History of phage

Frederick W. Twort is the director of the Brown Institute in London. Twater tried to find a vaccine virus variant for smallpox vaccine, which can replicate in living extracellular media. In one experiment, he inoculated a part of smallpox vaccine into a Petri dish containing nutrient agar. Although virus replication failed, bacterial pollutants grew rapidly on agar plates. Twater continued his cultivation and noticed that some bacterial colonies showed a "watery appearance" (that is, they became more transparent). When further cultured, such colonies can no longer replicate (that is, the bacteria are killed). Twater called this phenomenon transparent transformation. He went on to prove that infecting a normal bacterial colony with the principle of transparent transformation would kill the bacteria. This transparent entity can easily pass through ceramic filters and can be diluted by a million times. When placed on fresh bacteria, it will recover its strength or titer.

Twater published a short article describing this phenomenon, arguing that the explanation for his observation was the existence of a bacterial virus. Because of his service in World War I, Twater's research was interrupted. After returning to London, he didn't continue this research, so he didn't make further contributions in this field.

Meanwhile, Felix d'Herelle, a Canadian medical bacteriologist, is working at the Pasteur Institute in Paris. 1965438+In August 2005, a French cavalry squadron was stationed in Maisons-Lafitte, a suburb of Paris. A severe dysentery caused by Shigella caused a devastating blow to the whole army. Deherer filtered the patient's feces and quickly isolated Shigella from the filtered emulsion and cultured it. Bacteria keep growing, covering the surface of the Petri dish. De Herrell accidentally observed a transparent spot where no bacteria grew. He called these things milk spots or plaques. De Herrell tracked the whole infection process of a patient, observing when the bacteria were the most and when spots appeared. Interestingly, the patient began to improve on the fourth day after infection.

De Herrell called these viruses "bacteriophages", and then he invented a method that is still used in virology research. He diluted the plaque in a limited way to determine the concentration of the virus. His inference is that the appearance of spots indicates that the virus is a particle or microparticle. In his research, De Herrell also proved that the first step of virus infection is the attachment (adsorption) of pathogens to host cells. He proved this by mixing the virus with the host cell and precipitating it. He also proved that the virus does not exist in the supernatant. The virus attachment only occurs when bacteria are sensitive to the virus mixed with them, which indicates that the virus has a specific adsorption range for host cells. He also described the release of pyrolysis in clear modern terms. De Herrell is one of the founders of modern virology principles in many ways.

By 192 1 year, more and more lysogenic bacterial strains have been isolated, and it is impossible to isolate the virus from the host in some experiments. This makes Jules Bourdette of the Pasteur Institute in Brussels think that the infectious pathogen described by De Herrell is just a bacterial enzyme that promotes self-reproduction. Although this is a wrong conclusion, it is very close to the current view on prion structure and replication.

In the 1920s and 1930s, De Herrell devoted himself to exploring the application of his research results in medicine, but there was no result. The basic research at that time was often influenced by the strong personality of individual scientists in this field. Obviously, there are many different phages, some of which are lysogenic and some of which are lysogenic, but the relationship between them is still unclear. The important discovery in this period was Max Schlesinger, who proved that the maximum diameter of purified phage was 0. 1 micron and the mass was about 4x 10g. They are composed of protein and DNA in roughly equal proportions. At that time, no one clearly knew how to use this observation, but it had a great influence in the following 20 years.

Modern: 1938-1970

Max Delbruck is a physicist trained by Jiting University. His first job was at the Wilhelm Institute of Chemistry in Berlin, where he actively discussed the relationship between quantum physics and genetics with some researchers. Delbruck's interest in this field led him to invent the quantum mechanical model of genes. 1937 He applied for and won a scholarship to study at California Institute of Technology. As soon as he arrived at Caltech, he began to cooperate with another researcher, Emory Ellis. Ellis is studying a group of bacteriophages-T2, T4 and T6 (T- even bacteriophages). Delbruck soon realized that these viruses were suitable for studying virus replication. These phages are a way to explore how genetic information determines the structure and function of organisms. From the beginning, these viruses were regarded as a typical system to understand how cancer viruses and even sperm fertilized eggs and developed into new objects. Ehrlich and Delbrouck designed a one-step growth curve experiment. In this experiment, an infected bacterium released a large number of phages after a half-hour incubation period or an eclipse. This experiment defines the incubation period, that is, the time when the virus loses its infectivity. This has become an experimental example of this phage research group.

