Dna 2 Essay, Research Paper
In the last 10 years, there has been some scientific leaps concerning diabetes, and genetic engineering. The increasing incidence and diagnostic detection of diabetes worldwide coupled with changing trends in the food animal market stimulated people to seek alternative sources of insulin completely independent of an animal-gland source. (Ryan, 1986). The basic recombinant DNA process of formulating insulin was developed. DNA carries the genetic information that determines the fate of each cell. Recombinant DNA technology allows manipulation of bacterial host cells by insertion of genes for production of either the A chain or the B chain of the insulin molecule, or the entire proinsulin molecule. Prior to 1986, the A and B chains of the insulin molecule were produced by separate fermentations (growth of the genetically altered Escherichia coli under controlled conditions). The chains were purified and later combined by chemical techniques to produce insulin structurally and chemically identical to pancreatic human insulin. (Hyde, 1984)
As of 1986, human insulin began to be produced by a process which involves the enzymatic conversion of human insulin’s biosynthetic precursor, human proinsulin. The genetic coding for human proinsulin is inserted into the special E. coli bacteria which are then grown in a fermentation process to produce human proinsulin. With genetic engineering, new proteins are synthesized. They can be introduced into plants or animal genomes, producing a new type of disease resistant plants, capable of living in inhospitable environments. When introduced into bacteria, these proteins have also produced new antibiotics and useful drugs. Techniques of cloning generate large quantities of pure human proteins, which are used to treat diseases like diabetes. In the future, a resource bank for rare human proteins or other molecules is a possibility. For instance, DNA sequences which are modified to correct a mutation, to increase the production of a specific protein or to produce a new type of protein can be stored. This technique will probably play a key role in gene therapy. (Stwertka, 1982)
The insulin hormone is essential for a healthy and normal existence. The insulin molecule consists of two chains of linked amino acids, the A chain containing 21 amino acids, the B chain, 30. These chains are connected by two disulfide bridges (formed of two sulfur atoms each), while a third disulfide bridge stretches across several amino acids on the A chain. The connected chains are partially coiled and twisted into globular structure, a configuration essential for biological activity. Insulin s most prominent effect is to lower blood sugar, primarily by facilitating the uptake and use of glucose by muscle and fat cells and inhibiting the formation of new glucose by the liver. Insulin increases the storage of excess glucose in the form of glycogen. It also stimulates the storage of other energy forms (fat, protein) and inhibits the breakdown and use of these stored materials by the body. (Snyderman, 1998)
Insulin is the most important hormone in intermediate metabolism. When insulin is absent in the body, blood sugar levels rise; muscle and fat cells aren’t able to utilize glucose for energy. It signals the body that it s “hungry”, the liver then releases glycogen (a form of stored glucose). This further increases the blood sugar level. When blood sugar level reaches about 180 mg/dl, glucose begins to spill into the urine. A large amount of water is needed to dissolve the excess sugar, resulting in excessive thirst and urination. Without glucose for energy, the body begins to metabolize protein and fat. Fat metabolism results in the production ketones in the liver. Ketones are excreted in the urine along with sodium bicarbonate, which results in a decrease in the pH of the blood. This condition is called acidosis. To correct acidosis, the body begins a deep, labored respiration, called Kussmaul’s respiration. Left unchecked, a person in this situation will fall into a coma and die. (What is Insulin, 1998)
Insulin is produced by the pancreas that controls the level of sugar (glucose) in the blood and is used in the treatment of diabetes mellitus. The hormone is synthesized in beta cells, which are included in separated groups of hormone-secreting cells of the pancreas known as the islets of Langerhans. Insulin is secreted continually at varying rates and acts in fine tune with hormones (glucagon, catecholamines) that raise blood sugar to maintain blood sugar levels within very narrow limits (about 80 to 100 mg/100 ml of blood). The creation of insulin in the beta cells of the pancreas is a two step procedure. Beta cells first produce preproinsulin. Preproinsulin is cleaved to create proinsulin, which is further cleaved to produce equal amounts of insulin and C-peptide. By measuring the amount of C-peptide in the blood, scientists can determine the amount of insulin produced by the pancreas. A normally functioning pancreas can manufacture and release 40 to 50 units of insulin daily and have several hundred units available in pancreatic storage for release as needed. The pancreas usually stores about 200 units of insulin. The average basal rate for adults is one to two units per hour. After meals, secretion increases to four to six units per hour. (Britannica, 95)
The function of restriction enzyme is important in gene cloning. Each Restriction enzyme recognizes a short, specific sequence of nucleotide base, these regions are called recognition sequences and are randomly distributed throughout the DNA. The structure of DNA is a double helix, with about 10 nucleotide pairs per helical turn. Each spiral strand, composed of a sugar phosphate backbone and attached bases, is connected to a complementary strand by hydrogen bonding (non- covalent) between paired bases, adenine (A) with thymine (T) and guanine (G) with cytosine (C). Different bacterial species make Restriction enzyme that regonize different nucleotide. When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis, splitting of a chemical bond by addition of a water molecule, of the bond between adjacent nucleotides. (Ryan, 1986) The Type II restriction enzymes is used in cloning. The Type II Restriction enzyme is independent of its methylase. It cleaves DNA at specific sites within the recognition site. These enzymes have the ability to cut DNA at precise locations which enabled researchers to isolate gene-containing fragments and recombine them with other molecules of DNA. More than 2,500 type II R enzymes have been identified from a variety of bacterial species. These enzymes recognize about 200 distinct sequences, which are four to eight bases in length.
