Lecture #5 (Mobile DNA) Flashcards
Transposons
Mobile Genetic elements
- Important because drivers of genome plasticity
Transposons = tolls in biology
Elements can be transposed by different mechansims (Ex. movement of DNA or via RNA intermediate)
Mobile Genetic elements (Overall)
Common to all three domains of life (Prokytoes + Archea + Eukaryotes)
MGE = drivers of genomic plasticity (impact the genome structure + function + evolution)
Mobility can occur by an RNA intermediate or only DNA
Mobility can be stochastic or programs
MObile elements have been harnessed for experimental mutagensis or gene transfer
Transposition
Movement of discerete DNA element into diverse traget sites
- The discrete DNA that is moving = trasnpsoson
- NEW Place DNA goes = targte site
Two main types of tranpsosition
- Excision and integration - Donar site loses transpsone ; transposon goes to another place
- Replicative - Donor and target end with the transposon
Movement is mediated by proteins specific to a partcilar element
Important featire of transposons
Feature of tronspition = target site duplication
Arrow in the image - starst as one arrow thne have duplcation flamking the new site of the stransposons
- Flanking the transpsons is a small peice of the orginal DNA that now duplicated in the target DNA
- Transpsons = red lines –> flanking the transpons on both sides is a handful of duplciated nucelotodes
Called traget site duplication (consequence of the mechanism of transpostion
Effect of transposable elemts on genome
Transposable elemenys are potent sources of genetic diveristy
How:
1. Disruption of genes
2. Mobilization of genes within and amog chrosmosomes (Ex. bacetria - TE can mobilize and transfer AB resistence)
3. Alter gene expression by placing TE regulatory signlas near host genes (Ex. TE promoters + enhancers + splice sites + PolyA sites)
- Disrupt genes by having polyA transposon dropped into gene = causes termination
4. Substartes for homologous recombination
- Elements with multiple copies in the genome can be sites of HR –> cause scrambling of the genome
Barbra McClintock
Looked at the regulation of pigementation in corn
Found that she was mapping controlling elemenst that were hopping around (TE) and causing regulatory chnages
- Transposons reveal themselves by generating mutations
Haig Kazazian
Shows that transposon in humans are still causing some diseases
Studied an insertion in blood facor 8 (de novo mutation)
- Found insertion by using southern blot
FOUND that pateints had an insertion that was disrupting the gene
- Had transposition of non-LTR LINE elements that mobilized in pateints and caused disease
Showed that transposons remain active (0.3% of de novo mutations is due to mobilization of LINES)
- Many human diseases are now known to be caused by TE insertion
Fraction of de novo muttaions caused by transposon insertion
Fraction of de novo muttaions caused by transposon insertion varies between organisms (activity of TE vary between organism)
0.3% in humans (TE are 45% of the genome)
- Less mutation in humans
10% in mice (TE comprise 38% of genome)
> 50% in drosophilla (TE are 5.5% of the genome)
IN humans and mice you ahve 1 insertion in every 20-100 births BUT mos are inconsequential becase most of the genome is non-coding
Classifications of TE
TEs can be groups according to:
1. Element strcuture
2. Transposase strcture
3. Mechasnim of transosition
4. Effect of transopitiion on donor site
Grouping of TE according to element structure
- Transpose using only DNA only intermediates
- Transpose using RNA intermdiates
- Inlcudes Long terminal repeate elements (Ex. Retroviruses + retroviral-like elements) AND non-LTR elements
Distribution of the TE in different species
Prokaryotes - Have TN5/TN7 (DNA elements
- No retrotransposition in Prokaryites
vertebretes - Most of the TE are RNA class elements
- Have scares in the genome of DNA elements but none are active (Sequneces that are decated DNA transposons)
- Have 1 class of LINEs that are active in humans
DNA-only transposon
Sturcture (Image)
- Flanked by inverted repeats (Called TIR) -> TIR is duplicated and has inverse orientation (because they are inverted they look idetocal form the left or the right so when transposase is trying to recogze the TIR is looking at the same sequence context at the inverted repeats)
