Week 4 Flashcards
What are criteria for being a model organism?
Size, number of eggs/embryos, accessibility, speed of development, genetics, misexpression of genes, ease of manipulation
What makes a good model?
Simple diploid genetics (eukaryotics), Rapid generation time, controlled genetic crosses, generation of mutants (forward and reverse approaches, genetic map, amenable to transformation, genome sequence
Is there a correlation between genome size and number of protein-coding genes?
There isnt a correlation between the two
What was key about coding for model organisms with respect to the human genome project?
Model organisms were sequenced as part of the human genome project allowing for testing of tools to increase size of genome sequenced
How easy is it to access model organisms genome?
Complete genome sequences are deposited in public databases, which are easily accessible
What are the advantages in microbes as model organsisms?
They have very short reproductive cycles- (20 mins doubling time in E.Coli)
They are easily grown
The large numbers produced facilitate the rapid identification of rare mutation
What is the genome size and number of genes for Escherichia coli?
4.5 Mbp
4,500 genes
What is the genome size and number of genes for Saccharomyces cerevisiae (Brewer’s Yeast)?
16 Mbp
6,200 genes
What is the benefit of using Saccharomyces cerevisiae (Brewer’s Yeast)?
Eukaryotic, haploid, so cellular things can be applied to all eukaryotes like humans
What is the E.coli long-term evolution experiment (LTEE)?
Since 1988 Richard Lenski and group have had 12 parallel and independant bacterial populations allowed to grow and multiply
What have been the current outcomes of the LTEE?
By 20,000 generations in all populations grew approximately 70% faster than the ancestral strain
Six of 12 have had developed defects in their ability to repair DNA
1 strain has evolved the ability to citarte positve metabolism (ability to use a new source of respiration energy)
What is the genome size and number of genes of Caenorhabditis elegans (Roundworm)?
100 Mbp
20,000 genes
What is the genome size and number of genes of Drosophila melanogaster?
180 Mbp
15,500 genes
What is the genome size and number of genes Mus musculus?
3000 Mbp
20,000-21,000 genes
What are the key characteritics of Zebrafish (Danvio rerio)?
Fast early development
Many eggs, develop outside mother
F and R genetic screens are possible
Requires significant resources though
What is important when working with vertebrates and cephlopods?
Pre-register the methods, justification and number of animals and it needs to be independantly reviewed for animals welfare and ethics
Can forward genetics be done of Zebrafish?
Yes
How can forward gentics be done on Zebrafish?
Induce recessive mutations (m) in the male germ line (eg EMS)
Breed for at least two generations until homozygous offspring are observed
Analyse phenotype and clone the gene
What is the method for forward genetics on Zebrafish?
Male sperm has been mutagenised and bred with wild-type female
F1 individuals need to be selected and isolated
Each F1 will be heterozygous for a unique set of mutations
Some may result in a characteristic in m/m homozygoutes
Subsequent generations will need to be analysed
For recessive mutations only a small proportion of the F3 progeny (1/16) will display a mutant phenotype
What is Glofish?
The first GM pets - transgenic Danio rerio expressing colour varients of GFP and RFP
When did human and mice genomes diverge?
Around 85 mya
How many synthetic blocks are in both human mice genomes?
Human and mouse genomes comprise around 180 ‘synthetic blocks’ (genenomic blocks that contain the same DNA)
What has been conserved in both human and mouse genomes?
Conserved protein coding
Conserved transcriptional control sequences
What are key features of embyronic stem (ES) cells?
Stem cells- undifferentiated, can continue to divide
ES cells derived from inner cell mass (ICM) of blastocyst
Can be maintained indefinitely in undifferentiated state
Can be re-injected into blastocoel cavity of normal embryo and reincorporated into inner cell mass
How do you get ES cells from mice?
They need to be surgically removed
What can be done with ES stem cells when removed from mice?
They can be grown on mass in a culture
What is the function of Neomycin?
It is used as a positive selection marker ie to identify when the transgene has been integrated into the genome
What is the function of ganciclovir?
It is a negative selection marker due to it being converted to a toxic DNA precursor by the HSV thymidine kinase protein
What is the difference between the alleles during targeted mutagenesis?
Only one of the two alleles is inactivated by this process, so the cells produced are heterozygous
What happens during Homologous recombination of ES cells carrying a knockin mutant?
Homologous recombination –> Gene-targeted insertion –> Mutation in gene X –> Cells are resistant to G-418 and ganciclover
What happens during Nonhomologous recombination of ES cells carrying a knockout mutant?
