Editing Nirenberg and Matthaei experiment

{{Short description|1961 scientific experiment instrumental in deciphering the genetic code}}
[[File:J. Heinreich Matthaei and Marshall Nirenberg (29819142523).jpg|thumb|Nirenberg (right) and Matthaei at the [[National Institutes of Health]]]]
The '''Nirenberg and Matthaei experiment''' was a scientific experiment performed in May 1961 by [[Marshall W. Nirenberg]] and his post-doctoral fellow, [[J. Heinrich Matthaei]], at the [[National Institutes of Health]] (NIH). The experiment deciphered the first of the 64 triplet codons in the [[genetic code]] by using [[nucleic acid]] [[homopolymer]]s to translate specific [[amino acid]]s.

In the experiment, an extract was prepared from bacterial cells that could make [[protein]] without the presence of intact living [[cell (biology)|cells]]. An artificial form of [[RNA]] consisting entirely of [[uracil]]-containing nucleotides (''polyuridylic acid'' or poly-U) was added to the extract, causing it to form a protein composed entirely of the amino acid [[phenylalanine]]. This experiment cracked the first [[codon]] of the [[genetic code]] and showed that RNA controlled the production of specific types of protein.

== Background ==
Discoveries by [[Frederick Griffith]] and improved on by [[Oswald Avery]] discovered that the substance responsible for producing inheritable change in the disease-causing bacteria (''Streptococcus pneumoniae)'' was neither a protein nor a lipid, rather deoxyribonucleic acid ([[DNA]]). In 1944, he and his colleagues [[Colin Munro MacLeod|Colin MacLeod]] and [[Maclyn McCarty]] suggested that DNA was responsible for transferring genetic information. Later, [[Erwin Chargaff]](1950) discovered that the makeup of DNA differs from one species to another. These experiments helped pave the way for the discovery of the structure of DNA. In 1953, with the help of [[Maurice Wilkins]] and [[Rosalind Franklin]]'s [[X-ray crystallography]], [[James D. Watson|James Watson]] and [[Francis Crick]] proposed DNA is structured as a [[double helix]].<ref>{{cite book | title= iGenetics: A Molecular Approach, 3rd edition | author=Russell P. |year=2010| publisher= Pearson/Benjamin Cummings}}</ref>

In the 1960s, one main DNA mystery scientists needed to figure out was the number of bases found in each code word, or [[codon]], during [[transcription (genetics)|transcription]]. Scientists knew there was a total of four bases ([[guanine]], [[cytosine]], [[adenine]], and [[thymine]]). They also knew that were 20 known [[amino acids]]. [[George Gamow]] suggested that the genetic code was made of three nucleotides per amino acid. He reasoned that because there are 20 amino acids and only four bases, the coding units could not be single (4 combinations) or pairs (only 16 combinations). Rather, he thought triplets (64 possible combinations) were the coding unit of the genetic code. However, he proposed that the triplets were overlapping and [[Genetic code#Degeneracy|non-degenerate]]<ref name="Leavitt_craze">{{cite web |last= Leavitt |first=Sarah A.|title=Deciphering the Genetic Code: Marshall Nirenberg. The Coding Craze |url=http://history.nih.gov/exhibits/nirenberg/HS3_craze.htm |publisher=Stetten Museum, Office of NIH History |year=2004 |access-date=2009-10-05 |url-status=live |archive-date=9 February 2020 |archive-url=https://web.archive.org/web/20200209101250/https://history.nih.gov/exhibits/nirenberg/HS3_craze.htm}}</ref> (later explained by Crick in his [[Wobble base pair|Wobble concept]]).

