5. VL Flashcards
Drosophila melanogaster “the workhorse of genetics”
Why the fly?
- Easy and inexpensive to culture in laboratory conditions
- Very short life cycle (24h embryogenesis, 9 days until fertil adult, early embryonic development)
- Large numbers of externally laid embryos
- Simple genome
how does Gastrulation in Drosophila happen?
ventral furrow, germ-band extension (Keimbandverlängerung), mouthparts - thorax - abdomen
timetable of embryogenesis
clevage (stage 1-4; ~2:10h) blastoderm (stage 5; ~40min) gasturlation (stage 6-7; ~20min) germ-band elongation (stage 8-11; 4h) germ-band retraction (stage 12-13; 3h) head involution and dorsal closure (stage: 14-15; 2:40h) differentiation (stage: 16-17; 9h)
How many larvae stages and what do they have in common?
• head with acron with denticles bands on ventral side: • 3 thoracic segments • 8 abdominal segments • telson
Why (where) the fly os that easy to breed?
In your kitchen:
Adults lay their eggs on rotting fruit.
Larvae feed of decomposing fruit.
Females mate once and store sperm for the rest of their life
Characteristics of mature male
- continuous pigmentation of posterior end
- round shape of abdomen
- different genital apparatus
- sex comb on forelegs
What do you know about Drosophila’s genome?
- 4 chromosomes
- comparative genomics
- fully sequenced in the year 2000 (second multicellular genome to be sequenced)
- Genome size ∼ 180 Mb
- protein coding (~1500) and non-coding regions (~3000
- Half of D. melanogaster genes show alternative splicing
- Differential promoter-driven use of alternative first axons
- 45% of genes encode more than one protein isoform)
How would you create genetic crosses in Drosophila?
unmated females <8 hr old (virgins) of defined, genotype with visible marker
x
males of defined genotype with visible marker
What genetic tool in Drosophila is different as in other organisms and gives the fly an advantage as a genetic model organism?
Balancer chromosomes:
1. Dominant visiblemarker
2. Recessive deleterious mutations
3. Inversion break points
• keep homozygous lethal or sterile mutations from being lost
• prevent that of multiple alleles on the same chromosome become separated by recombination
On which websites do you find information about D. melongaster (or other flys)?
FlyBase, DorosphilaStockCenter
How can I identify genes involved in a developmental process?
males with mutagens (=sperms with mutagen) x wt females –> offspring with mutations
outcross offspring separately with wt (–> new genetic lines)
inbreed lines with new mutations
test homozygotes (if alive) for phenotype of interest
I have found some interesting candidates in my forward genetic screen. How can I find which gene is mutated?
Mapping
- Parallel complementation tests
- Recombination mapping
- Whole genome sequencing
1) Determine whether or not a given mutation is worth pursuing. (verfolgt werden muss)
2) Mapping process facilitates basic characterization.
3) Generation of useful reagents, like balanced strain and linkage to markers.
4) Scale down of sequencing efforts.
5) Great training in genetics.
What is the basic 2 point mapping?
a mutation in the gene of interest is mapped against a marker mutation
is used to assign mutations to individual chromosomes.
can also give at least a rough indication of distance between the mutation and the markers used.
who was the one, who created the first genetic map and when?
1913, Thomas H. Morgan and his students A. H. Sturtevant, C.B. Bridges and H.J. Muller made genetic linkage maps based on crossing over events
When should we start the diagnostic for linkage of 2 genes?
When two genes are close together on the same chromosome and they follow this general pattern in a test cross:
•Two equally non-recombinant classes totalling in excess of 50 %
•Two equally recombinant classes totalling less than 50 %
The farther two genes are apart…
… the more likely that a crossover will occur and the higher the proportion of recombinant products will be.
What does the recombination frequency (RF) reflect?
reflects the actual distances separating genes on a chromosome.
What fundamental developing questions can you answer while working with Drosophila?
- How are the body axes set up?
- Which mechanisms are involved in the patterning of the embryo?
- How is the animal body plan set up?
How does the patterning of Drosophila embryo takes place? (anterior - posterior patterning)
- maternal effect genes: laid down by female in the egg & provide first positional information on body axes
- gap genes: define formation of a block of segments
- pair-rule genes: define the parasegments in a double-segment periodicity
- segment polarity genes: refine pattern of parasegments
- homeotic selector genes: determine segment identity
what do you know in general about anterio-posterior patterning of the embryo?
- embryo patterned in a series of steps
- broad regional differences are established first
- then smaller developmental domains with a unique profile of gene activity are produced
- developmental genes act in a strict temporal sequence
- action of one set of genes activates another set and thus dictating next developmental stage
What do you know about maternal effect genes?
main body axes are established through morphogen gradients.
Morphogen gradients provide positional information along the a-p and d-v axes
Anterior group genes: bicoid, hunchback
Posterior group genes (formation of germ plasm): caudal, nanos Terminal group genes: torso
Dorsal group genes: gurken, spätzle, dorsal, toll
How does the specification of the antero-posterior polarity in the embryo takes place?
Bicoid protein provides an anteroposterior gradient of a morphogen
• maternal bicoid mRNA anchored at anterior end of egg
• fertilization & translation into Bicoid protein diffusion in posterior direction& gradient
• absolute prerequisite: syncytial blastoderm
• Zygotic genes are regulated by different concentrations of Bicoid
• dual function as transcription factor and regulator of mRNA translation
What is a morphogen?
Substance that differentially specifies the fate of cells by different concentrations
How was the concept of morphogens studied?
What is bicoid good for?
principle: „find it, loose it, move it“
bicoid gene breaks the symmetry along the anterior-posterior axis.
is necessary for the establishment of anterior structures because it produces a gradient
