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In the early part of the 20thcentury techniques were developed to examine the inner workings of the cell and much of the work was performed in bacteria due to their experimental accessibility. Before this period, the method of turning genetic information into the proteins that carry out cellular processes was completely unknown. Indeed, the chemical composition of the genetic material was being hotly debated. From Gregor Mendel's work with pea plants, the nature of inheritance and heredity was understood. However, it was unclear what molecule in the cell maintained and passed hereditary information on to subsequent generations. The leading contender for this role was protein, when in 1928 Fred Griffith discovered transformation in bacteria.
Griffith knew that when Streptococcus pneumoniae strains are injected into mice, they cause rapid deterioration and death. Griffith isolated strains of the bacteria that were no longer capable of producing an outer slime layer and appeared rough when grown on solid medium in contrast to the smooth colonies of the original isolate. When these rough strains were injected into mice, no illness resulted. Similarly, when a smooth microbe was heat-killed and then injected, the mice showed no signs of infection. A surprise was waiting for Griffith when he injected into mice a mixture of dead cells of a smooth isolate with live cells of a rough isolate: they died. When bacteria were isolated from these dead mice, they formed smooth colonies. Griffith hypothesized that the ability to create the slime layer was passed from the dead smooth cells to the viable rough cells, making them pathogenic again. The process became known as transformation.
Griffith was ridiculed, as most scientists believed his preparation were contaminated with viable smooth cells. It was not until 1944 that his student Oswald Avery and coworkers repeated the experiments of Griffith, reproducing his results and discovering that DNA was the material from the dead smooth cells that transformed the rough mutant. This was strong proof that the hereditary material in cells was DNA. Further experiments in the 1940's clearly established this observation with Joshua Lederberg discovering two more ways that DNA could be transferred between bacterial cells, conjugation and transduction. For more information on these experiments, see the chapter on Genomics and Genetics
In 1943 Beadle and Tatum reported experiments with the fungus Neuropsora crassa that eventually established the idea that each gene in the DNA typically codes for one protein (the one gene-one enzyme hypothesis). About 10 years later, Francis Crick, Rosalind Franklin, James Watson and Maurice Wilkins worked on experiments describing the structure of DNA and making predictions about how it was replicated. It was now clear that DNA stored the information for proteins and that proteins performed the many functions of the cell.
The important question now became, how does one convert the information in DNA into protein. A major contribution in understanding this puzzle was made by Paul Zemecnik and his laboratory who developed cell-free systems, first with rat liver and later using the bacterium E. coli. This allowed the study of translation in the test tube and he and other scientists used these systems to discover the important molecules involved in the process. An intense effort to describe these molecular events then ensued. The first insight came when Crick, Sydney Brenner and colleagues proposed the existence of transfer RNA (tRNA), a molecule that helps to create amino acid polymers based on nucleic acid sequence. Another critical player in the processing of information was revealed when Brenner, Francois Jacob and Matthew Meselson discovered that translation of genetic material into protein takes place on the ribosome and that the molecule being translated at the ribosome is RNA, not DNA. The next mystery was solved by Marshall Nirenberg and J. H. Matthaei when they developed methods to decipher the genetic code that dictates the correspondence of nucleic acids to amino acids. After the stunningly productive decade of the 1960's, the nature of the framework for the conversion of the genetic information into proteins was now understood and the basic mechanism has since been shown to be conserved across all biology. For more information on the process of transcription and translation, see the chapter on the Central Dogma
Table 1.7 lists important events in learning about the central molecular events in biology. Note that almost all of this work occurred in microorganisms.
