An interview with Manuel F. Varela and Ann Varela: Hamilton O. Smith—An American Microbiologist, Restriction Endonucleases, and the Nobel Prize

Dec 20, 2020 by

Hamilton O. Smith

I can still remember we were making a stock solution of orcein. It was in the hood; we were heating up the acetic acid in a flask. And he says, “OK, I’m going to add the orcein,” and he reached in and put it in, and it went “Poof!” ’cause we didn’t have boiling chips, and we had super-heated the solution, and the whole hood was covered with purple stain.”

—Hamilton O. Smith

I’ve always had kind of an inquiring mind. I always wanted to know how things worked.”

—Hamilton O. Smith

Michael F. Shaughnessy

1) When and where was Smith born, and what do we know about his early childhood?

Nobel Prize Laureate Hamilton O. Smith is credited for discovering one of the first restriction endonuclease enzymes. These proteins became necessary for gene cloning, a revolutionary advance in molecular biology. Because of Smith’s research investigations, molecular analysis of DNA became possible, and the field of recombinant DNA technology was enhanced. Hamilton Othanel Smith was born on the 23rd of August in 1931, in New York City, New York. Smith’s parents both started their careers as teachers at a high school in Panama City, Florida. Smith’s father, Bunnie Othanel Smith, went on to earn his doctorate in education at Columbia University in New York City. Although the Smith family split their time between New York and Florida for the next five years, memories made in the city were Smith’s fondest. Their parents entertained him and his older brother. The entertainment consisted of mathematical games and experiments conducted with a Gilbert chemistry set.

Smith’s family moved to Champaign-Urbana, Illinois, in 1937 when his father accepted a faculty position in the Department of Education at the University of Illinois. There, he spent the better part of his time teaching and writing. Smith’s mother, Tommie Naomi Harkey, was also writing, but not as prolifically as she desired. Despite her struggles, she remained ever encouraging to Smith and his brother Norman.

Music appreciation and performance were a significant part of Smith’s preteen years. He took piano lessons and was not a very motivated student until he heard Beethoven’s Pathetique Sonata on a vinyl recording performed by Arthur Rubenstein. From that moment on, he became more dedicated to his practicing.

Smith had many childhood friends who shared his interest in sports, music, chemistry, and electronics. He attended University High School, a college preparatory school. He graduated in three years due to some high school faculty, which allowed him to complete chemistry and physics during the summer term. Smith credits many of his high school teachers with positively influencing his achievements.

2) Hamilton Smith initially studied math and then moved to medicine—any explanation as to this switch?

Smith began his collegiate career at the University of Illinois as a mathematics major. He attended an inspiring seminar by George Wald on eye retina biochemistry. After reading a book about mathematical modeling of central nervous system circuits, gifted to him by his brother, Smith developed an interest in visual physiology. In 1950, Smith transferred to the University of California at Berkeley. While there, his goal was to apply to medical school. Smith earned his B.A. degree in Mathematics from the University of California at Berkeley in 1952.

Smith took his medical degree in 1956 from Johns Hopkins University. Within a year, he found himself called up in the Doctor’s Draft. Smith chose to enlist in the United States Navy and received a two-year assignment in San Diego, California. He then became head of the dispensary at the 11th Naval District Headquarters. Smith’s caseload was about 1,100 civilians and 500-600 military personnel. During this time, Smith took full advantage of idle time and kept current on medical issues. Smith read up on new research in human chromosomal irregularities. Genetics textbooks were also of interest to him.

Smith completed his service in 1959 and moved with his wife and one-year-old son to start his medical residency training at the Henry Ford Hospital in Detroit, Michigan. Smith made fair use of the medical library and acquired a new interest in phages. Smith began his research career in the lab of Myron Levine with a National Institutes of Health (NIH) postdoctoral fellowship. Levine’s lab was in the Department of Human Genetics at the University of Michigan in Ann Arbor. Smith studied the infectious stages of bacteriophage P22 C-genes, which regulated the lysogenic phases. Smith also studied the phage P22 gene called int, which conferred attachment of the virus to bacteria.

Once Smith completed his internship and residency, he joined the University of Michigan faculty in 1962. In 1967, Smith accepted an Assistant Professorship of Microbiology at John Hopkins, and then, in 1973, was promoted to Professor of Microbiology.

3) Restriction enzymes—what are they, and why are they important?

The restriction enzymes, also known as restriction endonucleases, are DNA-cutting proteins. These enzymes bind to DNA molecules at specific base-sequences and then cleave the DNA into pieces. These specialized DNA-cutting proteins protect bacteria from viral infection. The restriction enzymes also play vital roles in biotechnology and molecular biology. The restriction enzymes are critical for molecular gene cloning and recombinant DNA technologies.

