Students sometimes ask me how I set about to find a single-stranded-DNA-containing virus. How did I know where to look? Of course, single-stranded DNA wasn't the goal. The discovery was a result of serendipity - and belief in one's data. The goal was actually more ambitious - to understand the structure and life cycle of a virus in complete detail! In 1953, that was 'far out.' Biological research in thore days was much more a chancy contest with Naturc. a difficult ascent into the unknown with few obvious hand-holds and relatively primitive equipment. And, if successful. the vistas gained were the more uncxpected. the insights were the more satisfying. In 1953, I took six-months leave from my faculty position at Iowa State to learn the science and techniques of bacteriophage research in Max Dclbruck's laboratory at Caltech. 1 had already been engaged for several years in research with DNA. I thought I might be able to apply my knowledge of this field to the analysis of bactcriophage structure and replication. While in Delbruck's laboratory I came to realize that the principal bacteriophages then under the most intense study - the Tl-T7 series and, particularly, T2 and T4 - were, relatively. large and complex. Given the limited technical capabilities of the time, I felt it would be desirable to study the smallest known bacterial viruses, with the thought that their smallcr genetic cornponcnt (presumably DNA) would be more amendable to manipulation and analysis and that, correspondingly, their functional repertoire would be simpler and morc susceptible to dissection. I discussed this concept with Delbruck, who was not especially encouraging, but neither was he actively discouraging. A search of the literature disclosed two likely candidates, S13 discovered in England and 4x174 which had been discovered in Paris in 1944(l). The evidence suggesting that these viruses were small was suggestive, but tenuous. Large plaques suggested a high diffusion ratc. Penetration through filters and a small cross-section for inactivation by ionizing radiation similarly suggested minute dimensions. After my return to Iowa State, I establishcd a phage laboratory. Fortunately T was able to obtain samples of both S13 and 4x174 from laboratories, respectively in England and France. Research upon any new virus requires its 'domesti-
cation' - the establishment of suitable host cells, of optimal growth and storage conditions, of procedures for growth and purification of the virus on an adequate scale, et al. In my early research, 4x174 appeared to be significantly more stable than did S13, so efforts were concentrated on the former. During this period (1954-1956) we acquired the first analytical ultracentrifuge in Iowa, and it provided our first mcans to assay the purity of our virus preparations. It also provided a sedimentation coefficient for the virus (114S), which, again, was suggestive of a small size. An early electron microscope was available on the campus. When we had achieved a centrifugally pure preparation of q5X3I undertook to learn the operation of the microscope and the current techniques of specimen preparation, so as to 'see' the virus. Fields of uniform, approximately spherical particles, about 30 nanometer diameter. confirmed both the small size of CpX and the purity of the preparation. The preparation of nucleic acid from the virus provided our first hint of an anomaly. Chemical tests proved that i t was DNA and not RNA. (Of course, no RNA-containing phage were known at that time,). But the sedimentation coefficient of the DNA ( 2 4 ) implied (for ordinary double-stranded DNA) a molecular weight of some 16 million daltons. Such a DNA, with a length of some 8 microns, could hardly be accommodated within a virus of the observed dimensions. At this time, I was offered a faculty position at Caltcch and moved my laboratory there with a consequent - fortunately brief - hiatus in research. At Caltech 1 quickly set up a light-scattering instrument to determine both the molecular weight and radius of gyration of the @Xvirus and its DNA. The virus proved ~ daltons and, from to have a particle weight of 6 . 2 lo6 the scattering dissymmetry, a diameter of 29 nanometers. The DN A content of the virus, mcasured by chemical test, appeared to account for 25.5% of the mass, suggesting a DNA mass of 1 . 6 ~ 1 0daltons. ~ Lightscattering measurements of the DNA provided a very similar molecular weight of 1.7X lo6, demonstrating that there was one molecule/virus particle. Lightscattering also provided a dimension: a radius of NaCI). This was gyration of 44 nanometers (in 0 . 2 ~ much too small for a nativc double-stranded D N A of that molecular weight and was indicative of a more flexible, approximately random coil structure (consistent with the sedimentation coefficient). Furthermore, variation of the ultraviolct absorption of @XDNA with temperature and upon treatment with formaldehyde was also indicative of a single-stranded or denatured form of DNA as opposed to the conventional doublestranded form. Demonstration uf the formaldehyde effect upon the absorption of the DNA in the intact virus confirmed that this property was not a consequence of some modification during extraction. All of this accumulating evidence suggested more and more strongly that I had here an unusual form of DNA.
