LECTURE 10 & 11 (developmental genetics) Flashcards
What’s forward genetics?
pheny-gene
you have a phenotype and you want to study what’s the gene that causes that specific phenotype
What’s reverse genetics?
gene-pheny
you start with a given gene and see what’s the phenotype that you might get if you disrupt the gene.
How can we get new phenotypes?
you can use mutagens.
What was the 1st mutagen that was ever used?
nitrogen mustard (alkylating agent) used in chemotherapy
What are induced mutations?
mutations caused due to a mutagen that you insert to make some changes in the phenotype
What are natural variants?
mutations that are selected by natural selection (restrictsd to alleles that exist already in a pop)
how can developmental mutants be found? (6 steps)
- mutagenize an org,
- select a mutant,
- classify mutation,
- map mutation,
- validate gene,
- study mechanism of action
What’s EMS?
ethylmethanosulphate (BASE MODIFIER) adds ethyl group to O-6 position of guanine and then it pairs with thymine, it can cause loads of different mutations
Why is developmental genetics complicated to study?
because different animals have different developmental mechanisms and if we link it with genetics it’s even more complicated
Which experiment in developmental genetics got a nobel price?
isolation of 15 embryonic lethal mutations of C.elegans (forward genetics screens to identify mutants)
Appart from drosophila, which other is good model organism for developmental genetics?
C.elegans because it has been found morphological mutations; different body movements for different mutations
how can embryonic phenotypes be found in drosophila?
females were crossed with male mutants for several rounds until the mutation becomes homozygotic then have a look at the seq of those mutants
How can embryonic phenotypes be screened for lethal mutations?
you need to use balance chromosomes otherwise they don’t survive and you have no organisms to look at because lethal mutants all die.
why is the finding of embryonic phenotypes easier with hermaphrodites?
because in the 1st generation will be heterozygous for the mutation of interest and will segregate in 1/4 to next generation–> we’re certain about it
Once mutagenesis has been done in the organism, how do we get from the phenotype to the mutated gene?
- see if mutant breeds true
- get rid of unwanted mutations
- identify type of mutation
- complementation test
- modifier screens
- mapping
types of mutations when mutagenize an organism
- loss of function:
null: complete loss of function
hypomorph: output decreases
haploinssuficient: 1/2 output workd - gain of function:
hypermorphic: loads of output
neomorphic: change of function
antimorphic: one output affects another output
How can we determine the null phenotype gene?
null phenotype is a complete loss of function, to tell what happens when the gene of interest isn’t there we can use genetic analysis normally the null phenotype was one that was previously highly expressed so we can investigate the previous ones.
What is the complementation test for?
can be used to know whether two alleles correspond to the same gene
What does trans configuration tells?
that there’s complementation
What’s are the exceptions of the complementation test?
intragenic complementation and extragenic non-complementation.
What’s intragenic complementation?
it’s when two mutations in the same gene complement
what’s extragenic non-complementation?
when two mutations in two different genes do not complement (trans)
Why would we using mapping ?
it’s in order to map a mutation produced by a mutagen e.g EMS
How do we carry mapping for a mutation?
we need to cross the mutant (we performed mutagenesis to it) with the wild type. You get heterozygous progeny and the mate F1 with F1 and due to recombination we get 3/4 WT 1/4 mutants. now we isolate this mutant, perform DNA extraction and do PCR. We need to do a BSA by adding a marker.
What is bulked sergeant analysis?
Bulked segregant analysis (BSA) is a technique used to identify genetic markers associated with a mutant phenotype.
What can the marker of BSA show?
- bias towatds mutant parent band if mutation is linked to the marker
- no bias is shown to either parent band if mutation unlinked to the marker
Is mapping easy?
no, very long process
What is new sequencing technologies?
A technique to make mapping faster.
What are modifier screens for?
they’re enhancers and suppressors they’re used to identify interactors of these genes. .In a modifier screen, an organism with a pre-existing phenotype is selected. Thus the screen is designed to isolate mutants in which the pre-existing phenotype of interest is enhanced or suppressed.
