( 'hapter 22 



CLONES; TRANSFORMATION; 

 STRAND RECOMBINATION IN VITRO 







i k present understanding of 

 the mechanisms involved in 

 the biological replication of 

 DNA in vivo (Chapter 21 ) has been very 

 significantly advanced by experiments using 

 the DNA as well as the DNA polymerase of 

 bacteria. Electron microscopic examination 

 of bacteria reveals a nuclear region within 

 which it is possible to detect a chromosome- 

 like structure composed of DNA uncom- 

 bined with basic protein (p. 275). The 

 DNA within the bacterial nuclear body is 

 similar to typical chromosomal DNA in the 

 following ways: A = T and C - G; primary 

 and secondary organization; mechanism of 

 synthesis; and molecular integrity. There- 

 fore, it seems justified to consider bacterial 

 DNA as being primarily chromosomal DNA, 

 and, despite the chemical simplicity of this 

 structure, to use the term "chromosome" in 

 discussing bacteria. 



Since bacteria contain chromosomal DNA, 

 one expects them also to contain chromo- 

 some-type genes according to the hypothesis 

 — for which much indirect support has al- 

 ready been presented — that DNA is genetic 

 material. How suitable are bacteria as ex- 

 perimental material for the study of genetics? 

 The electron microscope reveals that each 

 Escherichia coli cell contains one to four 

 nuclear areas — usually two or four (Figure 

 22-1 ) — and that no nuclear membrane is 

 present. Although the morphological mech- 

 anism of nuclear division is still unknown, 

 a duplication of DNA occurs for each nu- 

 292 



clear body division, and it can be concluded 

 that daughter nuclear bodies are genetically 

 identical, just as they are after a typical 

 mitosis. 



Clones 



After nuclear-body replication, the bacte- 

 rium divides to produce daughter bacteria. 

 This method of increasing bacterial cell num- 

 ber is an asexual process called vegetative 

 reproduction. Starting with a single bac- 

 terium, continuous vegetative reproduction 

 results in a population of cells called a clone; 

 barring mutations, all members of a clone 

 are genetically identical. If mutation occurs 

 during clonal growth, the mutant is trans- 

 mitted to all the progeny of the mutant cell, 

 thus producing a genetically mosaic clone 

 whose proportion of mutant individuals 

 varies, depending upon the time the muta- 

 tion occurred and the relative reproductive 

 potential of mutant and nonmutant cells. 

 (All cells of a sexually-reproducing organ- 

 ism are also colonal in origin, except for 

 fertilization and meiosis and its products, 

 so that multicellular organisms can also be 

 mosaic for a mutant.) 



Consider the characteristics of bacteria 

 and their clones significant for a study of 

 mutation. The ease and speed with which 

 large populations of bacteria can be ob- 

 tained are of great advantage in mutation 

 studies. For example, under appropriate 

 culture conditions, E. coli divides about once 

 each half hour; in fifteen hours after thirty 

 successive generations have taken place one 

 cell produces a clone containing about ten 

 billion (K) 1 ") individuals. The number of 

 E. coli produced from a single cell after n 

 generations (or t hours) can be calculated 

 by the expression 2" (or 2 2t ) (Figure 22-2). 

 Space is no problem in working with bac- 

 teria since 10 10 individuals can readily be 

 grown in liquid broth in an ordinary test 

 tube. 



However, the small size of bacteria is a 