However, within six years of its publication in 1953. the double-stranded model of Watson and Crick had become dogma. Thus, we continued to try to force the data into a double-stranded model, perhaps one that was denatured so as to fit into the small virus coat, and one that was somehow inhibited from renaturation after extraction, Then, finally and definitively, I determined yuantitatively that the nucleotide composition of the viral DNA did not display the molar equivalence of G and C, and particularly of A and T, so characteristic of doublestranded DNA. The conclusion was unavoidable that we were dealing with a naturally single-stranded viral DNA genome. Published in 1959(2,3),the discovery of a singlestranded DNA in a virus was most unexpected and raised immediate questions as to its mode of replication. To resolve this, we pursued the viral DNA into the cell to ascertain its fate. One essential technique for this study was the development in my laboratory, largely by George Guthrie, of an assay for the infectivity of the free viral DNA? using bacterial protoplasts(4). Using this assay, together with isotopic techniques, we demonstrated that , once inside the infected cell, the single-stranded viral DNA was converted to a doublestranded form (called the 'replicative form'), which was then transcribed and replicated in the normal semiconservative mode('). Later in the infection the singlestranded progeny viral DNA was produced from the replicative form templates. Having available an intact and infective viral DNA led us to pose questions, which in turn led to another surprising result. In particular, we were curious as to the nature of the ends of this remarkable DNA. To investigate this, we studied the effect of highly purified exonucleases which could degrade DNA, nucleotide by nucleotide from either the S'-end or the 3'-end. Remarkably, in cxperiments conducted by Walter Fiers, then a post-doctoral fellow in my laboratory, these exonucleases released only a minute amount of mononucleotide and had no effect upon the infectivity of the DNA. In early discussion of these experimcnts, I had, jokingly, suggested that, possibly, there were no ends, that the DNA was a ring. Now, taking this concept seriously, I had to ask how we could demonstrate such a structure. (At that time clectron microscopy - the obvious method - was not yet applicable to DNA). In my first paper on 4X DNA. I had noted the presence of a second, distinct centrifugal component in our preparations, of variable proportion, sediinenting about 10-15% slower than the major DNA component. Subsequent infectivity studies had suggested that the relative specific infectivity of different DNA preparations correlated with the proportion of DNA in the leading component. Now it occurred to me that if the DNA were a ring, the once-cut DNA would be a discrete component, which would sediment some
10-15 % slower and which, in all probability, would not be infective. The explanation for our prcvious results and the design of a definitive experiment became obvious: Start with a DNA preparation, composed almost entirely of the faster-sedimenting component. Cut this, slowly, with a very low concentration of dcoxyribonuclease. Assuming a Poisson distribution, assay the average number of cuts per molecule by the decline in infectivity. at several time intervals. At each time point, determine, centrifugally, the amount of DNA remaining in the faster sedimenting component (uncut), the amount moved into the discrete. slowersedimenting component (once-cut), and the amount moved into disperse, slower-sedimenting material (twice-or more-cut). I derived the equations for the proportions of these components as a function of the number of cuts and Walter Fiers did the experiments. The results clearly confirmed the ring but, being inferential, were greeted with some skepticism. Alice Burton, another post-doctoral fellow in my laboratory. then applied the same principles to establish the ring character of the double-stranded replicative form DNA(9). Shortly after these experiments were completed, Prof. Kleinschmidt at UC Berkeley developed his shadowing method for observation of doublestranded DNA in the electron rnicroscopc. I took some replicative form DNA to his laboratory. The first field examined was rcplete with DNA rings of the expected circumference('*). 'Seeing was believing' and the skeptics were convinced. A few years later, Dr Freifelder developed a technique to observe smglestranded DNA in the electron microscope which confirined the ring structure of viral @X DNA("). Thcse experiments were possible becausc we had for the first time - an intact DNA molecule. All earlier studies of DNA had been performed with fragments. All of these results validated the choice of a small virus, 9x3174. as a favorable object of viral research and a source of a tractable DNA. The subsequent use of the $X DNA molecule as the subject, in Sanger's laboratory. for the determination of the first complete viral DNA sequence(") - and the revelations thus made available - further confirmed the value of that choice, made back in 1953.
