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Dale series Flashcards

1
Q

Gene)Expression)

A
  • A)gene)is)expressed)if)its)mRNA)sequence)is) found)in)the)cell.)
  • This)usually)means)its)protein)product)will)also) appear)in)the)cell)
  • It)is)the)differences)in)gene)expression)which) give)rise)to)different)cell)types.)
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2
Q

The)MEL)cell)

A
  • Murine)ErythroLeukemia)cells)
  • An)immature)red)blood)cell)line,)arrested)part) way)along)the)process)of)differenMaMon)
  • Produce)a)glycoprotein)which)binds)to)the) EPO)site,)so)growth)and)proliferaMon)is) independent)of)EPO)
  • ConMnuously)proliferaMng)
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3
Q

The)MEL)cell)

A

• Certain)chemical)agents)can)sMmulate)them)to) progress)further)through)the)proliferaMon) process.)
• One)example)is)DMSO)(dimethyl)sulfoxide))
• ATer)72)h)exposure)to)DMSO)the)cells)
become)more)differenMated.)

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4
Q

Common%methods%used%to% monitor%a%change%in%the%level%of%a% par5cular%protein:%

A
  • Enzyme)Linked)ImmunoSorbent)Assay)(ELISA)% • RadioImmunoAssay)(RIA))
  • Western)blot))
  • Enzyme)assay)
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5
Q

The)Transcriptome)

A
  • The)transcriptome)is)the)complete)set)of) transcripts)for)a)cell)under)a)defined)set) of)condiMons.))
  • This)set)reflects)both)the)diversity)of)the) sequences)and)the)relaMve)abundance.)
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6
Q

IsolaMng)RNA)

A
  • Before)you)can)measure)the)level)of)a) parMcular)mRNA)sequence)you)need)to) isolate)the)RNA)
  • RNA,)parMcularly)mRNA)is)designed)to)be) unstable!)
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7
Q

IsolaMng)RNA)

A
  • Rule%#1:%KILL%the%ribonucleases.)) • Rule%#2%KILL%the%ribonucleases.%% • Rule%#3%refer%to%rules%#1%&%2!!))
  • Most)methods)rely)on)lysing)the)Mssue)in)a) guanidine)isothiocyanate)soluMon)(chaotropic)) with)a)reducing)agent.)This)will)denature) ribonuclease)(not%permanently)%
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8
Q

IsolaMng)RNA)

A
  • Once)the)Mssue/cell)sample)has)been)lysed/ homogenised)you)need)to)separate)the)RNA)from) the)other)polymers,)parMcularly)protein)and)DNA!)
  • Phenol:chloroform)extracMon)followed)by)ethanol) precipitaMon))
  • TRI)reagent)is)a)combinaMon)of)the)guanidine)and) the)phenol)
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9
Q

Measuring)the)level)of)a)specific) mRNA)sequence)in)the)transcriptome:)

A
  • Northern)blot)
  • Real)Mme)PCR)
  • RNase)ProtecMon)assays)
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10
Q

HybridisaMon)

A
  • A)process)whereby)a)nucleic)acid)sequence) base)pairs)to)a)complementary)sequence)from) another)source,)producing)a)hybrid)double) stranded)structure.)
  • The)nucleic)acid)sequence)can)be)either)RNA) or)DNA)in)either)case.)
  • HybridisaMon)needs)condiMons)which)promote) base)pairing,)washing)later)with)increased) stringency.)
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11
Q

HybridisaMon)

A

• CondiMons)which)promote)base)pairing:) – high)ionic)strength)
– lower)temperatures)
– neutral)pH)
• CondiMons)which)disrupt)base)pairing:) – Lower)ionic)strength)
– Higher)temperatures)
– High)pH)

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12
Q

Real)Mme)PCR)