Smith, Thomas J. Kelly, Jr., and Kent W. Wilcox discovered the first type II restriction enzyme, HindII, and the sequence of its cleavage site on DNA in 1970. See Figure 82.

Figure 82. Restriction enzyme HindII and its sequence-specific cleavage sites on DNA. The small arrows indicate the HindII endonuclease DNA cutting sites.

In the 1980s, Smith’s research team discovered HhaII. They then determined a high-resolution protein structure based on X-ray diffraction studies of the HhaII crystal bound to DNA.

Many of these restriction enzyme proteins are made by microbes for protection. Bacteria readily produce restriction enzymes to digest invading bacteriophage DNA molecules. Digesting foreign phage DNA by restriction enzymes breaks up the rogue nucleic acids into useless pieces. Phage DNA that is busted up inside a host bacterium cannot proceed with infection. The digested phage genomes cannot undergo lytic or even lysogenic phases in a bacterium. The bacteria can remain phage-free as long as it has protective restriction enzymes on hand.

Molecular biologists and investigators from many different scientific specializations use molecular gene cloning technology. The cloning of genes requires a working knowledge of restriction enzymes.

The process of molecular gene cloning involves several steps. See Figure 83.

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Figure 83. Molecular gene cloning method.

Specific organisms will harbor a gene that is desired for cloning. The genes may be chosen for their ability to produce useful products. The foreign DNA carries the target gene, and such DNA is harvested from the organisms. Additionally, cloning vector DNA in the form of circular plasmids are used. Then, both the foreign DNA (containing the target gene to be cloned) and the cloning plasmid DNA are digested with one or two restriction enzymes. Because the restriction enzymes cut DNA at staggered locations, the ends of the generated DNA fragments will have exposed bases that can reanneal to complementary sequences. These cut-up DNA trimmings are known as “sticky ends” because of their complementary base-pairing properties.

The restriction enzyme digestion generates sticky-ended fragments of the foreign DNA, one portion containing the desired target gene for cloning. Meanwhile, the restriction enzyme digest of the cloning plasmid vector will linearize the circular DNA, also generating sticky ends.

The collection of foreign DNA fragments with its target gene will be mixed with the linearized cloning vector DNA. Because of their sticky ends, each foreign DNA fragment will anneal to one molecule of plasmid DNA. Next, a DNA ligase enzyme is used to connect the annealed pieces covalently. Once glued together, the DNA has been officially recombined, and the newly made molecules are referred to as recombinant DNAs. The recombinant DNA molecules are then introduced into bacteria by the transformation method. The transformants harboring the recombinant DNA molecules are then screened for the clone’s presence that holds the desired gene.

Another beneficial outcome from using the restriction enzymes includes the production of so-called restriction maps. These maps are frequently presented in diagram form, and they indicate the locations of restriction enzyme cutting sites on DNA. The restriction maps can generate cloning strategies or create chimeric molecules between different organismal origins.

Restriction enzymes can be used for DNA mutagenesis. A gene with two restriction sites targeted for mutation can be digested with the restriction enzyme. The fragment is taken way, and the two ends are ligated. The result is a deletion of a significant portion of that gene. A gene treated in such a fashion, with much of its genetic information taken away, will likely be destroyed. The deleted gene’s effects can be studied. Knowledge of the gene, such as gene regulation and its gene product, can be examined more closely.

Chimeric proteins with novel functions can be produced with knowledge of their genes’ restriction sites. These novel chimeras can possess desirable biological processes, such as fuel production, chemotherapies, new vaccines, etc. The possibilities are seemingly endless.

An additional practical technological advancement that arose from the restriction enzyme discovery was the mapping of genomic DNA. Genome mapping proved advantageous for advancing the various genome projects. From the restriction maps of genomes, molecular biologists could then determine the entire genomes’ DNA sequences. Viral genomes were quickly restriction mapped and later sequenced. One of the first genomes of a living organism to be mapped and sequenced in this way was Smith’s Haemophilus influenzae bacteria.

Soon, restriction enzymes were used to map and sequence genomes for other organisms, such as fungi, algae, plants, animals, and even humans. These various genome projects all contain maps characterized by the restriction sites on their genomic DNA. The restriction maps can be used to study the evolutionary relationships between groups of living and non-living organisms. Such knowledge of phylogenetic history can help predict what may become of particular species in the future.

In modern times, forensic science technology employs restriction maps to identify individuals involved in crimes, such as victims and their perpetrators. The method is called restriction fragment length polymorphism (RFLP). Each individual harbors DNA regions of highly repetitive genetic elements on their genomes. These repeating sequences will bear unique restriction sites and the numbers of their restriction sites. Thus, an RFLP analysis of a dead crime victim, a crime scene weapon, and a suspect can be conducted. The RFLP data will then help to identify the victim and convict or exonerate a suspect.

