Molecular Heredity

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    We need to know the form of the hereditary factor, the gene, and how it operates to produce the trait that it encodes in the chromosome. That means that we need to know the chemical structure of the gene, how it gets reproduced when the chromosome carrying the gene splits, and how it produces the chemicals and organizes them to manifest the trait that it controls. Today we know that the discovery and exploration of deoxyribonucleic acid (DNA), an on-going process that will not be complete for many years to come, will fulfill that need.

    But how did the original basic discoveries get made? Nobody can see an atom. To look into a cell and figure out what all the features in it do, to extract information from shadows dancing in a microscope, puts the human mind to a severe test. Like a detective solving the mystery posed by a murder, chemists and biologists had to gather evidence and draw inferences from it. But just as a detective must ask the right questions, scientists must carry out the right experiments. And, as we shall see, sometimes the most revealing experiments had to wait for other discoveries to be made in other fields of science.

    As a chemical, DNA was discovered in 1869 by a Swiss physician, Johan Friedrich Miescher (1844 Aug 13 – 1895 Aug 26), who discovered it in the nuclei of cells (leucocytes) that he had obtained from pus in discarded surgical bandages that he got from a nearby hospital. Once he obtained the nuclei from the cells, he purified them, subjected them to an alkaline solution, and then acidified the result. He found that he had a precipitate that he called nuclein. Upon further chemical analysis, he found that his nuclein contained phosphorus and nitrogen, but not the sulfur that he had expected. Indeed, the discovery was so unusual in that respect that the editor of the journal to which he submitted his paper actually repeated the experiments himself before publishing the paper in 1871. Of course, at that time nobody suspected the role that DNA plays in heredity.

    Around 1910 Phoebus Aaron Theodore Levene, M.D. (1869 Feb 25 (born Fishel Aaronovich Levin) – 1940 Sep 06) discovered that Miescher’s nuclein contains adenine, guanine, thymine, cytosine, and a phosphate group (a phosphorus atom hitched to four oxygen atoms). At first he believed that the four bases connected to each other in a ring by way of four phosphate groups. He then discovered deoxyribose in 1929 (he had discovered ribose in 1909) and determined that the components of DNA (DeoxyriboNucleic Acid) were linked together in the order phosphate - sugar - base to form units that he called nucleotides. That was good work as far as it went, but Levene’s hypothesis that DNA formed rings was wrong, which explains why he believed that DNA could not store genetic information: in his hypothesis the DNA molecule was just too small.

    While the chemists worked to figure out the composition and structure of DNA, the biologists continued to do their part in working out the basis of heredity in an organism. Frederick Griffith (1879 ? ? – 1941 Apr ?) made the basic discovery concerning the power of DNA to control the phenotype of a life-form. At the beginning of the Twentieth Century Friedrich Neufeld (1869 Feb 17 – 1945 Apr 18) used the solubility of pneumococci in ox bile to discover the existence of three different types of those bacteria. Applying that discovery, Griffith worked to create a vaccine against pneumonia, which had killed many people during the influenza pandemic of 1918. He used two strains of Streptococcus pneumoniae, one called smooth because it comes coated in a slimy polysaccharide capsule that protects it from the human immune system and the other called rough because it lacks that capsule and, thus, does not cause pneumonia. In 1928 Griffith discovered that when he heated a sample of the smooth strain to kill the bacteria and injected the killed bacteria into mice, the mice did not get sick. But, he found, if he mixed dead smooth bacteria with live rough bacteria and injected the resulting mixture into mice, the mice got sick and died. When he examined the bacteria drawn from the dead mice, Griffith found that the previously harmless rough bacteria had acquired capsules and had thereby become virulent. He explained that discovery by hypothesizing that what he called a transforming principle (which we now identify with DNA) had migrated from the dead smooth bacteria into the live rough bacteria and given them the ability to grow capsules.