A
  • Normal)PCR)is)a)great)photocopier!))
  • It)is)only)semi_quanMtaMve.)
  • If)you)could)monitor)the)amount)of)product) aTer)each)cycle)then)you)would)have)a) quanMtaMve)method.)
  • Sybr)green)is)a)dye)that)binds)to)double) stranded)DNA)ONLY)(not)single)stranded))and) fluoresces)at)a)specific)wavelength)when) bound.)
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13
Q

Real)Mme)PCR)

A
  • Shine)a)laser)beam)at)the)excitaMon) wavelength)of)Sybr)green)aTer)each)cycle)and) measure)the)fluorescence)
  • The)amount)of)fluorescence)is)proporMonal)to) the)amount)of)PCR)double)stranded)product)
  • Measure)the)number)of)cycles)needed)to) achieve)a)pre_set)amount)of)fluorescence)
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14
Q

Real)Mme)PCR)

A
  • The)more)of)the)sequence)in)the)original) mRNA,)the)fewer)number)of)cycles)needed)to) achieve)the)pre_set)level)or)threshold.)
  • Compare)to)a)reference)sequence,)oTen)18S) RNA.)
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15
Q

QuanMfying)Real)Mme)PCR)

A
  • Each)cycle)is)a)doubling)of)the)PCR)product)
  • If)there)is)16)Mmes)more)of)sequence)A)than) sequence)B,)it)would)take)4)more)cycles)for) sequence)B)to)achieve)the)same)pre_set) amount)of)PCR)product)than)sequence)A.)
  • Ct)is)the)#)cycles)to)reach)a)pre_set)level)of) product)(fluorescence))
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16
Q

Normalising)the)data)

A
  • How)do)you)know)you)have)the)same)amount) of)cDNA)in)each)sample?)
  • It)is)difficult)to)quanMfy)your)original)RNA) sample)to)the)level)of)accuracy)required)for) PCR)
  • Is)the)reverse)transcriptase)as)efficient)for) each)sample?)
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17
Q

Normalising)the)data)

A
  • SoluMon:)include)an)internal)standard.)
  • A)sequence)that)doesn’t)change)under)the) condiMons)you)are)treaMng)the)cells)
  • We)are)using)18S,)the)smaller)rRNA)species) which)is)found)in)the)40S)ribosomal)subunit.)
  • It)is)largely)constant)per)cell.)
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18
Q

Normalising)the)data)

A
  • To)normalise)your)data)to)18S)start)by)finding) the)relaMve)abundance)of)your)sequence)of) interest)to)the)18S)for)the)same)sample)at)the) same)diluMon.)
  • Ct)for)the)sequence)of)interest)_)Ct)for)18S)
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19
Q

Processing)the)data)

A
  • Step)1:)average)the)duplicates)(if)they)are) good))
  • Step)2:)check)that)the)Ct)for)the1)in)8)diluMon) is)about)+3)larger)than)the)undiluted)Ct.)
  • Step)3:)normalise)the)data)to)18S)and)average)
  • Step)4:)determine)the)fold)change)) (2^ΔCtC_CtD))))
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20
Q

Primers)

A
  • The)specificity)of)the)reacMon)is)dependent)on) the)choice)of)primers)
  • Are)some)primers)more)efficient)than)others?) • Will)this)affect)the)comparison?)
21
Q

The)Amplicon)

A
  • The)amplicon)is)the)whole)secMon)of)DNA) copied…the)amplified)PCR)product)
  • In)real)Mme)PCR)it)is)quite)short)(80)–)120)bp) …definitely)not)full)length)
  • We)are)not)trying)to)clone)the)product,)just) produce)a)unique)product)to)quanMfy)
  • Check)by)melt)curve)and)gel)
22
Q

What$is$RNA$edi(ng?$

A

• the$altera(on$of$the$base$sequence$of$the$ RNA$transcript$a;er$transcrip(on$
Considera(ons:$
– Is$it$restricted$to$mRNA?$
• Why$would$you$need$to$change$the$base$ sequence?$

