A Dip in the Gene Pool C'mon in! It's complementary! tN Today's Episode... Col in the Act This I'il guy contains about 3.2 picograms of DNA. That's its C-value. This is enough DNA to stretch 1 meter and is sufficient for about six million genes!! But by other estimates, there are only 50,000 - 100,000 genes in the human genomelll!!! So what's all that extra DNA for???? How's it organized??? That's the C- VALUE PARADOX CsCl density gradients tell Us that there ‘ore multiple types of DNA Denatured DNA Under conditions that permit reannealing, satellite DNA renatures more rapidly in the cell based on GC content is more dense than double-stranded DNA than mainband DNA \ 7 1) Native DNA Denatured DNA Renatured DNA 5 T GC is more dense Let's than AT Double-stranded DNA ||Q has an open crystaline || U 716606 structure. It is less a 0.00098 (%46¢) dense than single- t Cre stranded DNA! i Move it ouch t a Crea a 7 t Yer stp sn eReannealment is a function of how quickly homologous But by waiting long enough (time) or strands can find each other. Y 9 long enough (time) 8 A by increasing the initial DNA Tv $2 concentration (Co)... Be Gof Fett Fg “Ge A a K Just old me, O87 verses Je ¢C 1 T T ‘ a* A There's no one |= else ike you, Babe! The more repetetive a sequence i the mmore rapidly will it find a partner! even unique sequences will reanneal! This is a CoT curve for a simple genome (E. coli). ‘ As concentration and time increase, ‘ wo. SS DNA begins to reanneal. cot is, ' ‘At a high enough Cot value, plotte 1 virtually all DNA reanneals! ona log t me Mere What we want is the point scale! Yh 201 7 cat which 50% of the DNA (the grap! has reannealed "e single-stranded DNA g sp tema Triagy oT STE STEED : CoT} is the CoT value at which 50% of the DNA ‘ has reannealed! For E. coli, CoT} = 9*. That's oo its complexity! ee a ot a bu ty a “*CoT},_is dependent upon the expermental conditions, which may differ from lab to lab! Within ¢ oT (concentration x time) fas cana Deemer ener tne at ese raat oT} decreases with decreasing genome size! Relative to. coli, the other DNA's are more repetetive. 100% 4 polyA/T ms2 v4 E. coli 3x10" 9x10° & 10* 10° 10° 107 10 10! 10° CoT (concentration x time) A 5 differen DNA's ———————B A=2xB CC azaxc A=10xD A=1000xE “The CoT} is a mecsure of complexity Complexity is the number of base pairs per repeating Unit E. cols genome contains only 1 unique Sequence Tr hes only repeating unl Werisbeepars eg Thus in terms of total genomes, 1 Ib of DNA E is 1,000 x more repetetive than 1 Ib of DNA Al ths egret, Do fre thet hs umber an he wth ‘ipermentl eatin Tre The vake relate To ther DAA eatIf we use E. coli as Putting it all together, we get. an internal control Cott Complexity in a Cot experiment, E.coli 9x10 42x 10° then... T4 3x10" 14x10" Cot} X Complexity X MS2. 8x10° = 3.7.x 10° CT EEE Polya/T 2x10° 9.310" Cool! S now, what ; about eukaryotes? a A Hypothetical Eukaryotic CoT Curve From the density work, we know that there are C-value = 7 x 10° base pairs Youll! That's 4 at least two , different classes sox Complex : of DNA! > So, let's take a look ata \ eukaryotic on y CoT curve! 10) aie 1G tte 0), eee10 ane 10 e100, Cee 10 MeO, ° OK! com- Ok! Let's get a gripl! 100% ponent Lis wheeze obviously I'm good enough! I'm highly itivel smart enough! I know pean “We can find the mid-point of the dip and determine its CoT# value I can do this! a First, we know i “The upper flat part of the curve means none “ that there 1 of the highly repetetive DNA hos reanrealed we ' ( ( are three 1 “When the curve flattens after the first dp ofthe highly repetitive DNA must have classes of 1 Feamedled DNA 1 1 1 BOL we can multiply the CoT 3 Since we know the % at midpoint by % to get the of DNA in each component. Lo adjusted CoT? values. OK! We get the CoT} at the midpoint of each dip! DNA _ Character CoTiof Midpoint % Total Adjusted Cot Complexity 1 Highly Repetitive 1.3 x 10° 25% 3.25x104 340 2 Middle Repetitive 19 30% 0.57 6x 108 3 Unique 6.3 x 10? 45% 283.5 3 x 108 Cot$X__ Complexity X Wi onveah rete: inthis experiment, CoT} of E.coi= 4 GthEcl * 42x10Whoa! Reality Check herell Do those numbers actually mean anything? EFSE3 \ Well, if the unique sequences are 3 x 108 bp long and represent 45% of the DNA, then the entire genome can be estimated by the equation 3 x 10° / 0.45 = 6.6 x 10° Hey!! the C-value (bone-head chemical calculation is 7 x 10°! Not baaadlll! ..and if we take 7 x 10° and multiply it by 0.45, we get 3.15 x 10°! Therefore, the bone-head chemical method and the sophisticated kinetic method totally agree! Cool! You should be able to do the same thing for 1&2 and also 1 approximate the 2 C-val...covops! u 3 DNA Complexity _ =% 340 1360 6x10° 2x 10° 3x10° 6.6 x 10° *C-Value = 7 x 10° Oh Yeah! I Forgot! land 2 are repetitive! We calculated the number of bases per repeating unit - not the total number of bases in that component! If we know the length of the repeat and the total amount = pna Chemical complexity Kinetic Number of Repeats DNA C-Value (C-Value x %) Complexity _(Chemical/Kinetic) of DNA in each 1 7x10° 175x108 340 500,000 component, we can Siento 2.1.x 10° 6x 10° 350 figure out how many 3. 7x10 3.15 x10® = 3x 10° 1 times each sequence repeats itself! cnn 78 a Beeman errs Perens eens ‘sequences used for? How are they 1 seer, 3151308 tp ang 1210 op ete 0 oes if So, there you have it! An overview of the entire genome! Pretty cool, huh? 40 p repeated 800000 tines organized? Don't 1 Ask! Pleasel Not Now!