    In 1934 Torbjörn Oskar Caspersson (1910 Oct 15 – 1997 Dec 07) and Einar Hammersten (no dates), working in Sweden, discovered that DNA has the structure of a polymer. Because polymers can achieve great lengths, biologists now had a molecule that could encode vast amounts of information. In the period 1937 - 1939 Caspersson also participated in work that showed ribonucleic acid (RNA) richly represented in cells that are producing proteins, which discovery implies that RNA plays an important role in protein synthesis in cells.

    In 1937 William Thomas Astbury (1898 Feb 25 – 1961 Jun 04) received a well-prepared sample of DNA from Professor Caspersson and he found that it produced a diffraction pattern when a beam of x-rays passed through it. That fact implied that DNA has a regular (i.e. repeating) structure and Astbury inferred from his measurements that the structure repeated every 2.7 nanometers and that the bases lay flat in stacks spaced 0.34 nanometers apart (the modern figure for the B-form of DNA is 0.332 nanometers). This work was based on the discovery made in the spring of 1912 by Max Theodor Felix von Laue (1879 Oct 09 – 1960 Apr 24), who discovered that x-ray diffraction reveals clues to the atomic structure of substances made of repeating units of molecules. When a collimated beam of x-rays passes through such a substance some of the x-rays will get diffracted out of the beam by regularities in the substance and form spots on photographic film. From their measurements of the placement of the spots on the film, scientists can infer facts about the structure of the substance.

    Meanwhile, during the 1930's, biologists continued to work at extending Griffith’s experiment. Martin Henry Dawson (1896 Aug 06 – 1945 Apr 27) made the first improvement, so modifying the experiment that he could carry it out entirely in test tubes (in vitro) instead of in mice (in vivo). James Alloway (no dates) first obtained crude aqueous solutions of the transforming principle in 1933 and Colin MacLeod (1909 Jan 28 – 1972 Feb 11) worked from 1934 to 1937 to produce purer versions of those solutions. Maclyn McCarty (1911 Jun 09 – 2005 Jan 02) took over the work in 1940 and completed it. With those purified solutions and the ability to carry out Griffith’s experiment in vitro, those researchers then determined what molecule corresponds to the transforming principle by exposing it to enzymes that break up proteins and RNA. None of the enzymes they tried interfered with the action of the transforming principle on the bacteria, none except a crude preparation of desoxyribonucleodepolymerase, which they obtained from animal sources. The one enzyme that brought the transforming principle to a dead halt was the one that tears up DNA. Thus by 1943 Oswald Avery (1877 Oct 21 – 1955 Feb 02), Colin MacLeod, and Maclyn McCarty had proven and verified that DNA corresponds to Griffith’s transforming principle.

    In 1952 Alfred Hershey (1908 Dec 04 – 1997 May 22) and Martha Chase (1927 ? ? – 2003 Aug 08) used a virus called the T2 phage and colonies of the infamous bacteria Escherichia coli to prove and verify the Avery-MacLeod-McCarty experiment. They used a technique that only became available through the invention of the nuclear reactor in 1942, using radio-nucleides to mark the subjects of their experiment. They grew T2 phages in a solution containing phosphorus-32, exploiting the fact that phosphorus exists in DNA but not in any of the twenty amino acids that go into making proteins. They then allowed the phages to infect a colony of E. coli, then broke up the bacteria and separated the components. Radioactivity from the phosphorus appeared only in the bacteria’s innards and not in their protein shells. Next the experimenters grew their phages in a solution containing sulfur-35. Sulfur goes into the amino acids cysteine and methionine, which go into the production of proteins, but not into DNA. When they again allowed the phages to infect colonies of E. coli and broke up the bacteria, they found the radioactivity in protein shells and not in the bacteria’s innards. These results indicated that DNA was the part going into each bacterium and using the cellular machinery to make copies of the phage. Thus Hershey and Chase verified that DNA does indeed carry genetic information.

    What we usually regard as the main event in molecular genetics came in 1953, instigated by Rosalind Elsie Franklin (1920 Jul 25 – 1958 Apr 16) when she and her student, Raymond Gosling, made much improved x-ray diffraction pictures of DNA. One of those pictures in particular inspired James D. Watson (1928 Apr 06 – ?) and Francis Crick (1916 Jun 08 – 2004 Jul 28) to work out the double helix structure of DNA. This incident gives us a beautiful example of serendipity in science. In 1951 Crick, who had begun his studies in physics and only later shifted his attention to biology, helped develop a mathematical description of the pattern of spots that would result from x-rays passing through a material made of helical molecules. When he saw one of Franklin’s x-ray diffraction pictures, he recognized the pattern and then he and Watson worked out the structure of DNA.