- TIR function - recognized by transposase and drives the movement by the encoded ransposase
Mechanism of movement:
1. Cut and paste
2. Replicative (Nick and paste)
Autonomaus Vs. non-autonmous DNA tranpsosons
IF the transpons encodes its own trasnpsases = autonomous
- Transposaes = acts on the TIR
Non-Automous = does not encode its own transposase
IF the Non-Autonoous share the same TIR sequence as the autonoumous - transposase ca act on the non-autonouis elememnts because of the shared sequence
- Transposase from the autonmous elemnt can act on the non-autonmous elememt evcause they share the same TIR (SHare red sequence)
Long terminal repeat (LTR) transposons
Type of RNA transposons
- LTRs includes retroviruses and retroviral elements
Transposon cycle = involoves alternations between RNA and DNA copies of the element
- LTR = supports the conversion of RNA to cDNA –> cDNA is integrated into the host genome –> LTR then support transcription of the integrated DNA into another RNA copy
Structure of LTRs
Defined by the present of direct repeats (LTR) (flanked by LTR)
- Repeats are NOT inverted (go in the same dorection)
Has encoded proteins that allow for transposition of elements as they cycle between RNA and DNA
- LTR supoorts the conversion of the DNA copy to RNA copy then tp cDNA –> cDNA is integrated into the host genome
Proteins encoded:
1. GAG - DNA/RNA binding proteins
2. PRT - Protease
3. RT/IN - reverse transcriptate and Integrase
Retroviruses vs. retroviral elements
Structre of the LTR transposones = very similar to retroviral structures (Ex. HV)
Rertoeviruses - have etxracellular trasmission from cell to cell
- Env enocdes surface glycoprotein
- Have additional proteins that allows the retrovirus to move between cells
Retroviral elements = ONLY intracellular (Within one cell)
Non-LTR elements
2nd type of RNA intermediates
Includes LINE and SINEs
LINES:
- 20% of genome
- Automnous
- Encodes 2 proteins (ORF1 - chaparone ; ORF2 - Endonuclease/RT)
SINES:
- Non-Autonomous (rely on the RT in LINE elements to transpse)
- Shorts (~200 bases)
- 13% of human genome
- Example - Alu elements
- Have A and B –> RNA polymerase 3 promoter
DNA transposon superfamilies
DNA transposon superfcmailies = used for genome engineerring + reserach
Bacteria:
- IS10 –> Inserstion sequnece distributed in genome ; has inverted repeats (Arrows) that are recognized by tranpsoase
TN10 - DNA transpsons flanks genes (flanks Tet10 gene) –> Called “composite”
Image - has lists of DNA ransposon superfamilies in Bacteria + Euk
DNA cut and past elements
Image - Insersation sequences = IS elements (Orange = transposon)
- Have inverted repeats flakning transposase gene
IN composite bacterial transposons have rpeates falnkning the transposase IS10 gene AND have the IS elements flanning other genes (Ex. flanking Tn10)
- Can flank a drug resistent gene
- If the transposon insets in one place and then inserts in a second place at some distance from the first element = forms a composite element that carries eveyrthing between the two sites
Effect of composite elements
NOW the transpsons can move in differenet way
Ex. Have recognize the arrows on ine one transponson to give only IS10 transposition (Ex. transpson on the left or on he rght) OR can recognize one arrow in the elemnt of the left and one arrow on the element on the right = have Tn10 transposition
Major way to get AB resistnce in bacteria - captures genes i a selective pressure that can be mobilized by recignziing the ends of transposons
DNA cut and Paste Stradegey
Major way to transpose in DNA TE
Tranposase binds (pairing activates DNA cleaving) - Transposase recognizes inverted repeats –> brings the ends together –> Cleaves the ends –> THEN Excision event that expsoes a 3’ OH at each of the ends of the transposon –> NOW both ends have a free 3’OH that can engage in nucleophilic attack of target –> resukts in iserttion and repair
Cleaving of traget DNA in DNA cut and Paste Stradegey
Often NOT making a blunt cleavage