Nonhomologous recombination –> Random insertion –> No mutation in gene X –> Cells are resistant to G-418 but sensitive to ganciclovir
What is the first stage in generating mice with homozygous ‘knock outs’?
Inject ES cells into blastocoel cavity of early embryo
Surgically transfer embryoes into pseudopregnant female
What are the possible progeny of the eggs injected into females?
They are either black or have a chimeric colour
What happens to the chimeric coloured mice?
They are selected for crosses with wildtype black mice, this may result in heterozygous offspring. This will onlu happen if transgenic ES cells contribute to the germ line
What are the percentage of brown mice will contain the transgene?
Only 50%
How can you tell which 50% contain transgene?
Molecular screening to identify X-/X+ heterozygotes using PCR
What do you do with the X-/X+ heterozygotes?
They are mated with other heterozygotes
What are the percentage of the He x He are homozygotes recessive?
25% which can screened using PCR
What is the ecological breakdown Drosophila melanogaster?
Order- Diptera
Family- Drosophilidae
Genus- Sopophora (Not Drosophila)
What is the relationship between Drosophila and Humans?
It has a commensal relationship with humans
Where is Drosophila Melganogaster found?
Found on every continent expect Antarctica
What are advatanges of using Drosophila as a model organism?
They are easy to care for, many offspring, short generation time and well studied genomes
What is a key feature of fly wings?
Hind wings have been reduced to a stumpy pair of haltereres which act as stabilisers allowing for rapid change in movement
How many days will it take for a Drosophila Melanogaster take to fully develop eg egg to adult at 25 degrees C?
10 days
How long after egg laying can a egg hatch?
Less than a day
What happens when the fly egg hatches?
The fly becomes a instar which undergoes a series of malts across a couple days until it becomes 3rd instar stage
What happens when a fly reaches its 3rd instar stage of development?
Its called the wandering stage, so it will move around until it finds a suitable place to form a pupae
What happens when the pupae forms?
They undergo a process called total holometabolism in which the entire maggot is broken down and rebuildt to become an adult
What initiates and controls Drosophila metabolism?
Gene expression shifts that initiate Drosophila metamorphosis mediated by the hormone ecdysone
What happens during metamorphosis?
Larval tissues breakdown, adult tissues develop from 19 imaginal discs (tissue-specific progenotor cells)
How quickily after hatching can Drosophila Melanogaster reproduce?
In about 6 hours
What do wild Drosophila feed on?
Yeast, bacteria and rotting plant matter
What do lab Drosophila feed on?
Standardised diets consist of cornmeal/yeast/agar
Where do Drosophila lay their eggs in labs?
Adult flies lay their eggs on the food surface, larvae hatch and burrow into the food
What happens to the wandering third instars?
Wandering third instars crawl out and pupate on the sides of vials
What does the Drosophila genome consist of?
4 pairs of chromosomes, one sex chromosome, two standard autosomes, one ‘dot’ chromosome
What determines sex in Drosophila?
This is regulated by an X chromosome counting mechanism that ‘senses’ the dosage of X chromosome. It is not determined by the participation of the Y chromosome
What is the sex of XO flies?
They are male due to the decreased X dosage
What is the sex of XXY flies?
XXY flies are female due to having 2 X chromosomes
What happens to the hormones in flies with XX chromosomes?
Sex lethal (SXL) is produced
This allows for the production of TRA by binding to the gene preventing the alternate splicing from taking place which forms TRA-2
TRA acts a splice enhancer allowing for the splicing of double sex (DSX) female (DSX^F) to occur
What is MSL-2 and what does it do?
Male sex lethal and cause hyperactivation of the X chromosomes in males
When and by who was the first chromosome physical map of Drosophila created?
It was created by Calvin Bridges in 1935
What are polytene chromosomes?
They are special types of chromosomes that are found in some insect’s excretory cells eg Salivary glands in Drosiphila. They are caused by a chromosome rapidly dividing creating hundreds of copies without dividing. these can be seen under an optical microscope
What happens if you add a chemical dye to polytene chromosome?
This gives rise to unique banding patterns for each chromosomal region; these banded regions were numbered by Bridges and standardised as a map coordinated that are still used today
What dis Calvin Bridges do to map cooridinate Drosophila chromosomes?
They divided each chromosome up into equal parts and each of those parts had 6 subparts, with each subpart having a letter
What is the function of dividing each chromosome into parts and subparts?
They could assign phenotypic changes to changes in each part and subpart and be able to point to where the gene change happened and could demostrate that this can be heritable
What is the amount of protein-coding genes predicted by the latest genome assembly?
13,920 genes
What percentage of those genes produce proteins?
72%
How many genes can produce more than 1 protein?
