Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
© 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com book review The 1966 Phage and the origins of molecular biology6, edited by John Cairns, Gunther Stent, and James Watson, offered a genealogy of molecular biology with Delbrück as founding father. The book’s implicit message that molecular biology derived from the phage group drew fire from some. John Kendrew argued that structural biologists (such as those in his unit at Cambridge) had also played a crucial role in the founding of molecular biology7. Gunther Stent subsequently offered a conciliatory view of the origins of molecular biology that combined the ‘informational’ school of phage genetics with the ‘structural’ school of X-ray crystallographers, a meeting of the minds that was conveniently personified by Watson and Crick8. This enlarged family tree still did not satisfy everyone; Origins of molecular biology: A tribute to Jacques Monod9, stressed the early importance of French microbiologists. Nonetheless, historical interpretations that emphasized these various ‘origins’ tended to demarcate molecular biology firmly from pre-existing fields, especially biochemistry (Delbrück famously disparaged biochemistry). By all these accounts, Hershey makes an odd revolutionary. He is described by friends as a “biochemist’s biochemist” as well as a pioneering molecular geneticist. His research defied any dichotomy between informational and structural approaches, and he retained a skeptical attitude towards the supremacy of nucleic acids. Attention to Hershey as one of the original molecular biologists complicates the story, showing the limitations of views that emphasize a single approach, dogma, or charismatic leader. By portraying a trailblazing biologist who combined structural, biochemical, and genetic methods in his quest to understand life at the molecular level, We can sleep later cautions us to keep a broad view of molecular biology’s past — and its future. Angela N. H. Creager is in the Department of History and Program in History of Science, Princeton University, Princeton, New Jersey 08544, USA. email: [email protected] 1. As quoted in Judson, H.F. The eighth day of creation: The makers of the revolution in biology, 275 (Simon and Schuster, New York; 1979). 2. Hershey, A.D. & Rotman, R. Genetics 34, 44–71 (1949). 3. Hershey, A.D. & Chase, M. J. Gen. Physiol. 36, 39–56 (1952). 4. Avery, O.T., MacLeod, C.M. & McCarty, M. J. Exp. Med. 79, 137–158 (1944). 5. Hershey, A.D., ed. The bacteriophage lambda. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1971). 6. Cairns, J., Stent, G.S. & Watson, J.D. Phage and the origins of molecular biology. (Cold Spring Harbor Press, Cold Spring Harbor, New York; 1966). 7. Kendrew, J. Sci. Am. 216, 141–43 (1967). 8. Stent, G.S. Science 160, 390–395 (1968). 9. Lwoff, A. & Ullmann, A. Origins of molecular biology: A tribute to Jacques Monod. (Academic Press, New York; 1979). history Phage facts Often the simplest experiments lead to the most remarkable insights. So it was with the famous fluctuation experiments of Luria and Delbrück and the Waring blender experiments of Hershey and Chase for which they were awarded the Nobel Prize in Physiology or Medicine in 1969. While the results of these experiments are permanently etched into every first year biology student’s brain, it is worth recalling what was known at the time these experiments were conducted and the conclusions the authors drew. In the early 20th century, the role of chance mutations in the genetic variation of higher organisms was generally accepted. However, bacterial cultures seemed to be plastic. Exposed to adverse conditions, cultures of bacteria could give rise to resistant variants that remained resistant “to the action of the virus even if subcultured through many generations in the absence of the virus”1. At the time, many scientists believed that the “virus by direct action induced the resistant variants”1. Others believed that the “resistant bacterial variants are produced by mutation in the culture prior to the addition of virus. The virus merely brings the variants into prominence by eliminating all sensitive bacteria”1. These two alternative theories — induced immunity and selection of spontaneous mutations — were rigorously tested both theoretically and experimentally by Max Delbrück and Salvadore E. Luria, respectively, in 1943. The two hypotheses lead to different predictions regarding the distribution of resistant bacteria in a series of parallel cultures. The hypothesis of acquired or induced immunity predicts that the number of mutants in each culture would be clustered around the mean with little variation between cultures. According to the selection hypothesis, resistant bacteria could arise at any point in the life of the culture. This would result in very different numbers of mutants per culture and thus the variability from culture to culture would be high. Luria found that in every experiment the “fluctuation of the numbers of resistant bacteria is tremendously higher than could be accounted for by the sampling errors, in striking contrast to the results of plating from the same culture and in conflict with the expectations from the hypothesis of acquired immunity”1. Luria and Delbrück