- Image - cut at the G on the top and bottom strand –> in dsDNA trasnpons the left 3’OH will bind tio the bottom strand G and the right 3’OH binds to the top starnd G –> Need to fill in the ssDAN (seen in blue arrow)
Have 2 nicks (grey in image –> creates two gaps - 3’OH on the right aattcks the gap on the left forming a covaelent bond + same with the left side –> after attck have repair
- END have target site duplication
How do you get target site duplication
Target site duplication is characteristic of all transpons is a fucntion of activation of the traget tp receive the liberated transposn (BECAUSE the endonuclease cleave is staggered)
Stagering cleavge of the target = yeilds traget site duplicaion
In image - staggered cut is seen in the grey atrics
What do all exiscion pathway yeild
ALL pathways for excision yeild a 3’OH
- 3’OH generation mechansim depends on family
Generate 3’OH by:
1. Cleaving both strands (seen in B/C)
2. Nick –> have 3’OH attack the yellow arrow at the LTR –> genrates a haipin flanking donor (A)
3. Nick on transposon hat genrates a 3’Oh on the LTR –> genetes a hairpin on the transpson that needs to be opened to liberate the 3’OH for subseqenent attack
ALL 3 yeilds a free 3’OH that can be used for nucleophilic attack on free DNA
What cataylyzd dsDNA break
Transposase = cleave dsDNA (does so in a highly regulated way)
- DNA breakage by transposase is catylyzed in trans
Transposase needs to make sure that the reaction ONLY happens when there is a fullly assembled transposase complex –> HOW???
- Transpoases makes sure that it can’t one end of the transposon first and then the second INSTEAD the transposase structures makes sure the cut happens in a concerted way
One unit binds to one end of the transpons and the otehr units bonds to the other end BUT the complex does trans catalysis where the catalytic domain bound to one end of the transpon cleaves the other end of the transposon and vica versa
- Transposase is inactive until it has a fully formed cmplex where both ends of thee transposon have ben assembed into the transposase dimer
RAG and VDJ recombination
VDJ recombination - creates diveristy in immune system using RAG recombinase
- RAG is related to transposase
To get diveristy - have a set of V segments and a set of D segments –> In a B cell one V segment is joined with 1 D segement –> generates diversity
- Done at the DNA level (irreversible reorginzation of B cell genome)
VDJ recombination (process)
V segment joining the D segment occurs with RAG recombinase
- Have nicking to generate the 3’O at the junction with V and D segments –> attck of the 3’OH on second strand to generae hairpines in V and D segments –> liberate the dsDNA spacer (black) –> resolve the haorpin to generate the VD fusion that will become part of the AB or the TCR
- Hairpins = similar t HAT tranpsons (Nick to genrate a 3’OH in flanking DNA) –> attack t genrate harpin in flanking DNA liberating the transposon
Topology of the reaction looks the same as DNA element transpoition - hae black between the V and J (black can be thought of as a transponson –> raction liberates the transposon and joins the flanking V and D sequences together
- Have ancestral relationship beween transpositio and generting diveristy in immune system
Types of DNA transpotion
Start - Have donar DNA with the transposon AND a target –> CAN then do two different paths:
1. Cut and paste –> orginla sequence DNA might be repaired (Close circle) or might disappear
2. NIck and paste –> Duplication of tranlsocaton and co-integrate intermediate
- Co-integrate intermiediate –> transposon has been duplcated and the donar DNA and the target DNA are joined to one another
Replicative nick and transposition
2nd mechanism of DNA transposition - involoves duplication of translacation
- End have transposon in the initial site AND the target site
Overall - Replication –> fill in –> duplicate –> have duplicated transposon and the two molecules are joined
Start - Plamsid with Mu (transposon) –> Mu transposase makes a nicl at each 3’ end of Mu + Nick target DNA in offset way (NOT free transpon being only nicked on 1 strand not ddDNA break) –> 3’OH mMu ends atatck target DNA at staggard positions (3’OH from donaor joins the target DNA making 1 side linked to the donar DNA) –> begning of replication of Mu (begin replicaton from flanking traget DNA) (Blue arrows) –> completion of rpelciation of Mu (fill in and copy acros transpson) –> two copies of Mu link donar and target DNA
