Look for mutants, mapping, isolate canidate genes, sqeunce genes.
sevenless photorecptor is required for phototaxis towards UV light. What can we do to isolate mutants?
Used UV light. Wildetypes are attracted to the UV.
Sevenless is the lack of?
the R7 photorecptor
What do modifier screens do?
They help identify additional genes with pleiotropic effects.
What led to the discovery of the Ras and importanrt Ras regulators
Modifier screens
What can happen by incerting P elements into a genome?
Mutants could be created
Steps to identifying mutant gene.
1) verify sequence the gene and identify 2) transgene reque 3) Targeted mutant, compare to original mutant
DNA endonuclease; enzyme that cuts DNA, not-sequence specififc.
CRISPR/Cas9
What is an issue with using CRISPR?
It can break both DNA strands. Once a repair is made it causes a framshift.
What can be used to repair a DNA strand after using CRISPR
NHEJ Non-homologous end Joining. Smoothes the ends of the cut ends.
How do we study the genetic control of development?
Find interesting developmental patterns, make random mutants, use classi mendelion recombination mapping, use whole genome sequencing to find DNA sequence, and then verify canidate genes with transgene resuce and target mutations with CRISPR.
What are the ways that we can sudy gene expression: Where? When?
Situ Hybridization, Immunostaining, RNA sequencing, and Reporter transgenes
Uses synthetic acids to detect mRNAs in tissue sections. Easy to make thye probe and applicable to nearly all organisms and tissues. However poor image esolution and usually just one gene
Situ hybridization.
Uses antibodies to detect proteins in tissue section. Good image resolution and can use on 2-3 proteins at a time. However more expensive antibodies subject to availability, difficult to custom make.
Immunostaining.
Uses next generation sequencing technology. Seqencing all mRNAs from dissected tissue sample. Analyze all genes imn a single experiment. Data are quantitative. Howver, lose spatial information. limited by dissection technique.
RNA sequencing.
Uses recombinant DNA engineering in vitro to fuse the enhancer of gene of interest to someling like GFP. Live imaging. nopt dependent on nucleic acid or antibody probe. relatively easy. However only works for common lab organisms.
Reporter transgenes
Cause cegments to develop with the wrong identity.
homeotic
What is the size of the whole human genome?
3x10^9 bp or 1 meter
How much DNA do you need for sequencing?
4.5x10^10 cells population = 7.9x10^9
How do we find protein coding genes.
TATAAA We can also look for open reading frames.Average protein will have 100-200 amino acids.Finding a Start codon that will have groupings that go on for a long time before a stop codon would indicate a protein.
We can then compare the cDNA with the regular DNA and can find the similarities.
reverse transcriptase
How do we know what genes do?
We compair them to genes in similar organisms.
who Observed transformation of R form to S form in a test tube. Fractionated the killed S form and isolated the transforming principle and find that it is DNA. They then took the transforming principle and deactivated each of the macromolecules, removed proteins with protease, RNA with RNase, etc. and tested each for transformation. They always saw transformation except when the DNA was destroyed. This supported their idea that DNA was the transforming principle although they still received arguments that there could still be trace amounts of protein if their protease enzyme wasn?t very active and the transformation was due to protein activity
Avery, MacLeod and McCarty
who Used phages, one set grown in radioactive phosphorus which was incorporated into the phage protein and another set grown in radioactive sulfur which was incorporated into the phage DNA. They added each to bacteria cultures, after a time they used a blender to knock the phages off the bacteria and centrifuged to separate them. The phage ghosts stayed in the supernatant while the bacteria cells were in the pellet. They found that radioactive phosphorus was in the cell meaning that the phages injected DNA into the cells, so this was the transforming material.
Hershey and Chase
who used DNA crystallography.Showed that DNA was spiral shaped. Gave the spacing between repeating units, the length of a full helical turn, and the diameter of DNA
Rosalind Franklin
By examining many organisms, found that ratios between certain bases remains constant in all the organisms. Similar amounts of C and G, and similar amounts of A and T lead to the idea of complementary base pairing.