References AND BOULGA~OV, N . (1935). Clasiificalion et idcntification deb rgphi-phages. C. R. SOC.B i d . Puvii 199, 1270-1272. 2 SINSHLIMLR, R. L. (1959). Purifica~ion and properties of bactei-iophage 4x174. J . 1\40!. B i d 1 37-42. 3 SIUSHEIMER, R. L. (1959). A single-stranded deoxy-ribonucleic acid frum bacteriophage @X174.J. Mol. B i d . 1 , 43-53. 4 GUTHIUT, G. D. A K D SINSH~IILIFR, R . L. (1960). Inkclion nf protoplasts oi EF;c.chrvzdiiaCuli hy suhviral particleb of bacteriophage 4x174. J. Mol. B i d . 2. 297-305. 5 SIKSHFIMER. R. L.. SI1ARMAN. B.. NAGLLK, C'. A Y D GIITHRIE, S. (1Y62). '1he process ol infection with bacteriophage 4x174. I Evidence for a 'Replicative Form'. J . i M d . B i d . 4. 132-160.
1 SERTIC.V.
6 FIEKS, W. AND SINSHETkrER. R. L. (1962). The structure ol the DNA of bacteriophagc pX174. I The action of exopolynucleo~iddsel.J . Mol. Uiol. 5 , 4008-419. 7 FIERS, W. A N D SINSHWMRR. R. I.. (1962). I h e 5tructure of the DNA of bacteriophage +X174. 1I Thermal inaclivatiun. 1.Mol Uiol. 5 , 420-4113. 8 FIERS,W. ~ N DSINSHEIMER, K. L. (1962). The structure of the DNA of bacteriophage ~/1X174.I11 Ultracentrifugal evidence tor a ring sti-ucturc..I. Mol. Bid. 5 , 424-435. 9 BIJRTON, A. NU SIUSIIBIM~K. R. L. (lY63). PTocess of infection with 4x174: Effect nf exonuclcases on the replicative form. Science 142, 962-963. 10 KI ELNSCHMIUT, A. K., B L K I U N A., .khD SINSHEIMER, R . L. (1963). Electron microscopy 01 the replicative form of the DNA of bactcriophage $X174. Science 142. 961.
11 FKEI~ELDER. D.. KI~FINS~HMIDT, A. K. AND SINSHEIMER, K. 1. (196.4). Elcctroii microscopy of single-btranded DNA. Circularity of DNA of bactcriophage chXl74. ,Scrrnre 146. 254-255 12 SANCER.F., COUI.SON. A. R . , FKLLDMAN. T., AIR. G. M., BARRELL. B. G., BROWN;N. L.. FIDDES, J . C., HLIICHISOY, C. A , . 111. SLOCOMBE, P. M. AND SMTTH.M. (1978). 'The nucleotide sequence of bactcriophasc 4x174. J. Mol. Biol. 125, 225-246.
Robert L. Sinsheimer is at thc Department of Biological Sciences, University of California, Saiita Barbara, CA 93106, USA.
