Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Molecular Genetics and Regulatory Systems
Modification of Cellular DNA Structure
Transformation and Conjugation in Bacteria

Transformation refers to the process by which Genetic information is transferred from one Cell to another in the form of free DNA. In this process, a double-stranded DNA fragment penetrates a recipient cell that is in a specific physiological state enabling it to take up foreign DNA (such Cells are termed competent). If the transferred fragment is homologous to the recipient's DNA, the latter's chromosome is rapidly altered through Crossing-over.

The Mechanism of transformation allows living Bacteria to incorporate not only chromosomal fragments, but also Plasmids. In Section 2.3, we learned that a plasmid is defined as a DNA molecule that exists and replicates independently of the bacterial chromosome. Plasmids typically consist of relatively small circular molecules with a molecular weight on the order of 10⁶—10⁸ (Fig. 6.15). Plasmid-mediated transformation is one of the primary techniques in Site-Directed Mutagenesis, discussed in Section 6.3.

While plasmids generally do not perform essential Functions under normal cellular growth conditions, they can confer several advantageous traits under specific environments. It has been established, for example, that plasmids known as R-factors (resistance factors) confer Antibiotic Resistance in bacteria.

Closely related to plasmids are episomes—DNA molecules that can either integrate into the host cell chromosome or exist independently of it. One of the best-known Examples of an episome is the F factor (fertility factor), which characterizes E. coli cells during conjugation. During this process, cells carrying the F factor (F⁺ cells) transfer it to cells lacking the F factor (F^- cells); occasionally, a portion of the F⁺ cell chromosome is also transferred to the F^- cell. R-factors can be transmitted in a similar manner. However, certain F⁺ mutants, designated as Hfr, behave differently: rather than simply infecting an F^- cell with the F factor, they transfer a large segment of their chromosome, which subsequently undergoes crossing-over with the F^- cell's chromosome to generate genetically modified strains.

Numerous experiments have demonstrated that the chromosome of an F⁺ cell enters an F^- cell linearly as a single strand. If the process is interrupted at various time intervals, F^- cells will contain chromosomal segments of varying lengths. Subsequent comparison of recipient cell populations containing different amounts of chromosomal material from F⁺ cells makes it possible to map the arrangement of genetic material on the F⁺ cell chromosome. This exact approach was used to generate detailed genetic maps such as the one shown in Fig. 6.16. This map indicates the relative positions of genes characteristic of various mutants. For example, the symbol thr designates a Gene typical of a mutant that grows only in the presence of Threonine. Deciphering many such designations can be found in Watson's monograph (reference [7] in Chapter 2).

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FIG. 6.15. Electron micrograph of plasmid pSC101 (X230,000). This circular DNA molecule, which exists and replicates independently of the bacterial chromosome, served as a crucial tool in early recombinant DNA experiments. (Micrograph courtesy of Dr. S. N. Cohen.)