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In the early part of the 20^thcentury techniques were developed to examine the inner workings of the cell and researchers performed much of the work 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, we understood the nature of inheritance and heredity. 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 he injected the rough strains 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 awaiting Griffith when he injected a mixture of dead cells of a smooth isolate with live cells of a rough isolate into mice: 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 was 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 transformation 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 ten years later, Francis Crick, Rosalind Franklin, James Watson, and Maurice Wilkins worked on experiments describing the structure of DNA and hypothesized about its replication. 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 significant contribution in understanding this puzzle were the cell-free systems developed by Paul Zemecnik and his laboratory. The cell-free systems first used rat liver and later the bacterium E. coli. He and other scientists used these systems to study translation in the test tube, discovering many of 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). This molecule helps to create amino acid polymers based on the nucleic acid sequence. Brenner, Francois Jacob, and Matthew Meselson revealed another important part of the puzzle when they discovered that the ribosome was the site of translation and that the molecule it translated is RNA, not DNA. Marshall Nirenberg and J. H. Matthaei solved the next mystery by 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, we now understood the framework for converting genetic information into proteins. Further work demonstrated that this basic mechanism is 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.

Work on microbes paves the way to taking control of the genome

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. Little did Dr. Escherich know at that 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:

1. E. coli is easy to cultivate. It can grow on a wide variety of media, at temperatures ranging from 20-45 °C, in the presence or absence of air.
2. E. coli grows quickly. Under normal laboratory conditions, it goes through a complete life cycle and doubles its population about every 30 minutes. With this rapid growth rate, it is possible to generate large populations in just a few hours. Researchers can set up most experiments one day and observe results the next.
3. It can survive storage for long periods of time without losing viability using relatively simple preservation techniques.
4. It was available. There is an element of arbitrariness that this microbe came to serve such a vital role in biology. Many other bacteria probably could have filled this position, but E. coli was the one chosen.

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. Scientists later found that one class of plasmids encoded resistance genes that would enable 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 a 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, some viruses 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 could cleave 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 restriction enzymes could break any DNA into fragments, and by mixing that with a plasmid digested similarly, it was possible to create recombinant molecules. Moving the plasmid into a microbe and growing it on a 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 he 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 led to the isolation of a microbe, Thermus aquaticus, capable of growth at 85 °C.

Organisms capable of growing at this high 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 is extremely heat stable, surviving temperatures of 95 °C and copying DNA at 72 °C. This enzyme allowed the next 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 heating and cooling cycles. However, the cycle denatures the polymerase during each heating step so that the procedure would require massive amounts of prohibitively expensive enzymes. 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 several 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 project was equivalent at the time of proposing a mission to the moon, technically possible but with an unpredictable value. Scientists estimated the project's initial cost 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 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.

Due to the ease of DNA sequencing, there are now over 100,000 sequenced genomes. 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 use of sequence data to investigate biological problems has resulted in the development of the fields of genomics and bioinformatics. In their infancy, these fields are already producing astonishing findings that accelerate the progress of evolutionary biology, immunology, bacterial pathology, bacterial physiology, and cancer treatment. Table 1.8 lists important events in understanding the genome.

Let'send 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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