cation' - the establishment of suitable host cells, of optimal growth and storage conditions, of procedures for growth and purification of the virus on an adequate scale, et al. In my early research, 4x174 appeared to be significantly more stable than did S13, so efforts were concentrated on the former. During this period (1954-1956) we acquired the first analytical ultracentrifuge in Iowa, and it provided our first mcans to assay the purity of our virus preparations. It also provided a sedimentation coefficient for the virus (114S), which, again, was suggestive of a small size. An early electron microscope was available on the campus. When we had achieved a centrifugally pure preparation of q5X3I undertook to learn the operation of the microscope and the current techniques of specimen preparation, so as to 'see' the virus. Fields of uniform, approximately spherical particles, about 30 nanometer diameter. confirmed both the small size of CpX and the purity of the preparation. The preparation of nucleic acid from the virus provided our first hint of an anomaly. Chemical tests proved that i t was DNA and not RNA. (Of course, no RNA-containing phage were known at that time,). But the sedimentation coefficient of the DNA ( 2 4 ) implied (for ordinary double-stranded DNA) a molecular weight of some 16 million daltons. Such a DNA, with a length of some 8 microns, could hardly be accommodated within a virus of the observed dimensions. At this time, I was offered a faculty position at Caltcch and moved my laboratory there with a consequent - fortunately brief - hiatus in research. At Caltech 1 quickly set up a light-scattering instrument to determine both the molecular weight and radius of gyration of the @Xvirus and its DNA. The virus proved ~ daltons and, from to have a particle weight of 6 . 2 lo6 the scattering dissymmetry, a diameter of 29 nanometers. The DN A content of the virus, mcasured by chemical test, appeared to account for 25.5% of the mass, suggesting a DNA mass of 1 . 6 ~ 1 0daltons. ~ Lightscattering measurements of the DNA provided a very similar molecular weight of 1.7X lo6, demonstrating that there was one molecule/virus particle. Lightscattering also provided a dimension: a radius of NaCI). This was gyration of 44 nanometers (in 0 . 2 ~ much too small for a nativc double-stranded D N A of that molecular weight and was indicative of a more flexible, approximately random coil structure (consistent with the sedimentation coefficient). Furthermore, variation of the ultraviolct absorption of @XDNA with temperature and upon treatment with formaldehyde was also indicative of a single-stranded or denatured form of DNA as opposed to the conventional doublestranded form. Demonstration uf the formaldehyde effect upon the absorption of the DNA in the intact virus confirmed that this property was not a consequence of some modification during extraction. All of this accumulating evidence suggested more and more strongly that I had here an unusual form of DNA.
However, within six years of its publication in 1953. the double-stranded model of Watson and Crick had become dogma. Thus, we continued to try to force the data into a double-stranded model, perhaps one that was denatured so as to fit into the small virus coat, and one that was somehow inhibited from renaturation after extraction, Then, finally and definitively, I determined yuantitatively that the nucleotide composition of the viral DNA did not display the molar equivalence of G and C, and particularly of A and T, so characteristic of doublestranded DNA. The conclusion was unavoidable that we were dealing with a naturally single-stranded viral DNA genome. Published in 1959(2,3),the discovery of a singlestranded DNA in a virus was most unexpected and raised immediate questions as to its mode of replication. To resolve this, we pursued the viral DNA into the cell to ascertain its fate. One essential technique for this study was the development in my laboratory, largely by George Guthrie, of an assay for the infectivity of the free viral DNA? using bacterial protoplasts(4). Using this assay, together with isotopic techniques, we demonstrated that , once inside the infected cell, the single-stranded viral DNA was converted to a doublestranded form (called the 'replicative form'), which was then transcribed and replicated in the normal semiconservative mode('). Later in the infection the singlestranded progeny viral DNA was produced from the replicative form templates. Having available an intact and infective viral DNA led us to pose questions, which in turn led to another surprising result. In particular, we were curious as to the nature of the ends of this remarkable DNA. To investigate this, we studied the effect of highly purified exonucleases which could degrade DNA, nucleotide by nucleotide from either the S'-end or the 3'-end. Remarkably, in cxperiments conducted by Walter Fiers, then a post-doctoral fellow in my laboratory, these exonucleases released only a minute amount of mononucleotide and had no effect upon the infectivity of the DNA. In early discussion of these