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Abstract/Keywords: Bacterial&# 65279; NatureS Smallest Syringe: &# 65279; Abstract/Keywords: Bacterial viruses play an important role in the development of molecular biology. Thousands of different phages have been isolated and almost every one is uniquely suited to the investigation of a different biochemical or genetic event. With the experiments of dHerelle, Twort and the Phage group, the understanding of phages processes have grown throughout this century. Because of the many differences existing from phage to phage and the extensive information pertaining to this virus, my report does not focus on a specific bacterial virus but more of a general overview.
Bacteriophage (or phage), DNA, lytic cycle, lysogenic cycle, host cell (bacteria), phage therapy. Objectives: It is proved that bacterial cells, like many other cells are susceptible to viral infections. Once an ample amount of viruses get buried in a host cell, they have the ability to dictate the reproduction of other viruses. With the efforts and interests of many scientists in the twentieth century, our sense of what viruses do and how they act has heightened. Because viruses are so diverse and much information is written on the topic, my efforts were concentrated on the bacterial viruses named bacteriophage. Phages were the topic of interest during the early 1900 s, but when antibiotics was regularly practiced, phage therapy was out of the question.
Despite intensive work by drug companies, no new antibiotics have been discovered in the past 30 years. One result among many is the renewed interest in bacteriophage therapy. This paper is written primarily to put phage in historical and ecological context as well as explore its increasing popularity towards future applications. Background: The discovery of clear patches developing in cultures of bacteria was first made at the beginning of this century.
These areas are now called plaques and the clearing phenomenon which was shown to be due to an infective agent, was called the Twort- dHerelle phenomenon after the two discoverers. The infective agent was proved to be a virus, and named bacteriophage (bacteria eater) by dHerelle. We can distinguish these phage by the types of plaques they produce. A century ago, Hanging reported that the waters of the Ganges and Jumna rivers in India had marked antibacterial action which could pass through a very fine porcelain filter; this activity was destroyed by boiling.
He particularly studied the effects on Vibrio cholerae and suggested that the substance responsible was what kept cholera epidemics from being spread by ingestion of the water of these rivers. However, he did not further explore the phenomenon. The actual and noted discoveries were really made when Edward Twort and Felix dHerelle independently reported isolating filterable entities capable of destroying bacterial cultures and of producing small cleared areas on bacterial lawns (plaques), seemingly implying that discrete particles were involved. Today, they are jointly given credit for the discovery. It was dHerelle, a Canadian working at the Pasteur Institute in Paris, who gave them the name bacteriophages and who made them a major part of his lifes work. DHerelle, a largely self-trained microbiologist, had just spent 10 years in Guatemala, Mexico and Argentina.
There, he dealt with epidemics of dysentery, yellow fever and a coffee-killing fungus, isolated a bacterium from dying locusts to use in controlling locust plagues, and explored several interesting fermentation challenges all good preparation for his later work with phages. In his spare time, he was doing research with dysentery patients a frequent problem in wartime France. From the feces of several of these patients, he isolated a filterable anti- Shiga microbe which multiplied through many serial passages on its host bacterium, and which could produce tiny clear circles on a plate of this Shiga bacillus. DHerelle went on to carefully characterize bacteriophages as viruses which multiply in bacteria and worked out the details of infection by various phages of different bacterial hosts under a medley of environmental conditions, always working to combine natural phenomena with laboratory findings, to better understand immunity and natural healing from infectious disease.
At the Ninetieth Annual Meeting of the British Medical Association in Glasgow featured a very interesting discussion between dHerelle, Twort and several other distinguished scientists of the day on the nature and properties of bacteriophages. The main issue at that time was whether the observed bacteriologic principle was an enzyme produced by bacterial activity or a form of tiny virus with some sort of life of its own, as claimed by dHerelle; this controversy continued for many years, splitting the rapidly-growing community of people working with phages. DHerelle summarized the early phage work in a 300 -page book The Bacteriophage. He wrote classic descriptions of plaque formation and composition, infective centers, the lysis process, host specificity of adsorption and multiplication, the dependence of phage production on the precise state of the host, isolation of phages from sources of infectious bacteria and the factors controlling stability of the free phage some of which is included in my report. He quickly became fascinated with the apparent role of phages in the natural control of microbial infections.
He noted for example the frequent specificities of the phages isolated from recuperating patients for their own disease organisms and the rather rapid variations over time in their phage populations. He thus worked throughout his life to develop the potential of using properly selected phages as therapeutic agents against the most devastating health problems of the day. However, he initially focussed on simply understanding phage biology. Thus, the first known report of successful phage therapy came not from dHerelle but from Bruynoghe and Main, who used phage to treat staphylococcal skin infections. After a year at the Pasteur Institute of Saigon, dHerelle returned to tight physical conditions, personal conflict and intellectual controversy at the Pasteur Institute in Paris.
