Sunday, June 2, 2019
History of Genetic Engineering
History of Genetic engine roomGenetic engineering is a deliberate modification of the char stand foreristics of an organism by manipulating its divisortic material. This chapter describes how earn carried out between 1970s and 1980s produced technologies that researchers now riding habit to manipulate the genetic material of organisms.Key concepts coveredRecombinant- deoxyribonucleic acid engineering science is a technology in which genetic material from one organism is introduced into another(prenominal) organism and then replicated and expressed by that other organism.Gene sequencing is the process of ascertain the precise localise of nucleotides within a deoxyribonucleic acid molecule.Recombinant- deoxyribonucleic acid technology has been used to make insulin and other valet de chambre proteins for medicine.Recombinant DNAThe prospect of recombinant DNA emerged from twain advances in biochemistry(1) Discoveries of restriction enzymes that act as scissors to cut molecule s of DNA at specific nucleotide sequences and(2) Discoveries of DNA ligases enzymes that forge molecular bonds.Creation of First Recombinant DNA (1972)In 1972, capital of Minnesota Berg (1926- ), a biochemistry professor at Stanford University, make upd the first recombinant DNA molecule. He first isolated the DNA molecules from two different organisms, the SV40 monkey virus and a bacteriuml virus known as Lamdba bacteriophage (or phage ). Using a cut-and-splice method, he created sticky checks in the DNA of both viruses. Then he joined them together with DNA ligase.Invention of Recombinant DNA (rDNA) Technology (1973)Recombinant-DNA technology is a technology in which a rDNA plasamid is introduced into bacteria and then replicated and expressed by that bacteria.It was invented through the work of Herbert W. Boyer (1936- ), Stanley N. Cohen (1935- ), Paul Berg, and Janet Mertz (1949- ).After Berg created the first recombinant DNA molecules in 1972, Boyer and Cohen took Bergs wor k a step further by introducing the rDNA plasmid to E. coli bacterial cells.A plasmid is DNA, found in bacteria, that is separate from and can replicate independently of the bacteriums chromosomal DNA. The phenomenon of transformation permits the rDNA plasmid to be introduced into and expressed by E. coli cells. The bacteria containing the rDNA plasmid grow on petri dishes to form tiny colonies. But in a typical procedure, only 1 in about 10,000 bacteria cells takes up the rDNA plasmid. The rDNA plasmid must contain a selectable gene so that they can be efficiently picked up from the culture. This can be done by utilise a drug-resistance gene to make the rDNA plasmid resistant to antibiotics such as tetracycline. Adding tetracycline to the culture will ensure that only the bacteria with the rDNA plasmids survive.In 1974, at the urge of Standford Universitys patent forwardice, Boyer and Cohen filed a patent for recombinant DNA technology.Asilomar ConferencesPotential dangers of re combinant genetic engineering emerged even before Berg published his landmark 1972 paper. Although the SV40 virus was plan to be harmless for kind, Borg was concerned about the prospect of an altered form of the virus spreading through a common bacteria. So he deferred grammatical constituent of his research program, and did not insert the recombinant virus into bacterial cells as he originally planned.In 1973, Berg organized a small conference at Asilomar, California to address the growing concerns about gene-manipulation technology. In 1974 Berg published a widely discussed letter on the potential dangers of recombinant DNA research. Subsequently, a moratorium on research in 1975 (Asilomar II) provided time for regulations to be devised and put into effect in 1976.Gene Sequencing, Gene Splicing, and Reverse TranscriptionGene SequencingGene sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method that is used to deter mine the order of the four bases A, G, C, and T in a chain of DNA.Frederick Sanger (1918-2013), a biochemist in England, is a pioneer of sequencing. He has received two Nobel prizes one for the sequencing of proteins (in 1958), the other for the sequencing of DNA (in 1980).In the early 1950s, Sanger had solved the sequencing of a protein use a sequence of degradation reactions. A protein is made up of a sequence of amino acids strung into a chain. To identify the sequence of a protein, Sanger would arrest off one amino acid from the end of the chain, dissolve it in solvents, and identify it chemically. He would repeat the degradation and identification process until he reached the end of the protein.In the mid-1960s, Sanger switched his focus from protein to DNA. But his methods that had worked so well for proteins didnt work for