- having joining of the duplicated transposon
- NOW NO transposon being liberated by dsDNA break INSTEAD there is a nick on both strands making a 3’OH BUT orginal DNA stays attatched to the transposons
- Still get targte DNA repeats because ofset cleavage
Co-integrate resoluion
Co-integrate needs to be resolved in ordeer to get two olecules (orginal and target DNA –> BOTH have a copy of the transposon)
Co-integrae resolution occurs using a conservative site-specific recombination
- Resolved using resolvase
Resolvase - classss of transposons encide BOTH transposases and Resolvase (RES)
- RES - drives recombination between the two copies to liberate he co-integrate into two molecules
- Resolvase = catylyzes recombination between 2 RES sites
LTR elements
Example - Ty1 in yeast
ALL have similar porteins:
1. GAG - nucleic acid binding
2. PR - proteases
3. RT - reverse transcriptase
4. RH - RNase H
5. Integrase
6. ENV - envelope –> ONLY in retroviruses
RT/IN = critcal
LRT retrotranspon Lifecycle
LTR retrotransposons = intracellular propegation
Start - Have integrated copy of the element in the genome –> Transcribe an RNA copy –> RNA cop goes to the cytoplams –> RNA copy is tranlated –> Proteins are assmbled into a virus or a vrius like particle containing the RNA and retrotransoposon functional proteins Ipartcile has RT converting RNA to cDNA) –> cDNA is integrated back into the genome at a new location
- Lifecyle of a retrotransposon using an RNA intermediate
Retrotranspson Life cycle vs. Retrovirus life cycle
Retrotranspon life cyle = looks very similar to retrovirus lifecyle (Ex. HIV) BUT vrius can bud off to go to anotehr cell and integrate into the genome of another cell
Image - Retrovirus = Intercellular propegation
LTR elements transcript
Promter element drives a transcript that nitiates downstream of promoter = generates a partial transcripts
- Going from R-R in transcript
- Transcript lacks U3 on left and U5 on the right BUT the stcuture of the element allows restoration of the full element from the transcript
Even though trucates at the 5’ and 3’ ends you still have ALL of the elements in the transcript (because what is lost on one repeat is found in the otehr reapt ; stil have both U3 and U5)
Because you still have all of the elements you can make the full cDNA
- Explains why the LTR are flnked by the LTR
Lifecyle of transposons
Start - generate a partial cDNA at the 5’end –> degrade the transcript that was copied into DNA –> have a hop of the patial cDNA (R elememnt on one side can hybridzie and prime off the other side) –> copy the rest into DNA
THEN have a second hop that allows for completion
- Hopping creates a full LR on 1 side –> THEN the full LTR is hoped over to the other side –> Get full cDNA
Have complex set of moevemnt in RT setthat gets the peices of genetic infomration recopied to both eneds
- ALL happening in the virus or the viral like particle (compact area where isolmerization cen be engineered)
- Complucated conversion of retroviral RNA tp cDNA that regertates the full LTRs in the cDNA to preserve a complete and fucntional retroviral genome
Integration of cDNA intermediates of LTR elements
After conversion of the RNA to a full length cDNA that has 3’OH (NOW it is the same as DNA transposon that was liberated)
Can have integration (same as Cut and paste transposons) –> cleave the traget site with staggered cite –> generate gaps –> Inserte the cDNA into new site –> fill in (blue arrows)
- CREATES target site duplication
- Integrase = catyklzyes the cut and paste insertion
- When have hop = have evidence of 5 BP being duplicated on eitehr side of the new retrotransposon
DNA only transposons vs. Retroviral integrases
Have structural similarity of DNA only transpsons and retroviral integrases
- Catylytic cores contain spatially clustered DDE or DDD residues which bind Mg (Mg is essential for transposition)
Integrases (in retroviruses/retrotransposons) = carrying out a similar reaction to the cut and paste transosase
LINE