[[Seymour Benzer]] in the late 1950s had developed an assay using phage mutations which provided the first detailed linearly structured map of a genetic region. Crick felt ''he'' could use mutagenesis and genetic recombination phage to further delineate the nature of the genetic code.<ref>{{cite journal| doi= 10.1016/j.cell.2007.02.029| author= Yanofsky C.| year = 2007|title = Establishing the Triplet Nature of the Genetic Code| journal = Cell |volume = 128| issue= 5 | pages = 815–818| access-date = 2018-01-24| url = http://www.cell.com/cell/pdf/S0092-8674(07)00253-X.pdf | pmid= 17350564| doi-access = free}}</ref> In the [[Crick, Brenner et al. experiment]], using these phages, the triplet nature of the genetic code was confirmed. They used [[frameshift mutation]]s and a process called [[mutation#Classification of mutation types|reversions]], to add and delete various numbers of nucleotides.<ref>{{cite journal| doi= 10.1038/1921227a0| author=[[Francis Crick|Crick FH]], [[Leslie Barnett|Barnett L]], [[Sydney Brenner|Brenner S]], Watts-Tobin RJ |title=General nature of the genetic code for proteins | url = https://profiles.nlm.nih.gov/SC/B/C/B/J/_/scbcbj.pdf |journal=Nature |volume=192 |issue= 4809|pages=1227–32 |date=December 1961 |pmid=13882203 |bibcode=1961Natur.192.1227C| s2cid=4276146}}</ref> When a nucleotide triplet was added or deleted to the DNA sequence the encoded protein was minimally affected. Thus, they concluded that the genetic code is a triplet code because it did not cause a frameshift in the reading frame.<ref>{{cite journal| doi = 10.1073/pnas.48.4.666| author = Matthaei, H.J., Jones, O.W., Martin, R.G., and Nirenberg, M.W. Vol. 48 No. 4 | title = Characteristics and Composition of RNA Coding Units | journal=Proceedings of the National Academy of Sciences of the United States of America | volume = 48 | pages =666–677 | year = 1962| pmid = 14471390| pmc = 220831| issue = 4 |bibcode = 1962PNAS...48..666M| doi-access = free}}</ref> They correctly concluded that the code is degenerate (multiple triplets can correspond to a single amino acid) and that each nucleotide sequence is read from a specific starting point.<ref name="Judson1996">{{cite book | title=The Eighth Day of Creation: Makers of the Revolution in Biology| author=Judson H.| year=1996|location= Cold Spring Harbor| publisher=Cold Spring Harbor Laboratory Press}}</ref>

==Experimental work==
[[Image: 06 chart lg.jpg|thumb|200px| One of Nirenberg's laboratory notebooks]]
In order to decipher this biological mystery, Nirenberg and Matthaei needed a [[cell-free system]] that would build amino acids into proteins. Following the work of [[Alfred Tissieres]] and after a few failed attempts, they created a stable system by rupturing ''[[E. coli]]'' bacteria cells and releasing the contents of the cytoplasm.<ref>{{cite journal| author= Matthaei H. and Nirenberg | title = Characteristics and Stabilization of DNAase-Sensitive Protein Synthesis in ''E. coli'' Extracts | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 47| issue = 10 | pages = 1580–1588| year = 1962|doi=10.1073/pnas.47.10.1580 | bibcode = 1961PNAS...47.1580M | pmid=14471391 | pmc=223177| doi-access = free}}</ref> This allowed them to synthesize protein, but only when the correct kind of RNA was added, allowing Nirenberg and Matthaei to control the experiment. They created synthetic RNA molecules outside the bacterium and introduced this RNA to the ''E. coli'' system. The experiments used mixtures with all 20 amino acids. For each individual experiment, 19 amino acids were "cold" (nonradioactive), and one was "hot" (radioactively tagged with <sup>14</sup>C so they could detect the tagged amino acid later). They varied the "hot" amino acid in each round of the experiment, seeking to determine which amino acids would be incorporated into a protein following the addition of a particular type of synthetic RNA.