4) Smith shared the Nobel Prize with two other colleagues—who were they, and what discoveries or things did they explore or investigate or discover?

Drs. Daniel Nathans and Werner Arber shared the 1978 Nobel Prize with Hamilton Smith for their investigations dealing with the restriction endonucleases and their relevance to molecular genetics. Their work with restriction enzymes would apply to all disciplines of biological, biochemical, cell biological, and biomedical sciences. Their discoveries would enhance the progress of recombinant DNA technology and biotechnologies.

Hamilton O. Smith discovered the first so-called type II restriction enzyme, called HindII, from Haemophilus influenzae. He soon found additional DNA-cutting enzymes and studied their molecular, structural, and biochemical properties. Smith’s restriction enzyme work in 1970 was part of the inspiration for the molecular biological investigations conducted by Dan Nathans.

Daniel Nathans was born in Wilmington, DE, in 1928 on the 30th of December. Nathans had attended the University of Delaware to garner his undergraduate studies. Nathans took his M.D. degree from Washington University at St. Louis, Missouri. At Johns Hopkins, Nathans studied SV40, the Simian virus-40, known as an animal cell transforming agent, causing tumors. Nathans had envisaged that the restriction endonucleases could be used to map the DNA genome of SV40. Working with Stuart Adler, Kathleen Danna, and George Sack, Nathans cut up the SV40 genome into various pieces using various restriction enzymes available then. Next, he deduced their original order along the genome’s path by overlapping the DNA fragments. This new genetic mapping approach permitted a revolutionary advance in recombinant DNA technology. With new genetic maps in hand, Nathans and others created gene-deletion mutations in viral genomes. Later, point mutations were targeted to restriction-enzyme-based DNA-cutting sites. Nathans died on the 16th of November in 1999.

Werner Arber was born in Gränichen in Switzerland on the third day of June in 1929. In 1953, Arber took his undergraduate degree from the Swiss Federal Institute of Technology, formerly Swiss Polytechnical School. In 1958, Arber defended his Ph.D. thesis at the University of Geneva. After completing postdoctoral training under Giuseppe (Joe) Bertani at the University of Southern California, Arber became a faculty member of molecular genetics at the University of Basel. Arber had become interested in a bacterium’s ability to withstand the onset of bacteriophage infection. He reasoned that the restriction enzymes attacked invading phage DNA, permitting the host bacteria to live. Arber worked with his graduate student Daisy Roulland-Dussoix. They demonstrated that the degradation of foreign DNA phage P1 or λ in Escherichia coli cells required the DNA-modifying and restriction enzymes to do so. These studies provided a pathway for new technological developments towards gene mapping and recombining various DNA elements with each other.

5) Molecular genetics—what exactly is this—and why is it important? 

The field of molecular genetics aims to study the flow of genetic information from generation to generation, examine the genetic code moving from DNA to RNA to protein, and study the effects of expressed genes and gene expression regulation mechanisms. Let us consider each of these molecular genetics areas individually.

The flow of genetic information to subsequent generations of living organisms involves the copying of that information first. The process is known as DNA synthesis or replication. The microorganisms will harbor DNA replicating machinery. Such biological machinery can accurately make a copy of the original template DNA sequence. Of the numerous proteins involved in DNA replication, individual components constitute proof-reading mechanisms. These mechanisms ensure accurate DNA copying and are devoted to providing an exact copy for the next generation. Occasionally, the incorporation of nucleotides may be in error, producing new variations in the base sequences in the newly copied DNA. The base mutations can, hence, produce new gene variants. The base and gene variants may be examined more closely by restriction enzyme site alterations. Thus, the restriction enzymes have greatly facilitated the advancement of this particular area of molecular genetics.

The molecular genetics field also entails the study of RNA synthesis, also known as transcription. The central biological machinery involved in transcription is RNA polymerase. This enzyme can read the code on the DNA template and attach corresponding ribonucleotides to form RNA chains. Often transcription is the first stage of gene expression. Protein synthesis, also known as translation, is the second stage in the gene’s natural expression. Thus, gene expression entails transcription and translation. Transcription mechanisms make RNA, and translation systems make protein.

Gene expression systems can be regulated. One mode of gene expression regulation can occur at the level of transcription. There have been many transcription regulation mechanisms discovered and studied by investigators. One type of gene activity regulation takes advantage of promoter elements near the gene’s transcription starting site. This location is “upstream” of the transcription start site. These upstream promoters have binding sites on DNA for the RNA polymerase. Once bound to the template DNA, the RNA polymerase commences the synthesis of RNA. Another regulatory component is a group of proteins called sigma. These sigma proteins can bind to RNA polymerase and affect its binding behavior to promoters on DNA. A third regulatory element is called an enhancer. These enhancer regions are often distantly located from the gene’s location on DNA. Enhancer-binding proteins will mediate their effect on transcription and can boost the gene expression programs for specific genes.

