Genes determine hereditary traits, such as the color of our hair or our eyes. They do this by providing instructions for how every activity in every cell of our body should be carried out. For example, a gene may tell a liver cell to remove excess cholesterol from our bloodstream. How does a gene do this? It will instruct the cell to make a particular protein. It is this protein that then carries out the actual work. In the case of excess blood cholesterol, it is the receptor proteins on the outside of a liver cell that bind to and remove cholesterol from the blood. The cholesterol molecules can then be transported into the cell, where they are further processed by other proteins.
The Physical Carrier of Inheritance
While the period from the early 1900s to World War II has been considered the "golden age" of genetics, scientists still had not determined that DNA, and not protein, was the hereditary material. However, during this time a great many genetic discoveries were made and the link between genetics and evolution was made.
Friedrich Meischer in 1869 isolated DNA from fish sperm and the pus of open wounds. Since it came from nuclei, Meischer named this new chemical, nuclein. Subsequently the name was changed to nucleic acid and lastly to deoxyribonucleic acid (DNA). Robert Feulgen, in 1914, discovered that fuchsin dye stained DNA. DNA was then found in the nucleus of all eukaryotic cells.
During the 1920s, biochemist P.A. Levene analyzed the components of the DNA molecule. He found it contained four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit (nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to the sugar. He (unfortunately) also erroneously concluded that the proportions of bases were equal and that there was a tetranucleotide that was the repeating structure of the molecule. The nucleotide, however, remains as the fundemantal unit (monomer) of the nucleic acid polymer. There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine (A), and those with thymine (T).
During the early 1900s, the study of genetics began in earnest: the link between Mendel's work and that of cell biologists resulted in the chromosomal theory of inheritance; Garrod proposed the link between genes and "inborn errors of metabolism"; and the question was formed: what is a gene? The answer came from the study of a deadly infectious disease: pneumonia. During the 1920s Frederick Griffith studied the difference between a disease-causing strain of the pneumonia causing bacteria (Streptococcus peumoniae) and a strain that did not cause pneumonia. The pneumonia-causing strain (the S strain) was surrounded by a capsule. The other strain (the R strain) did not have a capsule and also did not cause pneumonia. Frederick Griffith (1928) was able to induce a nonpathogenic strain of the bacterium Streptococcus pneumoniae to become pathogenic. Griffith referred to a transforming factor that caused the non-pathogenic bacteria to become pathogenic. Griffith injected the different strains of bacteria into mice. The S strain killed the mice; the R strain did not. He further noted that if heat killed S strain was injected into a mouse, it did not cause pneumonia. When he combined heat-killed S with Live R and injected the mixture into a mouse (remember neither alone will kill the mouse) that the mouse developed pneumonia and died. Bacteria recovered from the mouse had a capsule and killed other mice when injected into them.
Hypotheses:
1. The dead S strain had been reanimated/resurrected.
2. The Live R had been transformed into Live S by some "transforming factor".
Further experiments led Griffith to conclude that number 2 was correct.
In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty revisited Griffith's experiment and concluded the transforming factor was DNA. Their evidence was strong but not totally conclusive. The then-current favorite for the hereditary material was protein; DNA was not considered by many scientists to be a strong candidate.
The breakthrough in the quest to determine the hereditary material came from the work of Max Delbruck and Salvador Luria in the 1940s. Bacteriophage are a type of virus that attacks bacteria, the viruses that Delbruck and Luria worked with were those attacking Escherichia coli, a bacterium found in human intestines. Bacteriophages consist of protein coats covering DNA. Bacteriophages infect a cell by injecting DNA into the host cell. This viral DNA then "disappears" while taking over the bacterial machinery and beginning to make new virus instead of new bacteria. After 25 minutes the host cell bursts, releasing hundreds of new bacteriophage. Phages have DNA and protein, making them ideal to resolve the nature of the hereditary material.