The key first experiments were done with poly-U (synthetic RNA composed only of uridine bases, provided by [[Leon Heppel]] and [[Maxine Singer]]<ref name="Nirenberg_1961">{{cite journal| doi= 10.1073/pnas.47.10.1588|author1=Nirenberg, M.W. |author2=Matthaei, H.J. |name-list-style=amp | year = 1961 |title = The Dependence Of Cell- Free Protein Synthesis In ''E. coli'' Upon Naturally Occurring Or Synthetic Polyribonucleotides | journal = Proceedings of the National Academy of Sciences of the United States of America |volume = 47| issue=10 | pages = 1588–1602|bibcode = 1961PNAS...47.1588N | pmid=14479932 | pmc=223178|doi-access=free}}</ref>). At 3 am on May 27, 1961, Matthaei used phenylalanine as the "hot" amino acid. After an hour, the control tube (no poly-U) showed a background level of 70 counts, whereas the tube with poly-U added showed 38,000 counts per milligram of protein.<ref name="Leavitt_polyU">{{cite web |last= Leavitt |first=Sarah A.|title=Deciphering the Genetic Code: Marshall Nirenberg. The Poly-U Experiment |url=https://history.nih.gov/exhibits/nirenberg/HS4_polyU.htm |publisher=Stetten Museum, Office of NIH History |year=2004 |access-date=2020-04-09|url-status=live |archive-date=9 February 2020 |archive-url=https://web.archive.org/web/20200209101327/https://history.nih.gov/exhibits/nirenberg/HS4_polyU.htm}}</ref><ref name="Nirenberg_1961"/> Subsequent experiments showed that the 19 "cold" amino acids were not necessary and that the protein product had the biochemical characteristics of polyphenylalanine,<ref name="Nirenberg_1961"/><ref name="Nirenberg_2004">{{cite journal |doi=10.1016/j.tibs.2003.11.009 |author=Nirenberg, Marshall |year=2004 |title=Historical review: Deciphering the genetic code – a personal account |journal=Trends in Biochemical Sciences |volume=29 |issue=1 |pages=46–54|pmid=14729332}}</ref> demonstrating that a chain of repeated uracil bases produced a protein chain made solely of the repeating amino acid phenylalanine. While the experiment did not determine the number of bases per codon, it was consistent with the triplet codon UUU coding for phenylalanine.

In analogous experiments with other synthetic RNAs, they found that poly-C directed synthesis of polyproline. Nirenberg recounts that the labs of [[Severo Ochoa]] and [[James Watson]] had earlier done similar experiments with poly-A, but failed to detect protein synthesis because polylysine (unlike most proteins) is soluble in [[trichloroacetic acid]]. Further, using synthetic RNAs that randomly incorporated two bases at different ratios, they produced proteins containing more than one type of amino acid, from which they could deduce the triplet nature of the genetic code and narrow down the codon possibilities for other amino acids.<ref name="Nirenberg_2004"/> Nirenberg's group eventually decoded all the amino acid codons by 1966,<ref name="Judson1996"/> however this required additional ingenious experimental methods (see [[Nirenberg and Leder experiment]]).

==Reception and legacy==

In August 1961, at the International Congress of Biochemistry in Moscow, Nirenberg presented the poly-U experiments &ndash; first to a small group, but then at [[Francis Crick]]'s urging, again to about a thousand attendees. The work was very enthusiastically received, and Nirenberg became famous overnight.<ref name="Fee_public">{{cite web |last= Fee |first=E. |title=Profiles in Science: The Marshall W. Nirenberg Papers. Synthetic RNA and the Poly-U Experiments, 1959-1962 |url=https://profiles.nlm.nih.gov/spotlight/jj/feature/syntheticrna |publisher=National Library of Medicine |year=2000 |access-date=9 April 2020|url-status=live |archive-date=10 April 2020 |archive-url=https://web.archive.org/web/20200410023158/https://profiles.nlm.nih.gov/spotlight/jj/feature/syntheticrna}}</ref><ref name="Nirenberg_2004"/> The paper describing the work was published the same month.<ref name="Nirenberg_1961"/>