He soon accepted an offer to move to the Netherlands, where he was provided better conditions for his work with the recovery from infectious disease and the properties of bacteriophages, published his first book and a number of papers, and received an honorary MD degree. In 1925, he became a health officer for the League of Nations, based in Alexandria, Egypt, with special responsibility for controlling infectious disease on ships passing through the Suez Canal and during some of the major Muslim pilgrimages. Phage therapy and sanitation measures were the primary tools in his arsenal to deal with major outbreaks of infectious disease throughout the Middle East and India. Throughout this period, he continued publishing on his research and clinical trials and assisting others who were willing to do so with phages and consultations, often undertaking extended travel at his own expense. One of the most extensive trials of phage therapy he helped set up was the Bacteriophage Inquiry of 1927 - 1936, which led to what seems to be convincing results, endorsed by refined committees yet still left many skeptics of phage therapy; these studies deserved closer investigation. In 1928, dHerelle was invited to Stanford to give the prestigious Lane Lectures; his discussion of The Bacteriophage and its Clinical Applications was published as a monograph.
He gave many lectures for medical schools and societies as he crisscrossed the country. He then went on to Yale to take up a regular faculty position yet he continued to spend summers in Paris working with the phage company he had established there, run by his son-in-law, in response to strong demands for phage preparations with careful quality control. He returned permanently to Europe in 1933, spending much time the following two years in Tiflis, Georgia, helping to set up an international Bacteriophage Institute there, as discussed further below. From early on, one major practical use of phages was for bacterial identification through a process called phage typing the use of patterns of sensitivity to a specific battery of phages to precisely identify microbial strains.
This technique takes advantage of the fine specificity of many phages for their hosts and is still in common use around the world. The sophisticated ability of phages to destroy their bacterial hosts can also have a very negative commercial impact; phage contaminants occasionally spread havoc and financial disaster for the various fermentation industries that depend on bacteria, such as cheese production and fermentative synthesis of chemicals. Phage therapy was tried extensively and many successes were reported for a variety of diseases, including dysentery, typhoid and paratyphoid fevers, cholera, and pyogenic (pus-producing) and urinary-tract infections. Phages were poured directly into lesions, given orally or applied as aerosols or enemas. They were also given as injections intradermal, intravascular, intramuscular, intra duodenal, intraperitoneal, even into the lung, carotid artery and pericardium. The early strong interest in phage therapy is reflected in the fact that some 800 papers were published on the topic between 1917 and 1956.
The reported results were quite variable. Many physicians and entrepreneurs became very excited by the potential clinical implications and jumped into applications with very little understanding of phages, microbiology or basic scientific process. Thus many of the studies were poorly controlled, many of the failures were predictable and some of the reported successes did not make much scientific sense. Often, uncharacterized phages at unknown concentrations were given to patients without specific bacteriological diagnosis, and there is no mention of follow up, controls or placebos.
Much of the understanding gained by dHerelle was ignored in this early work, and inappropriate methods of preparation, preservatives and storage procedures were often used. On one occasion, dHerelle reported testing 20 preparations from various companies and finding that not one of them contained active phages. On another occasion, a preparation was advertised as containing a number of different phages, but it turned out that the technician responsible had decided it was easier to grow them up in one large batch than in separate batches. Not too surprisingly, checking the product showed that one phage had out competed all the others and this was not, in fact, a polyvalent preparation. This was the origin of the phage T 7, whose RNA polymerase now plays a major role in biotechnology. In general, there was no quality control except in a few research centers.
Large clinical studies were rare and the results of those few were largely inaccessible outside of Eastern Europe. In 1931, an extensive review of bacteriophage therapy was commissioned by the Council on Pharmacy and Chemistry of the American Medical Association. In evaluating this report, it is important to realize how little was yet known then about bacteriophages. In fact, their first conclusion was Experimental studies of the lytic agent called bacteriophage have not disclosed its nature. Dherelle's theory that the material is a living virus parasite of bacteria has not been proved. On the contrary, the facts appear to indicate that the material is inanimate, possibly an enzyme.
