Gga 15: Genetic Screens For Developmental Genes

Front 1

Are mutations in the 'toolkit genes' often lethal?

Back 1

Yes.

Front 2

What is the problem for a lethal recessive mutation?

Back 2

Homozygotes die, and if heterozygotes aren't selected, the gene can be lost completely.

Front 3

What is a balancer chromosome?

Back 3

Modified chromosomes used for genetically screening a population of organisms to select for Heterozygotes.

Front 4

What process do balancer chromosomes prevent?

Back 4

Crossing over during metaphase I.

Front 5

What is the structure of the balancer chromosome relative to the normal chromosome?

Back 5

The middle sequences (not the first and last) are flipped around (reversed) in the balancer chromosome.

Front 6

What are the two distinguishing features of a balancer chromosome (what major things do they encode)?

Back 6

le - a recessive lethal mutation. If two balancers are present, the organism will die. If heterzygous, it will survive.
Cy - A dominant marker causing curly wing phenotype. Allows quick detection of balancer chromsome.

Front 7

Do the normal and balancer chromosomes lie top and tail?

Back 7

Yes.

Front 8

Why is this important when it comes to crossing over?

Back 8

When crossing over occurs and material is exchanged, the centrosome is also transferred. This results in one chromosome without centromeres and one with two centromeres.

Front 9

When crossing over occurs in top and tail normal and balancer chromosomes, does the sequence of nucleotides change?

Back 9

No, just the centromere arrangement.

Front 10

What combinations of the balancer chromosome and normal chromosome (if it is recessive lethal) will result in death?

Back 10

Homozygous balancer or homozygous normal (heterozygous is ensured).

Front 11

How can we make sure the genotype is heterozygous?

Back 11

Through the dominant curly wing marker (heterozygotes will have curly wing)

Front 12

What is done to a genome at the start of a mutagenesis screen?

Back 12

Mutated by exposure to mutagens (EMS or X Rays)

Front 13

What is the general principle of a mutagenesis screen (in terms of chromosomes and inheritance)? Parents.

Back 13

Mother: 1 normal chromosome and 1 balancer chromosome.
Father: 2 normal chromosomes (with mutagen influence)

Front 14

What is the general principle of a mutagenesis screen (in terms of chromosomes and inheritance)? F1.

Back 14

Females: 1 normal chromosome and 1 balancer chromosome.
Males: One mutant chromosome and 1 balancer chromosome.

Front 15

What is the general principle of a mutagenesis screen (in terms of chromosomes and inheritance)? F2.

Back 15

Females: One mutant chromosome and 1 balancer chromosome.
Males: One mutant chromosome and 1 balancer chromosome.

Front 16

What is the general principle of a mutagenesis screen (in terms of chromosomes and inheritance)? F3 possible offspring.

Back 16

Dead balancer homozygotes.
One mutant chromosome and 1 balancer.
Homozygotes for mutated chromosome (showing phenotype, can be measured; usually lethal)

Front 17

What is the earliest we can see the mutation? Why is it different in Drosophila?

Back 17

F2. F3 in Drosophila since they have 2 sexes.

Front 18

When does the maternal effect occur?

Back 18

When the phenotype of an organism is determined by the genotype of its mother.

Front 19

Why do maternal effects occur?

Back 19

Since the mother provides a certain mRNA sequence or protein to the oocyte.

Front 20

If the maternal effect gene is present in the mother, when will the effect of the gene be seen?

Back 20

F2

Front 21

What genotype must the mother have for a maternal effect gene to be passed on?

Back 21

Homozygous recessive

Front 22

What is a zygotic effect gene?

Back 22

A gene that is expressed from the zygote's own genome (instead of the maternal genome)

Front 23

When will mutations be seen that originate in the zygote genome?

Back 23

Immediately

Front 24

Can the zygote be homozygous recessive for a maternal effect gene and live if the mother is heterozygous?

Back 24

No.

Front 25

What is transposon tagging?

Back 25

When transposons are amplified inside a biological cell by a tagging technique.

Front 26

What organism can be used as an example of transposon-induced mutation?

Back 26

Snapdragon

Front 27

What is the gene responsible for pigment in the flower cells? Which genotype produces which colour?

Back 27

Pallida gene. Pal+ produces red cells, Pal- produces white cells.