45%
What is EMS?
Ethyl methanesulfonate
What is the process of foward genetics in Drosophila?
Males eat food with mutagen
Developing sperm (and other cells) each have new mutations
Cross to wildtype females
Offspring each carry different mutations
Outcross offspring seperately
Make inbred lines of new mutations
Test homozygotes (if alive) for phenotype of interest
What was the work by the Heberlein lab with genes to do with alcohol tolerance and alcoholims?
They did the forward genetics approach and exposed them to ethanol vapours and pick the flies that where different or particually receptive. The genes found where barfly and tipsy
What was the similarity to alcohol between drosophilia and humans?
The neural responses have a basic similarity with humans
What are the 2 types of transposons?
Reterotransposon (Class 1)
DNA transposon (Class 2)
How do Reterotransposons works?
They create RNA copies of themselves which then get converted into new DNA strands which can be inserted back into a different location in the original DNA strand
How do DNA transposons work?
They create DNA intermiades of themselves which then bind to the newly created DNA strand during replication
How many families of transposons are in the Drosophilia melanogaster genome?
~150 families of transposons
Why are TEs deleterious?
They can be inserted into genes or cause chromosomal rearrangements
What is important amount P elements with the idea of transgenics?
The P element helped form the basis transgenics
What are P elements?
They are naturally occuring Class 2 transposon
What was one of the earliest transgenics experiments using P elements in Drosophilia?
They had a mutant white- eye coloured fly.
To determine the white- eye caused white eye colour rather than the dominant red, they added a transgene carrying white+
The transgene’s had the white+ gene, flurescent marker which is flanked by the required P elements
If the gene gets restored and the phenotype does this allows for understanding of thats genes function
When does the P element need to be introduced?
This needs to be done very early in syncutium mass of the embryo development
When an embryo is a few hours old multiple DNA replication events have occured but no cell membranes have formed. This forms G0 fly
What is the key feature of fly G0 with a transgene?
It is a mosaic so most are wildtype with some containing the transgene. So it most likely wont express the mutant gene
What happens to the G0 to determine whether it has uptaken the gene?
G0 is bred with another knockout fly in order to create G1 which some of them should express the transgene
What is GAL4?
A gene that codes for the GAL4 protein. This protein can bind to repetative DNA elements UAS enhancer.
What is the UAS?
A promoter that only works when GAL4 protein binds to it
What is GAL4/UAS fly?
The breeding of a fly with an enhancer before GAL4 which has been bred with a fly with UAS and the gene of interest
What sort of experiments can you do with these experiments?
If you want to to know where a gene was expressed, you would get a fly with the promoter region of gene of interest and bred witg a UAS GFP fly. The resulting flies will only produce GFP in the required region
If you want to find the gene function of something later in life but not as an embryo or maggot as it is required for survival. You can use the appropriate enhancer GAL4 along with UAS RNAi for a gene to only be functional during early development allowing for phenotype change later in life
What would a UAS-GFP reporter line with no GAL4 look like?
Little to no GFP in any of the stages from L3 ro 4-7 day adult
Autofluorescence is more prevalent in the L3 larae but not in the L3 body wall or at anyother stage
What would a UAS-GFP reporter line with MHC look like?
High levels of expression through out as seen with constant green glow. When an adult the gene is concentrated in the muscle tissue. This is due to MHC being a muscle tissue driver
What would a UAS-GFP reporter line with DJ694 look like?
Little expression in the L3 stage
Not a lot in early pupae
By day 2 it is expressed in the gut and abdomen.
DJ694 is a muscle gene for muscles in the gut and adbomen
What would a GAL4 with UAS Cas9 look like?
A specific gene knockout at a required location and stage of development
How many Gal4 lines are there?
Around 9000
How many UAS lines are there?
6000 lines
How many balancer lines are there?
400 lines
How did Thomas Morgan use drosophila to win a nobel prize in 1933?
Uncovered the role played by chromosomes in heredity
How did Hermann Joseph Muller use drosophila to win a nobel prize in 1946?
Used X-ray irradiation to increase mutation rates in fruit flies
How did Edward B Lewis, Christiane Nusslein-Volhard and Eric F Wieschaus use drosophila to win a nobel prize in 1995?
To understand genetic control of embryonic dvelopement
How did Richard Axel use drosophila to win a nobel prize in 2004?
The understanding of how the olfactory system operates
How did Jules A Hoffmann use drosophila to win a nobel prize in 2011?
For his research on the activation of innate immunity
How did Jeffrey C Hall use drosophila to win a nobel prize in 2017?
Uncovering the molecular mechanisms that control circadian rhythms
