Gierer & Schramm's FIRST Experiment with TMV

    In 1957 these two researchers published a disturbing article that contradicted that DNA was the genetic material. Their work progressed through various steps. The first was inventing a procedure for the isolation of nucleic acids. Even at the opening of the 21st Century their method is in large part adhered to. It is based on treating disassembled cellular or viral "soups" with phenol (carbolic acid; the same stuff that Lister had used a century earlier to sterilize patients and operating rooms). To the "soup" is added an equal volume of buffered phenol and this is shaken violently for a few seconds to form a colloidal solution before being subjected to centrifugation. The semi-miscible phenol then separates to the bottom with an overlying aqueous phase. At the interface is a great deal of denatured glop - proteins, lipids, etc., but in the aqueous phase are the organism's small molecules AND nucleic acids. These could be separated from each other by virtue that macromolecules such as DNA and RNA precipitate with either 70% ethanol or trichloroacetic acid (TCA)

    Secondly, they turned their attention to an agricultural disease called tobacco mosaic virus (TMV). When the plant - ether tobacco or tomato - is infected, the leaves get brown spots on them, and the centers of the spot deteriorate to the point of leaving only a weblike patchwork of veins. By simply scraping browning areas, the two came up with suspensions of virions (whole viruses). To prove their were the real thing, they swabbed these onto leaves and brown spots soon appeared. Their scrapings were indeed infectious - the real thing!

    When they subjected their viral preparation to their phenol-extraction method, they, of course, obtained an aqueous phase and found that ethanol caused a flocculant precipitate to form. When filtered out and redissolved in buffer, these particular viral macromolecules were readily degraded by RNase and not by DNase - a very powerful indication that TMV possessed no DNA, but instead used RNA as its genetic material.

    An associated lab corroborated this finding by growing the virus on leaves that were perfused with either C-14-uradine or H-3-thymidine. This other lab group found that the thymidine did NOT become part of the new TMV particles, but that new virions became very radioactive with C-14, indicating that uracil - hence RNA - was central in the viral particles.

    Thus, importantly, TMV's genetics was confirmed as based on RNA from two different directions - always nice to do in science!

    Meanwhile back in Gierer and Schrammland, they found a method for more delicately separating the viruses into their protein and RNA components so that in one test tube they had TMV-protein, and in a second test tube they had TMV-RNA. These were swabbed onto leaves both separately and combined.

    Now comes one of the amazing manipulations in all of biology. Look at the three leaves here and what was applied to them.

What TWO meanings are shouting back at you?-!