DNA. Proteins are chemically structured such that amino acids can be serially snapped off the chain moreover with DNA, no such tools existed.In 1971 , Sanger devised a gene-sequencing technique using the copying reaction of DNA polymerase. At first, the method was inefficient and erring because the copying reaction was too fast. In 1975, He made an ingenious modification. He doctored the copying reaction with a series of chemicals variants of A, C, G, and T -that were still recognized by DNA polymerase, but slowed down its copying ability. On February 24, 1977, Sanger used this technique to reveal the full sequence of phi X 174 (or X174) bacteriophage.Gene SplicingIn 1977, scientists discovered that most animal (and animal virus) proteins were not encoded in long, continuous stretches of DNA. They were split into modules, interrupted by regions called introns that do not hold protein-encoding information. By dissever the genes into modules, a cell could generate more combination of messages out of a single gene. When a DNA with introns is used to build RNA the introns have to be removed from the RNA message. This phrase for the process is called gene splicing or RNA splicing.Reverse TranscriptionIn 1970, David Baltimore (1938- ) and Howard Temin (1934-94), two virologists, discovered an enzyme that could build DNA from an RNA template. They called the enzyme reverse transcriptase. Using this enzyme, every RNA in a cell could be used as a template to build its synonymic DNA.The production of proteins from recombinant DNA represented a crucial transition in the history of medical technology. To see to it the impact of this transition from genes to medicine we need to understand the nature of drugs.Nearly every drug works by binding to its target and enabling or disabling it turning molecular switches on or off. To be useful, a drug must bind to its switches but to only a selected set of switches. Most molecules can barely achieve this level of specificity but proteins have been designed explicitly for this purpose. Proteins are the enabler and disablers, the regulators, the gatekeepers, the operat ors, of cellular reactions. They are the switches that most drugs seek to turn on or off.Proteins are thus poised to be some of the most potent and most discriminating medicines in the pharmacological world. But to make a protein, one needs its gene and here recombinant DNA technology provided the crucial link. The cloning of human gens allowed scientists to manufacture proteins and the synthesis of proteins opened the possibility of targeting the millions of biochemical reactions in the human body. Proteins made it possible for chemists to throw in on previously impenetrable aspects of our physiology. The use of recombinant DNA to produce proteins thus marked a transition not just between one gene and one medicine, but between genes and anovel universe of drugs.Founding of Genetech (1975)In 1975, Robert Swanson (1947-99), a venture capitalist, approached Herb Boyer with a proposal to starting a company that would use gene-cloning techniques to make medicines. Boyer was fascinate d. His own son had been diagnosed with a potential growth disorder, and Boyer had been gripped by the possibility of producing human growth hormone, a protein to treat such growth defects. leash hours subsequently they met, Swanson and Boyer had reached a tentative agreement to start such a company with seed moneys from venture firms. Boyer called this company Genentech a condensation of Genetic Engineering Technology.Synthesis of Insulin (1978)Purified animal-sourced insulin was the only type of insulin available to diabetics until genetic advances occurred later with medical research. The amino acid structure of insulin was characterized in 1953 by Frederick Sanger. The protein was made up of two chain (A and B) one larger and one smaller, cross-linked by chemical bonds.Boyers plan for the synthesis of insulin was simple. He did not have the gene for human insulin at spend no one did but he would build it from scratch using DNA chemistry, nucleotide by nucleotide, triplet u pon triplet. He would make one gene for the A chain, and another gene for the B chain. He would insert both the genes in bacteria and trick them to synthesizing the human proteins.. He would purify the two protein chains and then stitch them chemically to obtain the U-shaped molecule.But Boyer was cautious. He treasured an easier running play case before lunging straight for insulin. He focused on another protein somatostatin also a hormone, but with little commercial potential. To synthesize the somatostatin gene from scratch, Boyer recruited Keiichi Itakura and Art Riggs from the City of Hope in Los Angeles. Swanson was opposed to the whole plan. He wanted Boyer to move to insulin