Hamilton O.Smith Nobel Prize Winner in Medicine in 1978 - Coursework Example

Nobel Prize Laureate in Physiology or Medicine of (1978)

Short Biography

 Hamilton O. Smith date of birth is 23rd August the year 1931 in the city of New York, U.S.A. His father was called Bunnie Othanel Smith while his mother’s name was Tommie Naomi Harkey. Both his parents were distinguished academicians. At the time Hamilton’s elder brother, Norman, was born, his father was an assistant professor at the University of Florida in Gainesville. His father later went to New York to Columbia University to pursue a doctorate in education. It was during the same period that Hamilton was born. Then his father was appointed in the Faculty of Education at the University of Illinois where he spent his entire career life. Hamilton's mother was a school teacher (Nobel.org, 2016).

Hamilton was introduced to an academic lifestyle by his parents since his early life. His father and mother would teach him and his brother slight Gilbert chemistry set and some mathematical equations. Hamilton’s grew up in Champaign-Urbana, Illinois. Even though at the time of growing up in Urbana the country faced a great depression and the Second World War, his academic life continued to take shape. His father would keep writing and reading thereby inspiring Hamilton. On the other hand, his mother taught him the virtue of respect and being creative in life.

Hamilton enrolled in a French class and pursued music during these pre-teen years. He later enrolled at University High School. He completed his high school in a record three years due to the influence of is teachers who laid down a solid academic foundation for his life.

He later enrolled at the University of Illinois, majoring in Mathematics though it did not interest him. However, his interest in Biology was aroused when his brother gave him a book describing central nervous system circuits which were modeled mathematically. The author of the book was Rashevsky. In 1950, he moved to Berkeley to the University of California where his interest in biology, cell physiology, and biochemistry was further enhanced.

He later joined the Medical School at the Johns Hopkins University in Baltimore, Maryland. He graduated from the medical school in 1956. He proceeded to do his internship at the Barnes Hospital located in St. Louis. Hamilton met Elizabeth Anne Bolton, during the internship period. Together they have four sons and a daughter.

Hamilton enrolled to the Navy in San Diego for two years. Due to the free time in the Navy, he started to research on human chromosomal irregularities. After completing his Navy attachment, he moved to Detroit in Michigan at the Henry Ford Hospital. He got a postdoctoral fellowship of N.I.H in 1962 and started his research job at the University of Michigan, Ann Arbor. There he met Myron Levine who was studying the phage. Together, they discovered the int gene, which is a gene that controlled the attachment of prophage. In 1967, he published the work on imperfect transducing particles that are formed from the int mutant prophage.

According to Nobel.org (2016), Hamilton joined the Johns Hopkins in 1967. He was appointed as an assistant professor in the Department of Microbiology. His work on the research includes genetic regulation in eukaryotes and prokaryotes, modification and restriction enzymes, the enzymology during genetic recombination, and the bacterial transformation mechanism. In the period of 1975 and 1976, he did work on histone gene sequence and arrangement with Birnstiel Max from Zurich University in Switzerland.

In 1978, Hamilton earned a Nobel Prize in Physiology or Medicine jointly with Nathans Daniel and Arber Werner. In 1995, he guided a team that sequenced the pioneer bacterial genome using Haemophilus influenzae at The Institute for Genomic Research. He also directed the team in Celera Genomics on the human genome, the group at J. Craig Venter Institute that created the synthetic bacterium. In 2003, they assembled the Phi X 174 bacteriophage. Hamilton is currently the director at the Synthetic Genomics which researches on eco-fuels which use microorganisms and recombinant algae. Additionally, he left a legacy at John Hopkins where the Hamilton Smith Award for Innovation Research is awarded annually to distinguished individuals in the field of research on enzymes.

