Enzymology primer for recombinant DNA technology
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Sign In Register. Staff Pick. The Dutch House. How Powerful We Are. Macca's Makeover. Australian Pocket Oxford Dictionary. DNA is the molecule of heredity, and the procedure used for preparing rDNA is referred to as genetic engineering. The biotechnology industry and much of modern medicine, basic research, and agriculture depend on the use of rDNA. The discovery of restriction endonucleases, enzymes that reproducibly cleave double-stranded DNA molecules at specific sequences, paved the way for the development of rDNA technology.
Restriction endonucleases are produced in bacteria as a defense mechanism of that bacterium to restrict the growth of invading bacterial viruses; they act by destroying viral DNA. The enzymes hydrolyze or "cut" specific sites within a DNA molecule. Smith for the discovery and investigation of restriction endonucleases. The cuts made by these enzymes often leave single DNA strands with sticky ends due to the asymmetry of the cut made to a double-stranded molecule and the tendency of the bases in DNA to form hydrogen bonds with complementary bases on another strand.
Scientists realized that these enzymes could serve as a powerful tool for manipulating DNA in a controlled way. When scientists became concerned about whether this new technology posed risks to humans and the environment, there was an unprecedented and temporary suspension of experiments using rDNA.
In , a conference was held in Asilomar, California, to assess such risks, and it was determined that most rDNA work should continue as long as appropriate safeguards were in place. That committee, composed of scientists, physicians, ethicists, and legal experts, has met regularly since that time. When a DNA carrier, called a vector, and a targeted DNA sample are treated with the same restriction enzyme, the resulting fragments are left with matching, or complementary, sticky ends.
Some vectors are designed to induce protein synthesis from the information in the inserted genes. Other vectors are designed to deliver large segments of DNA to specific cells. Essentially every area of biological research has been affected by the use of rDNA technology. Transgenic animals into which DNA from another species has been inserted have been bred to expand the study of human biochemical processes and diseases. Transgenic mice that are highly susceptible to breast cancer or Alzheimer's disease have furthered the understanding of those diseases. Modern medicine is inextricably linked with rDNA technology.
Because cDNAs represent only the portions of eukaryotic genes that are transcribed into the mRNA, cDNA clones are particularly useful for analysis of gene expression and cell specialization. The existence of a cDNA is also evidence that the gene is active, or transcribed, in the cells or tissues from which the mRNA was isolated. Such information can be used to compare gene activities in healthy versus diseased cells, for instance. Frequently the simpler sequence of a cDNA is easier to analyze than the corresponding genomic sequence since it will not contain noncoding, or intervening, sequences introns.
Another advantage of cDNA is that generally the sequence does not include enhancers or regulatory sequences to direct their transcription. As a result, they can be combined with other regulatory systems in the clone to direct their expression.
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Genome sequencing projects typically generate sequence information from many different cDNA clones. To make a library, the thousands of different mRNAs are first harvested from the cell of interest, and cDNA is made using reverse transcriptase. The cDNA is then cloned into plasmids, and introduced into bacteria. Under the right conditions, each bacterium will take up only one cDNA.
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The bacteria are then grown in Petri dishes on a solid medium. A library therefore consists of a mixed population of bacteria, each carrying one type of cDNA. To find the bacterium containing a particular type of cDNA, one can either search for the gene itself with a nucleotide probe or for its protein product with an antibody. Screening a library depends either on having a probe bearing part of the nucleotide sequence or an antibody or other way of recognizing the protein coded by the gene.
Screening by nucleotide probes labeled with radioactive or chemical tags for detection depends on base pair complementarity between the single-stranded target DNA and the probe DNA; this allows the label to mark the cell with the desired cDNA. Screening by labeled antibody depends on binding of the antibody to the protein encoded by the gene.
Literally thousands of cloned genes have been isolated this way from libraries of many different species. One of the most powerful observations in biology is that the same or similar gene sequences can be isolated from different species, ranging from bacteria to humans. Human insulin was the first medicine to be created through recombinant DNA technology. Insulin is a protein hormone produced by the pancreas that is vital for regulation of blood sugar. In the disease insulin-dependent diabetes mellitus IDDM , the immune system attacks and destroys the insulin-producing cells. A person with IDDM requires daily injections of insulin to control blood sugar.
Before , insulin was isolated from pigs or other animals. Animal insulin has a slightly different amino acid sequence from the human form. In the early s, recombinant DNA technology was used to splice the human insulin gene into bacteria, which were grown in vats to make large amounts of the human protein. Recombinant human insulin was the first recombinant drug approved for human use. Since then more than two dozen other drugs have been created in this way, including growth hormone , blood clotting factors, and tissue plasminogen activator, used to break up blood clots following a stroke.
Gene sequence similarities indicate that all living organisms have descended from shared common ancestors, back to the beginning of life. Cloned DNA can also be incorporated into the genomes of multicellular organisms to create a transgenic organism. This makes possible a new approach to designing genotypes by adding genes gene-coded functions to species where those genes functions do not exist.
