Lecture #9 - Drosophilla 1 Flashcards
Why is Drosophilla a good model
- Many genes and biological processes are conserved in flies (Ex. 75% of human disease causing genes have homologs in Drosophila meaning we can swap human homologs)
- Inexpensive
- Easy to maintain
- Small
- Short life cycle
- Produce many offspring (females lay 100 eggs per day)
- Simple genome (Have four chromosomes = designing genetic crosses is simple)
- Array of sophisticated genetic tools are available
Answer - E
Can store dead embypres in the freezer BUt they would not be alive after
Drosophila genome
15k protein-coding genes
Higher gene density than humans
Drosophilla Life cycle
Have a short life span (40-50 days)
Four stages in Drosophila development - egg + larva + pupa + adult
Life cycle - Egg is fertilized –> after 24 hours it will undergo embryonic development and hatch into a larva –> The larva subsequently undergoes three molting phases called instars (instar 1 –> 2 –> 3) –> Next, the pupa enclosed in a pupal case undergoes metamorphosis and the body structure is rearranged and the adult structure
- Instar 1 and 2 = last one day vs. the third instar = last two days
- Adult flies begin mating about ten hours after eclosion (emergence from pupal case)
- Life cycle is dependent in temperature
What type of females are used in genetic crosses
Need to use female virgins when setting up a genetic cross to ensure parentage of the progeny
- Important because females can store sperm from males
Drosophila – Tissues + development + brain functions
There is a lot of conservation of development in drosophila and humans –> A lot of the foundational understanding of development came from drosophilla
Example - Can stain for different features of the embyroe
- Top image – patterning genes that set up the body plan (early embryo)
- 2nd image – Trachea
- 3rd image – Muscles (Figured out how cell-cell fusion works by studying flies )
- 4th image – Nervous system in flies
- Image in middle – Larave neuromuscular junction (study in flies)
- Right image – dopamine neurons in brain in adult (study brain using flies)
Circadian rythms + flies
Circadian rhythms = discovered in flies –> work in fruit fly led to nobel prize in understanding the central clock that regulates fly and human circadium rhythms (molecularly conserved)
- Molecular conservation = led to understanding of basic biology
Polytene chromosomes
Polytene chromosmes = Giant chromosomes formed by multiple rounds of DNA replication without cell division (DNA is replicated 1000s of times)
Polytene chromosomes are found in certain cells in flies
Use - Early fly geneticists used polytene chromsomes to decipher the structure and arrangement of the genome
- PolyT chrosomes = how poeple made maps of chromosomes
Drosophilal chrosome cytological maps
Able to look at PolyTene chromosomes by squashing the cells and flattening the nucleus content (including the chromsomes)
- Looking at PolyT chromosome = gave people link between chromsomes and where genes are in position along the chromosome
Bottom part of image – see PolyTene chromosome
- See dark and light areas –> shows areas with more or less DNA condensation (The pattern is reproducible across flies)
- Image shows bands 20-40 on second chromosome left arm
- Have pictures for all chromosomes in flies
TOP picture = shows the bands on all arms of the chromosome (X has 0-20 ; 2 has 21-60 ; 3 has 61-100 )
ANSWER – 3R (because 94 is between 81 and 100 – look at last slide)
Can do FISH for all genes and they will map to band on chromosome (green signal)
- Upper micrograph = have 94e1 THEN do FISH and see a fluorescent band
Sexing Flies
Male (XY):
1. Smaller
2. Black and rounded abdomen
3. External genitalia
4. Have sex combs (patches of dark bristles on the foreleg for mating)
Female (XX):
1. Larger than males (BUT could have mutations that change body size)
2. Internal genitalia (no Sex combs)
3. Striped and pointed abdomen
Y does NOT determine maleness –> INSTEAD the lack of a second X chromosome determines maleness
Drosophila chromosomes
Drosophila have 4 pairs of chromosomes
- Chromosome 1 = the sex chromosome
- Chromosomes 2-4 = autosomes
Chromosome 4 – dot chromosomes (very small ; 2% of genome)
Chromosome 2 and 3 = have their centromere positioned in the center which seperates each chromosome into a right arm and a left arm (Each arm carries about 20% of the fly’s genes)
Drosophilla genome
132 million base pairs (1/20 the size of humans)
15k gene
Gene density is much higher than the human genome
Karyotype - Shows have 2 pairs of chromsome 2 + shows chrosmome 3 i+ shows the X chrosmome in orange + shows chromsome 4 as two dots
Do we care about chromosome 4
Often don’t care about chromsome 4 in crosses because has so few genes
- Often don’t include when writting genotypes of flies
BUT Chromosme 4 does HAVE essential genes
- Hard to resrecah things on chromosome 4 because it has a lot of heterochromatin
Genetic Nomenclature
Wild type = wt or +
Gene name and genetic elements is Italicized (Curly)
- Genes in flies are given the name of the mutant phenotype
Gene symbole is itallized (abreaviate name of gene) - Ex. Cy
Dominant mutations are capitalized (Ex. Dr or Cy)
Recessive mutations are written in lowercase (Ex. vg or hh )
Allele of gene uses a super script (allele name is written as a super script)
- Example - W^a –> allele of white gives rise to a pale orange eye color (allele = apricot)
- Example 2 – hh^8 –> 8th allele of hh gene (allele name is superscript)
Writting a genotype
Overall - the genotype of each chromosome is indicated separately
Chromosomes are separated by semicolons
- Chr 1 ; Chr 2 ; Chr 3
Each homologous chromosomes are separated by a line