Major Scientific Achievements and Impacts

Modification and Restriction in Bacteria

In the 1950s, Human and Luria, and Weigle and Bertani made some observations on phage which grew on two kinds of bacteria (Nathans et al, 273-293, Luria and Mary, 557). The Phage grew poorly, they were restricted, on one type of bacteria but grew very well in the other set of bacteria.A curious observation occurred when some phage moved out of restriction and grew well on the new host. It was as if they had developed a particular modification on the host which protected it from the host's restriction. The kind of biochemical source of the discovery stayed unknown till Werner Arber and his team showed that the modification on the host particular occurred on the DNA of the phage, while the restriction was due to the degradation of the DNA of the phage ( Dussoix and Arber, 37-49). Arber, in 1965, hypothesized that the restriction enzymes that are site-specific existed. He proposed that the modification was due to the production of DNA methylases that is host specific. Therefore, the idea of R-M, that is, restriction and modification system within bacteria had 2 same enzymes that have a limitation on endonuclease which identifies sequences of short nucleotide and DNA that is cleaved, and an enzyme modifier which identifies the original sequence and changes it to defend it against cleavage. Similarly, the DNA of the host cell would be offered protection, unlike the DNA that is foreign and incoming with inappropriate alteration which will be cleaved and destroyed. In 1968, Arber and Linn discovered an activity in E. coli extracts with properties. Additionally, Yuan and Meselson did some research using a restriction endonuclease that is highly-purified and obtained from E.coli K. By use of gradient centrifugation of sucrose; the final confirmed the notion that their enzyme was cleaved phage that was unmodified λ DNA resulting to many remains although the DNA that was modified remained pure. The distinguished enzymes’ feature was its ATP, S-adenosylmethionine, and Mg2+. They made assumptions that the enzyme was acting on the λ DNA at fixed locations, but they did not prove the idea by use of the fragmented species using the analysis by sucrose gradient. This observation was attributed to the nature of the restriction enzymes which are not DNA specific sites cleaver. This Class I

Enzymes are multimeric, complex proteins which require S-adenosylmethionine, Mg2+, and ATP, as the cofactors which function as modification methylases and restriction endonucleases. The enzymes are specific sites recognizers within the DNA and cleave erratically at a significant distance from the place of recognition. Therefore, they are not good for DNA analysis as the enzymatic tool.

Discovery of the Cleavage and Site Precise Restriction Endonuclease in Haemophilus

influenzae Rd

In, 1968, Hamilton and Kent Wilcox explored recombination of genes in Haemophilus influenza Rd which had been introduced earlier by Roger Herriot. Their experiments used the Nucleotide Sequence Specificity-Restriction Endonucleases viscometer to measure the sensitive of endonucleolytic cleavage in DNA using cell extracts (Smith and Wilcox, 379-391).Anotherset of experiments was on the donor DNA recovery from cells later from uptake. They used a page P22’s DNA which was a bacterial virus, in an experiment. To their astonishment, they did not recover the DNA foreign to the cells. They thought that it was due to restriction or their viscometer’s experience. The next day, they set-up two viscometers, one had Haemophilus DNA while the other had P22 DNA. Nest, cell extracts was added to each set-up. It was noted that the P22 DNA was less viscous while the DNA of Haemophilus was steady. They thought that they had discovered an active restriction enzyme which required Mg2+ as a cofactor.

They proceeded to show that the purified enzyme degraded only duplexP22 DNA to fragments of a length of about 100 base pairs. The DNA of Haemophilus existing in the same reaction mixture remained in its state. Zero free nucleotides and nil nicks were observed. Consequently, they discovered that the enzyme was an endonuclease which resulted to double-strand breaks and foreign DNA-specific. They later proved the cleavage was site-specific.

Recognition Site sequencing

In 1968, Hamilton and Kent started sequencing efforts. The method included marking the DNA’s 5’-termini through radioactive phosphorus by use of ATP-labeled as 32P gamma and T4 polynucleotide kinase trailed by digestion with DNase of the pancreas and either exonuclease I to produce terminal Dinucleotides, orvenom phosphodiesterase to produce terminal nucleotides. They started sequencing by use of phage T7 DNA’s restriction enzyme digests. During their initial experiment, they treated it with alkaline phosphatase to do away with the terminal 5’-phosphoryl category in the fragmented cleavage to get labeling. The DNA cleavage chain resulted to 3’-hydroxyl, 5’- phosphoryl termini. They scrutinized thenucleotides’ terminal. They discovered that the labeling of 32P terminal appeared in dAMP and dGMP, proving the enzyme- specific nature.

