Welcome to the Genome. Michael Yudell
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that results in the body’s inability to break down a substance called alkapton (today we know that alkaptonuria is caused by a mutation in the HGD gene on chromosome 3, which impairs the body’s ability to break down the amino acids phenylalanine and tyrosin). By studying familial patterns of this disease, Garrod came to infer that the missing enzyme was a problem of inheritance; most of the children with the defect were born to parents who were first cousins. (20) This “shallow” gene pool made the emergence of this recessive trait more likely.
Four decades later at Stanford University, biochemist Edward Tatum and geneticist George Beadle refined Garrod’s observations, suggesting in 1941 that one gene codes for one enzyme, a theory that was a cornerstone of molecular biology for more than five decades. They were awarded a Nobel Prize for their discovery in 1958. (21) Although DNA itself was coming to be known to be the stuff of heredity, enzymes and other proteins, it was turning out, were essential to the successful operation of the cell and therefore of the organism. If hereditary information was carried on DNA, then the different classes of proteins are, in large part, heredity’s workhorses, delivering instructions for many of life’s intricacies at the beck and call of the DNA molecule itself.
Work at the cellular level, with its varied goals, was less directed, for example, than the search for the structure of DNA. Some scientists were busy taking the cell apart to determine how DNA replicated, others learning how proteins were synthesized, and still others inquiring about the nature and function of proteins. In fact, Arthur Kornberg carried out his Nobel Prize‐winning discovery of the protein in bacteria that controls DNA replication without Watson and Crick’s work in mind. Perhaps what Kornberg himself called his “many love affairs with enzymes” distracted him from the broader goings‐on in molecular biology. “The significance of the double helix did not intrude into my work until 1956,” Kornberg wrote, “after the enzyme that assembles the nucleotide building blocks into a DNA chain was already in hand.” (22)
Kornberg’s discovery, once known as DNA polymerase or Kornberg’s enzyme and now known as DNA polymerase I, catalyzes the addition of nucleotides to a chain of DNA (other DNA polymerases were discovered later, and were in turn known as polymerases II, III, etc.). In other words, DNA polymerase is the mechanism by which DNA clones or copies itself. Working with the bacteria E. coli, a bacteria that is usually beneficial to the function of the human digestive tract, Kornberg showed that the enzyme DNA polymerase was able to synthesize a copy of one strand of DNA. With a single strand of DNA in a test tube, the presence of DNA polymerase served as the catalyst (or initiator) for DNA replication. These experiments revealed only that the synthesized DNA was true to Chargaff’s rules, having the correct ratio of As to Ts and Cs to Gs. (23) Kornberg’s results did not, however, reveal the sequential arrangement of nucleotides, nor was it known at this time whether this laboratory model was what actually happened in living organisms. (24)
It later turned out that Kornberg’s polymerase was not the key polymerase in DNA replication; DNA polymerase III was. Scientists who questioned the function of Kornberg’s polymerase in live organisms were only partially correct; polymerase I’s role was still found to be vital, playing a key role in chromosome replication and DNA repair. (25) Over the next two decades the approaches pioneered by Kornberg and his associates resulted in the discovery of a broad array of enzymes and other proteins important in the replication of DNA and the translation of proteins. An intriguing aspect of these discoveries is that polymerase enzymes do not need to be in cells to work. Biochemists used this feature of polymerase to develop methods to take proteins out of cells and coax them to activate in test tubes. The other enormously important result of Kornberg’s work was that scientists now had a laboratory reagent—the DNA polymerase itself—that could be used in a test tube to replicate DNA.
RESEARCH MILESTONE 4: SEEING GENES
Sanger’s sequencing of insulin’s amino acids, the cracking of the genetic code, and Kornberg’s work on DNA polymerase were all technologies that would someday lead to the sequencing of a whole genome. But the ever‐increasing knowledge of the molecular basis of inheritance could not reach its full potential for both scientific and biomedical research without techniques to sequence genes quickly and accurately. So we now turn from deciphering the interiors of the cell to technologies that capitalized on these discoveries and enhanced our ability to see the most fundamental mechanisms of heredity. By the 1970s laboratories around the globe were focused on finding ways to better characterize, at the molecular level, genes and their component parts.
Oxford University biologist Edward Southern revolutionized molecular biology in 1975 with a method that came to be known as the Southern blot. (26) Southern blots allowed geneticists to locate and look at DNA and genes within a genome by capitalizing on the following characteristics of DNA. First, DNA is a negatively charged molecule; thus when electricity is present, it can hitch a ride on a current—it migrates to the positive terminal in an electric field.
Second, DNA molecules are small and can be separated by passing them through a porous gel made from either agarose (extracted from seaweed) or acrylamide (a synthetic polymer). The size of the DNA fragment, the strength of the current, and the concentration of acrylamide or agarose in the gel mixture dictate how fast molecules will pass through it. In fact, the concentration of an acrylamide gel can be adjusted to such a fine degree that DNA molecules of one base pair difference in length can be distinguished. Third, one fragment of DNA can be used to find another. This process, known as DNA hybridization, activates one strand of a double helix to search for the other strand, to reform hydrogen bonds and make a new double helix.
Hybridization doesn’t have to be perfect; only 60–70% of the two strands of a helix must match for the two strands to stick together.
Southern created a technique whereby a small piece of an organism’s genome can be arranged by size along an agarose gel. The technique then involves the transfer of DNA from gels onto nitrocellulose membranes. To detect where a specific gene is, a fragment of the gene of interest is labeled with radioactivity and then hybridized (attached) to the DNA on the nitrocellulose. Radioactive molecules hybridizing to the DNA on the filter emit particles that react with photographic film and can therefore be seen as a dark spot on the film. With his blot, Southern solved the problem of finding a genetic needle in a genomic haystack. Techniques were later developed to similarly isolate RNA molecules (jokingly named Northern blotting) and protein molecules (named Western blotting). (27)
Figure 2.4 DNA hybridization occurs when a single strand of a double helix finds a complementary strand to form a new double helix.
Credit: Exhibitions Department, American Museum of Natural History
RESEARCH MILESTONE 5: COPYING DNA
During the 1970s scientists improved upon the Southern blot and other gel electrophoresis methods. Southern’s method required a tremendous amount of DNA and thus a tremendous amount of laboratory labor. It also lacked the precision to see the location of individual bases. To get around this shortcoming, scientists developed methods to amplify or clone (meaning simply to copy) DNA.
Found in bacteria, the small, circular extrachromosomal DNA molecule known as a plasmid often times carries genes that confer antibiotic resistance to bacteria. Under normal conditions a plasmid can facilitate genetic exchange between different bacterial strains: A gene fragment from one bacterium is carried to and inserted into a chromosome by a plasmid. In 1973 scientists discovered that if you biochemically insert a target stretch of DNA into a plasmid and put the plasmid into a bacterial cell, such a cell makes thousands and perhaps millions of copies of the plasmid and hence the attached DNA. (28) This procedure, incorporated into sequencing technology, made it easier to make large amounts of a desired stretch of DNA. There are, however, two serious shortcomings of the use of plasmids. First, bacterial plasmids must be cultured. This is time‐consuming. Second, plasmids can take up only a small piece of DNA efficiently. If the DNA stretch picked up by a plasmid is too large,