Front 28

In the flower of the Snapdragon plant, there can be both white and red pigments (and therefore cells). Why is this?

Back 28

A transposable element can move into the Pallida gene, inactivating it, but can also move out, activating it. This is very unstable, which is why many of the cells vary in colour.

Front 29

What is the name of the transposable element in question? How does it inactivate the gene?

Back 29

Tam3, disrupts the open reading frame.

Front 30

What external factor influences the action of Tam3 (its incorporation into the Pallida gene)?

Back 30

Temperature

Front 31

If a snapdragon flower is grown at 15 degrees produces as much red and white cells, and another flower grown at 25 degrees produces only white cell, in which flower is Tam3 more actively transposing?

Back 31

The flower at 15 degrees.

Front 32

Can Tam3 cause mutations in other genes too?

Back 32

Yes (including developmental genes)

Front 33

Parent Snapdragons grown at 15 degrees will produce which genotypes and at what frequency? M1.

Back 33

+/+ and m/+ (m for mutant), but the m/+ will be rare.

Front 34

Why will m/+ be rare?

Back 34

Since transposable elements will only effect very few genes

Front 35

Self pollination of the progeny from M1 will produce which genotypes at what frequency? M2.

Back 35

+/+ and m/m (m/m only seen in large screen because transposon must have affected both alleles)

Front 36

How is the Floricaula mutation caused in Snapdragon?

Back 36

Transposable elements again!

Front 37

What happens in the Floricaula (flo) mutation?

Back 37

Flowers aren't produced, and are replaced by leafy structures.

Front 38

How can the Tam3 clone be used?

Back 38

As a probe in Southern Blotting

Front 39

If the flo mutation is caused by a Tam3 insertion, what can we infer?

Back 39

There must be Tam3 DNA inserted into the FLO gene.

Front 40

What is the problem with this?

Back 40

There are many (10-30) Tam3 insertions throughout the Snapdragon genome.

Front 41

How can we distinguish between different Tam3 inserts?

Back 41

By using a restriction enzyme, which will cut the Tam3 and another part of the genome (outside the transposon), and Southern Blotting. Different sized fragments will be produced, and we can distinguish them.

Front 42

How can we find the Tam3 insert that is different in the wild type and Floricaula?

Back 42

Run both the flo and wild type mutation on the Southern Blot, and look for different bands.

Front 43

What does the Floricaula gene with the Tam3 transposon look like? What is the size of the gene?

Back 43

Tam3 in middle, with the Floricaula gene around it. 7.5kb.

Front 44

What can the sequence be used for once it has been cloned?

Back 44

Identify where the gene is using in situ hybridisation of labelled RNA probes.

Front 45

How is in situ hybridisation carried out?

Back 45

Take gene cDNA (for example, agamous) and clone it into a plasmid vector.

Front 46

How is the cDNA incorporated into the plasmid?

Back 46

Inverted orientation, so that the strand synthesised is the antisense (complementary) to the transcribed mRNA.

Front 47

How is the gene cloned once the cDNA is in plasmid?

Back 47

Plasmid linearised (by cutting the plasmid), RNA Polymerase added and binds to the RNA Pol binding site, and transcribes the DNA into mRNA.

Front 48

What would the produced mRNA do with the original mRNA of the gene?

Back 48

Hybridise with it.

Front 49

What can a probe be used for in this context? How?

Back 49

Detect where the antisense RNA binds. Modified UTP (modified by adding a molecule to the end of it, molecule can be recognised by specific antibodies)

Front 50

How is in situ hybridisation carried out? Step 1 (why?)

Back 50

Fixed in formaldehyde to cross link the RNA with the proteins and thus fix it in place.

Front 51

How is in situ hybridisation carried out? Step 2.

Back 51

The sample is incubated with labelled an antisense AG RNA probe.

Front 52

What does the RNA probe do in the sample? Where does hybridisation occur?

Back 52

It hybridises to the complementary AG mRNA. In cells where the gene is active.

Front 53

What are the 4 things we can do with a gene once it is cloned?

Back 53

Modify the gene and create a transgenic organism.
Can test effects of mis expressing the gene
Can characterise the regulation of a gene
For lethal mutants can restore gene and withdraw functions at different stages of development.