In 1952, Alfred D. Hershey and Martha Chase conducted a series of experiments to determine whether protein or DNA was the hereditary material. By labeling the DNA and protein with different (and mutually exclusive) radioisotopes, they would be able to determine which chemical (DNA or protein) was getting into the bacteria. Such material must be the hereditary material (Griffith's transforming agent). Since DNA contains Phosphorous (P) but no Sulfur (S), they tagged the DNA with radioactive Phosphorous-32. Conversely, protein lacks P but does have S, thus it could be tagged with radioactive Sulfur-35. Hershey and Chase found that the radioactive S remained outside the cell while the radioactive P was found inside the cell, indicating that DNA was the physical carrier of heredity.
The Structure of DNA
Erwin Chargaff analyzed the nitrogenous bases in many different forms of life, concluding that the amount of purines does not always equal the amount of pyrimidines (as proposed by Levene). DNA had been proven as the genetic material by the Hershey-Chase experiments, but how DNA served as genes was not yet certain. DNA must carry information from parent cell to daughter cell. It must contain information for replicating itself. It must be chemically stable, relatively unchanging. However, it must be capable of mutational change. Without mutations there would be no process of evolution.Many scientists were interested in deciphering the structure of DNA, among them were Francis Crick, James Watson, Rosalind Franklin, and Maurice Wilkens. Watson and Crick gathered all available data in an attempt to develop a model of DNA structure. Franklin took X-ray diffraction photomicrographs of crystalline DNA extract, the key to the puzzle. The data known at the time was that DNA was a long molecule, proteins were helically coiled (as determined by the work of Linus Pauling), Chargaff's base data, and the x-ray diffraction data of Franklin and Wilkens.
DNA is a double helix, with bases to the center (like rungs on a ladder) and sugar-phosphate units along the sides of the helix (like the sides of a twisted ladder). The strands are complementary (deduced by Watson and Crick from Chargaff's data, A pairs with T and C pairs with G, the pairs held together by hydrogen bonds). Notice that a double-ringed purine is always bonded to a single ring pyrimidine. Purines are Adenine (A) and Guanine (G). We have encountered Adenosine triphosphate (ATP) before, although in that case the sugar was ribose, whereas in DNA it is deoxyribose. Pyrimidines are Cytosine (C) and Thymine (T). The bases are complementary, with A on one side of the molecule you only get T on the other side, similarly with G and C. If we know the base sequence of one strand we know its complement.
DNA Replication
DNA was proven as the hereditary material and Watson et al. had deciphered its structure. What remained was to determine how DNA copied its information and how that was expressed in the phenotype. Matthew Meselson and Franklin W. Stahl designed an experiment to determine the method of DNA replication. Three models of replication were considered likely.
1. Conservative replication would somehow produce an entirely new DNA strand during replication.
2. Semiconservative replication would produce two DNA molecules, each of which was composed of one-half of the parental DNA along with an entirely new complementary strand. In other words the new DNA would consist of one new and one old strand of DNA. The existing strands would serve as complementary templates for the new strand.
3. Dispersive replication involved the breaking of the parental strands during replication, and somehow, a reassembly of molecules that were a mix of old and new fragments on each strand of DNA.
DNA Sequencing
The process of determining the order of the nucleotide bases along a DNA strand is called sequencing. In 1977, 24 years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method and the chemical degradation method. Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis, described earlier.
The DNA to be sequenced, called the template DNA, is first prepared as a single-stranded DNA. Next, a short oligonucleotide is annealed, or joined, to the same position on each template strand. The oligonucleotide acts as a primer for the synthesis of a new DNA strand that will be complementary to the template DNA. This technique requires that four nucleotide-specific reactions—one each for G, A, C, and T—be performed on four identical samples of DNA. The four sequencing reactions require the addition of all the components necessary to synthesize and label new DNA, including:
- A DNA template
- A primer tagged with a mildly radioactive molecule or a light-emitting chemical
- DNA polymerase, an enzyme that drives the synthesis of DNA
- Four deoxynucleotides (G, A, C, and T)
- One dideoxynucleotide, either ddG, ddA, ddC, or ddT
To read the newly generated sequence, the four reactions are run side-by-side on a polyacrylamide sequencing gel. The family of molecules generated in the presence of ddATP is loaded into one lane of the gel, and the other three families, generated with ddCTP, ddGTP, and ddTTP, are loaded into three adjacent lanes. After electrophoresis, the DNA sequence can be read directly from the positions of the bands in the gel.
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