In 1969, Hamilton worked on the dinucleotide standards necessary for terminal dinucleotides identification. He also organized the exonuclease supply from the polymerase preparation of the DNA. He proceeded to show that the terminal dinucleotide was pApA or pGpA, proving the cleavage specificity again. In the same period, Hamilton was joined by Thomas J. Kelly, Jr. and togetherframed on how to work on the approach. They used the isotope 3 3P. T7 DNA which was homogenously labeled as 33P-cleaved using the endonuclease that was restricting, 5’-terminally marked as the next isotope, designated as 32P, was digested to oligomers by DNase of the pancreas. Then the products were portioned per the length using the chromatography of ion-exchange. The oligonucleotides of individual size class were analyzed electrophoretically. At the trimer and dimer level, 33P uniformly and two 32P-terminal, species that were labeled were obtained. The trimer and dimer organisms got eluted using an electrophoretic strip, and then digestion was carried out by venom phosphodiesterase resulting to the identification of the 33P-labeled nucleotides. It confirmed that the 5'- terminal dinucleotide was pPu-A, while trinucleotide was pPu-A-C. To account if the cleavage is “staggered” or "even," they developed a three-sequence arrangement. They used a micrococcal nuclease in releasing the 3’-terminal dinucleotide monophosphate which complemented the 5’-terminal dinucleotide. Therefore, the enzyme identified the 2-fold six nucleotides that were rationally symetical in the sequence of (5’) G-T-Py↓ Pu-A-C (3’). .... (3’)C-A-Pu ↑Py -T-G(5’)... and resulted in an even cleavage that was duplex, shown using arrows. The enzyme was predicted to cleave once per 1024 base pairs by judging the projected occurrence of the sequence in a random DNA.

Methylases in H. influenzae Rd Modification

In 1970, Hamilton and Paul carried out a study of methylases of the DNA in H. influenzae Rd. They first recognized that H. influenzae Rd contained a minute content of methylated bases in their DNA, 5-methylcytosine occurred per8 000 bases, while N-6-methyladenine occurs per 280 bases. Theydiscovered that most of the methylation seemed to be distinct from R-M systems and some methylases seemed present. It was indicated by Arber that most DNA methylation in E. coliwas not related to the R-M systems, 5% was involved in E.Coli. They thus implemented the universal method which was designed to show the whole cells' DNA methylases. They extracted a basic cell's proteins which they took for chromatography using phosphocellulose and analyzed for its ability in transferring methyl groups from the marked S-adenosylmethionine on the DNA of Salmon sperm, T7 DNA.

Using the way, they detected four DNA adenine methylases. The T7 DNA was shielded from cleaving by the enzyme that restricted; resulting in the destruction of the enzymatic Four DNA adenine methylases were detected. It was noted that one of te methylase offered protection to the T7 DNA against cleaving resulting to destruction by pre-digestion for the enzymatic site in the DNA of the salmon sperm. The observations indicated that the methylase and the restriction enzyme shared a mutual recognition site of the DNA. They then analyzed 5' and 3' closest neighbors to the 3H-methylated adenine products as a result of enzymatic activity. The output gave them an incomplete arrangement for the methylase, which was the nucleotide, 5', Pu-A-C, which rhymed with their sequence for the restriction enzyme.

During the research, an unexpected and additional observation in methylase studies was noted. It was pointed out that during the experiment on salmon sperm, the ability of the methyl acceptor was lost during the other methylases of the DNA. The methylase of interest was different from the restriction enzyme. It suggested that there was a restricting enzyme contained in the preparation of the endonuclease.

Their understanding was acknowledged by the parting of the restriction enzymesby separation of the two limit events in the laboratory of Nathan, and by a message from K. Murray from Scotland. Murray stated that he had purified the enzyme that was new and identified its site sequence recognitions, S' pA-A-G-C-T-T. This was good information to Hamilton as he knew that in T7 DNA, it was only one enzyme that was active.

The Search for the New Restriction Enzymes

The enzymes were noticed to be useful tools for the analysis of DNA when the group of Nathans used cleavage-site- specific restriction endonucleases in the SV40 tumor analysis. Nathans work provoked the search for differing-specificities enzymes. Hamilton’s work on the Haemophilus restriction endonuclease culminated in the discovery of enzymes which are readily detected by a bacterium could also be detected by biochemical means. The idea was supported by the invention of gel electrophoresis for DNA analysis on fragments due to restriction cleavage (Danna and Nathans, 2913-2917). As Sharp et al., (3055-3063) mentions, the ethidium bromide invention, was the fluorescent stain of DNA. The discovery led to the widespread studying of the restriction endonucleases. Richard J. led the drive in isolation of the enzyme.

The Process of Recognition in Nucleotide Sequence

Hamilton noted that most R-M system sites for recognition contain 2-fold rotationalsymmetry. There are two fundamental recognition mechanisms for the sites; Recognition in symmetrythat uses contacts in bilateral symmetry site and recognition in asymmetry by use of a set of nonsymmetrical contacts. For sites with a single strand, the asymmetric mechanism is only which may apply.