Bacterium uses the Restriction enzyme as a form of defense mechanism. Restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. It is used to defend against bacterial viruses called bacteriophages, or phages. A phage infects a bacterium by inserting its DNA into the bacterial cells so that it might be replicated. The Restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. In the bacterial cell, Restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensable tools of recombinant DNA technology. (Watson, 1981)
Gene cloning ranks as one of the most significant accomplishments involving recombinant DNA. This procedure has enabled researchers to use E. coli to produce virtually limitless copies of donor genes from other organisms, including human beings.
To perform gene cloning, researchers first use a class of bacterial enzymes: restriction endonulease to remove a fragment of double-stranded DNA, containing the genes of interest, from a donor. Restriction endonucleases can be thought of as “biological scissors”; each of these enzymes cleaves DNA at a specific site defined by a sequence of four or more nucleotides. (figure 2) Once the desired DNA fragment has been removed from the donor cell, it must be inserted into the bacterial cell. This is done by first inserting the donor DNA into a plasmid, one of the small, circular pieces of DNA that are found in E. coli and many other bacteria. Plasmids generally remain separate from the bacterial chromosome, although some plasmids do occasionally become incorporated into the chromosome, but they carry genes that can be expressed in the bacterium.
Plasmids generally replicate and are passed on to daughter cells along with the chromosome. By treating a plasmid with the same restriction endonuclease that was used to cleave the donor DNA, it is possible to incorporate the foreign DNA fragment into the plasmid ring. This can occur because the restriction enzyme cleaves double-stranded DNA in such a way as to leave chemically sticky end pieces. (figure 1) Thus, It is possible for the sticky-ended fragment of foreign DNA to attach to the complementary sticky ends of the cut-open plasmid ring. This laboratory procedure, called “gene splicing,” is the major operation of recombinant DNA technology. The molecular biologist then uses the plasmids as vectors to carry the foreign gene into bacteria. This is accomplished by exposing bacteria to the plasmids. (Wayne, 1997)
Plasmids are highly infective. Therefore, many of the bacteria will take up the particles; to insure maximum uptake the bacteria are often treated with calcium salts, which makes their membranes more permeable. Picking out the correct gene. The incorporation of the plasmids into the bacterial cells transfer the genes of one species into the genome of another. As a result of the high infectivity of plasmids and the rapid growth of E. coli, investigators can quickly culture large numbers of bacteria, many of which will have incorporated the foreign human DNA. (As many as 1×109 bacteria can grow in one millilitre of medium overnight). Researchers can select the bacteria that contain the foreign DNA by attaching to the fragment of DNA a gene that gives resistance to an antibiotic such as tetracycline. By treating the culture with tetracycline, all bacteria that have not incorporated the gene for resistance will be killed. The remaining cells can be grown in enormous numbers, most of which will contain the cloned fragment of foreign DNA. (screening) The cloned DNA can be removed from the bacterial culture as follows. First, the bacteria are broken apart and the DNA content is separated by centrifugation. The DNA fraction is then heated, which causes the double-stranded molecules to separate into single strands. Upon cooling, each single strand will hybridize, to another single strand to which it is complementary (adenine opposite thymine, cytosine opposite guanine). The fermentation step is stopped by heat sterilization to eliminate any possibility of contamination. The connecting peptide is then enzymatically cleaved from the human proinsulin to produce human insulin. Sophisticated purification techniques and sensitive analytical procedures, including chromatography and computerized are used to assure the purity of human insulin.
Although gene therapy is still experimental, in other ways genetic research already has changed how medicine is practiced. This is because of the genetically engineered drugs that are now available through biotechnology. Take, for example, the treatment of diabetes. In the past, the only way to get insulin for diabetics was to process it from pigs and cattle. Then researchers learned how to make insulin by cloning the human gene that carries the instructions for making insulin. Cloning and other techniques of genetic engineering have had many positive results. Genetic engineering has helped increase the supply of medical products and lower their costs. It has resulted in new drugs being created. Another benefit of genetically engineered materials is their purity. This is important, since there have been cases in the past where medical products processed from animals or human donors carried disease.