|1928||Frederick Griffith discovers transformation in bacteria and establishes the foundation of molecular genetics.|
|1941||George Beadle and Edward Tatum develop the one-gene one-enzyme hypothesis.|
|1943||Salvador Luria and Max Delbruck demonstrate that inheritance in bacteria follows Darwinian principles.|
|1944||Oswald Avery, Colin MacLeod, and Maclyn McCarty show that DNA is the hereditary material.|
|1946||Joshua Lederberg and Edward L. Tatum discover a second method of gene transfer in bacteria: conjugation.|
|1952||Joshua Lederberg and Norton Zinder find a third method of DNA transfer in bacteria using bacteriophage: transduction.|
|1952||Alfred Hershey and Martha Chase suggest that only DNA is needed for viral replication.|
|1953||Francis Crick, Maurice Wilkins and James Watson describe the structure of DNA.|
|1954||Paul Zemecnik develops a cell-free system for translation using rat liver|
|1954||Francis Crick, Sydney Brenner, and colleagues propose the existence of RNA that helps to transfer the information in DNA into protein (tRNA).|
|1956||Paul Zemecnik discovers tRNA in his cell-free system|
|1957||Francois Jacob and Elie Wollman show that the chromosome of E. coli is circular.|
|1959||Peter Mitchell proposes the chemiosmotic theory in which a molecular process is coupled to the transport of protons across a biological membrane. He argues that this principle explains ATP synthesis, solute accumulations or expulsions, and cell movement.|
|1959||Arthur Pardee, Francois Jacob, and Jacques Monod show that lactose induces β-D-galactosidase the catabolic enzyme that begins the degradation of the sugar.|
|1960||Arthur Kornberg demonstrates DNA synthesis in cell-free bacterial extracts and later shows a specific enzyme complex catalyzes the synthesis of DNA.|
|1960||Francois Jacob, David Perrin, Carmen Sanchez and Jacques Monod propose a mechanism for the for control of bacterial gene expression in an organization they call the operon.|
|1960||Paul Zemecnik and Robert Lamberg develop a bacterial cell-free system using E. coli|
|1961||Marshall Nirenberg and J.H. Matthaei observe that a synthetic polynucleotide, composed only of a string of the base uracil, directs the synthesis of a polypeptide composed only of phenylalanine. This begins the quest to unravel the genetic code that translates DNA into protein.|
|1961||Sydney Brenner, Francois Jacob and Matthew Meselson show that ribosomes are the site of protein synthesis and that RNA carries messages from the DNA to the ribosome.|
|1968||Lynn Margulis proposes that endosymbiosis has led to the generation of mitochondria and chloroplasts from bacterial progenitors.|
|1970||Howard Temin and David Baltimore independently discover reverse transcriptase, a radically different way to alter genetic information in cells.|
|1975||Thomas Cech and Sidney Altman independently show that RNA, and not just protein, can serve directly as a reaction catalyst.|
One final thread we will trace is the rise of molecular microbiology and the technique of genomics. This pathway begins in 1885 with the seemingly unimportant isolation of a common intestinal microorganism by Theodor Escherich. The microbe is eventually renamed Escherichia coli in his honor. It could not have been understood at the time, but this bacterium would play a central role in our understanding of life. E. coli achieved this position because it has several properties that made it invaluable as an experimental system:
Several key discoveries laid the foundation for future developments in molecular biology. The first of these was the discovery of autonomously replicated pieces of DNA separate from the bacterial chromosome. plasmids, as Joshua Lederberg called them, were capable of carrying genes that could be regulated and moved from one microbe to another independently of the bacterial chromosome. The ability of some plasmids to move between different cells meant that different bacteria could rapidly inherit genes carried on these plasmids. It was found that one class of plasmids encoded resistance genes that would enable the bacteria to grow in the presence of an antibiotic. It later became clear it was possible to select for the presence of these plasmids by demanding growth of the bacterial strain on medium containing the antibiotic to which the plasmid encoded resistance.
A second discovery that greatly impacted modern genetic engineering was that bacteria have their own parasites. As mentioned earlier, there are viruses that infect and kill bacteria, just as viruses infect animals and plants. The analysis of bacterial viruses was a major avenue to understanding the bacteria themselves.
For example, investigation of the ability of some viruses to grow well on certain strains of bacteria and not others lead to the discovery of restriction-modification systems in bacteria. These systems allow bacteria to recognize and destroy foreign DNA and consist of two parts. A modification enzyme that labels DNA as belonging to the bacterium and a restriction enzyme that recognizes a certain 4-8 base pair sequence in the DNA and cuts it in two if it has not been modified. The landmark paper of Hamilton Smith and Kent Wilcox showed that restriction enzymes were capable of cleaving double-stranded DNA into discrete pieces. The authors correctly hypothesized that the enzyme was recognizing a sequence of DNA and cutting it.