Edwin Chargaff
Loss of function mutations
Amorphic, or null, Hypomorphic, Halopinsufficiency, Antimorphic
full loss of function
Amorphic
partial loss of function, protein production is insufficient for normal funcion.
hypomorphic
dominant loss of function mutation; haploid- a single wild type gene copy makes ? the amount of product but that is not enough to produce the wild type phenotype. One wild type gene is insufficient to avoid a mutant phenotype
Haploinsufficiency
Gain of function mutations
Hypermorphic, neomorphic
Increases gene function
Hypermorphic
New gene function
neomorphic
One or more sets of chromosomes has a different copy numbner than others
Aneuploidy
2n
Euploidy
2n-2
Nullisomy
2n-1
Monosomy
2n+1
Trisomy
Most organisms can tolerate aneuploidy in the sex chromosomes because of _________. Will have mild consequences.
x-inactivation
Meiotic non-disjunction causes
aneuploidy
Non disjunction in the 1st meiotic division results in all
aneuploid dametes
Non disjunction in the 2nd meiotic division results in
two haploid and two aneuploid gametes
Mitotic nondisjunction during the 1st mitotic division of an XX cell results in
one Xo male and one XX female
A variation in number of chromosome sets
Polyploidy
produces an odd gamete ratios leading to odd mendelian ratios. (assuming no crossover 1:4:1) which in turn lead to odd phenotypic ratios.
Tetraploid heterozygote
Which of the following is not a reason that makes the drosophila eye a good model for studying developmental biology?
The eye being required for survival
When a large number of mutant flies are produced, scientist interested specifically in eye development need to come up with tricks to quickly identify rare eye mutants. What might be such a trick?
They can use behavioral screen, such as a sesitivity to UV light.
A hypomorphic allele of sevenless reults in about 50% of ommatida lacking the R7 photoreceptor. In a suppressor screen, you isolate an allele of the ras gene. Flies that are homozygous for the hypomorphic sevenless allele and are also heterozygous for the ras suppressor mutation have mostly normal photoreceptors. The ras suppressor mutation by itself as a heterozygote has no phenotype (look like normal) What kind of mutation do you think the ras allele is?
A weak hypermorph
Scientists used the sevenless regulatory region to express transgenic RasG12v, which is hypermorphic mutant allele of the ras gene. These transgenic animals have ommatidia that have excessive cell proliferation, resulting in a roughb eye phenotype. They used this mutant to perform a modifier screen to find more genes that function in the sevenless pathway. What do you think would happen if, instead of using transgenic apporach that limits RasG12V to photoreceptors, scientists tried to create mutant flies that had the G12V mutation directly incorporated into the native locus?
Since ras is widely used to control cell proliferation in many tissue types, this might result in flies that have pleotrophic effects and might not be useable. (dead)
Once candidate mutant genes are identified through classical genetic means, geneticists willm use various techniques to verify whether they have identified the correct gene responsible for the mutant phenotype, How is CRISPR/Cas9 used for this pupose?
It is used to create targeted mutations in candidate genes- scientists can compare whether the targeted mutation has the same phenotype as the mapped mutant.
Transgenic reporter genes use the regulatory sequencesof genes of interest fused to a reporter gene such as GFP. This technology can be adpated to all organism, even those poorly studied. T/F
F
In the classic nussein -Volhard/Weischaus experiment, scientists screened for embryonic lethal mutation in drosophila. what was the rationale for screening for such mutations?
They were interested in genes that controlled embryonic development.
When screening for embryonic lethal mutations, at what generation would you be able to tell is an embryonic lethal mutation had been indentified.
F3
When screening for embryonic mutations that map to chromosome II, what is the purpose of using the Curly (Cy) mutation, which also maps to chromosome II?
It allows you to directly identify which animals are heterozygous for a lethal mutation
Which of the following is a plausible explanation for why homeotic transformations in Drosophila occur?
A change to a regulatory sequence of a homeodomain gene causes the gene to be mis-expressed in the wrong body segment.