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J. Biol. Chem., Vol. 281, Issue 7, 7, February 17, 2006
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Classics
Purification of the Alanine-, Valine-, Histidine-, and Tyrosine-acceptor Ribonucleic Acids from Yeast
(Apgar, J., Holley, R. W., and Merrill, S. H. (1962) J. Biol. Chem. 237, 796–802)
Nucleotide Sequences in the Yeast Alanine Transfer Ribonucleic Acid
(Holley, R. W., Everett, G. A., Madison, J. T., and Zamir, A. (1965) J. Biol. Chem. 240, 2122–2128)
Robert William Holley (1922–1993) was born in Urbana, Illinois. In 1938, he enrolled at the University of Illinois where he majored in chemistry and received his B.A. in 1942. He then took up graduate studies with Alfred T. Blomquist at Cornell University and was awarded a Ph.D. in organic chemistry in 1947. He would have finished sooner, but his research was interrupted by the war during which he spent 2 years (1944–1946) with Vincent du Vigneaud at Cornell University Medical College, assisting in the first chemical synthesis of penicillin. Some of du Vigneaud's research was featured as a Journal of Biological Chemistry (JBC) Classic (1).
After completing his graduate work, Holley did a year of postdoctoral work at Washington State College (now University) with Carl M. Stevens. He then went to Cornell University as Assistant Professor of Organic Chemistry at the Geneva Experiment Station. In 1948, he became assistant professor at the New York State Agricultural Experiment Station, a branch of Cornell, in Geneva. He was promoted to associate professor in 1950, full professor in 1964, and was chairman of the department in 1965 and 1966.
In 1955, Holley went to the California Institute of Technology on a Guggenheim Memorial Fellowship to work with James Bonner, who was the author of a previous JBC Classic (2). There he started to investigate protein synthesis and the chemistry of nucleic acids. Toward the end of his year away from Cornell, Holley began to look specifically at the structure of transfer RNA (tRNA). Back at Cornell he set out to isolate an individual tRNA for chemical study. This is the subject of the two JBC Classics reprinted here.
Holley's first problem was to find a fractionation technique that could be used on tRNA. He settled on Lyman Creighton Craig's countercurrent distribution technique, which was discussed in a previous JBC Classic (3). In collaboration with Jean Apgar and Susan H. Merrill, Holley adapted the countercurrent distribution procedure into an applicable method for the fractionation of tRNA. This method is presented in the first JBC Classic. Using the modified countercurrent distribution technique, Holley, Apgar, and Merrill purified alanine, valine, histidine, and tyrosine tRNA.
Using purified alanine tRNA, Holley and his colleagues set out to determine its sequence. However, the amount of alanine tRNA they could isolate was very limited, even when they used a large countercurrent apparatus and a modified solvent system to increase the solubility of the RNA. During his 3 years of work on the structure of the alanine tRNA, Holley used a total of only 1 g of highly purified material, which he isolated from approximately 200 g of bulk yeast tRNA, which in turn was obtained by phenol extraction of approximately 140 kg of commercial bakers' yeast.
To determine its sequence, Holley cleaved the 77-nucleotide alanine tRNA polynucleotide chain into 16 fragments, identified the small fragments, and then reconstructed the original nucleotide sequence by determining the order in which the small fragments occurred in the RNA molecule. This is the subject of the second JBC Classic. Specifically, Holley, George A. Everett, James T. Madison, and Ada Zamir first used pancreatic ribonuclease to cleave the RNA chain next to pyrimidine nucleotides and then used takadiastase ribonuclease T1 to cleave the RNA chain at guanylic acid residues. They isolated the resulting fragments by ion-exchange chromatography. The components of dinucleotide fragments were then identified by chromatographic and electrophoretic properties and spectra. Larger fragments were digested with snake venom phosphodiesterase and sequenced. The determination of the structures of all the fragments took approximately 2
years.
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In 1968, Holley joined the Salk Institute as a resident fellow where he began to study the molecular factors that regulate growth and multiplication of cells. He discovered that the concentrations of peptide and steroid hormones determine the rate of cell division. Holley also studied the effects of nonhormonal factors, such as certain sugars and amino acids, on cell proliferation and identified growth inhibitors. He remained at the Salk Institute as an American Cancer Society Professor of Molecular Biology until his death in 1993.
Holley was a member of the National Academy of Sciences and the American Academy of Arts and Sciences. He received the Albert Lasker Award in Basic Medical Research in 1965, the Distinguished Service Award of the U. S. Department of Agriculture in 1965, and the U. S. Steel Foundation Award in Molecular Biology of the National Academy of Sciences in 1967.1
FOOTNOTES
1 All biographical information on Robert W. Holley was taken from Refs. 5 and 6. 
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