Porth's Essentials of Pathophysiology, 4e

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Genetic Control of Cell Function and Inheritance

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according to size by electrophoresis. The single-stranded DNA is then transferred to nitrocellulose paper, baked to attach the DNA to the paper, and treated with a series of radioactive probes. After the radioactive probes have been allowed to bond with the denatured DNA, radiog- raphy is used to reveal the labeled DNA fragments. When used in forensic pathology, this procedure is applied to specimens from the suspect and the forensic specimen. Banding patterns are then analyzed to see if they match. With conventional methods of analysis of blood and serum enzymes, a 1 in 100 to 1000 chance exists that the two specimens match because of chance. With DNA fingerprinting, these odds are 1 in 100,000 to 1 million. When necessary, the polymerase chain reaction (PCR) can be used to amplify specific segments of DNA (see Chapter 14). It is particularly suited for amplifying regions of DNA for clinical and forensic testing proce- dures because only a small sample of DNA is required as the starting material. Regions of DNA can be amplified from a single hair or drop of blood or saliva. GeneTherapy Although quite different from inserting genetic mate- rial into a unicellular organism such as bacteria, tech- niques are available for inserting genes into the genome of intact multicellular plants and animals. The adeno- viruses are promising delivery vehicles for these genes. These viruses are ideal vehicles because their DNA does not become integrated into the host genome; however, repeated inoculations are often needed because the body’s immune system usually targets cells expressing adenovirus proteins. Sterically stable liposomes also show promise as DNA delivery mechanisms. This type of therapy is one of the more promising methods for the treatment of genetic disorders such as cystic fibrosis, certain cancers, and a number of infectious diseases. Two main approaches are used in gene therapy: transferred genes can replace defective genes or they can selectively inhibit deleterious genes. Cloned DNA sequences are usually the compounds used in gene ther- apy. However, the introduction of the cloned gene into the multicellular organism can influence only the few cells that get the gene. An answer to this problem would be the insertion of the gene into a sperm or ovum; after fertilization, the gene would be replicated in all of the differentiating cell types. Even so, techniques for cell insertion are limited. Not only are moral and ethical issues involved, but these techniques cannot direct the inserted DNA to attach to a particular chromosome or supplant an existing gene by knocking it out of its place. To date, gene therapy has been used successfully to treat children with severe combined immunodeficiency disease (see Chapter 16) and in a suicide gene transfer to facilitate treatment of graft-versus-host disease after donor lymphocyte infusion. RNA InterferenceTechnology One method of gene therapy focuses on the previ- ously described replacement of missing or defective

genes. However, several genetic disorders are not due to missing genes, but to faulty gene activity. With this in mind, some scientists are approaching the problem by using RNA interference (RNAi) to stop genes from making unwanted disease proteins. RNA interference is a naturally occurring process in which small pieces of double-stranded RNA (small interfering RNA [siRNA]) suppress gene expression. Scientists believe that RNAi may have originated as a defense against viral infections and potentially harmful genomic invaders. In viral infec- tions, RNAi would serve to control the infection by pre- venting the synthesis of viral proteins. With the continued refinement of techniques to silence genes, RNAi has already had a major impact on molecular biology. For example, it has given scientists the ability to practice reverse genomics, in which a gene’s function can be inferred through silencing its expres- sion. Increasingly, pharmacologic companies are using RNAi to identify disease-related drug targets. There also is considerable interest in harnessing RNAi for therapeutic purposes, including the treatment of human immunodeficiency virus (HIV) infection and hepatitis C. Before this can occur, however, the therapeutic methods must be shown to be safe and effective, and obstacles to delivering RNAi into targeted cells must be overcome. It is difficult for RNA to cross the cell membrane, and it is quickly broken down by enzymes in the blood. ■■ The genome is the gene complement of an organism. Genomic mapping is a method used to assign genes to particular chromosomes or parts of a chromosome.The most important methods used are family linkage studies, gene dosage methods, and hybridization studies. Linkage studies assign a chromosome location to genes based on their close association with other genes of known location. Dosage studies involve measuring enzyme activity to determine if both members of a gene pair are present and functioning normally. Hybridization studies involve the fusion of human somatic cells with those of a different species to extraction of specific types of mRNA used in the synthesis of complementary DNA strands. The complementary DNA strands, labeled with a radioisotope, bind with the genes for which they are complementary and are used as gene probes. ■■ Genetic engineering has provided the methods for manipulating nucleic acids and recombining genes (recombinant DNA) into hybrid molecules that can be inserted into unicellular organisms study gene location on chromosomes. ■■ Recombinant DNA studies involve the SUMMARY CONCEPTS

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