11—
Iowa State—
At Full Speed

It was a rare privilege. Robert Oppenheimer and I were having lunch. He had come to Ames to deliver the first Frank Carlson Memorial Lecture, to honor his former student. We spoke of many things: the Institute for Advanced Study, which he now headed; the state of theoretical physics; the international situation; his long-standing interest in Hindu philosophy. He was curious as to the state of biophysics. I avoided mention of his recent "trial" at the AEC and his loss of security clearance—in my view a gross miscarriage of justice. He seemed to bear no rancor and his general mood was philosophical, detached, with an eye to the long view of events. I was captivated by his use of language—eloquent yet precise, almost poetically phrased.

I did not fully comprehend his subsequent lecture, which concerned calculation of the self-energy of the electron and discussed more recent refinements of work that Frank Carlson had initiated as Oppenheimer's student. But the luncheon discussion was a glimpse of true and wide-ranging brilliance.

After Caltech, my return to Ames was bittersweet—a homecoming, yet a letdown. Almost immediately, I went East to the Cold Spring Harbor Conference, at which the double helix structure for DNA was formally presented by Watson and Crick. After Caltech and Cold Spring Harbor, Ames seemed quiet—and, now, isolated.

As quickly as possible, I established my phage research. The bacterial viruses provided enticing access to a wealth of important biological problems. With them, genetic structure and function, gene replication

and mutation were all open to investigation with the several techniques we had been developing. These techniques were not perfect; they were limited and often tedious, but they would take us a long way. The full fruition of this was to come later with the research into the small bacteriophage f X174, but for the present I undertook to analyze the nucleic acids of the well-known T-series of phages.

There was a quiet moment of triumph when I observed my first phage plaques in Ames. My facilities were not comparable, but I had transplanted the phage technology from Pasadena to Ames.

To our astonishment, when we isolated the DNA from the T2 and T4 bacteriophages, we found that it could not be quantitatively digested to mononucleotides by the methods that had been successful with all other DNAs studied. It had been known that these particular viral DNAs had an unusual pyrimidine, 5-hydroxymethylcytosine, which replaced the usual cytosine. However, the resistance to the enzymatic digestion proved to be due to the presence of yet another, unsuspected, modification, a previously unknown pyrimidine. This was a glucose-substituted 5-hydroxymethylcytosine, which inhibited the enzymes. The extent of glucose substitution was 60 percent in T2 and 100 percent in T4; analysis of the progeny of phage crosses from mixed infections with T2 and T4 suggested interesting genetic interactions.

Many laboratories were working with T2 and T4 and these curious modifications of the viral DNAs spawned a variety of research projects, but I was soon diverted in other directions. As the research with f X progressed, it became more interesting and attracted an ever-increasing share of my attention.

After my experience at Caltech, I realized the importance of being informed of the most recent work in the field, which was now beginning to advance much more rapidly. Most of the research would of course ultimately appear in publications, but only after a delay of six months to a year. I had been a regular attendant at the summer Gordon Conferences; I now joined the American Society of Biological Chemists to be able to participate in its meetings, but these also were annual events. I maintained contact with colleagues I had met at Caltech. Also, by now some of our work had been published. This resulted in invitations to lecture at various universities and these visits broadened my network.

Through these activities, I became aware of the growing availability of support from the National Institutes of Health and the National Science Foundation. This possibility met a critical need. Having dem-

onstrated that the RNA of the tobacco mosaic virus could be isolated as an intact molecule, and accepting the new dogma that nucleic acids were invariably the genetic material, I was led to the concept that the RNA of the virus might by itself be sufficient to induce infection, without any involvement of the protein component. Indeed, we were able to demonstrate infectivity with purified RNA preparations. However, per molecule, the RNA was about 1/1000 as infective as the intact virus. By chemical means, we could not exclude the possibility of residual protein equivalent to a 1 percent intact virus contamination of our RNA preparation. We needed a means to purify the RNA more completely.

This could be done in an ultracentrifuge, which could separate molecules by size. But we had none; indeed, there were none in the state of Iowa, and since they cost twenty-five thousand dollars, the campus could not provide one for us. I therefore applied to the NSF for an equipment grant, and in due time received the funds and installed the first ultracentrifuge in Iowa.

Then, just as we set out to use this instrument to provide data to verify our concept that the RNA itself was infective, a paper from the Virus Laboratory at Berkeley announced their results, indicating the infectivity of TMV RNA. Competition in the field, and from better-equipped laboratories, was clearly becoming more intense. We were not entirely satisfied with the quality of the evidence in the published paper and undertook a series of experiments that definitively correlated the infectivity in the RNA preparation with the sedimentation of the RNA in the ultracentrifuge. Nice, even elegant, work but in effect a gloss.

There had been only the most rudimentary studies of X-ray diffraction patterns from RNA. Having in hand this large, homogenous, biologically active RNA led us to undertake such studies. Of course, we hoped to find another striking regular structure such as the double helix. Unfortunately, as we now know, most RNA does not assume such structures. While we obtained some valuable information about nucleotide and internucleoude dimensions in the RNA, no dramatic structure resulted.

Because of the progress of our work and the gradual change in its emphasis toward more functional aspects, I began to attract graduate students from the biochemistry program, which was formally located within the Department of Chemistry. To facilitate this interaction, I was given a joint appointment with the Department of Chemistry. Having this dual affiliation had some advantage but, as I soon found out,

also involved me in twice as many committee meetings and departmental disputes. But I enjoyed the personal contacts and could work within another domain to seek to increase the number of faculty doing work on the problems of what was coming to be called "molecular biology." I needed more colleagues, both for direct interaction and for their networks of professional acquaintance.

In the years 1953 to 1960, Max Perutz and John Kendrew develop the isomorphous replacement method of X-ray diffraction analysis and apply it to obtain the first detailed three-dimensional structures of the proteins myoglobin and hemoglobin. These structures incorporated the earlier models of Pauling and Corey but also provided specific details, down to atomic dimensions, essential to our understanding of the functions of the proteins.

As the genetic material, DNA must be accurately reproduced at each cell division. This reproduction is accomplished by a set of enzymes, DNA polymerases, which therefore play an essential role in heredity. In 1956 Arthur Komberg isolates the first DNA polymerase, making it possible to study DNA synthesis outside a cell.

The hereditary information of DNA is contained in the sequence of its nucleotide subunits. This information is used in the cell to specify the sequences of amino acids in proteins. Translation from nucleotide sequence to amino acid sequence is accomplished in a complex chain of reactions, in which a key role is played by "transfer RNA" molecules. These molecules, with the help of enzymes, recognize both a nucleotide sequence and its cognate specific amino acid. Mahlon Hoagland and Paul Zamencik first describe transfer RNA in 1957.