Further studies on restriction enzymes led to the birth of genetic engineering. Janet Mertz and Ronald Davis made restriction enzymes into tools for the manipulation of DNA when they showed that many of the breaks these enzymes made in the DNA produced single-stranded complementary ends. Stanley Cohen, Annie Chang, Robert Helling and Herbert Boyer then demonstrated that any DNA can be broken into fragments with restriction enzymes and by mixing that with a plasmid digested in a similar manner, it was possible to create recombinant molecules. Moving the plasmid into a microbe and growing it on selective medium could produce any desired amount of these recombinant molecules.
Another discovery was "brewing" in the western United States. In the mid 1960s, Thomas Brock was on a trip to Yellowstone and became intrigued by the mats of green, brown and pink material that were found in many of the hot springs. Brock was sure that these were living communities of microorganisms, yet the springs were at temperatures near boiling. Subsequent research proved his hypothesis correct and lead to the isolation of a microbe, Thermus aquaticus, capable of growth at 85 °C.
Organisms capable of growing at this high a temperature were unheard of at the time and the discovery spawned two major developments. First, work with T. aquaticus and other unusual microbes by Woese and many others lead to the discovery of the Archaea. Second, the enzyme responsible for replicating DNA in T. aquaticus was found to be extremely heat stable, surviving temperatures of 95 °C and copying DNA at 72 °C. This enzyme allowed the following important breakthrough. In the early '70s, H. Gobind Khorana and colleagues had suggested a method to amplify DNA in a test tube. As they envisioned it, the method used short oligonucleotides, DNA polymerase and repeated cycles of heating and cooling. However, the polymerase was killed during each heating step, so massive amounts of prohibitively expensive enzyme were required. When Kary Mullis reinvented the method in 1985, the thermostable polymerase of T. aquaticus was readily available, which made the procedure wildly easier and cheaper to apply. He termed it polymerase chain reaction (always referred to as PCR) and it has become a pivotal technique in many areas of science, from detecting bacteria in the environment to the analysis of evidence at crime scenes.
In 1977 Walter Gilbert and Fred Sanger independently developed methods for determining the exact sequence of bases in DNA. These techniques became immediately useful in determining the sequence of numerous important genes being investigated in countless laboratories.
Further refinement has made DNA sequencing more efficient. It became efficient enough that in 1985 Robert Sinsheimer convened a meeting of a number of biologists active in genetics and gene mapping. Out of this meeting came a proposal to sequence the entire three billion base pairs of the human genome. This was equivalent at the time of proposing a mission to the moon, technically possible, but with unpredictable value. The initial cost of the project was estimated to be $10 a base pair or a staggering 30 billion dollars. There was resistance from a significant portion of the scientific community, fearing that the project would siphon funds away from other worthy scientific projects. However, the clarity of the goal ignited the imagination of Congress and the public, creating unstoppable momentum for the project. After about a decade of effort, a preliminary draft of the entire genome was released in the spring of 2001, earlier than projected and dramatically under budget. The project itself drove the development of sequencing technology, allowing the determination of millions of base pairs of sequence. Due to advances in DNA sequencing technology costs have dropped to far below a fraction of a penny per base at large sequencing facilities.
Along with the human genome, hundreds of other organisms have already been sequenced and there will be thousands of sequenced genomes before the decade is out. These include the genomes of many pathogenic bacteria, the mouse, the fruit fly and the nematode Caenorhabditis elegans, the last being an experimental model for development in eukaryotes. The analysis and application of this sequence data to investigate biological problems has resulted in the development of the field of of genomics and bioinformatics. In their infancy, these fields are already producing astonishing findings that are accelerating the progress of fields such as evolutionary biology, immunology, bacterial pathology, bacterial physiology and cancer treatment. Table 1.8 lists important events in understanding the genome.
