Requiem for an Apparatus

by Roberto
 

Fig.1. Autoradiograph of a two-dimensional fractionation (cellulose acetate pH 3.5, 3000 volts, 2 hr. DEAE paper pH 1.9, 1500 volts, 4 hr) of a ribonuclease T1 digest of 16S ribosomal RNA. Source

Have you ever seen the type of apparatus that Carl Woese and colleagues used to obtain data to support their conclusion that Bacteria and Archaea were so distant phylogenetically as to merit being different domains of life? They compared RNA sequences, but how were the sequences obtained? The experimental approach, now defunct, is worth revisiting.

 

I was inspired to write this post after re-reading Elio's 2012 post "Requiem for a Machine" where he describes the by-then-idle Spinco Model E ultracentrifuge. The Model E, "the pride of research departments," the machine used by Meselson and Stahl to demonstrate semiconservative DNA replication, had been largely superseded by diverse electrophoresis apparatus by 1970. When I arrived at UCSD in 1975, the once-revered Model E lay gathering dust, abandoned in a basement hallway. Who needed to determine the sedimentation coefficient of a macromolecule when you could sequence it? Well, almost sequence it. Protein sequencing, yes. DNA sequencing, not yet developed. RNA sequencing? Yes... but just barely. Being quite keen on the evolving methodology of RNA sequencing, I did a rotation in John Abelson's lab where I got a chance to learn how.

 

I'll spare you much of the detail of how we obtained the RNAs to be sequenced. Suffice it to say that if the RNA was made in vivo, cells had to be labeled with radioactive phosphorus (³²PO₄). If the RNA was made in vitro, the starting materials included radioactive nucleotide triphosphates. And yes, I did spend hours in the cold room purifying E. coli RNA polymerase. With radioactive RNA in hand, we proceeded with partial or complete enzymatic digests of the RNA with diverse ribonucleases. This we followed with separation and characterization of the resulting oligonucleotide fragments by two-dimensional paper electrophoresis. That was just the beginning. Each of the oligonucleotide spots had to be eluted from the paper and then subjected to further enzymatic digests and further two-dimensional paper electrophoresis. In the end, you would have a catalog of oligonucleotides and the trick was to get enough information to put together the puzzle of the entire sequence. RNA sequence was a gargantuan undertaking. Getting the sequence of a tRNA could signify a PhD thesis. The sequence of the entire 16S ribosomal RNA? Not a chance, at best a long catalog of oligonucleotides. And yet, from such catalogs, Woese and co-workers were able to establish a universal phylogeny that completely changed our view of the natural history of life on Earth.

 

Fig.2. Plexiglass tank (65 x 50 x 15 cm; about 50-liter volume) for two-dimensional paper electrophoresis. Electrodes connected to a power supply are immersed in buffer (clear). The entire paper is immersed in Varsol (stippled) which is cooled by tap water flowing through steel coils. The plexiglass rack used to support the paper is illustrated to the right. Source

Let me describe the apparatus I got to use in Abelson's lab to carry out such electrophoresis, very much what all RNA sequencers of those days used. By today's standards, it was definitely crazy. We applied the radioactive sample to long strips of cellulose acetate for the first dimension. Once separated, the fragments were separated onto very large sheets of DEAE paper for the second dimension. To fit the large pieces of paper, the plexiglass electrophoresis tanks were quite large, holding upwards of 50 liters. Buffers made up about half that volume. The other half? What was needed was a water-immiscible solvent that would not conduct electricity so that all the current would go through the paper, moving the RNA fragments along. The solution: many gallons of volatile and highly flammable organic solvents, varsol or mineral spirits for example. The stuff you might douse charcoal with to jump-start your grill. We immersed the strips or sheets of paper, loaded with radioactive RNA fragments, through the solvent so that their ends would reach the aqueous buffer. We then subjected them to kilovolts of electricity, generating enough heat that the solvent had to be cooled to prevent it from reaching its flash point. This was an apparatus that required the utmost respect and great care in its use. It's no wonder such things are no longer a common sight in labs. The moment easier and safer sequencing techniques became available, two-dimensional paper electrophoresis units went by the wayside. Yet, despite their obvious hazards, they were key for obtaining one of the most remarkable results in molecular biology of last century: Woese's universal phylogeny.

 

What is the message here? Experimental approaches come and go. It's the nature of the scientific enterprise. Whatever is your favorite instrument today, don't get too attached. It may be mothballed in the not-so-distant future. But also, somewhere in your mind, keep an archive of archaic methods that might be worthwhile resurrecting someday, hopefully in safer versions than in the past.

 

 

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