    Shortly after that success the physicist George Gamow (Georgiy Antonovich Gamov; 1904 Mar 04 – 1968 Aug 19) suggested that in order for DNA to encode the instructions for assembling proteins from amino acids, the four bases (adenine, cytosine, thymine, and guanine) must come in trios, because the third power of four is the smallest power of four that will cover all twenty of the amino acids that go into the proteins found in living organisms. Crick noted that Gamow’s suggestion inspired his further work on elucidating the structure of what biologists came to call codons.

    In 1957 Crick laid out the "Central Dogma" of molecular biology. He stated that biological information can only go from nucleic acid (either DNA or RNA) to protein and never from protein to nucleic acid. He might have better called it the "Central Doctrine" of molecular biology, but, being an atheist, he likely did not know the distinction between dogma and doctrine (or perhaps he did and intended his label as a joke).

    The next year Matthew Meselson (1930 May 24 – ?) and Franklin Stahl (1929 Oct 08 – ?) put the final touch on the proof of DNA as a doubly helical molecule. They conducted their experiment with nitrogen-15, which is a little more than seven percent heavier than the more common nitrogen-14, and E. coli and demonstrated that the replication of DNA follows a semi-conservative pattern and neither the conservative pattern nor the dispersive pattern.

    In their experiment Meselson and Stahl grew E. coli for several generations in a medium containing nitrogen-15. When they extracted the DNA from the bacterial cells and centrifuged it in an aqueous solution of salt with a high density gradient, the DNA settled at the point at which its density equaled that of the salt solution: it wouldn’t go any lower in the solution because it would have been light enough to float and it wouldn’t have risen any higher because it would be too heavy and would sink. The DNA containing nitrogen-15 had a higher density (was heavier) than did the DNA containing nitrogen-14, so the two types could be separated. The experimenters could then create E. coli cells with only nitrogen-15 in their DNA. Those bacteria were put back into a growth medium containing only nitrogen-14 and were allowed to divide only once. The resulting DNA was then extracted from the cells and was compared to DNA containing only nitrogen-14 and DNA containing only nitrogen-15. They found that their new DNA had a density close to the density intermediate between the densities of the pure DNA containing only nitrogen-14 and pure DNA containing only nitrogen-15. Conservative replication would have produced equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), because conservative replication would occur by a strand of DNA simply organizing material along its surface into new DNA without incorporating any new material into itself. The experimenters thus excluded conservative replication from the possible means of DNA reproduction.

    However, the experiment gave a result consistent with both semi-conservative replication and dispersive replication. Semi-conservative replication, in which the DNA separates into two strands and each strand then accumulates a new partner, would result in double-stranded DNA with one strand of DNA containing only nitrogen-15 and one strand of DNA containing only nitrogen-14. Dispersive replication, on the other hand, would result in double-stranded DNA in which both strands contain mixtures of bases containing nitrogen-15 and nitrogen-14. Either of those possibilities would have appeared in the variable-density saline solution as DNA of a density intermediate between that of the pure nitrogen-14 DNA and the pure nitrogen-15 DNA.

    So the experimenters repeated the experiment, taking bacteria that had grown for several generations in a growth medium containing only nitrogen-15 and allowing them to divide twice in a growth medium containing only nitrogen-14. DNA extracted from those bacteria and centrifuged was found to consist of equal amounts of DNA of two different densities. One density corresponded to the density of DNA in which half of the nitrogen was nitrogen-15 and the other density corresponded to the density of DNA in which all of the nitrogen was nitrogen-14. This did not match the result that would have come from dispersive replication, which would have produced DNA of a single density. In addition, that density would have come out lower than the density of the DNA taken from bacteria that had been allowed to divide only once in the nitrogen-14 growth medium but still higher than the DNA taken from bacteria grown only in a pure nitrogen-14 medium, because the original nitrogen-15 DNA would have been dispersed evenly among all of the DNA strands. So the result confirmed that semi-conservative replication, in which half of the twice-divided bacteria would have one strand of the original nitrogen-15 DNA along with one of nitrogen-14 DNA, which fact accounts for the DNA of intermediate density, while the DNA in the other half of the bacteria would consist entirely of nitrogen-14 DNA: one set of DNA would have been synthesized in the first division and the other in the second division.