|1885||Theodor Escherich isolates a microbe from the colon that is later given the name Escherichia coli in his honor. This microbe later becomes the workhorse of molecular biology.|
|1897||Edward Buchner helps launch the field of enzymology by developing a cell extract from yeast that is able to ferment sugar to alcohol.|
|1952||Salvador Luria and Mary Human, and independently Jean Weigle, describe sensitivity in bacteriophage imposed by the host on which it was grown. The viruses are restricted to grow well only on specific strains of bacteria. This later leads to the study of bacterial systems of restriction and modification, and eventually the discovery of restriction enzymes.|
|1959||O. Sawada and others demonstrate that antibiotic resistance can be transferred between Shigella strains and Escherichia coli strains by plasmids.|
|1966||Jon Beckwith and Ethan Signer move the lac region of E. coli into another microorganism to demonstrate genetic control. It is quickly realized that chromosomes could be redesigned and genes moved.|
|1967||Waclaw Szybalski and William Summers develop the technique of DNA-RNA hybridization (mixing nucleic acids together and allowing them to base pair) to investigate the bacteriophage T7. This technique finds wide use in many experiments.|
|1967||Thomas Brock identifies Thermus aquaticus, a bacterium that grows at 85 °C. Heat-stable DNA polymerase is later isolated and used in PCR. Investigation of this organism also leads to the discovery of the domain Archaea.|
|1967||Werner Arber shows that bacterial cells have enzymes capable of modifying DNA by adding methyl groups at cytosines and adenosines. This methylation helps the cell identify its own DNA. Accompanying nucleases recognize these sites and cut the DNA if it is not methylated.|
|1970||Hamilton Smith and Kent W. Wilcox describe the action of restriction enzymes, discovered by Arber, by the purification of one of these enzymes from Haemophilus influenzae.|
|1972||Janet Mertz and Ronald W. Davis establish that the RI restriction enzyme from Escherichia coli cuts at a specific site on the DNA. They also reveal that the cleaved ends of the DNA are complementary, opening the way for cloning.|
|1972||Paul Berg creates the first recombinant DNA molecule from viral and bacterial DNA.|
|1973||Stanley Cohen, Annie Chang, Robert Helling, and Herbert Boyer develop the process of gene cloning.|
|1975-1976||The Asilomar Conference is convened to discuss possible problems associated with gene cloning. A one-year moratorium is suggested, as well as guidelines for cloning research and for genetic engineering.|
|1977||Walter Gilbert and Fred Sanger independently develop methods for determining the sequence of DNA.|
|1980||The U. S. Supreme Court rules that microorganisms altered in the laboratory can be patented.|
|1982||U. S. Pharmaceutical manufacturer Eli Lilly markets the first genetically-engineered human insulin.|
|1983||Jeff Schell and Marc Van Montagu, Mary-Dell Chilton and colleagues move genes into plants.|
|1988||Kary Mullis uses a heat-stable enzyme from Thermus aquaticus to establish PCR technology.|
|1992||The entire sequence of one of the sixteen chromosomes of the yeast Saccharomyces cerevisiae is determined.|
|1995||Craig Venter, Hamilton Smith, Claire Fraser, and colleagues at TIGR elucidate the first complete genome sequence of a microorganism, Haemophilus influenzae. In the ensuing years many laboratories have produced sequences for dozens of microbes including many important pathogens and those of industrial or environmental importance.|
|2000||The human genome project begun in 1990 finished a working draft of the entire human genome.|
Let's end this chapter with a defense of fundamental scientific research. That is, research without an obvious applied goal or application. If you look at the initial results generated in many of these important analyses, the research would have seemed esoteric and inconsequential. Is the analysis of microbes in the colon important? Who cares why a virus can grow in one strain of bacteria and not another? Why should the government support Tom Brock's isolation of microbes from the hot springs of Yellowstone National Park? What use is knowing the DNA sequence of a virus or a bacterial operon? Yet, each of these insights began a thread of inquiry that changed the world. The take-home message is that basic research creates insights and applications beyond the imagination of even the scientists performing the work and should be supported.
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