23
Q

The$first$examples:$trypanosomes$
• The$inser)on$or$dele)on$of$Us$creates$a$ translatable$mRNA$
• The$first$and$quintessen(al$example$

A

Trypanosoma*(the%italic%comments%in% parentheses%are%mine)$
• Trypanosoma%brucei$is$a$species$of$salivary$ trypanosome$(parasi5c%protozoan)$which$causes$ African$trypanosomiasis,$known$also$as$sleeping$ sickness$in$humans$and$nagana$in$animals.$T.% brucei$has$tradi(onally$been$grouped$into$three$ subspecies:$T.%b.%brucei,%T.%b.%gambiense$and$T.%b.% rhodesiense,$the$first$of$which$is$unable$to$infect$ humans.$However,$rare$cases$of$T.%b.%brucei,$ Animal$African$Trypanosoma,$have$been$ reported$to$infect$a$human.[1]$from$Wikipedia$• Transmission$of$T.%brucei$between$mammal$hosts$is$usually$ by$an$insect$vector,$the$tsetse$fly.$T.%brucei$parasites$ undergo$complex$morphological$changes$as$they$move$ between$insect$and$mammal$over$the$course$of$their$life$ cycle$(similar%to%malaria).$The$mammalian$bloodstream$ forms$are$notable$for$their$variant$surface$glycoprotein$ (VSG)$coats,$which$undergo$remarkable$an(genic$varia(on,$ enabling$persistent$evasion$of$host$adap(ve$immunity$and$ chronic$infec(on.$T.%brucei$is$one$of$only$a$few$pathogens$ that$can$cross$the$blood$brain$barrier.$There$is$an$urgent$ need$for$the$development$of$new$drug$therapies$as$current$ treatments$can$prove$fatal$to$the$pa(ent.$

24
Q

Kinetoplast$$

A

• In%trypanosomes,%a%group%of%flagellated% protozoans,%the%kinetoplast%exists%as%a%dense% granule%of%DNA%within%the%large%mitochondrion.% Trypanosoma%brucei,%the%parasite%which%causes% African%trypanosomiasis%(African%sleeping% sickness),%is%an%example%of%a%trypanosome%with%a% kinetoplast.%Its%kinetoplast%is%easily%visible%in% samples%stained%with%DAPI,%a%fluorescent%DNA% stain,%or%by%the%use%of%fluorescent%in%situ% hybridiza5on%(FISH)%with%BrdU,%a%thymidine% analogue.$

25
Q

Structure$of$the$Kinetoplast$

A

• The%kinetoplast%contains%circular%DNA%in%two%forms,%maxicircles%and% minicircles.%Maxicircles%are%between%20%and%40kb%in%size%and%there% are%a%few%dozen%per%kinetoplast.%There%are%several%thousand% minicircles%per%kinetoplast%and%they%are%between%0.5%and%1kb%in%size.% Maxicircles%encode%the%typical%protein%products%needed%for%the% mitochondria%which%is%encrypted.%Hereinliestheonlyknown* func6onoftheminicircles7producingguideRNA(gRNA)to decodethisencryptedmaxicircleinforma6on,typicallythrough* theinser6onordele6onofuridineresidues.%The%network%of% maxicircles%and%minicircles%are%catenated%to%form%a%planar%network$ that%resembles%chain%mail.%Reproduc5on%of%this%network%then% requires%that%these%rings%be%disconnected%from%the%parental% kinetoplast%and%subsequently%reconnected%in%the%daughter% kinetoplast.%This%unique%mode%of%DNA%replica5on%may%inspire% poten5al%drug%targets.$

26
Q

The$maxicircles$

A
  • There$are$about$50$linked$together$ (catenated)$
  • They$code$for$the$usual$mitochondrial$ suspects;$components$of$electron$transport$ and$oxida(ve$phosphoryla(on$which$are$ embedded$into$the$mitochondrial$membrane$
  • 12$of$the$18$genes$do$not$contain$start$or$stop$ codes.$
27
Q