Genetically modified organisms GMOs created by modifying a gene or adding one from another species frequently offer the most direct way to improve the way people use organisms for food or chemistry. One example of a GMO is the development of "golden rice," designed to reduce blindness caused by vitamin A deficiency in rice-consuming areas of the world. To convert rice endosperm into a beta-carotene-rich food, a transgene was constructed with the genes required for beta-carotene production and inserted into rice cells. The transgene consists of a cDNA for phytoene synthase, from a daffodil flower library, plus other sequences.
Rice with these extra genes show a rich "golden" color from the beta-carotene that accumulates in the rice grain.
If golden rice can be bred into commercial strains and enough can be provided into the diet to reduce the incidence of vitamin A — related blindness, current agitation against GMO crops may evolve into enthusiasm for their application. Recall that cDNAs do not contain introns. Comparing a cDNA sequence with its corresponding DNA sequence on a chromosome the genomic sequence reveals the locations of introns in the genomic sequences. In the more complex eukaryotes the same genomic region may correspond to several different cDNAs.
This reveals the existence of alternative splicing, in which different sets of exons are used to make separate mRNA transcripts from one gene region. This expands the diversity of the protein, encoded by a single gene to include slightly different protein forms, called isoforms. Tissue-specific regulation of splicing indicates that these isoforms contribute important nuances to creating developmental differences between tissues.
Genomic DNA libraries have also proved invaluable for isolating genes that are poorly expressed that is, make little mRNA and for mapping disease-causing genes to specific chromosomal sites. The vectors used in genomic libraries are designed to incorporate greater lengths of cloned DNA than plasmids can carry. The first of these vectors was the lambda bacterial virus, which could hold an insert of 15 kb, followed by the cosmid, a hybrid between a plasmid and a phage a virus that infects bacteria with a DNA insert size of 45 kb.
Development of linear yeast artificial chromosomes YACs , which include a yeast centromere , origin of replication, and ends telomeres , which successfully grow in the yeast Saccharomyces cerevisiae , carry clones of kb to more than 2, kb. Subsequent development of bacterial artificial chromosomes BACs that contain kb of insert DNA and are relatively easy to culture has put genomic cloning within reach of almost every molecular biology laboratory.
Enzymology primer for recombinant DNA technology
Clones are harder to work with as they get larger. BACs provided one route to sequencing the human genome, where their large capacity was critical. All the different genome sequencing projects start with a large number of BAC clones for that species, subclone 1 kb fragments of the DNA from each BAC into plasmids, and determine their sequence using high-speed machines. Computer-based comparisons of the results then assemble the nucleotide sequences into a coherent order by aligning the regions where they overlap.
A library of genomic DNA contains many clones with inserts that partially overlap each other because random breakage of chromosomal DNA is used to produce fragments for cloning. The order of fragments in the original chromosome can be determined by "chromosome walking. The two clones are then compared, and the nonoverlapping end of the second clone is subcloned for use as the next probe. In this way, a "walk" is carried out over many steps to identify adjacent DNA on the same chromosome, allowing the fragments to be placed in sequence.
A series of sequential, partially overlapping clones is termed a "contig" for contiguous sequence ; the goal of genome mapping is to make a separate contig for all the DNA clones from one chromosome a continuous covalent molecule. Contigs made large genome sequencing feasible since a minimum number of BACs could be chosen from their order in the map. Locating a human disease gene on a chromosome map is now equivalent to locating the gene approximately on a contig and the DNA sequence map.
This speeds gene identification through cloning the gene and determining what protein the gene encodes. The positional approach is important for single-gene Mendelian disease traits that are well known clinically but not at a biochemical level. Cystic fibrosis CF was one such disease. It is the most common severe autosomal recessive disorder among European populations and their descendants in the New World. Patients suffer from mucus accumulation and frequent bacterial infections in their lungs.
In the United States , CF patients are the single largest group receiving transplants to replace damaged lungs. However, the clinical studies failed to determine which gene product is defective in the patients. Extensive studies on families with CF led to identification of the causative gene on chromosome 7. Initially recombination studies placed the gene within a small region of the chromosome of approximately one million base pairs. Starting at DNA clones from both ends of this region, the researchers used chromosome walking to clone all of the interval; several candidate genes were identified within the region but rejected as the cause of CF.
Finally, one gene was identified within these clones that had the right properties: It was normally expressed in the lungs but not the brain, and it encoded a protein that made sense for the cause of the disease. In addition, patients with CF had specific mutations in this gene.
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The functional CF gene encodes a chloride channel transmembrane regulatory protein CFTR that controls transport of certain ions in and out of epithelial surface cells. The most common mutation encodes a CFTR protein that is missing one amino acid and cannot reach its site of function in the cell membrane. As a result, ions become too concentrated inside the cell, and water moves in.
The result is dried secretions , such as very sticky mucus.
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Gene therapy to add a functional copy of the CFTR to lung cells has not been successful, in part because the patients develop an immune response to reject the vector, and, in some cases, the normal protein. Mild improvements have been short-lived, or affect only small patches of cells in the respiratory tract. Alternative approaches to better understanding the physiology of the disease to direct drug design seem more viable.
To this end, a mouse model with an inactivated CFTR gene is used to test potential drugs. Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York : Garland Publishing, Felsenfeld, Gary.