3 LINE families related by sequence BUT only 1 is active
- LINE elements are responsible for most RT of the genome (Ex. resonspible for retrotransposition of SINEs + generation of Psudeogenes)
- RT can be used to mobilize nay RNA
- 6 kb
Has promoter at 5’ end - Polymerase 2 promoter (regulates transcription)
- Transcript made is controled by regulatpry element upstram gives ful length transcript
- Regulated by its own promoter
LINE = has RNA binding proteins (ORF1) and RT/EN for copying RNA and DNA(ORF2)
SINE elements
Example Alu elements
Derived from small cellular RNA (related tp tRNA and 7S RNA)
Have Pol 3 promoter
Looks like a line structure becase full length transcript and PolyA tail at the end
- Have similar mechansim as LINE to move
ORF enzymes that move LINE can also mobilize ANY RNA that is Polyadenylzted = can move SINE
- DNA transposons = sequence drives reconiztion in a specific way BUT with Non-LTR trasnposons the mechasnim dpeends on the presence of PolyA in the transcript
Transposition of Non-LTR elements
non-LTR transcribes full length RNA (have polyA tail on the RNA) -> endonuclease clip T rich target site (Nicked T in target site pairs with polyA) –> 3’OH serves as a primer for RT reaction that can copy the entor element to ssDNA (LINE RNA is the template) –> have a second nick on the target site (two green lines)–> DNA synthesis is complete using the ssDNA as a template –> make dsDNA –> RNA is degraded and the repair outs cDNA into the new site
- Second cut = on the right image (have the cdNA on the other DNA – get rid of the blue RNA)
Origin of Introns
Introns in bacteria = self-splicing introns
Group 2 mobile introns (structure at the top)
- intron between 2 exons in prokaryotes = self splicing RNA that arises from endoed proteins
Have transcription –> get premRNA –> translate the protein –> have RME protein – proteins allow splicing of the introns out to make leyreyet with the introns on the complex (intron wrapped around RME) –> THEN exons are spliced together
NOW the intron can reintergrated into a new site –> reverse splice the leriet –> makes a hybrid molecule where 1 strand has picked up the RNA copy of an intron –> have traget site primed RT –> get cDNA –> get new ontrons that mobilize in the middle of a gene
Recombination between inverted repeats
Recombination requires homology)
Repeats inverted with respect to each other will generate an inversion following reombination
- What is between will end up being inverted
Image - A, B, C atatched to the left blue and red and DEF atatced to the right blue and red –> have recombination –> get FED
- Yelds inversion of everything betweeb the two elements
recombination between diect reaptes
Direct repeats (heads to tail with respect to each other) –> generate excision/deletion
In image - D is attatched to the left blue which joins with the right red (right red is atched to F and E) -> end up with FED
- ONE of the circles will be lost
Resolution of P1 replicate intermediate by Cre
Cre = resolvase
Cre-Lox recombination
Have Cre recogniztion site –> Cre resolvase cuases reombination (makes a recombiantion event between the two elements)
Have 2 elements with the sequence Cre recognizes –> Cre will catylyze homologous exchnage between the two
Synaptic substrates for Cre-mediated recombination
Based on the crystal structure - should end up with Left blue and pink RED BUT they actually align in oppsoite orientation so he right blue atatches to he right red and the left blue atatches tp the left red
In Crystal structure he reaptes (substares for HR) = no alligned doirectley ; substartes are inverted with respect to one another (weird recombination
- - Macheasnim for recombinase passes through holiday junction (looks like recombitoral substrate before reoslutiion into he two recombined DNAs)
Model for Cre-mediated recombination
Model for Cre-mediated recombination by means of a Holliday junction
Nick one strand on each dsDNA –> attack tyrosine –> give 3’P-Try and a 5’OH –> exchnage 5’OH ends between duplexes –> 5’ OH and the P-Try become coveletley linked ( 5’OH is free for nucleophilic attach by the p-Try –> getnerase the covaelnt bond) –> strands link –> isomerozation event –> second strand exchnage