Why$the$U$inser(ons?$

A
  • These$start$and$stop$codons$(and$open$ reading$frames)$are$created$by$the$inser(on$ (or$dele(on)$of$Uridines$at$discrete$sites$
  • They$also$change$the$sequence$of$the$pep(de$ coded$
  • Start:$AUG,$stop:$UAA,$UAG,$UGA$
28
Q

But$what$of$the$minicircles?$

A

• They$produce$guide$RNA$(gRNA)$
• small$40$–$80$nucleo(de$RNA$molecules$that$ contain$the$sequence$informa(on$required$to$ recruit$the$RNA$edi(ng$machinery$needed$to$ make$the$precise$inser(ons$and$dele(ons.$
You$have$been$introduced$to$guide$RNA$with$ CRISPR.$This$system$endeavours$to$exploit$ the$use$of$gRNA$when$repairing$“indels”$

29
Q

Why$go$to$all$this$trouble?$

A
  • It$has$been$proposed$that$the$trypanosome$will$ at$some$stages$in$its$life$cycle$need$a$func(onal$ mitochondrion$(when%living%in%the%insect%midgut)$ so$it$will$need$CoxII$and$all$the$mito$friends.$
  • When$it$lives$in$the$blood$stream$it$behaves$ metabolically$like$a$red$blood$cell,$deriving$its$ energy$from$glycolysis$only….suppressing$the$ Krebs$cycle$and$ox$phos.$$
  • The$RNA$edi(ng$changes$support$this$theory$
30
Q

The$diversity$of$RNA$edi(ng$within$ Trypanosomes$

A
  • Not$all$mitochondrial$genes$are$edi(ed$and$ even$within$the$ones$that$are,$some$are$ edited$only$at$the$5’$end$while$others$are$ edited$throughout$the$sequence$
  • What$is$edited$in$one$trypanosome$species$is$ not$edited$in$another.$
  • The$restricted$edi(ng$of$some$sequences$may$ have$arisen$by$retrotransposon$events.$
31
Q

The$role$of$the$oligo$U$tail$

A
  • Seems$to$me$to$be$analogous$to$the$oligo$dT$ tail$on$cDNA…binds$to$a$purinecrich$region$ just$upstream$from$the$edi(ng$site$
  • Stabilises$the$gRNA:RNA$hybrid$and$stops$the$ fragments$floa(ng$away$once$the$edi(ng$ begins$
  • Removes$secondary$structure$in$the$mRNA$ making$the$edi(ng$sequence$more$accessible.$
32
Q

C－》U$conversions$

A

• The$quintessen(al$example$is$Apolipoprotein$B$
• The$liver$protein$is$huge,$4563$amino$acid$ residues$which$makes$its$mol.$Wt.$a$staggering$
~450,000.$This$is$known$as$ApoB100$(100%%of%coding% sequence)%
• The$small$intes(ne$ApoB$is$about$half$the$size,$ ApoB48$(48%%of%the%coding%sequence)%
• They$have$different$roles$in$the$acquisi(on$and$ delivery$of$dietary$fat.$

33
Q

Apolipoprotein$B:$ApoB100$

•

A

The$liver$version,$ApoB100,$is$an$integral$ component$of$VLDLs$and$LDLs$(the$very$lipid$ laden$lipoproteins$that$deliver$lipid$from$the$liver$ to$cells$via$the$bloodstream)$and$the$ApoB100$ allows$the$LDL$par(cle$to$dock$at$the$LDL$ receptor$on$cells.$The$cells$can$then$take$up$ cholesterol$from$within$the$LDL$par(cle.$
• The$lipid$carried$on$these$par(cles$is$either$ dietary$in$origin$or$has$been$synthesised$in$the$ liver$

