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ID 16395

Animation18: Bacteria and viruses have DNA too.

Description:
Joshua Lederberg worked with bacterial genetics while Alfred Hershey showed that DNA is responsible for the reproduction of new viruses in a cell.
Transcript:
Hello, I'm Joshua Lederberg. In 1945, I was a graduate student in Edward Tatum's lab when I read Avery's paper on the transforming ability of DNA. I became very excited by the possibilities. How was the DNA getting from one bacterium to another? One possibility is that the bacteria mate and physically exchange DNA — the bacterial equivalent of sex! My supervisor, Edward Tatum, made mutations in the bacteria, Escherichia coli (E. coli), to further study the "one gene, one enzyme" phenomenon. Like Neurospora, E. coli can normally synthesize all the nutrients it needs. For example, E. coli has enzymes that bind and convert precursor molecules into essential nutrients like amino acids methionine (MET), proline (PRO), and threonine (THR), as well as the vitamin biotin (BIO). The mutant strains Tatum made were unable to synthesize some of these nutrients. For example, Mutant#1 had two genetic mutations (met- and bio-), which made it unable to synthesize the amino acid methionine (MET) or the vitamin biotin (BIO). It was still able to make all the other amino acids and vitamins. On the other hand, Mutant#2 had two genetic mutations (pro- and thr- ), which made it unable to synthesize the amino acids proline (PRO) or threonine (THR). It could make all the other amino acids and vitamins. These mutant strains can grow on agar plates if the right supplements are added to the media. Of course, we need a microscope to see a single bacterium, but on agar plates what we "see" are bacterial colonies. Each colony contains thousands of genetically identical bacteria, because each colony starts from one bacterial cell that divides multiple times. Since Mutant#1 could make what Mutant#2 needed and vice versa, I used these two mutants to test for genetic exchange. First, I mixed the two mutant strains and grew them together in culture medium containing all four supplements. After the two strains had grown together for some time, I spread them onto a culture plate with no supplements, and let them grow overnight. The spreading technique isolates individual bacterial cells. Therefore, any cell that survives must have all the genes needed to make all four nutrients. The survivors would reproduce and I'd see it as a visible colony on the plate. After the two strains had grown together for some time, I spread them onto a culture plate with no supplements, and let them grow overnight. The spreading technique isolates individual bacterial cells. Therefore, any cell that survives must have all the genes needed to make all four nutrients. The survivors would reproduce and I'd see it as a visible colony on the plate. As I hoped, when I examined the plate the following morning, I saw that a few bacterial colonies had grown on the unsupplemented plate. The only way that any bacteria could grow on this unsupplemented plate is if one mutant had "donated" a copy of its genes to the other. I calculated that this exchange occurred in about one in every 10 million bacteria grown together in the flask. I named this process of gene exchange conjugation, and believed it had to occur through direct contact between bacteria. Later, William Hayes found that conjugation always occurs between bacteria of different "mating types" — the bacterial equivalent of sexes. First, a bridge, or pilus, forms between the two bacteria. Then, genes move through the pilus from the "+" mating type to a "—" mating type. Gene transfer can be interrupted by shaking bacterial cultures using a blender. The agitation breaks the pilus, which connects the mating pair. By interrupting mating at increasing time intervals, more genes are transferred. The order of some bacterial genes was determined using this method. These experiments showed that bacteria mate and exchange genes, much like plants and animals. This convinced scientists that bacteria can be used as models for looking at gene function in higher organisms. Hello, I'm Alfred Hershey. While Lederberg was doing his work on bacterial genetics, a group of us at Cold Spring Harbor Laboratory were studying bacteriophage genetics. Bacteriophage, or phage for short, are viruses that specifically attack and infect bacteria. Phage rely on bacteria to reproduce. We knew from electron micrographs that during infection, phage attach to bacteria by their tails. We assumed that after attaching, genes are pumped into the bacterial host, which then direct the bacterium's enzymes to replicate new phage particles. We set out to determine exactly what caused the "transformation" of bacteria into a phage-producing factory. Could it be that, as suggested by Avery's work, phage DNA was a "transforming principle?" In 1952, my colleague Martha Chase and I decided to test these ideas. From previous chemical analyses, we knew that DNA is high in phosphorus (P) atoms but has no sulfur (S). Conversely, proteins contain sulfur atoms, but have no phosphorus. So knowing this, we used radioactive phosphorous ( P) or sulfur ( S) to selectively label phage DNA and protein. We then designed an experiment to test which component entered the bacteria for infection. In two parallel experiments, we combined the radiolabeled phage with bacteria that were not labeled. We waited long enough for the phages to attach, and then disrupted the attachment by mixing the culture in a Waring blender. Next, we spun the samples in a centrifuge to separate the phage from the bacteria. Because the bacteria are larger and heavier than phage, the bacteria collect at the bottom of the test tube as a pellet, while the phage stay in suspension in the supernatant. Let's first examine the results from the S samples. We saw that the S label stayed with the suspended phage and not the pellet of bacteria. The new phage made by these infected bacteria did not contain radioactive sulfur. Therefore, the phage coat, which is made of protein, was not used inside the bacteria to make new phage particles. When we looked at the 32P samples, we found that 32P always pelleted with the bacteria. Moreover, new phage made by these infected bacteria contained radioactive 32P. Therefore, phage DNA was used inside the bacteria to make new phage particles. The phage coat is just the package that delivers the phage DNA into the bacteria. We concluded that the phage DNA alone carries the instructions needed to replicate phages inside the bacteria. So, DNA is the genetic material.
Keywords:
joshua lederberg, precursor molecules, alfred hershey, bacteria escherichia coli, genetic mutations, mutant strains, bacterial genetics, edward tatum, amino acids, neurospora, e coli
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