In retrospect, the proof that phages are viruses looks solid and it is hard to see how they could have come to this conclusion, which clearly impacted all of their other findings. These included: Since it has not been shown conclusively that bacteriophage is a living organism, it is unwarranted to attribute its effect on cultures of bacteria or its possible therapeutic action to a vital property of the substance. While bacteriophage dissolves sensitive bacteria in culture and causes numerous modifications of the organisms, its lytic action in the body is inhibited or greatly impeded by blood and other bodily fluids. The material called bacteriophage is usually a filtrate of dissolved organisms, containing, in addition to the lytic principle, antigenic bacterial substances, products of bacterial growth and constituents of the culture medium.
The effects of all these constituents must be taken into consideration whenever therapeutic action is tested. A review of the literature on the use of bacteriophage in the treatment of infections reveals that the evidence for the therapeutic value of lytic filtrates is for the most part contradictory. Only in the treatment of local staphylococcic infections and perhaps cystitis has evidence at all convincing been presented. This assessment clearly had a strong influence on the investment of the medical community in exploring phage therapy seriously, at least in the United States.
Points are raised which still need to be considered, particularly in terms of the many trials described there in animals or humans which seemed to show little or no success and in terms of such potentially confounding explanations of the successes as the apparent strong stimulation of natural immune mechanisms by the bacterial debris in the lysates used. Then in the 1940 s, the new miracle antibiotics such as penicillin became became widely available, and phage therapy was largely abandoned in the western world. Even though the popularity of phages as treatment decreased, one man and his college believed that more research needed to be done. The phage group was an informal group of scientists at different universities who worked together and separately on bacterial viruses, or phage. The groups spiritual leader was, a charismatic and brilliant German physicist-turned-biologist. The phage groups origins lie in the December, 1941 meeting of the American Physical Society, in Philadelphia.
Delbruck, then at Vanderbilt University, liked to keep up with theoretical physics, though he no longer practiced. In Philadelphia he met an Italian national who was not a physicist, but was interested in physics. The two began talking about phage and went up to Luria's lab at the College of Physicians and Surgeons in New York. They did a 48 hour bout of experiments and cemented nearly a decade of collaboration. Delbruck planned to go to Cold Spring Harbor in the summer, to attend the annual Symposium and to stay on to do experiments for the rest of the summer. He invited Luria to come, Luria accepted, and the phage group was born.
In the summer of 1942, Delbruck and Luria brought more phage workers to Cold Spring Harbor. The two men worked on the resistance of phage to sulfa drugs, the state-of-the- art way to battle bacterial infection. They showed that phage were resistant to sulfa drugs. In 1943 Delbruck invited Luria to do some experiments at Vanderbilt.
They arranged a simple experiment to test the occurrence of spontaneous acquisition of bacterial resistance to phage infection. Delbruck and Luria took samples of bacteria from small populations and from large ones. They diluted the samples and transferred them into containers of equal size, so that population numbers would be roughly equal the only difference was the size of the parent population. They then infected the samples with phage. Normally, a certain percentage of bacteria will acquire a resistance to phage infection. Delbruck and Luria showed, however, that the variation within bacterial samples drawn from small populations was much larger than that within samples drawn from large populations.
In other words, by putting the bacteria through what a population geneticist would call a bottleneck, Delbruck and Luria showed that the probability of acquiring resistance was due to an event occurring at the time of bacterial replication, not the time of separation of the populations. They had demonstrated the existence of mutation in bacteria, thus firmly establishing the phage / bacteria system as a valid one for studying the nature of the gene. This experiment, which came to be known as the fluctuation test, was the basis of many of the experiments preformed and taught by phage workers throughout the 1940 s and profoundly influenced the current and subsequent generations of phage biologists. In 1943 the third key member of the group was added. Hershey was working with phage at Washington University in St. Louis, under phage pioneer J.
J. Bronfenbrenner. Delbruck became interested in Hershey's work and invited him to Vanderbilt to do some experiments together. In the summer of 1944 Delbruck drafted the Phage Treaty. In it he dictated that all phage workers should focus on a group of phage species, the T-series, and E. coli strain B in a nutrient broth at 37 degrees C.
According to TF Anderson, the T series phages were chosen because they were well-behaved, i. e. , they give easily countable plaques, and the strains of B that are phage-resistant can be freed of the phage to which they are resistant. Delbruck's intent was to standardize the experiments done by the growing numbers of phage workers, so that their results would be comparable. The next year, he would take a much more drastic step toward unifying the growing field of phage genetics That first summer Delbruck and Luria demonstrated the mutual exclusion principle.
They plated out bacteria with two different strains of phage, alpha and gamma. Each type of phage has a characteristic and highly regular time course; nearly to the minute, it will cause the bacteria to burst, or lyse, at a fixed time after infection. The alpha phage had a much faster lyse time than the gamma. When allowed to compete for the bacteria, however, the slower gamma phage always won. This showed that only one type of phage could infect a given bacterium, to the exclusion of the other. Delbruck and Luria compared this to the fertilization membrane an ovum forms after it is penetrated by a single sperm.
