MOLECULAR BIOLOGY CASE STUDY
Introduction
Insulin deficiency in humans is a common and serious pathologic condition. The major function of insulin is to counter the concerted action of a number of hyperglycemia-generating hormones and to maintain low blood glucose levels. Because there are numerous hyperglycemic hormones, untreated disorders associated with insulin generally lead to severe hyperglycemia and shortened life span. In the past, the insulin used for treatment of diabetes mellitus was derived from animal pancreata. Recently, however, human insulin produced by the recombinant DNA process has come into use because some patients develop immunity and sensitization against animal insulin, thus limiting its effectiveness ( 2000).
Recombinant DNA technology has revolutionized biology and is having an ever-increasing impact on clinical medicine. Much has been learned about human genetic disease from pedigree analysis and study of affected proteins, but in many cases where the specific genetic defect is unknown, these approaches cannot be used. The new technology circumvents these limitations by going directly to the DNA molecule for information. Manipulation of a DNA sequence and the construction of chimeric molecules, so-called genetic engineering, provides a means of studying how a specific segment of DNA works. Through this way, diabetes mellitus can be treated.
Problem Description
Diabetes is an example of a human disorder which is caused by the absence or malfunction of a protein normally synthesised in the body. This disorder can be treated by supplying the patient with the correct version of the protein. Insulin, synthesized by the b-cells of the islets of Langerhans in the pancreas, controls the level of glucose in the blood. An insulin deficiency manifests itself as diabetes mellitus. The problem lies in obtaining sufficient quantities of protein (initially insulin was purified from cadavers and animal pancreas products).
Possible Solutions
Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA. The complete DNA sequence of many plasmids is known; hence, the precise location of restriction enzyme cleavage sites for inserting the foreign DNA is available. Plasmids are smaller than the host chromosome and are therefore easily separated from the latter, and the desired DNA is readily removed by cutting the plasmid with the enzyme specific for the restriction site into which the original piece of DNA was inserted (2000).
The plasmid is cut across both strands by a restriction enzyme, leaving loose, sticky ends to which DNA can be attached. Special linking sequences are then added to the human cDNA so that it will fit precisely into the loose ends of the opened plasmid DNA ring. The plasmid containing the human gene, also called recombinant plasmid, is now ready to be inserted into another organism, such as a bacterial cell.
The recombinant plasmids and the bacterial cells are mixed up. Plasmids enter the bacteria in a process called transfection. With the recombinant DNA molecule successfully inserted into the bacterial host, another property of plasmids can be exploited – their capacity to replicate. Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies. When the bacteria divide, the plasmids are divided between the two daughter cells and the plasmids continue to reproduce. With cells dividing rapidly (every 20 minutes), a bacterium containing human cDNA (encoding for insulin, for example) will shortly produce many millions of similar cells (clones) containing the same human gene.
E. coli cells that are capable of forming colonies must either contain the vector without a chromosomal DNA insert or a vector with a chromosomal DNA insert. If the correct ratio of chromosomal DNA to vector DNA is used, rarely does a vector have two distinct pieces of chromosomal DNA inserted into it.
The inserted DNA should be cloned ‘in frame’ which means that the DNA sequence must be cloned in a way that the reading frame of the gene is maintained. Cloning vectors can incorporate many different features including transcription and translation signals. To clone into a vector and have the protein of interest be expressed from the regulated promoter, the DNA sequence must be cloned so that the reading frame of the gene is maintained.
Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used. The resulting offspring from mammalian clones have to arise from sexual reproduction.
Another example of a human protein that has been cloned and is produced in bacteria is the antibody. In 1976 it became possible to ‘immortalise’ single antibody-producing cells (from mice); such clones would be ideal for us, but very tantalisingly, this method has never worked well for human cells. In the present decade, however, the ability to clone the antibody genes has proved a promising alternative. If they are sampled from a suitable source, they can be cloned, identified and then mass-produced in bacteria, so that many single antibodies can be compared with each other (1998).
The coding regions of DNA, the transcripts of which ultimately appear in the cytoplasm as single mRNA molecules, are usually interrupted in the eukaryotic genome by large intervening sequences of noncoding DNA called introns. The function of the intervening sequences, or introns, is not clear. One proposed hypothesis is that they may serve to separate functional domains (exons) of coding information in a form that permits genetic rearrangement by recombination to occur more rapidly than if all coding regions for a given genetic function were contiguous. Such an enhanced rate of genetic rearrangement of functional domains might allow more rapid evolution of biologic function (2000).
Individuals with diabetes mellitus have the disease as a result of a genetic defect leading either to failure of insulin production in the pancreas or to failure of the target tissue to respond to insulin (1994).
Heredity has been recognized for many years as playing an important role in the etiology of a number of diseases affecting man. Diabetes mellitus in all its many forms is one of these diseases. Diabetes mellitus is not a single disease but is a collection of diseases having in common an abnormal glucose-insulin relationship. It has long been known to have a strong genetic component. Current estimates of the number of genetic mutations that are thought to be responsible for the development in diabetes mellitus range from 20 to 100, depending upon the definitions used for the disease (1994).
Conclusion
The advances in molecular biology have paved way for diseases and disorders to be treated in new ways that was not possible before. One of these diseases is diabetes mellitus which is basically a disorder from lack of insulin produced by the body. By cloning the genes that are responsible for the production of insulin, diabetes can be helped suppressed.
Credit:ivythesis.typepad.com
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