BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 31. GENE REARRANGEMENTS: RECOMBINATION, TRANSPOSITION, AND CLONING

The subject of this chapter is Gene rearrangement through the movement of large DNA segments. First, we discuss The process of genetic recombination, in which a new DNA molecule is generated by the breakage and reunion of DNA strands. The probability of genetic recombination is substantially increased in the presence of extensive regions of Homology between interacting DNA molecules. Recombination intermediates have been isolated, and more recently, The enzyme catalyzing the reciprocal exchange of DNA strands has been characterized. Next, we discuss transposition—the movement of a gene from one chromosome to another or from one site to another within the same chromosome. Unlike general recombination, transposition does not require extensive regions of homology. In prokaryotes, the presence of so-called insertion sequences, or IS elements, imparts mobility to unrelated DNA fragments, ensuring their joining. Recombination and transposition have played a vital role in evolution by driving The Emergence of new genomes. At the end of the chapter, we examine the construction of novel gene combinations in vitro and their expression in host Cells. Genes can be covalently joined to plasmid and viral DNA using Restriction Endonucleases and DNA ligase. Such recombinant DNA molecules can replicate and be expressed in suitable host cells. In addition, we discuss The Significance of Gene cloning and its potential Structure/179.html">Practical Applications. Research on recombination and transposition, as well as The Development of cloning Methods, is proceeding at an exceptionally rapid pace. These efforts are giving rise to powerful new METHODS FOR STUDYING genomes, providing deeper insights into the mechanisms of their evolution and expression.

31.1 Genetic Recombination is Based on the Breakage and Reunion of DNA Strands

Genetic recombination produces a DNA molecule whose sequence derives in part from one parental molecule and in part from the other. Studies of E. coli cells infected with a mixture of T4 phages labeled with ³²P and bromodeoxyuridine have provided insight into the molecular nature of recombinant molecules. The buoyant density of bromouracil-labeled DNA is significantly higher than that of ³²P-labeled DNA, allowing parental molecules to be separated from each other and from recombinant molecules by cesium chloride (CsCl) density gradient centrifugation. The results of centrifugation experiments showed that, following infection with the phage mixture, recombinant molecules contain both ³²P and bromouracil. The structure of the hybrid molecules depended on whether DNA Synthesis occurred during their formation. In the absence of DNA synthesis, the ³²P-DNA within the recombinant molecules was not covalently linked to the bromouracil-labeled DNA. Upon heating above the melting Temperature of the double-helical molecules, the hybrid dissociated into light and heavy components. The fragments of parental molecules in this hybrid are held together by base pairing; therefore, this intermediate is termed a joint molecule (Fig. 31.3). If DNA synthesis did occur, single-stranded gaps in the joint intermediate were filled by DNA polymerase I, and the ends were joined by DNA ligase. The resulting recombinant molecule could no longer be separated into light and heavy components because the DNA segments were covalently linked. These experiments demonstrated that single-stranded DNA regions are intermediates in genetic recombination and that Enzymes participate in this process.

Class="center">Fig. 31.1. Transfer of Genetic information from one E. coli Cell to another. This electron micrograph shows two E. coli cells connected by a pilus during conjugation. DNA is transferred through the pilus from the donor cell to the recipient cell

Fig. 31.2. Electron micrograph of a circular DNA molecule containing genes for resistance to multiple Antibiotics. Such Plasmids (R factors, from resistance) confer resistance to various agents upon cells, which can be transferred to other cells

Fig. 31.3. Joint intermediate of T4 phage DNA recombination in infected Bacteria. ³²P-labeled DNA is shown in red, and bromouracil-labeled DNA in blue