Axis of Symmetry

Symmetric Recognition

Asymmetric Recognition

FIGURE1-The two unique ways for which modification and restriction of enzymes may relate with a symmetrical nucleotide sequence by a rotationality that is two-fold to achieve recognition. In the model of symmetric recognition, the protein contains subunits which are arranged in a rotational symmetry that is two-fold, and each similar half interrelates with n/2 nucleotides. In the model of asymmetry, the protein is presumed to be a structure with an asymmetry and must interlink with a minimum of one nucleotide at respective base-pair position for the maximum n nucleotides (From Hamiton, 1978)

It was noted that most restriction endonucleases seem to need sites that are duplex, as earlier confirmed by Hind11 (14). Some enzymes, for example, HinfI, Mbo1, SfaI HaePIII, and HhaI act gradually on DNAs with double strands.(Blakesley et al, 421-422) but that was thought to be because of the development of short duplexes (Blakesley et al, 7300-7306). It was possible that the greatest number of restriction enzymes use the mechanism of symmetric recognition. That was due to several explanations. In the first explanation, because hemimethylated spots are produced during replication, the process of recognition obligates the responsiveness to methylation on whichever strand. That was achieved by bilateral, protein-DNA that is symmetric and links at the positions of methylation inside the site that is duplex. Secondly, it is cheaper to stipulate a site of protein monomer that recognizes n/2 bases rather than one identifying n bases. Lastly, the EcoRI endonuclease occurs as tetramers and dimers of a lone 28,500-dalton subunit and cleaves the two strandsof a site that are duplex in a single obligatory event, and under physiological situations (Rubin and Modrich, 7265-7272). Therefore, the tetrameric or dimeric structure is the rule for other enzymes.

Modification Methylases recognize the sites in a way that is not like enzymes that restrict. Several the enzymes acted on sites with straight strands indicating the recognition process of asymmetry. Some of the enzymes were capable of’ working on sites with only single strand, implying a process of asymmetric recognition. That observation suggested that the discriminatory interactions involved only the strand’s base.

Initially, it was proposed that restriction endonucleases which recognized a site that was symmetrical depended on the site’s structure. Hamilton acknowledged the lac operator or repress studies and analyses of the base pairs’ structure, which showed that the minor and major helical DNA’s groove was adequate in biased interactions.

Hamilton ad Michael Mann chose the restriction enzyme in their first study with site (5’) pG-C-GJC. This was due to the simplicity and the natural synthesis in the alternating polymer form. They chose the modification of the bases’ chemically to get to know how the clusters in the main and minor grooves which help in recognition. They measured the catalytic activity effects. He used the Seeman’s analysis for data interpretation. The writers compared the numerouspossible sites for links in the minor and major base pair grooves against the protein-DNA discrimination.

The GC base pair, in the HhaI site, is shown in FIGURE. 2. The minor and major grooves are imagined as separated into central and outer regions. The major central groove has six guanine atoms while the hydrogen is attached to the cytosine amino which is N4. The exterior main groove has the N7 guanine atom and the C5 cytosine hydrogen atom. The middle groove that is minor has the 2-Amino, N2, guanine group. The exterior small groove bears N3 guanine atom and 02 cytosine atom. The last is the hydrogen atom attached to the 2-amino guanine set in the site of HhaI is shown in FIGURE 2with potential interactive atoms.

FIGURE 2- Probable minor and major groove protein-DNA which discriminates interfaces positions in the HhaI recognition site of the restriction endonuclease. A. A stereo chemical drawing of a G.C pair of the base in DNA. Probable atoms for the interface are

shown. B. A draft of a part of helical DNA HhaI site contained. The pairs of the base

are marked by horizontal bars. The observation is overhead the key groove and estimated

Interacting atoms positions are shown (From Hamilton, 1978 with the arrows added by current author).

The HhaI modification methylase movesgroups of methyl from S-adenosylmethionine

Into the five-location of the cytosines located inside, in the site of Hhuland Shields from Hhul endonuclease cleavage. (Mann and Smith, 483-493). Hamiton inferred that the contacts between DNA and protein might take place in the two-outer central groove duplex site positions. They also showed that methylation presented on the five-position of the exterior cytosines prevent cleavage by R~Hhal. Hence, the enzyme closely fits these.

They then concluded that the N7 position methylation of any G particles in the location exhibited cleaving protection. The effect was likely steric, and theysuggested that the enzyme meticulously fitted the external major groove site. They summarized that methylation in either of the 8 sites of the exterior portions forming the central groove inhibited cleaving. They then looked at the minor groove connections by looking at the activity on the alternating Poly, dI-dC. Inosine has an H-atom in the 2-amino category guanine position in the minor middle groove. Therefore, a base pair of dI-dC is like a base pair of dA-dT in the small groove. The R.HhaI slices alternating d I - d C proficiently, therefore, the 2-amino group plays no role during the discrimination of G.C to A.T base pairs. Consequently, it was noted that the minor middle groove was not occupied. Therefore, they concluded that the Hhul endonuclease occupied the main groove and stems all biased contacts from outside primary and central groove location's group Smith (455-462).