    In this way Meselson and Stahl confirmed Crick’s Central Dogma. At the same time their work laid the foundations for further experiments with DNA.

    Having thus identified the specific chemical that encodes hereditary information and laid out its basic structure, biochemists then turned their attention to figuring out how DNA encodes hereditary information. George Gamow had already shown that in a system with four basic units at least three of those units would be necessary to manifest a code that could distinguish among the twenty amino acids that go into producing proteins. In 1961 Francis Crick and Sydney Brenner (1927 Jan 13 – ?) and others performed the experiment that proved and verified Gamow’s hypothesis. They demonstrated that three bases of DNA encode one amino acid. The experiment also shed light on the nature of gene expression and of certain mutations.

    Brenner had already shown that all over-lapping codes in DNA are impossible. That led Crick to propose the existence of what we now call transfer RNA, which bears an anti-codon to mediate between a codon on the DNA and the resulting protein. The transfer RNA hypothesis, in turn, formed the basis for Crick’s Central Dogma.

    The triplet nature of protein coding came clear through the Crick-Brenner experiment, which also demonstrated the existence of frameshift mutations, mutations caused by insertion or deletion of a base, thereby shifting the rest of the string relative to the "start reading here" codon. In an English text if we carry out such a mutation, then the instruction to "Loose the dogs of war" might become "Lose the dogs of war" (not a bad mutation there, actually). In English a frameshift mutation might still yield a meaningful statement, but in DNA it generally does not. Crick and Brenner exploited that fact to measure the length of the DNA codon.

    In the experiment Crick, Brenner, and their colleagues used proflavin to induce mutations in the T4 bacteriophage gene, rIIB. Proflavin causes mutations by inserting itself between DNA bases, which insertion typically results in the insertion or deletion of a single base pair in the DNA. The experimenters then isolated the mutated viruses and studied their responses to the mutations.

    Crick and Brenner found that their mutant viruses could not produce a functional rIIB protein because the insertion or deletion of a single nucleotide (DNA base) caused a frameshift mutation. The transfer RNA could not read the instruction properly, if at all. Mutants with two or four nucleotides inserted or deleted also came out nonfunctional. However, the experimenters found that they could make the mutant strains functional again by using proflavin to insert or delete a total of three nucleotides. In order for the transfer RNA to do its job, the number of bases in the instruction string had to be a whole multiple of three. This proved and verified the proposition that the genetic code uses a codon of three DNA bases, each codon corresponding to an amino acid.

    The next step, of course, was to determine what each of the sixty-four different possible codons codes for. What amino acid goes with which codon?

    In 1960 Marshall W. Nirenberg (1927 Apr 10 – ?) teamed up with J. Heinrich Matthaei (1929 ? ? – ?) at the National Institutes of Health to begin the process of answering that question. They began by answering a question that Nirenberg had asked himself: "Is DNA read directly to protein?" At that time biologists had established that DNA resides in the cell nucleus and that the synthesis of proteins occurs in the cytoplasm, the liquid in which the nucleus floats. In the light of those two facts Nirenberg’s question became more specific: Does DNA itself leave the nucleus or does it produce some intermediate molecule that leaves the nucleus? The question itself suggested a very clever experiment.

    Nirenberg and Matthaei produced a cell-free system from E. coli by stripping the outer membrane from the bacteria, thereby producing a soup of cytoplasm with the organelles and other cell structures intact and still functional. When they put DNA into their cell-free system nothing happened. But when they put RNA into another cell-free system, the system produced proteins that had not previously existed in the cytoplasm. In this way Nirenberg and Mattaei showed the machinery that made Crick’s Central Dogma possible. But their experiment pointed the way to doing much more.

