What Grains Tell...

Pic­tures Con­sid­ered #23

Note. Trans­fer­ring our more than 2,000 posts from one plat­form to an­other, Word­Press, is a cum­ber­some task. Thus, many of our posts still con­tain bro­ken links. We will fix this one by one. We ask for your pa­tience.

by Christoph

In #2 X of our 'Pic­tures Con­sid­ered' se­ries Elio had con­si­der­ed the iconic au­tora­di­ograph by John Cairns show­ing an in­tact cir­cu­lar Es­cherichia coli chro­mo­some. We had a brief dis­cussion in the com­ments to that post whether Cairns' au­tora­di­ograph proves – in ad­di­tion to cir­cu­lar­ity – bidi­rec­tional repli­ca­tion of the E. coli chro­mo­some and con­cluded: No, it doesn't. In an ad­den­dum, Elio gave credit to the work that ac­tu­ally proved bidi­rec­tional repli­ca­tion and showed a pic­ture. Well, that pic­ture was not the one I had in mind, and it took blow­ing lay­ers of dust from piles of very old pa­pers to fi­nally get hold of it *). Here we are: #23 (Fig­ure 1).

Fig­ure 1. Au­tora­di­ograph of a chro­mo­some of E. coli la­beled with thymine-³H (5 Ci/mmole) for sev­eral min­utes, be­gin­ning at the time of ini­ti­a­tion of the DNA repli­ca­tion cy­cle, and then for 8 min­utes with thy­mi­dine-³H (50 Ci/mmole). The ori­gin of chro­mo­some repli­ca­tion is lo­cated in the re­gion of the light, cen­tral au­tora­di­ographic im­age pro­duced by the thymine-³H. The denser, long im­ages at both ends were pro­duced by denser ra­dioac­tiv­ity of thymi­dine-³H added there­after. Repli­ca­tion forks are lo­cated at the two ends of the au­tora­di­ograph. The two daugh­ter du­plexes are dis­cernible, the un­la­beled parental DNA pre­sum­ably ex­tends from the two repli­ca­tion forks. Ex­po­sure time:6 weeks. Bar: 60 µm. Source

Prescott & Kuem­pel give a de­tailed de­scrip­tion of their method which bears the typ­i­cal smell of ex­pert work in the "wet lab". Next to the la­bel­ing pro­ce­dure they de­scribe the cru­cial step: "Spread­ing (of DNA) is done on mi­cro­scope slides coated with a thin layer of gelatin ap­plied as a sub­bing so­lu­tion. DNA re­leased from cells with N‑lauroylsarcosine ad­heres much bet­ter to subbed slides than to clean glass. A drop (ap­prox­i­mately 5 µl) of cells is placed at one end of the subbed slide. If bac­te­ria are be­ing used, the cell wall must first be di­gested with lysozyme. An equiv­a­lent amount of lysing so­lu­tion is then placed ad­ja­cent to the drop of cells. We have achieved the best ly­sis and spread­ing us­ing a 2% so­lu­tion of N‑lauroylsarcosine con­tain­ing 0.01 M EDTA and buffered at pH 8.1 with 0.01 M Tris. The two drops are al­lowed to fuse, and the mix­ture is left undis­turbed for 10 ‒15 min­utes at room tem­per­a­ture to per­mit ly­sis of cells or spher­o­plasts and re­lease of high mol­e­c­u­lar weight DNA. The cell lysate is then spread over the mi­cro­scope slide with a sec­ond mi­cro­scope slide in the same man­ner that a blood smear is made. This is ac­com­plished by hold­ing the sec­ond or "spread­ing" slide at an an­gle of ap­prox­i­mately 15° to the subbed slide and slowly draw­ing the "spread­ing" slide up to the lysate un­til the lysate uni­formly fills the acute an­gle be­tween the two slides. The "spread­ing" slide is then slowly pushed in the op­po­site di­rec­tion down the length of the slide, spread­ing the lysate be­hind it. The slides should then stand up­right un­til dry." De­spite their use of thymine/thymidine at ex­tremely high spe­cific ra­dioac­tiv­ity (³H at 50 Ci/mmole!) ex­po­sure of the slides for autoradio­graphy took up to ten weeks – which they con­sid­ered 'rea­son­able'. And while fol­low­ing the lines of grains on an au­tora­di­ograph un­der the micro­scope was rou­tine, ac­tu­ally find­ing the ~1% of chro­mo­somes dis­played in an in­ter­pretable fash­ion was te­dious. The en­tire pro­ce­dure was in a way the fore­run­ner of today's sin­gle-mol­e­cule ex­per­i­ments.

Fig­ure 2. The Prescott&Kuempel au­tora­di­ograph from Fig­ure 1. (top) and a schematic rep­re­sen­ta­tion of ex­pected la­bel dis­tri­b­u­tion for bidi­rec­tional vs. uni­di­rec­tional replica­tion. By the au­thor

The clever idea of Prescott & Kuem­pel for show­ing bidi­rec­tional repli­ca­tion was to ex­pose their cells to two pulses of la­bel­ing with dif­fer­ent ra­dioac­tive in­ten­si­ties: the first pulse with lower ra­dioac­tiv­ity at the (approxima­te) time point of ini­ti­a­tion of repli­ca­tion, the sec­ond, a stronger one, shortly af­ter. They rea­soned that this should la­bel the repli­ca­tion ori­gin weakly and – much more in­tensely – the repli­ca­tion fork(s), as shown schemat­i­cally in Fig­ure 2. With­out any doubt, Fig­ure 1 demon­strates that repli­ca­tion pro­ceeds in a bidi­rec­tional fash­ion from the chro­mo­so­mal repli­ca­tion ori­gin of E. coli.

As in­for­ma­tive – and es­thet­i­cally pleas­ing – this autoradio­graph is, it was an ex­per­i­men­tal 'dead end' with re­spect to map­ping the ex­act lo­ca­tion of the repli­ca­tion ori­gin on the E. coli chromoso­me. This was achieved later by ge­netic map­ping and cloning of minichro­mo­somes – se­lec­table plas­mids that de­pend on a cloned repli­ca­tion ori­gin for main­te­nance – in a fierce com­pe­ti­tion be­tween the labs of Yuki­nori Hi­rota and Wal­ter Messer, which cul­mi­nated in their si­mul­ta­ne­ous pre­sen­ta­tion of the com­plete oriC se­quence of E. coli  at the XLIII Cold Spring Har­bor Meet­ing in 1978.

*) This is, ad­mit­tedly, the In­di­ana-Jones-ver­sion. Re­al­ity was more pro­fane: it only took some googling to find the PDF on the jour­nal home­page. How­ever, it il­lus­trates the poli­cies of the pub­lish­ing com­pany that they charged $ 38.12 (incl. tax) for the 1‑time down­load of a 40 year-old pa­per.