The experiment ushered in a furious race to fully crack the genetic code. Nirenberg's main competition was the esteemed biochemist Severo Ochoa.  Dr. Ochoa and Dr. Arthur Kornberg shared the 1959 Nobel Prize in Physiology or Medicine for their previous "discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid."  However, many colleagues at the [https://www.nih.gov/ National Institutes of Health (NIH)] supported Nirenberg, aware that it may lead to the first [[Nobel Prize]] by an intramural NIH scientist. [[DeWitt Stetten Jr.]], the NIH director who first hired Nirenberg, called this period of collaboration "NIH's finest hour."<ref name="Leavitt_polyU"/><ref name="Davies2001">{{cite book | title=Cracking the Genome: Inside the Race to Unlock Human DNA| author=Davies K.| year=2001| location=New York|publisher=The Free Press}}</ref><ref>{{Cite web|title=The Nobel Prize in Physiology or Medicine 1959 |url=https://www.nobelprize.org/prizes/medicine/1959/summary/|access-date=2020-10-16|website=NobelPrize.org|language=en-US}}</ref>

Indeed, "for their interpretation of the genetic code and its function in protein synthesis," Marshall W. Nirenberg, Robert W. Holley, and Har Gobind Khorana were awarded the 1968 [[Nobel Prize in Physiology or Medicine]].<ref>{{Cite web|title=The Nobel Prize in Physiology or Medicine 1968|url=https://www.nobelprize.org/prizes/medicine/1968/summary/|access-date=2020-10-16|website=NobelPrize.org|language=en-US}}</ref>  Working independently, Dr. Holley (Cornell University) had discovered the exact chemical structure of transfer-RNA, and Dr. Khorana (University of Wisconsin in Madison) had mastered the synthesis of nucleic acids.<ref name=":0">{{Cite journal|last1=Shampo|first1=Marc A.|last2=Kyle|first2=Robert A.|date=2004|title=Stamp Vignette on Medical Science: Marshall W. Nirenberg—Nobel Laureate in Physiology or Medicine|url=https://www.mayoclinicproceedings.org/action/showPdf?pii=S0025-6196%2811%2962759-6|journal=Mayo Clinic Proceedings|volume=79|issue=4|pages=449|doi=10.4065/79.4.448|pmid=15065607|via=Mayo Foundation for Medical Education and Research|doi-access=free}}</ref>  Dr. Nirenberg showed - excluding nonsense codons - every combination of a triplet (i.e. a codon) composed of four different nitrogen-containing bases found in DNA and in RNA produces a specific amino acid.<ref name=":0" />

''The New York Times'' said of Nirenberg's discovery that "the science of biology has reached a new frontier," leading to "a revolution far greater in its potential significance than the atomic or hydrogen bomb." Most of the scientific community saw these experiments as highly important and beneficial. However, there were some who were concerned with the new area of [[molecular genetics]]. For example, [[Arne Tiselius]], the 1948 Nobel Laureate in Chemistry, asserted that knowledge of the genetic code could "lead to methods of tampering with life, of creating new diseases, of controlling minds, of influencing heredity, even perhaps in certain desired directions."<ref name="Fee_public2">{{cite web |last= Fee |first=E. |title=Profiles in Science: The Marshall W. Nirenberg Papers. Public Reaction |url=https://profiles.nlm.nih.gov/spotlight/jj/feature/publicreaction |publisher=National Library of Medicine |year=2000 |access-date=9 April 2020|url-status=live |archive-date=9 April 2020 |archive-url=https://web.archive.org/web/20200409213318/https://profiles.nlm.nih.gov/spotlight/jj/feature/publicreaction}}</ref>

In addition to the Nobel Prize, Dr. Nirenberg has received the Molecular Biology Award of the National Academy of Sciences and the Biological Science Award of the Washington Academy of Sciences (1962), the Paul Lewis Award of the American Chemical Society (1963), the Department of Health, Education, and Welfare Medal, along with the Harrison Howe Award of the American Chemical Society of USA, in America (1864).<ref name=":0" />

==See also==
*[[Crick, Brenner et al. experiment]]
*[[Nirenberg and Leder experiment]]

==References==
{{Reflist}}

==External links==
* "[http://history.nih.gov/exhibits/nirenberg/index.htm Marshall Nirenberg: Deciphering the Genetic Code]" – Office of NIH History and Stetten Museum

{{History of biology}}

[[Category:Genetics experiments|Nirenberg and Matthaei experiment, The]]
[[Category:History of genetics]]
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