    By this time researchers at The National Institutes of Health had begun synthesizing artificial nucleotides that each consisted of one base repeated over and over: they simply treated the bases as monomers and polymerized them. In May of 1961 Nirenberg and Matthaei obtained a sample of artificial RNA made solely of uracil, a nucleotide that only occurs in RNA and corresponds to thymine in DNA. They then added this synthetic poly-uracil RNA to a cell-free system along with an enzyme that tears up DNA, so that the system would produce no additional proteins other than that coming from their synthetic RNA.

    In the next step they added the twenty amino acids known to exist in living organisms to the mix. In each sample they tested, only one of the amino acids carried a radioactive tag, a different amino acid carrying the tag in each of twenty samples. They then removed the polypeptide that the RNA had produced from their samples and checked it for radioactivity. They found that the extract containing the radioactively labeled phenylalanine produced the radioactive polypeptide. Thus they knew that they had found the genetic code for phenylalanine: UUU (three uracil bases in a row) on RNA will pick up phenylalanine and add it to whatever molecule it is producing. Thus began the process of deciphering the codons of the genetic code.

    Nirenberg pressed on. Within a few years, he and his research team had performed similar experiments and found that three-base repeats of adenosine (AAA) added the amino acid lysine to polypeptides, triple-cytosine (CCC) added proline, and triple-guanine (GGG) added glycine. Over time Nirenberg’s group identified fifty-four of the sixty-four codons with their amino acids. By thus deciphering the genetic code, Nirenberg and his co-workers created what James Watson has called "The Rosetta Stone of Life".

    The next breakthrough came when Phillip Leder (1934 Nov 19 – ?), a postdoctoral researcher in Nirenberg's lab, developed a method for determining the genetic code on pieces of transfer RNA. This greatly sped up the assignment of three-base codons to amino acids so that 50 codons were identified in this way.

    In 1964 Nirenberg and Leder began an experiment meant to shed additional light on the triplet nature of the genetic code. The results of that experiment also allowed them to decipher most of the rest of the codons of the standard genetic code.

    In the experiment the researchers passed various combinations of messenger RNA through a filter, which contained ribosomes. Unique triplets of the DNA bases made specific transfer RNAs bind to the ribosome. By thus associating the transfer RNA with its specific amino acid, the researchers determined the base-triplet sequence on the messenger RNA that coded for each amino acid. This enabled the researchers to determine the amino acids associated with the codons that did not involve three repeats of the same base.

    Har Gobind Khorana (1922 Jan 09 – ?) performed experiments to confirm these results and he and his team completed the genetic code translation. With this, Khorana and his team completed nearly a century of work that established that the mother of all codes, the biological language common to all living organisms, is spelled out in three-letter words, each set of three nucleotides encoding a specific amino acid.

    Khorana used the knowledge that he gained through those experiments to become the first to synthesize oligonucleotides, strings of nucleotides that represent a kind of crude artificial DNA or RNA. Typically containing twenty or fewer bases, oligonucleotides are short strings of nucleotide polymer. Artificial production can create strings as long as 200 bases. Khorana also became the first to isolate DNA ligase, an enzyme that links pieces of DNA together.

    Khorana thus invented a means of producing custom-designed pieces of artificial genes that are now widely used in biology labs for sequencing, cloning and engineering DNA for new plants and animals. Automation and commercialization of the process now makes it possible for anyone to order a synthetic gene from any of a number of companies. All one needs to do is to provide the desired genetic sequence to one of the companies and that company will provide the oligonucleotide with the desired sequence in return.

    Thus, through well-planned observations and cleverly-constructed experiments, both chemists and biologists found the clues they needed to infer what causes heredity, where it resides in the cell, and what its structure looks like. Advances in other fields, such as physics, enabled researchers to look ever deeper into the nature of the hereditary unit. Having thus determined what that unit, DNA, looks like and developing the means to manipulate its structure, they began the ongoing process of determining how it works.

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