- END - have verticle excision in middle –> get hetrduplex of DNA at regions of strand exchnage
Transposase mediated DNA fragmentation process
TN5 uses cut and paste to carry out transpon reaction effeictely
TN5 = DNA trasnpson = 3’OH on excised transpon cleaves the target DNA
For tagmentation (fragmentation method_ –> purified transposases are loaded iwth linkers
- Linkers = grey that correspond to sequnce at the end of the T5 transponse (see little grey on the TN5 dimers)
- Sequence that the grey linker correpsonds = part of the short inverted repeats at th end of TN5 that permits loading into transposases
Transponsase takes the TN5 transonson end and attacks target DNA
- TN5 is loaded with short oligios that are stand in for transpson neds = meidates attck on DNA (get fusions of the oligios at the end of DNA sequences)
- Red/Blue = primer sites –> once shatter the genome the fragments are tagd at both ends by a peice of TN5 derived seuqnces and by the blue/red primers = can be aplcified by PCR to genertae a librar of all of the fragemnts in the genome
ATAC-Seq
Looks at chromatin structure
Process - targte DNA is chromatin –> TN5 transpsoase is loking for a traget site
- In heterochormatin the DNA is insensitive to TN5 BUT in open chromatin TN5 can attck
Frequencey that TN5 inserts at a given nucleotie = proxy for how open the chromatin is
How do you di it – have chromatin and TN5 with the oligios in a 1 tube –> Add chromatin –> do gap filling –> have fragments that can be sequnced
- Insertion sites in the sequence twll you what is open vs. closed
ATAC-Seq results
Image:
Green part (ATAX seq):
Y Axis - cut density (form sequences you can see what is 5’ end of the fragment)
X - Axis - loaction in genome
See peaks with high insertions = get map of open and closed chromatin states
Red Part - Accisibility of chromatin by DNAse (DNAse can digest open chromatin)
Red and green charts have the same pattern
- Peaks = acceisble DNA
Fineness of ATAC-Seq
Accsibility of tranposase allows you to map the exact position of the underlrying nucelsome
Chart:
Y Axis - fragment size
X -Axis = genomic position (MIdpoint of the DNA fragment (Fragemnt 100 nucletodes the midpoint is 50 and the 50 is plotted)
- Center of the nuclosome = 0)
How many of he sequneces have this nuclostie at 0 AND what wa sthe fragment length –> leads to a traingle disturbution where the density focuses at 0
- 0 ONLY have things that are 120 nucleotides long
- Get disrubtio of fragemnst where the midpoint and the fragment size creates V shape ; as fragment get shorter the only ragments in the library correspond to 0 (Vs. Larger fragmemts have midpoints in more places )
- Distubution of freqnece lengtsh tel you position of the nucleosome
Nucleosome mapping of ATAC-Seq
Nucleosomes can’t be hit by TN5
IF fragment was 200 nucleotides then the possible origin that 1 TN5 can cut includes many places far and wide from the nuclseosome = can get many 200 B fragmemts
IF fragment is 120 nucleotides (slice DNA wraping around the histones) = only wya to get is if cut at teh arrows irght next to the nucleosome
- Center fragments hows the center of the nuceleosome
Results in last chart - Result reflects accibility of TN5 around a single nucleosome
- TN5 200 nucloetide fragemnts ahv many possiblities to be made= midpoint can be anywhere acrps sthe whole regino BUT ONly way to genrate 120 is to cleave right before and irght after nucleosome = center of teh 120 fragment reflects the center o the nucleomes
Selection for a mutate Methods
- Selection - Impose sleection so you get the mutant you want
- Screen many mutatnt 1 by 1 –> recognize the one that is needed
- Paraell screen - Take all the mutatnts –> screen mutants –> everything survives but the 1
- Mutant is NOT in the final popultion
- Can see what was lost at the end
- Can see what is present at the begining but lost in the end
- Mutant is NOT in the final popultion
Selection using transposons
IF have libraru of trasposon mutatiions (have transpons in diffferent cells disrupting different genes)
Location of the transpsone:
1. Disrupts gene
2. Marks place in the genome
Can use a primer that is within he transpson (arrow on left) AND ligate a universal primer (right arrow) –> do PCR to generate a fragment for all transpson in library