34
Q

Apolipoprotein$B:$ApoB48$

A
  • The$small$intes(ne$version$forms$part$of$the$ chylomicron.$The$chylomicron$is$a$lipoprotein$$ formed$in$the$small$intes(ne.$Its$role$is$to$ carry$dietary$fat$absorbed$in$the$small$ intes(ne$and$deliver$it$to$the$body$via$the$ lympha(c$circulatory$system.$The$chylomicron$ remnants$then$return$to$the$liver$where$they$ are$repackaged$as$VLDLs.$
  • ApoB48$lacks$the$LDLR$binding$domain.$
35
Q

back$to$RNA$edi(ng….$

A
  • This$single$edi(ng$event$in$nucleo(de$6666$ converts$a$C$in$the$glutamate$codon$CAA$to$a$U$ making$the$codon$now$UAA,$thus$crea(ng$a$stop$ codon.$
  • It$is$carried$out$by$an$“editosome”$(we%have%Vomes%or%V somes%for%everything%now)%
  • The$reac(on$is$carried$out$by$a$specific$cy(dine$ deaminase,$APOBECc1$(APOlipoprotein$B$mRNAc Edi(ng$enzyme$Cataly(c$polypep(deF1)$
36
Q

How$is$this$reac(on$so$specific?$

A
  • Only$the$6666th$C$is$edited$which$is$exquisite$ specificity$and$only$in$the$intes(ne$
  • APOBEC$has$a$partner$ACF$(APOBEC$ Complementa(on$Factor)….it$needs$this$ protein$for$transport$to$and$from$the$nucleus.$
  • RNA$edi(ng$occurs$in$the$nucleus,$just$a;er$ transcrip(on.$
  • ACF$drives$APOBEC$to$the$nucleus$in$the$ intes(ne$but$not$the$liver.$
37
Q

Other$roles$for$APOBECs$

A
  • One$of$the$more$interes(ng$roles$of$these$ enzymes$is$to$limit$the$mobility$of$ retroviruses$probably$by$edi)ng$the$genomes$ of$these$viruses.$
  • Trouble$is$the$retroviruses$have$also$evolved$ strategies$to$get$around$this.
38
Q

An$example$from$HIV$

A

• APOBECc3G$has$the$poten(al$to$inhibit$HIV$
infec(ons,$however$$
• In$the$cat$and$mouse$game$of$survival$HIV$has$ evolved$a$protein$(encoded$on$a$very$small$ genome$termed$a$viral$infec(on$factor$or$Vif)$ that$ubiquinates$APOBECc3G$and$consigns$it$ to$destruc(on$by$the$proteosome.$$
• Vifcdeficient$HIV$strains$cannot$infect$T$cells$ and$so$this$has$become$a$possible$target$for$ AIDS$therapy.$

39
Q

Adenosine$deamina(ons:$A$!$I$

A

• The$most$common$A$!$I$conversion$is$carried$
out$on$tRNA$molecules.$$
• The$specific$deaminases$involved:$TadA$in$ bacteria$or$ADATs$in$eukaryotes…Adenosine$ Deaminases$Ac(ng$on$tRNA).$
• Other$A$!$I$conversions$in$animals$are$carried$ out$on$mRNAs$by$ADARs$(Adenosine$ Deaminases$Ac(ng$on$RNA)$

40
Q

Adenosine$deamina(ons:$A->I

A
  • Because$the$conversion$changes$the$ proper(es$of$the$base$and$alters$the$base$ pairing,$a$different$transla(on$product$will$ result.$
  • While$there$are$many$examples$I$thought$I$ would$focus$on$2$neurological$examples;$one$ of$the$brain$glutamate$receptors,$GluRc2$and$ one$of$the$serotonin$receptors,$5cHT2CR$
41
Q

Glutamate$

A
  • Apart$from$being$one$of$the$20$amino$acids$that$ make$up$proteins,$glutamate$is$a$very$abundant$ neurotransminer$in$the$CNS$and$brain….it$is$the$ major$excitatory$neurotransminer$of$the$brain.$
  • It$is$the$precursor$of$GABA,$the$major$inhibitory$ neurotransminer$of$the$human$brain.$
  • The$released$glutamate$crosses$the$synapse$ where$it$binds$to$glutamate$receptors$(GluR)$and$ mediates$the$post$synap(c$signal.$
42
Q