After the outbreak of World War II, Delbrouck stayed in the United States (at Vanderbilt University) and met Italian refugee salvador luria. Luria fled to the United States to study T 1 and T2 phage at Columbia University. They met at a meeting held in Philadelphia on February 28th, 1940, and planned an experiment at Columbia University for the next two days. These two scientists will recruit and lead more and more researchers to focus on using bacteria and viruses as models to understand the process of life. The key to their success was that in the summer of 194 1, they were invited to do experiments in the cold spring harbor laboratory. In this way, a German physicist and an Italian geneticist have been cooperating all over the United States during World War II to recruit a new generation of biologists, who later became known as the phage research group.

Shortly thereafter, tom anderson, an electron microscope expert at RCA Laboratory in Princeton, New Jersey, met Delbrouck. By March of 1942, they obtained a clear picture of phage for the first time. At about the same time, these phage variants were isolated and identified for the first time. By 1946, the first phage course was opened in Cold Spring Harbor Laboratory. 1947 in March, eight people attended the first phage conference. Molecular biology developed from these slow beginnings. The focus of this science is to study bacterial hosts and their viruses.

The following 25 years (from 1950 to 1975) was a fruitful period for virology research with phage. Hundreds of virologists have published thousands of papers, mainly covering three fields: (a) the research of T-even phage on lysogenic infection of Escherichia coli; (b) Study on the lysogenicity of λ phage; (c) Study on the replication and characteristics of several unique phages, such as Ф x174 (single stranded circular DNA), RNA phage, T7 and so on. They laid the foundation for modern molecular virology and biology. It is impossible for this paper to introduce all these scientific documents one by one, only to mention some selective key points.

From 1947 to 1948, it became popular to study the changes of phage infected cells by biochemical methods. Seymour Cohen studied lipids and nucleic acids with erwin chargaff at Columbia University, then studied tobacco mosaic virus RNA with Wendell Stanley, and majored in Delbrouck phage course at Cold Spring Harbor Laboratory from 65438 to 0946. He studied the effect of DNA and RNA levels in cells infected by phage by colorimetric analysis. These studies show that the synthesis of macromolecules in phage infected cells has changed greatly: (The net accumulation of RNA stops in these cells. Later, this became the basis for the discovery of various RNA, which proved the existence of messenger RNA for the first time. (b) DNA synthesis stopped for 7 minutes, and then resumed at a speed of 5 times to 10 times. (c) At the same time, studies by Monod and Wollman showed that the synthesis of inducible β -galactosidase (a cellular enzyme) was inhibited after phage infection. These experiments divide the incubation period of the virus into two stages: prophase (before DNA synthesis) and anaphase. More importantly, these results indicate that the virus may change the macromolecular synthesis process of infected cells.

By the end of 1952, two experiments had an important impact on this field. First, hershey and Chase used labeled virus protein (SO4) and nucleic acid (PO4) to track the attachment of phage to bacteria. They can use a blender to remove the protein shell of the virus, leaving only DNA related to infected cells. This enables them to prove that this DNA has all the information needed to regenerate a large number of new viruses. The isomorphism between hershey Chase's experiment and the new DNA structure elaborated by Watson and Crick a year later became the cornerstone of the molecular biology revolution.

The second experiment in the field of virology was conducted by G.R.Wyatt and S.S.Cohen in 1953. They found a new base, 5' hydroxymethylcytosine, while studying T- even phage. This newly discovered base seems to have replaced cytosine in bacterial DNA. This enabled scientists to study DNA synthesis in bacteria and phage infected cells for 10 years. The most critical research shows that viruses introduce genetic information into infected cells. By 1964, Mathews et al. proved that 5' hydroxymethylcytosine does not exist in uninfected cells, and it must be encoded by virus. These experiments put forward the early enzymatic concepts of deoxypyrimidine biosynthesis and DNA replication, and provided clear biochemical evidence that a new information can be encoded and expressed in infected cells. After detailed gene analysis of these phages, the genes encoding protein were confirmed, and the gene map was drawn, which made the concept more complete. In fact, the genetic analysis of T- even phage R Ⅱ and B cis-trans has become one of the most fully studied "genetic fine structures". Replication of viral DNA in vitro by phage mutants and extracts has made an important contribution to our contemporary understanding of how DNA replicates itself. Finally, through the detailed genetic analysis of phage assembly and the complementarity of phage mutant assembly in vitro, this paper expounds how organisms use the principle of self-assembly to construct complex structures. The genetic and biochemical analysis of bacteriophage lysozyme is helpful to explain the molecular characteristics of mutation, and phage mutation (amber mutation) provides a clear way to study the second site suppressor mutation at the molecular level. The circular arrangement of DNA and the redundant structure at the end can explain the circular genetic map of T-even phage.