- Red sequence tells you the location of the transposon (tells you the transpson is in the red gene)
- Sequce next to transpons = bar code that tells you the transpson is present and is disrupting the gene
- Amplification has the little peice of trasnposon + the gene
- Sequneces = can tel you where all of the transpsons are in the library
Selection using transposons process
Sequnce alll of the sequneces NEXT to the trasnpson –> can compare the beginingpool and the end poool and see which tranpsons vanished from the pool
Used in bactreia + yeast
Transposon mutegensis
TNC - generate a library of transpons that will land anywhere -> grow cells –> some trasnsons will be lost
- Lost transposons indicate essnetial genes
Borad insertion of transpons –> captore of insertion sites as a library –> sequence –> Comparison of insertion library before and after growth yeilds gene map
Transposon mutegensis results
Y Axis - set of transpson mutants
X Axis - genes
Each bar = transposon insertion site
- Red boxes = see have no transpsons –> lack of transpsons is because they are essential genes
- When the librar was grown the tranposons were dropped because the gene is essnetila and so the cells with transpons in that gene did nots survive (only one without them in the gene survived = those were seuqnced)
ALLOWS you to define the essential genes in an organism (genes that can’t tolerate insertions)
Transposon mutegensis part 2 results
X axis - libraru being propogated in control
Y axis - libraru being propegated with drug
Dits on the line = mutatnt that is uneffected by the condition (representation does not chnage when adding condition)
Abive line = mutant makes the cell resistent to the drugs (mutant is present 1000X with drug but only 100 without drug)
below line = things that are resitive to the drug
IN image - Red genes abov line are all involoved in mitocindral funtion
- Bottom image =two genes in TCA cyle are below line and rest of the genes are above the line = get model for the genes making sensitivity and the genes giving resistnce
DNA transposons in mice
Mice don’t have DNA transposon BUt in the genome there are scraes of them –> someone looked at all of the sequences and saw they were the same non-function element because they were mutated –> combined the sequences and derived the ancestral active sequnce –> manufactured an active verision
Active version = Sleeoing beurty (transposon that now functions)
Top sequence = active sleeping beuty tranpson
- Has promoter + polyA site + inverted repeats
- IF lands next to a gene it can drive transcription of gene BUT if it lands inside a gene it inactives the gene
- Genes are recognized by transposase (transposase is regulated by the Lox system)
Cre-Activate tranpson mutagensis
Experimnet - Mouse with transposon in genome
Two mice line (tranpsoase line and he transpon line –> progeny mice in certain tsisues (depending on where Cre is expressed the transposases becomes active = goes all over genome) –> Age mice –> tumors form (because transpons landed next to genes that they affect expression or into gene sthat they are dusruping) –> isolate DNA –> PCR off the ends of the transpons –> sequence idetofy the sites of transpoon insetion in tumors
- MIce = 1 line with the tranpsosae promoter in the Lox P system (Promter is not active = no tranposases = no tramsposition) ; 1 line with the transposne + has Cre recomniase in some tissues
- Get mutation in certain cells
Cre-Activate tranpson mutagensis results
Do PCR (red dot and green box are the primers) –> ampfy between the dot and the box =amplify the gene that the transposon inserted into
Compare tumors and derive a signature for insertions in genes that come up frequnetley (look for genes that are reprodusiibly mutated = gives finger print for diferent tissues for what genes might be mutated sin mice that predisoposes teh tissue to tumor formatino)
- Look at multiple tumors (look a the tranpsons that are shared between tumors of a specific type
- Get finger print of genes that are disrupted that give tumors
- Can change the tissue he recombinase is expressed in
Overall - making transposon library