directly. Genentech was living in borrowed space on borrowed money. Still Boyer convinced Swanson to give somatostatin a chance. In the meantime, two teams of of geneticist had also entered the race to make insulin. superstar at Harvard and the other one at UCSF.By the fall of 1977, they succeeded i n synthesizing somatostatin, and started focusing on insulin. At this time, the competition was fierce. The Harvard team had apparently cloned the native human gene out of human cells and were ready to make the protein. The UCSF team has synthesized a few micrograms of protein and were planning to inject the human hormone into patients.It was Asilomar that came to their rescue. Like most University laboratories with federal funding, the UCSF team was bounded by the Asilomar restrictions on recombinant DNA. In contrast, Boyers team had decided to use a chemically synthesized version of the insulin gene. A synthetic gene DNA created as a naked chemical fell into the gray zone of Asilomars language and was relatively save. Genentech, as a privately funded company, was also relatively exempt from the federal guidelines.In the summer of 1978, Boyer learned that the Harvard team was about to announce successful isolation of the human hormone gene. To his relief, the gene that the Harv ard team had cloned was not human but rate insulin. Cloning had made it easy to cross the barriers between species.By May 1978, Genentech had synthesized the two chains of insulin in bacteria. By July, the scientists had purified the proteins out of the bacteria debris. In early August, they snipped of the the attached bacterial proteins and isolated the two individual chains. On August 21, 1978, they joined the protein chains together in a test tube to create the first molecules of recombinant insulin. In September 1979, Genentech applied for a patient for insulin. The Genetech patent would soon become one of the most lucrative petents in the history of technology.Synthesis of mover VIII (1983)Hemophilia is a rare drawing disorder in which the blood doesnt clot normally.If you have hemophilia, you may bleed for a longer time than others after an injury. You also may bleed inside your body (internally), especially in your knees, ankles, and elbows. This bleeding can damage your or gans and tissues and may be life threatening.Hemophilia is caused by a single mutation in the gene for a crucial clotting gene in blood, called factor VIII, and, until the mid-1980s, was treated with injections of concentrated factor VIII. During 1982 and early 1983, an emergence of mysterious immunological collapse among patients with multiple blood transfusions pinpointed the cause of the illness to blood-born factor that had contaminate the supply of factor VIII -a virus called AIDS. Nearly all the HIV-infacted hemophiliacs from the initial cohort had died of the complications of AIDS.In the spring of 1983, Dave Goeddel (1951- ) at Genentech began to focus on cloning the factor VIII gene. Meanwhile, a team of researchers from Harvard, lead by Tom Maniatis (1943- ) and Mark Ptashne (1940- ), formed a company called Genetics Institute (GI) also joined the race.As with insulin, the logic behind the cloning effort was evident rather than purifying the missing clotting factor out of liters of human blood, why not create the protein artificially, using gene cloning? If factor VIII could be produced through gene-cloning methods, it would be virtually free of any human contaminants, i=thereby reading it inherently safer than any blood-derived protein.Genetech k invigorated that the factor VIII project would challenge the outer limits of gene-cloning technology. Somatostatin had 14 amino acids insulin had 51. Factor VIII had 2,350. To succeed, the gene cloners would need to use new cloning technologies Both the somatstatin and insulin genes had been created from scratch by stitching together bases of DNA. But factor VIII gene was far too large to be created using DNA chemistry. To isolate the factor VIII gene, Genetech would need to tpull the native gene out of human cells.Tom Maniatis of GI, found a solution he had pioneered the technology to build genes out of RNA templateds using reverse transcriptase, the enzyme that could build DNA from RNA. Reverse transcri ptase made it possible to clone a gene after the intervening stuffer sequences had been snipped off by the cells splicing apparatus.In April, 1983, both Genentech and GI announced that they had purified recombinant factor VIII in test tubes a blood-clotting factor untainted by human blood.The production of factor VIII from its gene broke an important conceptual ground. The fears of Asilomar had been perfectly inverted. And gene cloning had emerged as potentially the safest way to produce a medical product for human use.