- Maternal chr 1 / paternal Chr 1 ; Maternal chr 2 / paternal Chr 2 ; Maternal chr 3 / paternal Chr 3
Different alelles on the same chromosme are seperarated by commas
- +/+ ; Allele 1, Allele 2/ + ; +/+ (Allele 1 and 2 both on chromosome 2)
The sex chromosomes can be written in multiple ways
- Female = +/+ or x/x
- Male = +/ weird symbol or x/y
Answer – C –> has 2 domiannt mutations on chromsome 2 and a recessive mutation on chromsome 3 and it is male
ANSWER – B
Answer – C
w- = means have 2 X chromosomes (w-/w-)
Chr 2 = lowercase = recessive
Ch 3 = uppercase = Dominant
Answer E (Know it is E because hh is on the second chromsome (based on A) AND NOT D – because gig is on the 3rd chrosome (Based on A
w is mutant
- w- is an allele of w mutant
Shortcuts to write fly genotypes
Might not write out all of the chromosomes (if one chromosome does not have any alleles of note on it it won’t be written)
If a pair of chromosomes are homozygous THEN only one chromosome will be shown
Sometimes alleles are not separated with a comma
Sometimes allele names are not written out
Example – Cross a male that is heterzygous for Cy (on 2nd chromosome) and Heterzygous for dr (on the 3rd chromsome) TO females that is homozygoyz for eb (recceisve mutation on the 3rd chromsome)
Each offspring receives one chromosome from each homologous pair from each parent
Because the female is homozygous for every chromosome, there is only one combination of maternally inherited chromosomes –> +; + ; eb
Male can give 8 possible combinations (Can give + or Y on chromsome 1 ; can give Cy or + on chromsome 2 ; can give Dr or + on chrosome 3)
END - There are eight possibilities for the genotypes of the offspring (because 1 possible from female and 8 possible form male)
Calculating offspring ratios
Maternal gametes – w or +
Paternal gametes – w or Y
SLIDES = shows possible offspring
Punnet square = shows you the distribution of gamete
ANSWER – ¼
How to assign sex to the parent genotypes
Need to consider many things when thinking about which parent genotype will be male or female in the cross:
1. The chromosomal location of genes within the parental genotypes
2. 2 - If there needs to be meiotic recombination to produce the desired F1 progeny
3. If one of the parent genotypes has a limited amount of flies
4. If you are going to mutagenize the parents
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes
If the gene to be inherited by the F1 generation is on the X –> the parent with the X containing gene must be female
Example – IF you want progenicy with baltimore in genotypes
- When cross a fly that is homozygous for the gene Baltimore on the X chr and WT on the other chromosomes to a fly that is homozygous for the gene Maryland on the 2nd chr but WT on the other chromosomes –> the homozygous Baltimore fly must be the female to generate progeny with 1 copy of Baltimore in their genotype (see genotype on slide)
- WHY - If we were to make the homozygous Baltimore fly male, then only the the female progeny (NOT the male progeny) will have 1 copy of Baltimore in their genotype
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If there needs to be meiotic recombination to produce the desired F1 progeny
If there needs to be meiotic recombination to produce the desired F1 progeny
Only females can do meiotic recombination = you must use females parents
BOTH males and females can do mitotic recombination.
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If one of the parent genotypes has a limited amount of flies
If one of the parent genotypes has a limited amount of flies –> should make that the male parent
WHY - males do not have to be virgins prior to mating –> means it is easier to use males when there is a limited number of flies with that genotype
How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If you are going to mutagenize the parents
If you are going to mutagenize the parents you should mutagenize males
WHY – because males generate more gametes than females –> means that there is higher potentoal for more uniquley mutagenized progeny
Fowards genetics in Drosophilla
Fowards genetics – Go from phenotype to genotype
Uses:
1. Random point mutatiions (EMS or radiations)
2. Random Insertions (P-elements)
3. Enhancer or supressor screens
4. Deficicey kits
Reverse genetics in Drosophila
Reverse Genetics – Go from genotype to pheotype
Uses:
1. Gene Knockdown (RNAi)
2. Knockins/deletions (CRIPSR)
3. Overexpression (GAL-4-UAS)
4. Cell types specific expression (GAL-4-UAS and clonal analysis)
Generating Mutations in Flies
- Chemical mutagens (EMS) –> Makes Point mutations
- Pro – random + saturation + different types of muatnts
- Con – Slow gene identifications + large scale - Ionizing radiation (X-ray and gamma rays) –> Makes chromosome rearangments
- Pro – Random + gene deletions + duplication
- Con – Slow gene identification + no real saturatiion + inefficnet - Transposons –> Makes DNA insertiions (mostly hypomorphic in UTRs)
- Pro – fast gene identification + flexible scale
- Con – Non-random (hot spot) + no saturation - Homologous Recombination and CRIPSr/cas9 –> Makes trageted mutogensis of specfic
- Pro - Very efficient
- Con – Currentley ineffcincet for screening
Foward genetic Screen
Foward genetic Screen = unbiased screening for mutations producing a phenotype of interest
Example Fowards Genetics Screen
Goal – Screen for genes on second chromosome essential for photoreceptor function
- Fundemental for studying RAS – all unraveled by studying fly eye
Example Fowards Genetics Screen - What method would you use to generate mutation
ANSWER – EMS – want point mutations (most common)
- NOT CRSIP because not efficinet
- NOT trasnpons because would get large deletions/rearngemnmts