DISCOVERY OF PRIONS

The early investigations of prions were baffling. There had long been known a neurological disease in sheep called "scapies" because the crazed sheep would scrape themselves upon sharp rocks and barbed wire fences to the point that they would scratch their skin off. The histological specimens of the brains were highly abnormal, and it was soon discovered from using preserved brains that, while all other known organisms and viruses were killed by 1% formalin (1% formaldehyde gas in water), the infectious agents in these sheep brains were totally resistant to 10% formalin. Furthermore there was no evidence that any DNA or RNA existed in the infectious fractionations of these samples! Was this a case of something that could reverse-translate and perhaps also reverse transcribe? Where were the genes for this infectious agent? Thus matters stood for decades!

New technology - primarily Laemmli's invention of polyacrylamide gel electrophoresis (PAGE) - allowed closer inspection of a spectrum of a huge number of proteins in samples. In the case of these mysterious diseases we now call prion diseases, when normal samples were compared with those from individuals suffering from a prion disease, a new band showed up, as indicated here:

Soon someone got the idea to use a new technique called ELISA (Enzyme-Linked ImmunoSorbant Assay) to study these gels. Antibody was made against the new band and then an enzyme was covalently bonded to the highly specific antibody molecule.

Thus wherever the antibody attached to its antigen (a prion protein), the enzyme would be and would reveal itself if the proper colored reagents were flooded over the spectrum of bands of the electrophoretically separated proteins. Because it is better to do ELISA on very thin things, and gels are not all that thin, the bands had to be lifted out of the gel. Another brilliant technician suggested that were one to lay an electrophoresed gel on top of a nitrocellulose sheet (NC tenaciously adsorbs many macromolecules including proteins and ssDNA), and then somehow blot the bands from the gel onto the NC sheet, ELISA could be performed on the very thin sheet. This brilliant technician suggested that since an electrical field had moved the proteins down the gel, another field could likewise move the proteins out of the face of the gel and onto the NC sheet. In the next diagram, the anionic bands in the overlying gel are being subjected to a strong field that will move the proteins out the back of the gel and onto the NC sheet.

Then the NC sheet was sequentially treated through the several steps necessary for doing ELISA. When this was finished, a big surprise was revealed:

Not only did the new and suspected prion bands react with the ELISA reagents, but so did another band - AND in even the normal individual! Thus the two proteins in the diseased subjects shared sufficient antibody reaction to show "cross-reaction." This meant that those two proteins shared a lot of common amino-acid sequence. And it meant that since the normal individual had that sequence, it meant that the genetic material for this disease was in the normal animal. Are we all carrying the capacity for this disease? Frightening!

When the normal protein and the disease agent were isolated and submitted for molecular weight analysis using sucrose gradient centrifugation, these frustrating results occurred:

So let's take a closer look at this in a timed reaction. We want to do a "kinetics" study.

Here we see in the 0-time centrifuge tube only the normal because there is so little Mad Mink factor added that it cannot be seen. But as time goes on, the normal factor disappears and Mad Mink factor accrues. It is as if the normal was being converted to the disease agent! Here is a figure

We see that because the normal protein had a higher S-value than that of the disease factor, the normal factor must be more compact. It turns out that the normal protein consists of two parts that align themselves rather compactly. However, due to some rare event (cosmic ray?), the alignment can go askew destroying whatever critical function the origin protein possessed (growth factor?). What is more is that the misaligned protein can serve as a catalyst causing normal ones to misalign. Thus, slowly all the body's normal ones are converted to inertness, and there are no normal ones left to due their critical function.

So you see that the genes for this "disease" are in us all to begin with!

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