease in SHH
What is hypertelerism?
Gain of midline space due to an increase in SHH
What happens when there is a single point mutation in the ZRS enhancer?
Extra digits or preaxial polydactylyl
Are there digits present in the deletion of the ZRS which leads to a loss of SHH?
Yes, in the anterior because that was not dependent on SHH expression to begin with
What is a serpentized mouse?
A mouse that contains a degraded ZRS enhancer from a snake
Can a snake be rescued?
In theory it is more difficult in practice
