Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    original donor DNA and with subsequent growth of the bacteria the ratio increases in parallel with the total bacterial number. The marker carried by the original donor DNA has therefore become part of the recipient genome and is replicated in each division cycle. Five minutes after entry into a recipient, a length of donor DNA displays its original ability to transform a genetic marker; and this activity is multiplied with its subsequent replication. Before its original properties are regained at 5 minutes, however, the transforming DNA passes through an eclipse phase during which only a small part of its activity can be recovered. Following the protocol illustrated in Figure 1.2, Fox (1960) exposed p-nitrobenzoic acid-resistant pneumococci to transforming DNA carrying the streptomycin-resistance marker for 3 minutes, washed for 1 minute with DNAase to remove extraneous DNA molecules, and then reextracted DNA after varying times. To provide a control by which the activity of the donor (streptomycin-resistant) marker can be assessed, the ability of the reextracted DNA to transform for the recipient marker, p-nitrobenzoic acid resistance, was measured as well as its ability to transform for streptomycin-resistance. Immediately after exposure to DNA, the ratio of streptomycin-resistance to p-nitrobenzoic acid resis tance activity in the reextracted DNA was very low (about 5% of its ultimate level), but increased with longer periods of incubation before extraction to a maximum value between 5 and 10 minutes. Further incu bation then fails to promote any increase in the ratio. A donor transform ing DNA therefore loses its transforming activity immediately upon enter ing a recipient, but regains it during the 5 minute eclipse period; at the end of this period, the donor marker has become part of the recipient chromo some and so its activity relative to a recipient marker has reached a plateau. Venema et al. (1965a,b) reported a similar eclipse phase in B.subtilis. However, no eclipse was detected in H.influenzae in a study by Voll and Goodgal (1965). Changes in the state of transforming DNA after entry into a recipient suggest that only one strand of the donor DNA is utilized by the recipient. Lacks ( 1962) treated pneumococci with 32 P labeled transforming DNA for 5 minutes and then incubated the cells for 0, 3, 10 or 40 minutes before extracting the DNA from the recipients and examining the state of the label. The reextracted DNA was separated by chromatography on MAK (methylated albumin kieselguhr) columns into four classes: degraded fragments (oligonucleotides or nucleotides), sequences eluting together with RNA, single strands of DNA and duplex strands. Table 1.1 shows that immediately after entry, almost none of the radioactivity is present as duplex DNA; about half is in the form of degraded fragments, a small amount separates with RNA (due simply to the technical conditions of