In 1952, Alfred Hershey and Martha Chase, plus earlier findings from Avery and his colleagues proved that only the DNA of the bacterial virus and not its protein portion must enter the host bacterium to initiate infection, establishing again the fact that DNA was the universal genetic information for cells. Methodologies: These viruses, particularly their genetics, now form a complete study on their own. They have a 50 % nucleic acid content of both RNA or DNA. Most bacteriophage are classified according to host range, immunologic relationships and most importantly nucleic acid properties, and phage morphology. Bacterial viruses can be tables icosahedral, filamentous, non-contractile or contractile tails. Very few have envelopes.
Bacteriophages are composed of a head on top of a thin cylindrical core and covered by a sheathing coat. At the base of the core dwells six spider like legs which are used as a sort of anchor. Phages inject their DNA into a bacterium, cause it to be replicated many times over, package the newly replicated DNA in viral-protein coats and kill the bacterium; the progeny phages released from the cell go on to infect other susceptible host. It is important to note that the phage coat remains extracellular and taking no part in replication. With a hetero polymeric shape and organic compounds, bacteriophages are able to self- replicate. During the infection of bacteria, two things can happen.
The bacteria undergoes what we call lytic infection by virulent phages in which progeny phages are produced or it undergoes the lysogenic cycle, where temperate phages insert themselves into the host and their genome remains silent. During the lytic cycle the phage culminates with the host cell, bursting and releasing virions. The T series of phages are the most complex. They are large and lytic with one single molecule of DNA while most are double stranded. Basically, their bacteriophage DNA directs a program of events that produce around 100 new phage particles in about 20 min, at which time the infected bacteria lyses (bursts), releasing a new generation of phage. In the 1940 and 50 s, studies were more concentrated on the T 4 phage.
The genetics of the T 4 bacteriophage has been elucidated in some detail by using mutations, and it has been shown that there are over sixty genes involved in the production of a complete virus. The lambda phage has often been mentioned, the entire sequence of over 50, 000 base pairs is known and more than 50 genes have been identified on the genetic map. Among these are genes that encode the phage specific proteins that regulate transcription of the phages own DNA. From this detailed knowledge, much has been learned about how an integrated set of transcriptional controls is responsible for physiological choices in a cell containing lambda DNA.
During growth and reproduction, certain phages blend the host DNA into their own genetic material (DNA or RNA) and are able to pass on this DNA to other hosts. Bacteriophage that pick up specific regions on the bacterial genome are called special or restricted transuding phage. On the other hand those who attain genes at random are named general transuding bacteriophage. Defective phage are viruses who obtain too much DNA that there is no more room for replication of genetic material, therefore they can no longer kill the host but invade and transfer genes. But the big question is; what happens to the genetic material during this eclipse phase? In ultra-sections if infected bacteria certain events can be discerned thanks to the discovery of the electron microscope.
Within five minutes of infection the bacterial nucleus, at first compact and discrete, breaks up into granules that migrate to the periphery of the cell and disappear. Between five and ten minutes threadlike elements appear and coalesce into spherical packets. Around the 15 th minute particles resembling mature phage with head membranes and tails can be seen. If infected cells are broken open and the contents examined ten minutes it is sometimes possible to observe structures called doughnuts which are head membranes containing no nucleic acid, Tail sheaths, fibres and end plates may also be observed.
It is only during the latent period that the cell ruptures and releases the newly produced bacteriophages. Phages; like viruses can be neutralized by antibodies. When sufficient antibody molecules become attached to the sheath protein of the distal third of the tail in such a fashion as to prevent the process of virus attaching to the bacterium. Bacteriophages exhibit heritable traits, for example deciding its rate of growth, specificity for a certain type of bacterium or temperature range which can be used for identifying the strain of virus.
Furthermore, different strains of viruses can exchange genetic data only when the strains infect the same cell, proving that indeed viruses partake in a genetic process within the host cell. The knowledge that DNA was the controlling molecule of life brought forth suggestions that: (1) early evolution must have depended on the growth of a cell carrying adequate instructions in its DNA to grow (2) random variation led to changes in species (3) the reproduction of DNA from generation to generation causes it to function like its parent cell (4) the programmed uncoiling of the genetic donation in the DNA underlies the development of every new plant or animal Future work/Applications: Some phages are small in size, you could fit about 680, 000 phage on the thread of a pin. Today, they can be manipulated or reconstructed by a variety of recently developed tools. The molecules are easily isolated, unbroken in large amounts. They can be cut at a number of defined sites with restriction enzymes and the resulting fragments can be rejoined with one another or joined to foreign DNA segments to reconstitute the original molecule or make a hybrid molecule (made of a plasmid fused with foreign DNA).