concluded that the “resistance to virus is due to a heritable change of the bacterial cell which occurs independently of the action of the virus”1. In other words, the mutation was there before the virus was added. nature structural biology • volume 8 number 1 • january 2001 Later, when Alfred D. Hershey and Martha Chase began their experiments, little was known about the early steps of phage infection. What was known was that after the phage adsorbed to the bacteria there was a latent period of ∼10 minutes after which time infectious virus particles were made, ultimately leading to host cell lysis and phage release. Hershey and Chase reasoned that if they knew the fate of the viral protein and the nucleic acid at the beginning of phage infection they would understand more about the nature of those early steps. They labeled the phage with either 35S or 32P, allowed them to adsorb to the bacteria and then the cells were separated from the unadsorbed material by centrifugation. The cells were resuspended and the suspension was spun in a Waring blender to separate the phage from the infected bacteria. They found that ∼80% of the 35S label was removed from the bacteria whereas only ∼20% of the 32P label was removed. They concluded that “most of the phage DNA enters the cell, and a residue containing at least 80% of the sulfur-containing protein of the phage remains at the cell surface”2. Together with the 1944 experiments of Oswald Avery3 in which nucleic acid, not protein, was shown to have “transforming” properties, these findings persuaded most scientists that DNA carried the genetic information. Curiously, however, Hershey himself remained skep19 © 2001 Nature Publishing Group http://structbio.nature.com history © 2001 Nature Publishing Group http://structbio.nature.com tical. Hershey and Chase concluded their paper by saying “the sulfur-containing protein has no function in phage multiplication, and the DNA has some function”2. Talk about an understatement. More bons mots from and about Hershey can be found in “We can sleep later: Alfred D. Hershey and the origins of molecular biology” which is reviewed on page 18 of this issue. Boyana Konforti 1. Luria, S.E. & Delbrück, M. Genetics 28, 491–511 (1943). 2. Hershey, A.D. & Chase, M. J. Gen. Physiol. 36, 39–56 (1952). 3. Avery, O.T., MacLeod, C.M. & McCarty, M. J. Exp. Med. 79, 137–156 (1944). picture story A DNA wormhole The bacteriophage φ29 is a virus that infects the bacterium Bacillus subtilis. The mature φ29 contains a head region that encloses its genome and a tail region that contacts the cell surface to initiate the infectious cycle. In the late stage of the maturation process from which functional phage particles emerge, the phage genome — a linear double-stranded DNA of ∼19,000 base pairs — is packaged into a preassembled empty head (called the prohead). This process requires condensing the viral genome, which would span ∼6,400 Å if it were completely extended, into the phage head, which is only ∼500 Å in length. How the phage performs this remarkable task is not yet fully understood. At one end of the phage head is a small opening through which the viral genome enters and exits. A protein complex called the ‘connector’ — so named because the tail of the phage is connected to the head through this complex — sits at the opening and participates in the DNA packaging process. To understand the structural basis of how the connector regulates DNA packaging, Simpson et al. (Nature, 408, 745–750; 2000) have determined its high resolution structure. The connector complex consists of 12 identical subunits (top and middle panels, one monomer is colored red to illustrate how the subunits are assembled in the complex). The overall shape of the complex is like a bottle stopper (side view; middle panel) with a central channel (top 20 view; top panel). The narrowest part of the channel is ∼36 Å in diameter; a double stranded DNA could easily pass through the channel without any steric problems. To characterize how the connector fits into the phage head, Simpson et al. generated a three-dimensional reconstruction of the prohead from cryo-electron microscopy data and then fit the crystal structure into the electron density. The result shows that the narrow part of the connector protrudes outside of the viral shell (bottom panel, a thin section across the center of the prohead along the axis of the connector central channel is shown; the red mesh wire represents the electron density from the prohead and the DNA is modeled in the superposition for illustration purposes). This region of the connector interacts with a phage-encoded RNA (marked by green mesh wire) and possibly a viral ATPase (not present in this reconstruction), both of which had been shown to be important for DNA packaging. With the translocating DNA as the movable central shaft, the connector, the viral RNA and ATPase appear to constitute a ‘motor’, with the viral ATPase powering conformational changes in the connector complex. The energy of ATP hydrolysis is converted into a translational motion of DNA. This study thus provides a glimpse of the initial event in the DNA packaging process of bacteriophage φ29. Hwa-ping Feng Adapted from Simpson et al. nature structural biology • volume 8 number 1 • january 2001