Major impact of work to biological sciences/modern genetics

Hamilton and his team discovered the pioneer of what came to be known as the type II restriction enzymes. The enzymes were able to recognize a particular area of a sequence in a DNA and cleave at the precise site in the DNA. The discovery caused the type II restriction enzymes treasured subject in the field of DNA technology and consequent studies on DNA structure. Hamilton was appointed the scientific director of the Institute for Biological Energy Alternatives, designated as IBEA in 2002. He spearheaded research on the conceptualization of an artificial organism made up of one cell which was able to reproduce and surviving by itself.

The major aim of the investigation was to know the type of and number of genes required to support life. IBEA and TIGR combined to create the J. Craig Venter Institute. Hamilton Smith was appointed the leader of the research group on bioenergy and synthetic biology. Hamilton has made a great scientific impact on current genetics and here main working in the genetics field.

My Reflection

Harrison O. Smith’s revolutionary discovery impressed me given how limited technology and knowledge of DNA was there at his time of research. Restriction enzymes have continued to be a central focus in the study of DNA, and the way these enzymes have been used has significantly increased since Smith first discovered them in 1970. Today, restriction enzymes are used to identify specific genes that contribute to an organism's appearance. This has allowed scientists to research specific mutations in genes and has led to the thought that scientists could change genes one day.

According to Nobel.org (2016), Hamilton joined the Johns Hopkins in 1967. He was appointed as an assistant professor in the Department of Microbiology. His work on the research includes genetic regulation in eukaryotes and prokaryotes, modification and restriction enzymes, the enzymology during genetic recombination, and the bacterial transformation mechanism. In the period of 1975 and 1976, he did work on histone gene sequence and arrangement with Birnstiel Max from Zurich University in Switzerland.

In 1978, Hamilton earned a Nobel Prize in Physiology or Medicine jointly with Nathans Daniel and Arber Werner. In 1995, he guided a team that sequenced the pioneer bacterial genome using Haemophilus influenzae at The Institute for Genomic Research. He also directed the team in Celera Genomics on the human genome, the group at J. Craig Venter Institute that created the synthetic bacterium. In 2003, they assembled the Phi X 174 bacteriophage. Hamilton is currently the director at the Synthetic Genomics which researches on eco-fuels which use microorganisms and recombinant algae. Additionally, he left a legacy at John Hopkins where the Hamilton Smith Award for Innovation Research is awarded annually to distinguished individuals in the field of research on enzymes.

Major Scientific Achievements and Impacts

Modification and Restriction in Bacteria

In the 1950s, Human and Luria, and Weigle and Bertani made some observations on phage which grew on two kinds of bacteria (Nathans et al, 273-293, Luria and Mary, 557). The Phage grew poorly, they were restricted, on one type of bacteria but grew very well in the other set of bacteria.A curious observation occurred when some phage moved out of restriction and grew well on the new host. It was as if they had developed a particular modification on the host which protected it from the host's restriction. The kind of biochemical source of the discovery stayed unknown till Werner Arber and his team showed that the modification on the host particular occurred on the DNA of the phage, while the restriction was due to the degradation of the DNA of the phage ( Dussoix and Arber, 37-49). Arber, in 1965, hypothesized that the restriction enzymes that are site-specific existed. He proposed that the modification was due to the production of DNA methylases that is host specific. Therefore, the idea of R-M, that is, restriction and modification system within bacteria had 2 same enzymes that have a limitation on endonuclease which identifies sequences of short nucleotide and DNA that is cleaved, and an enzyme modifier which identifies the original sequence and changes it to defend it against cleavage. Similarly, the DNA of the host cell would be offered protection, unlike the DNA that is foreign and incoming with inappropriate alteration which will be cleaved and destroyed. In 1968, Arber and Linn discovered an activity in E. coli extracts with properties. Additionally, Yuan and Meselson did some research using a restriction endonuclease that is highly-purified and obtained from E.coli K. By use of gradient centrifugation of sucrose; the final confirmed the notion that their enzyme was cleaved phage that was unmodified λ DNA resulting to many remains although the DNA that was modified remained pure. The distinguished enzymes’ feature was its ATP, S-adenosylmethionine, and Mg2+. They made assumptions that the enzyme was acting on the λ DNA at fixed locations, but they did not prove the idea by use of the fragmented species using the analysis by sucrose gradient. Read More