What’s 2nd type mutagenesis for?
to allow viable double mutants
What’s a suppresor for un 2nd mutagenesis?
to make the mutation less severe
What’s an enhancer for in 2nd mutagenesis?
to make the mutation more severe.
Describe the life cycle of drosophila.
It’s a complete metamorphism, egg, larva pupa (transition from larva to adult) and adult
it’s a very fast process (few days)
What’s interesting about drosophila melanogaster genes?
they share 50% homologs with vertebrates
What are the most famous drosophila mutants?
Bithorax (T3–> T2) and anthennapedia (antena to feet)
these are called homeotic mutants
what are homeotic mutants?
phenomenon in which certain body parts transform to another body part.
What are hox or homeotic genes?
genes that determine the body plan of drosophila
How are homeotic genes determined in drosophila?
thanks to Ubx which is detected in posterior thoraces segments and anterior ambdiminal of the embryo this tells the organization of genes. there are other genes that determine other regions.
What if there’s a mutation in Ubx?
then Drosophila mutants with body parts where they shouldn’t be.
what’s the colinerality principle?
looking for mRNA levels theres a correlation of the positioning of certain genes in the chromosome with the site of expression
Which TF do hox genes encode?
they encode TF of homeodomain proteins
How many homeobox families are there?
around 20
what is an homeobox region?
the region of the hox gene.
what is pax6?
a homolog in mice that make eyeless mice
do hox genes in drosophila have homologs?
yes, hox genes have a lot of conservation; they’re found in many organisms and the homeobox regions are highly similar in some cases there are 60 aa fully conserved within spp.
Are box genes found in all animals?
they determine the linearity of a body and they have been found in all branches of metazoan tree; important for patterning.
Why is there so much diversity between animals if box genes are so much conserved?
because there’s evolution within the box genes thats very relevant for diversity.
are hox genes the same w/in all the flies?
no, there can be different modulations within different flies more or less ubx expression
What the gene DII?
it’s a gene that develops prolegs in caterpillar associated with development.
What’s forward genetic screens for?
it’s to identify developmental mutants for example looking at denticle mutations in drosophila embryos, the identity of the segment and the different segments are revealed by the denticles. allow the discovery of developmental genes that are imp for development in a unbiased way
What’s the anterior-posterior pattern?
found in drosophila, box genes are a key thing for patterning anterior and posterior parts.
How is the polarity done in drosophila?
to make sure that posterior matches posterior and anterior matches anterior, there are different genes that match polarity; maternal genes–> 1st ones determining polarity, gap genes–> divide embryos in regions and pair rule genes —> establish segment plan. and segment-polarity proteins –> set the boundaries
What are the 4 main things that establish polarity in drosophila?
- maternal genes: 1st ones to establish polarity
- gap gene: divide the embryos in regions
- pair rule genes: establish segment plan
- segment polarity proteins: set the boundaries.
What’s the most common maternal gene in drosophila?
bicoid; this gene is highly expressed in the anterior site but it does not act alone
nanos: reverse effect
What can occur if you’re a bicoid mutant?
there’re no gradient changes
How do maternal genes interact?
there’s interaction bt different maternal genes for example nanos acts in reverse to bicoid, but when bicoid acts, it inhibits the production of nanos and nanos protein inhibits hunchback TF
What are HOM-C?
homeotic gene clusters in mammals.
difference bt mammals and insects regarding Hox genes.
4 HOM-C in mammals located in different chr but are paralogous bc the order of genes in each cluster is very similar whereas in insects only one HOM-C
What does it mean that two HOM-C are paralogous?
it means that they show similarity in the order of genes in the cluster.
How many human genes associated with a heritable disease have a homolog in drosophila?
60 %
Whtat’s the genetic cascade for box genes?
maternal gap 1st pair rule 2nd pair rule segment polarity
(it defines regions little by little)
How does the genetic cascade for box genes work?
by loads of transcriptional regulation . combinatorial regulation ro drive the expression
do all hox genes take place along frontal basal polarity?
no, there are some that take place ventral dorsal obviously.