Another mode of gene expression regulation takes place at the level of translation. Here, numerous mechanisms have been discovered, and they vary significantly in their properties and players. One exciting cell molecular tool, called ribosomal frameshifting, involves Coronavirus. In this regulation mode, the ribosome binding sites on mRNA are moved to different locations. The ribosome binding to the new site starts the translation of a new protein.

One more level of gene expression regulation is called a post-translational modification. In this scenario, the protein that comes off the translational machinery is biochemically modified. These post-translational modifications can activate or inactivate the protein’s function, depending on the protein and the biochemical change type. These modifications take the form of attaching chemical adducts, like phosphates, acetyl groups, lipids, sugars, etc. The types of post-translation regulatory systems are extraordinarily diverse and represent significant areas of molecular investigation.

Repressors and inducers can regulate gene expression. One well-studied repressor is the famous lac repressor discovered by professors Jacques Mond and François Jacob and purified by Benno Müller-Hill and Walter Gilbert. Another example of a gene expression regulator is the equally famous lambda repressor isolated by Professor Mark Ptashne (see chapter 19). An example of a classic inducer is the allolactose molecule, a variant of the sugar called lactose. The allolactose binds to the lac repressor. Hence, the inducer-repressor complex cannot bind to the operator element on DNA. The RNA polymerase can, thus, attach to the promoter and permit induction of the transcriptional machinery. The expression of a gene is consequently allowed to proceed.

6) His years at Johns Hopkins—how productive were they?

Smith accepted a position and returned to Johns Hopkins in 1967. As described above, He did his Nobel Prize-winning work there in 1970. Smith also studied DNA methylases and nucleases from Haemophilus influenzae. Interestingly, these and other bacteria have developed ways to avoid self-digestion of their own genomic DNA. These microbes employ a series of DNA methylase enzymes that attach methyl groups to the bacterial genome’s specific bases. Smith studied these DNA methylase enzymes. Smith and Paul H. Roy purified DNA methylase enzymes from a bacterial strain called Rd of Haemophilus influenzae. Next, they identified the nucleotide sequences that the methylases recognized, bound, and methylated. See Figure 84. Later on, the crystal structure of a DNA methylase was determined. It was shown to be attached to the specific base sequence of DNA as predicted by Smith.

Figure 84. DNA methylase enzyme bound to its specific base sequence on DNA.

The DNA of methylated genomes are not cleaved by their bacterial restriction enzymes. Therefore, this bacterial methylation mechanism serves as a protection system and prevents endonuclease auto-digestion of the genome. Thus, the bacterium that makes a restriction enzyme is immune to its innate DNA-cutting effects, preventing its genome from destroying itself.

7) What is he currently working on or doing?

A Guggenheim Fellowship, which brought Hamilton O. Smith to the University of Zurich, was awarded to Smith in 1975. In 1994-5, he collaborated with J. Craig Venter at The Institute for Genomic Research (TIGR) to sequence Haemophilus influenzae by whole-genome shotgun sequencing and assembly. In July 1998, he joined Celera Genomics Corporation, where he participated in the Drosophila and human genome sequencing. In 2002 Smith became the scientific director at the Institute for Biological Energy Alternatives (IBEA) in Rockville, Maryland. He directed research on the generation of a synthetic single-celled organism capable of surviving and reproducing independently.

Figure 85. Hamilton O. Smith.

In 2006, a merger transpired among TIGR, IBEA, and several other centers to form the J. Craig Venter Institute. Smith became the leader of the synthetic biology and bioenergy research group. Smith served as the scientific director of the private company Synthetic Genomics, Inc., located in La Jolla, California, which he co-founded in 2005 with Craig Venter. Figure 85 shows Smith in 2006, enjoying life.

8) What have I neglected to ask?

Hamilton O. Smith was widely regarded. He was a recipient of a coveted Guggenheim Fellowship 1975-1976. In 1978, Smith shared the Nobel Prize for Physiology or Medicine with Werner Arber and Daniel Nathans to discover a new class of restriction enzymes that recognize specific sequences of nucleotides in a molecule of DNA cleave the molecule at that particular point. Smith is an elected member of the prestigious National Academy of Sciences. Smith is also a member of the Board of Applera (Celera Genomics, 1998-present).

For additional information regarding this ground-breaking molecular biologist and molecular geneticist, visit:

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