Porth's Essentials of Pathophysiology, 4e

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Genetic Control of Cell Function and Inheritance

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chromosomes of both species. Because these hybrid cells are unstable, they begin to lose chromosomes of both species during subsequent cell divisions. This makes it possible to obtain cells with different partial combina- tions of human chromosomes. The enzymes of these cells are then studied with the understanding that for an enzyme to be produced, a certain chromosome must be present and, therefore, the coding for the enzyme must be located on that chromosome. In situ hybridization involves the use of specific sequences of DNA or RNA to locate genes that do not express themselves in cell culture. Deoxyribonucleic acid and RNA can be chemically tagged with radioactive or fluorescent markers. These chemically tagged DNA or RNA sequences are used as probes to detect gene loca- tion. The probe is added to a chromosome spread after the DNA strands have been separated. If the probe matches the complementary DNA of a chromosome segment, it hybridizes and remains at the precise location (therefore the term in situ ) on a chromosome. Radioactive or fluo- rescent markers are used to find the location of the probe. Haplotype Mapping As work on the Human Genome Project progressed, many researchers reasoned that identifying the com- mon patterns of DNA sequence variations in the human genome would be possible. An international project, known as the International HapMap Project, was orga- nized with the intent of developing a haplotype map of these variations. One of the findings of the Human Genome Project was that the genome sequence was 99.9% identical for all people. It is anticipated that the 0.1% variation may greatly affect an individual’s response to drugs, toxins, and predisposition to various diseases. Sites in the DNA sequence where individuals differ at a single DNA base are called single-nucleotide polymorphisms (SNPs, pronounced “snips”). A haplo- type consists of the many closely linked SNPs on a single chromosome that generally are passed as a block from one generation to another in a particular population. One of the motivating factors behind the HapMap proj- ect was the realization that the identification of a few SNPs was enough to uniquely identify the haplotypes in a block. The specific SNPs that identify the haplotypes are called tag SNPs . A HapMap is a map of these hap- lotype blocks and their tag SNPs. This approach should prove useful in reducing the number of SNPs required to examine an entire genome and make genome scanning methods much more efficient in finding regions with genes that contribute to disease development. Recent improvements in sequencing technology (“Next-Gen” sequencing platforms) have dramati- cally reduced the cost of sequencing. The goal of the 1000 Genomes Project is to sequence the genomes of a large number of people and provide a comprehen- sive high resolution resource for human genetic varia- tion (www.1000genomes.org). It is anticipated that the HapMap Project and the 1000 Genomes Project will provide a useful tool for disease diagnosis and manage- ment. Much attention has focused on the use of SNPs

to decide whether a genetic variant is associated with a higher risk of disease susceptibility in one population versus another. Pharmacogenetics addresses the variabil- ity of drug response due to inherited characteristics in individuals. With the availability of SNPs, it may soon be possible to identify persons who can be expected to respond favorably to a drug and those who can be expected to experience adverse reactions. This would result in safer, more effective, and more cost-efficient use of medications. Recombinant DNATechnology The term recombinant DNA refers to a combination of DNA molecules that are not found together in nature. Recombinant DNA technology makes it possible to iden- tify the DNA sequence in a gene and produce the pro- tein product encoded by a gene. The specific nucleotide sequence of a DNA fragment can often be identified by analyzing the amino acid sequence and mRNA codon of its protein product. Short sequences of base pairs can be syn- thesized, radioactively labeled, and subsequently used to identify their complementary sequence. In this way, iden- tifying normal and abnormal gene structures is possible. Gene Isolation and Cloning The gene isolation and cloning methods used in recom- binant DNA technology rely on the fact that the genes of all organisms, from bacteria through mammals, are based on a similar molecular organization. Gene cloning requires cutting a DNA molecule apart, modifying and reassembling its fragments, and producing copies of the modified DNA, its mRNA, and its gene product. The DNA molecule is cut apart using a bacterial enzyme, called a restriction enzyme, that binds to DNA wherever a particular short sequence of base pairs is found and cleaves the molecule at a specific nucleotide site. In this way, a long DNA molecule can be broken down into smaller, discrete fragments, one of which presumably contains the gene of interest. Many restriction enzymes are commercially available that cut DNA at different recognition sites. The restrictive fragments of DNA can often be replicated through insertion into a unicellular organism, such as a bacterium (Fig. 5-12). To do this a plasmid , which is a cloning vector such as a bacterial virus or a small DNA circle that is found in most bacteria, is used. Viral and plasmid vectors replicate autonomously in the host bacterial cell. During gene cloning, a bacterial vec- tor and the DNA fragment are mixed and joined by a special enzyme called a DNA ligase . The recombinant vectors formed are then introduced into a suitable culture of bacteria, and the bacteria are allowed to replicate and express the recombinant vector gene. Sometimes, mRNA taken from a tissue that expresses a high level of the gene is used to produce a complementary DNA molecule that can be used in the cloning process. Because the fragments of the entire DNA molecule are used in the cloning pro- cess, additional steps are taken to identify and separate the clone that contains the gene of interest.