This reassembling is done by readily available enzyme of bacterial origin known as DNA ligases. These enzymatic molecules recognize the ends of DNA molecules and fuse them without any trace of assemblage. The newly formed genome is introduced in a bacterial cell, where they are replicated many times over and each hybrid molecule bears a separate progeny population identical to the originator. Why dont we use phage medicines?
As mentioned earlier, phage is specific to its bacteria yet antibiotics are multi tasked and work on several bacterium. It is preferable that an antibiotic is given first because we arent sure of the causative agent causing the disease when treatment is given yet diseases like Cystic Fibrosis almost always progress to Pseudomonas infection and could be treated with phage and also diseases like syphilis or tuberculosis. Secondly, as a doctor you are less likely to get sued prescribing the wrong antibiotic to a patient than prescribing a particular phage. And most importantly, before a drug or treatment goes on the market, it must undergo licensing and prove that the treatment in question is effective against the disease, more cost effective than other methods and the need for the treatment is there. Extensive clinical research and implementation of phage therapy continued to be carried out in Eastern Europe over the last 50 years.
The results of that work effectively complement the limited recent animal work in the West that is primarily cited in the recent articles, encouraging optimism that phage can indeed play an important role in dealing with infections involving increasingly drug-resistant microbes. We need to draw as much as possible on the largely-unknown body of knowledge that has accumulated in Poland, France and many parts of the former Soviet Union (FSU) as we again explore phage therapy, and to give credit where it is due for the many years of hard, careful work they have invested in the field. This paper is written primarily to put phage therapy in historical and ecological context and to explore some of the more interesting and extensive work in Eastern Europe, little of which has been inaccessible in english. Each kind of bacteria has its own phages, which can be isolated wherever that particular bacterium grows from sewage, feces, soil, even ocean depths and hot springs.
The process of isolation is easy. Just let the sample sit in an appropriate nutrient broth, separate off the liquid part, and pass it through a filter with pores so tiny that bacteria cant get through. Then mix it (at several different dilutions) with a culture of the bacteria in question. Spread a few drops on a block of appropriate nutrient medium which is made firm with agar taken from seaweed. The next day, one sees a dense covering or lawn of bacteria with round clear spots, called plaques.
Each plaque contains many million phage particles, all progeny of one phage which was immobilized there on the agar. That phage infected a cell, multiplied inside it, and caused it to burst. This released many phages, which infected nearby cells and repeated the process. One can stick a toothpick into one of these plaques, transfer it to a fresh culture of the bacteria in liquid medium, and grow up a homogeneous stock of descendants of that particular phage, whose properties can then be studied. Major discoveries made with bacteriophages: -conformation that genes are made of DNA -the nature of the genetic code -nature of a virus life cycle -messenger RNA -nature and types of gene mutation -DNA ligase -overlapping genes -co-linearity of gene and protein -characterization of insertion sequence, transposon's and invertible DNA segments -virus-mediated gene transfer between cells (now called gene therapy) Note: included in my report are articles pertaining to the subject, extracted from the Journal of Molecular Biology. Conclusion: DNA, the germinal substance of bacterial viruses, is also the essential heredity material of all living organisms and thus controls the activity of living cells of every kind.
Yet it is only in the virus that we can study its reproduction and function so easily. In a molecular sense, phages are the most studied virus yet several issues of DNA replication have not been resolved. As yet, most of the systems reconstructed in vitro do not start synthesis of DNA at the correct site or in the correct manner. In fact, how the start site is located is not clear in most cases and the initiation of DNA is vaguely understood. But phages, help us control bacterial diseases and help us identify some of the mysteries of genetic identity and function, the most fundamental substance of all living things. Bibliography &# 65279; web web web web web web web web American Scientific (readings).
The Molecular Life. New York: W. H. Freeman and Company. , 1985 Burnet, MacFarlane. Genes, Dreams and Realities. New York: Basic Books Inc. , 1971.
Darnell, James E. Molecular Cell Biology. New York: Scientific American Books Inc. , 1986. Judson, Horace Freeland. The Eighth Day of Creation. New York: Touchstone. , 1979.
Radestsky, Peter. The Invisible Invaders: the story of emerging age of viruses. Toronto: Little, Brown and Company, 1991.
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