In phage-infected cells, the synthesis of virus and cellular protein has changed obviously, which was discovered when sodium dodecyl sulfate (SDS)- polyacrylamide gel was used in early research. The results showed that there was a specific sequence of viral protein synthesis, which was divided into early protein and late protein. This short-lived basic regulatory mechanism finally found the ∑ factor that regulates RNA polymerase and gives gene specificity. The original data obtained from the study of phage infectivity revealed almost every aspect of gene regulation (transcription, RNA stability, protein synthesis, protein treatment).

Although the research on lysogenic phage has made such remarkable progress, no one can clearly explain lysogenic phage. This situation changed in 1949, when Andre Lwoff of Pasteur Institute began to study Bacillus megaterium and its lysogenic phage. By using a micromanipulator to divide a single bacterium as many as 19 times, no virus was released. When lysogenic bacteria dissolved from the outside, no virus was found. However, it often happens that a bacterium spontaneously dissolves and releases many viruses. Ultraviolet rays can induce the release of these viruses, which is an important discovery. This observation can outline the wonderful relationship between virus and host. By 1954, Jacob and Wollman of Pasteur Institute had reached an important research result, that is, the genetic hybridization between a lysogenic strain (Hfr, λ) and an insoluble receptor led to the induction of the virus. They call this process zygotic induction. In fact, the position of lysogenic phage or protophage in the chromosome of its host Escherichia coli can be mapped by standard interrupted mating experiment after genetic hybridization. This is one of the most critical experiments to understand lysogenic virus conceptually, for the following reasons: (a) The virus behaves like a bacterial gene on a bacterial chromosome; (b) It shows that the genetic material of the virus remains static in the virus due to negative regulation. When the chromosome is transferred from lysogenic donor bacteria to non-lysogenic recipient host, the genetic material of the virus is lost; (c) This helps to explain that Jacob and wolman realized that the induction of enzyme synthesis and phage production were the same phenomenon as early as 1954 ". These experiments laid a foundation for the properties of operon model and synergistic gene regulation.

Although 1953 expounds the structure of DNA and 1954 describes zygote induction, the relationship between bacterial chromosomes and viral chromosomes in lysogeny is still called attachment site, which can only be considered from these angles at that time. Later, according to the fact that the phage marker sequence is different from the replication or growth sequence, Campbell put forward the model of integrating DNA with bacterial chromosome λ, and realized the real close relationship between virus and host. This leads to the separation of negative regulatory genes or inhibitory genes of λ phage, which is a clear understanding of the immune characteristics of lysogens and one of the early examples of how to coordinate genes. Genetic analysis of λ phage life cycle is an important academic exploration in the field of microbial genetics. It deserves detailed study by all molecular virologists and biologists.

P22 of lysogenic phage such as Salmonella typhimurium is the first case of general transduction, and λ phage is the first case of special transduction. Viruses may carry cell genes and transfer them from one cell to another, which not only provides an accurate method of gene location, but also is a new concept in virology. With the more detailed study of bacterial genetic factors, it is obvious that from lysogenic phage research to appendages, transposons, retrotransposons, insertion elements, retroviruses, hepatotropic DNA viruses, viroids, viroids (also known as viroids, which refer to viruses wrapped in plant virus particles) and prions, all these make the relationship between the definition and classification of genetic information between viruses and their hosts blurred.

The genetic and biochemical concepts derived from phage research make it possible to further develop virology. The experience and lessons of bacteriolytic phage and lysogenic phage research are often re-learned and revised with the study of animal viruses.