The$GluR2$receptor$

A

A->I edi(ng$of$a$single$base$ in$exon$11$results$in$a$change$ in$the$amino$acid$sequence;$ Gln$(Q)$goes$to$Arg$$(R).$
$ This$significantly$reduces$the$ Ca2+$permeability$of$the$ GluR2$receptor.$
$ If$this$edi(ng$does$not$occur$ epilepsy$results.$

43
Q

Epilepsy$

A
  • Epilepsy$in$all$its$forms$has$one$common$ feature,$neuronal$hyperexcitability…..this$then$ leads$to$seizures.$$
  • This$hyperexcitability$can$result$from$ increased$intracellular$[Ca2+].$$
  • As$edi(ng$the$GluRc2$Q/R$site$reduces$Ca2+$ permeability$it$is$easy$to$explain$why$defec(ve$ edi(ng$of$this$site$causes$epilepsy.$$
44
Q

Serotonin$

A
  • Serotonin,$produced$from$the$amino$acid$ tryptophan$via$a$hydroxyla(on$and$ decarboxyla(on,$is$one$of$those$really$ important$neurotransminers$that$is$ implicated$in$mood,$circadian$rhythms,$ appe(te$and$even$sexual$behavior!$$
  • It$is$abbreviated$to$5$HydroxyTryptamine$(5$ HT)$
  • It$binds$postcsynap(cally$to$a$serotonin$ receptor$
45
Q

The$Serotonin$Receptor$

A
  • There$are$7$classes$of$serotonin$Receptor$or$5c HTR$5cHT1c7R$and$many$subclasses$e.g.$5cHT2CR.$ These$subclasses$are$dis(nguished$by$their$ response$to$agonists$and$antagonists.$$
  • There$are$13$dis(nct$genes$for$the$serotonin$ receptor$and$many$more$variants$of$the$protein.$$
  • Six$of$the$7$classes$of$5cHTR$(classes$1,$2,$4,$5,$6,$ 7)$are$Gccoupled$proteins$(7cTMS)$and$5cHT3R$is$ an$ion$channel.$$
46
Q

The$Serotonin$Receptor$

A

• Only$one$of$the$serotonin$
receptors,$5cHT2CR,$is$edited.$$
• This$edi(ng$is$quite$specific$ and$alters$the$func(onality$of$ the$receptor.$$
• This$receptor$form$was$the$ first$discovered$and$is$highly$ expressed$$throughout$the$ brain.$

47
Q

Edi(ng$in$detail$

A

• There$are$5$sites$for$edi(ng$(A$–$ E)$which$can$produce$a$number$of$ proteins$depending$on$the$extent$ of$edi(ng.$
•T he$more$edi(ng$the$lower$the$
cons(tu(ve$ac(vity$of$the$
GU2$ receptor.$The$fully$edited$form$
(VGV)$has$much$less$potency$than$ the$unedited$(INI)$form.$
•T he$fully$unedited$mRNA$however$ is$more$likely$to$be$spliced$at$GU1,$ giving$a$truncated$noncfunc(onal$ protein$than$the$edited$forms$
which$are$usually$spliced$at$GU2.$ $

48
Q

Edi(ng$Consequences$

A
  • Edi(ng$panerns$of$the$5HT2CR$have$been$ inves(gated$in$many$condi(ons:$ schizophrenia,$depression,$anxiety,$suicide$ vic(ms$and$obesity.$$
  • Suicide$vic(ms$show$markedly$altered$edi(ng$ panerns$in$the$prefrontal$cortex$
  • The$effect$of$edi(ng$on$appe(te$regula(on$ may$prove$the$most$interes(ng$

zBCHM (5 decks)

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