experimcnts, I had, jokingly, suggested that, possibly, there were no ends, that the DNA was a ring. Now, taking this concept seriously, I had to ask how we could demonstrate such a structure. (At that time clectron microscopy - the obvious method - was not yet applicable to DNA). In my first paper on 4X DNA. I had noted the presence of a second, distinct centrifugal component in our preparations, of variable proportion, sediinenting about 10-15% slower than the major DNA component. Subsequent infectivity studies had suggested that the relative specific infectivity of different DNA preparations correlated with the proportion of DNA in the leading component. Now it occurred to me that if the DNA were a ring, the once-cut DNA would be a discrete component, which would sediment some
10-15 % slower and which, in all probability, would not be infective. The explanation for our prcvious results and the design of a definitive experiment became obvious: Start with a DNA preparation, composed almost entirely of the faster-sedimenting component. Cut this, slowly, with a very low concentration of dcoxyribonuclease. Assuming a Poisson distribution, assay the average number of cuts per molecule by the decline in infectivity. at several time intervals. At each time point, determine, centrifugally, the amount of DNA remaining in the faster sedimenting component (uncut), the amount moved into the discrete. slowersedimenting component (once-cut), and the amount moved into disperse, slower-sedimenting material (twice-or more-cut). I derived the equations for the proportions of these components as a function of the number of cuts and Walter Fiers did the experiments. The results clearly confirmed the ring but, being inferential, were greeted with some skepticism. Alice Burton, another post-doctoral fellow in my laboratory. then applied the same principles to establish the ring character of the double-stranded replicative form DNA(9). Shortly after these experiments were completed, Prof. Kleinschmidt at UC Berkeley developed his shadowing method for observation of doublestranded DNA in the electron rnicroscopc. I took some replicative form DNA to his laboratory. The first field examined was rcplete with DNA rings of the expected circumference('*). 'Seeing was believing' and the skeptics were convinced. A few years later, Dr Freifelder developed a technique to observe smglestranded DNA in the electron microscope which confirined the ring structure of viral @X DNA("). Thcse experiments were possible becausc we had for the first time - an intact DNA molecule. All earlier studies of DNA had been performed with fragments. All of these results validated the choice of a small virus, 9x3174. as a favorable object of viral research and a source of a tractable DNA. The subsequent use of the $X DNA molecule as the subject, in Sanger's laboratory. for the determination of the first complete viral DNA sequence(") - and the revelations thus made available - further confirmed the value of that choice, made back in 1953.
References AND BOULGA~OV, N . (1935). Clasiificalion et idcntification deb rgphi-phages. C. R. SOC.B i d . Puvii 199, 1270-1272. 2 SINSHLIMLR, R. L. (1959). Purifica~ion and properties of bactei-iophage 4x174. J . 1\40!. B i d 1 37-42. 3 SIUSHEIMER, R. L. (1959). A single-stranded deoxy-ribonucleic acid frum bacteriophage @X174.J. Mol. B i d . 1 , 43-53. 4 GUTHIUT, G. D. A K D SINSH~IILIFR, R . L. (1960). Inkclion nf protoplasts oi EF;c.chrvzdiiaCuli hy suhviral particleb of bacteriophage 4x174. J. Mol. B i d . 2. 297-305. 5 SIKSHFIMER. R. L.. SI1ARMAN. B.. NAGLLK, C'. A Y D GIITHRIE, S. (1Y62). '1he process ol infection with bacteriophage 4x174. I Evidence for a 'Replicative Form'. J . i M d . B i d . 4. 132-160.
1 SERTIC.V.
6 FIEKS, W. AND SINSHETkrER. R. L. (1962). The structure ol the DNA of bacteriophagc pX174. I The action of exopolynucleo~iddsel.J . Mol. Uiol. 5 , 4008-419. 7 FIERS, W. A N D SINSHWMRR. R. I.. (1962). I h e 5tructure of the DNA of bacteriophage +X174. 1I Thermal inaclivatiun. 1.Mol Uiol. 5 , 420-4113. 8 FIERS,W. ~ N DSINSHEIMER, K. L. (1962). The structure of the DNA of bacteriophage ~/1X174.I11 Ultracentrifugal evidence tor a ring sti-ucturc..I. Mol. Bid. 5 , 424-435. 9 BIJRTON, A. NU SIUSIIBIM~K. R. L. (lY63). PTocess of infection with 4x174: Effect nf exonuclcases on the replicative form. Science 142, 962-963. 10 KI ELNSCHMIUT, A. K., B L K I U N A., .khD SINSHEIMER, R . L. (1963). Electron microscopy 01 the replicative form of the DNA of bactcriophage $X174. Science 142. 961.
11 FKEI~ELDER. D.. KI~FINS~HMIDT, A. K. AND SINSHEIMER, K. 1. (196.4). Elcctroii microscopy of single-btranded DNA. Circularity of DNA of bactcriophage chXl74. ,Scrrnre 146. 254-255 12 SANCER.F., COUI.SON. A. R . , FKLLDMAN. T., AIR. G. M., BARRELL. B. G., BROWN;N. L.. FIDDES, J . C., HLIICHISOY, C. A , . 111. SLOCOMBE, P. M. AND SMTTH.M. (1978). 'The nucleotide sequence of bactcriophasc 4x174. J. Mol. Biol. 125, 225-246.
Robert L. Sinsheimer is at thc Department of Biological Sciences, University of California, Saiita Barbara, CA 93106, USA.
