The paradigm shifting (one gene, one protein) experiment of Hozumi and Tonegawa (1976)

These 2 people, hozumi and tonegawa asi!all" de!ided to stud" the light !hain
The"re idea was that 2 genes differed in a !ell, the !ell produ!ing antiod" and all the other !ells
not produ!ing antiod"
#o the" de!ided to !onstru!t the dna from 2 different sour!es and used 2 more lines of !ell
antiodies
$asi!all" 2 more plasma !ell lines so the" would get a lot of dna
%nd the" also used emr"oni! li&er !ells
The" were tr"ing to demonstrate that something is going on in !ells is that pie!es of dna a!tuall"
!hange position
That was a totall" re&olutionar" idea e!ause it implies the di&ison and !utting of dna in somati! !ells
The" !ut the dna with restri!tion enz"mes the" will !ut the dna in !ertain sizes and "ou !an predi!t
the size of those fragments
The" go on and then the" a!!ept the dna, run the dna in ele!trophoresis and laeled the dna from the
2 different sour!es with radioa!ti&e !ompounds, one whi!h was for the ' and ( regions
The" !ut the dna and dis!o&ered that when the" used the ! region in the germline, the" see the
radioa!ti&it" in this position
)hen the" see the ! region in the antiod" produ!ing !ell dna the position has !hanged
That means the gene in this fragment is in a different fragment
Then the" too* the proe for the whole antiod" mole!ule and the" see the radioa!ti&it" is split into 2
fragments
)hen the" proe the dna !oming from the !ell, the" see the radioa!ti&it" is together
That+s the a!tual demonstration that 2 pie!es of dna were in 2 separate fragments ut then !am
together in that !ell during that produ!tion of antiod"
The" sol&ed the ,uestion-
The genome has no inta!t genes for .g we ha&e gene segments whi!h are separated
The ( and ' regions are in separate fragments and are put together during rearrangement pro!ess that
happens during !ell de&elopment
'on!lusions from Tonegawa+s wor*
/enome has no inta!t 0 light !hain gene
( and ' regions en!oded " different exons
/ermline 12% in $ !ells rearranges in ./0 lo!us to ring ' and ( exons !loser together
The" were stud"ing the light !hain, ut then the hea&" !hain was also studied
The" dis!o&ered the same was happening in all .gs
This pro!ess onl" happens in $ and T !ells, not in another !ell in our od"
There was paradigm shifting experiment e!ause it allowed to thin* 1 protein as eing made "
different genes that were a!tuall" shuffling to !reate
)e ha&e part of genes that are randoml" pi!*ed to e line*d together and !reate the enormous
dio&ersit" of our antiod" repoitoire
This pro!ess is li*e shuffling !ards and produ!ing something different out of it
3&iousl", the pro!ess is tightl" regulated
The ( region is !omposed of two different gene segments (( and 4), ( en!odes for most of the (
domain and the 4 segment is 5ust the end of the ( domain
That+s for the light !hain, the hea&" !hain has a additional gene segment
.n the hea&" !hain, we find more than 1 ( segment whi!h en!odes for the first 166 amino a!ids then
we ha&e a 1 segment and a 4 segment
The 4 segment is ,uite small and en!ode 5ust a few aa whi!h are part of '178, whi!h is the most
h"per&ariale region in the antigen inding site