Figure 515

The basis of gene transfection. (a) Bacterial restriction enzymes break the two strands of DNA at different points, producing ends with exposed bases that are complementary to each other. (b) A segment of DNA containing one or more genes from one organism (donor) can be inserted into the DNA of another organism (host) by using the same restriction enzymes to produce complementary breaks in the host DNA, to which the donor DNA can bind.

Vander et al.: Human Physiology: The Mechanism of Body Function, Eighth Edition

PART ONE Basic Cell Functions bases that is complementary to the exposed strand on the other side of the break. This produces DNA fragments with "sticky" ends that can undergo base pairing. If a particular fragment that contains a gene or gene segment of interest can be identified and isolated, it can be inserted into another molecule of DNA, allowing the exposed ends of the fragment to hybridize with the exposed ends of the DNA that have been treated with the same nuclease. An enzyme known as a ligase can then be used to covalently link together the cut ends, resulting in the insertion of the DNA fragment into a second molecule of DNA. This technique can be used to insert DNA from one organism into the DNA of another, a procedure known as transfection. The organisms into which the DNA has been trans-fected are known as transgenic organisms.

A major problem occurs at the point where fragments of DNA must be introduced into a living cell because large molecules, such as DNA fragments, do not readily cross cell membranes. To overcome this problem, DNA fragments are inserted into the DNA of viruses that are able to enter host cells, carrying the modified DNA with them.

Replication of the transfected DNA inserted into bacteria produces additional copies of the DNA, or cloned DNA, each time the bacterium divides, that can be isolated in sufficient quantities to determine its sequence. Bacterial DNA, however, does not have in-trons, and so bacteria lack the spliceosomes required to delete intron-derived segments from DNA. Thus, bacteria are unable to use the transfected intron-containing DNA of eukaryotic organisms to form protein. DNA segments lacking introns, which are known as cDNA, or complementary DNA, can be derived from the isolated spliced mRNA that lacks introns. Using a viral enzyme called reverse transcriptase, the isolated mRNA can serve as a template for the synthesis of a complementary DNA strand. This cDNA can be transfected into bacteria that can then use it to form protein.

The transfection of a human gene, in its cDNA form, into bacteria can be used to produce large quantities of human proteins. For example, the gene for human insulin can be transfected into bacteria where it is transcribed into the protein insulin, which can then be isolated from the transfected bacteria and made available for the treatment of some forms of diabetes in which the patients are unable to synthesize insulin (to be discussed in Chapter 18).

Another genetic engineering procedure used in the study of DNA includes the formation of transgenic animals, primarily mice, in which a particular gene in the reproductive cells has been inactivated or deleted, forming a knockout organism. The effects of the absence of the gene's expression can be observed in the offspring of these mice, which provides insights into the normal function of the absent protein. Transgenic techniques can also be used to form cells that overproduce a particular protein.

It is hoped that the techniques of genetic engineering will one day be able to selectively replace mutant genes in humans with normal genes and thus provide a cure for genetic diseases.

SUMMARY

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