See­ing What Isn't There

Pic­tures Con­sid­ered #6

by Elio (prompted by Christoph's sug­ges­tion)

Map­ping dele­tion mu­ta­tions by ge­netic tools is one thing, see­ing them dis­played along the length of a DNA mol­e­cule is quite an­other. But how can one see what isn't there? In 1968, Ronald Davis and Nor­man David­son did just that—visualizing dele­tion mu­ta­tions in the genome of col­iphage λ. They rea­soned that if the dou­ble-stranded DNA chro­mo­somes of wild type phage and of a dele­tion mu­tant were dis­so­ci­ated and al­lowed to an­neal to­gether, a dele­tion would show up in hy­brid chro­mo­somes as an (unan­nealed) sin­gle-stranded loop flanked by dou­ble-stranded (an­nealed) DNA re­gions. They set to work in the lab and suc­cess­fully con­firmed this idea.

A re­an­nealed DNA mol­e­cule show­ing two sites of dele­tion, "bushes" b₅ and b₂₁₁. Source

Un­der the elec­tron mi­cro­scope, such hy­brid DNA mol­e­cules were seen to be mostly or­di­nary dou­ble-stranded DNA punc­tu­ated by oc­ca­sional vis­i­ble struc­tures they called "bushes." Each bush was formed by the col­lapse of a loop of sin­gle-stranded DNA, thus likely rep­re­sented the lo­ca­tion of a dele­tion mu­ta­tion. Al­though the con­tour length of a bush could only be es­ti­mated, their lo­ca­tion along the DNA mol­e­cule could be ac­cu­rately mea­sured. Sure enough, the po­si­tion of the bushes cor­re­sponded to the lo­ca­tion of the dele­tions as es­ti­mated from re­com­bi­na­tion ex­per­i­ments. The frac­tion of the DNA deleted, as mea­sured in the pho­tographs, was con­sis­tent with the ob­served changes in the buoy­ant den­sity of the wild type and mu­tant phages in ce­sium chlo­ride den­sity gradients—a fur­ther con­fir­ma­tion of their in­ter­pre­ta­tion of the bushes. The au­thors de­vel­oped a bunch of con­trols to en­sure that they were not look­ing at ar­ti­facts. Thus, they could con­clude that ge­netic mea­sure­ments of dis­tances re­flected a phys­i­cal re­al­ity. On a gross scale, this had been known from the po­si­tion of the bands in the gi­ant sali­vary gland chro­mo­somes of Drosophila, but this study es­tab­lished that the gene chro­mo­some map cor­re­sponded to a phys­i­cal chro­mo­some on a mol­e­c­u­lar level.

In case you won­der why the au­thors didn't sim­ply se­quence the mol­e­cules, well, prac­ti­cal DNA se­quenc­ing didn't be­come avail­able for an­other five years or so. Once se­quenc­ing be­came a rou­tine pro­ce­dure, it ob­vi­ated the ne­ces­sity to come up with clever ap­proaches to many ba­sic ques­tions. Ah, well...

The pa­per ends with the fol­low­ing state­ment: Af­ter com­plet­ing this man­u­script we have learned that the tech­nique de­scribed herein has been in­de­pen­dently con­ceived and de­vel­oped by West­more­land, Szy­bal­ski, and Ris, who have ob­tained re­sults which are gen­er­ally sim­i­lar to ours. It hap­pened even then!

 

Ref­er­ence

Davis RW, David­son N (1968). Elec­tron-mi­cro­scopic vi­su­al­iza­tion of dele­tion mu­ta­tions. Proc Natl Acad Sci USA, 60 (1), 243−250. PMID 5241526