Mapping Integrated Lambda

Mapping Integrated Lambda

How do we know lambda phage associates with the chromosome when it lysogenizes a cell? Lambda lysogens could possess copies of the phage DNA freely floating in the cytoplasm. A problem generated by this mode of existence is that the molecules would be randomly inherited by daughter cells on division. As a result, the lower the average number of lambda molecules per cell, the higher the fraction of daughters that might fail to inherit any and therefore become nonlysogenic. To explain lambda’s low rate of spontaneously becoming nonlysogenic, which is about 10^{- 6} per doubling, cells would have to possess unacceptably large numbers of the phage genomes. A second method to ensure proper inheritance of lysogeny would be for a nonintegrated lambda to possess a special segregation mechanism. Several genetic elements use this mechanism for stable inheritance. These include the phage P1 and the F factor, both of which have been discussed earlier in other contexts. This mechanism does not require large numbers of the lambda genome to be present. The third and simplest segregation mechanism would be for lambda to attach to or integrate into the host chromosome. Then it would be replicated and segregated into daughter cells with the host chromosomes.

Elegant genetic experiments showed not only that lambda associates with the chromosome but that it associates with a specific site on the chromosome. Now, of course, a simple Southern transfer experiment could settle the issue. The original data suggesting that the lambda DNA was associated with a particular region of the chromosome came from mating experiments between lysogenic male bacteria and nonlysogenic female bacteria.

Figure 18.1 The transfer frequency of markers from lambda lysogens or non-lysogens. Marker X+ is located near the origin of transfer, and markers Y+ and Z+are transferred after the point of lambda insertion.

Transfer of genetic markers  lying between the origin of transfer and the gal region showed no differences between lysogenic and nonlysogenic males. The frequency of female recombinants incor-porating genetic markers transferred after the gal genes, however, revealed a discrepancy (Fig. 18.1). Their frequency was much lower if the DNA was transferred from lysogenic males than if the DNA came from nonlysogenic males. This can be understood as follows: if lambda is attached to the host genome in the vicinity of gal, then when it enters a nonlysogenic female, it finds itself in an environment lacking lambda repressor. As a result, the phage induces and proceeds through a lytic cycle analogous to infecting a cell. This process of a phage inducing upon its transfer into a nonlysogenic female is called zygotic induction. Its parallel for the lac operon leads to temporary induction of the lacZYA genes upon their introduction into cells deleted of the operon. Later as lac repressor accumulates, transcription of the genes shuts off.

A minor paradox is raised by the lambda results discussed above. The DNA is transferred to females in a linear fashion beginning at a particu-lar point. Therefore it would seem that if a genetic marker lying ahead of the integrated lambda were transferred to females, the lambda itself eventually would also be transferred. Why are such cells not killed by zygotic induction?

The resolution to the paradox is that the majority of recipient cells that receive a gal-proximal marker also do not also receive gal. The DNA strand being transferred frequently breaks and conjugation stops. Even though genetic markers lying near the origin are transferred at high efficiency, markers lying farther away, and therefore transferred later in the mating process, are transferred with substantially lower efficien-cies. Consequently, most cells receiving a marker proximal to lambda or gal never receive lambda or gal; so zygotic induction occurs in only a tiny fraction of the cells that receive markers transferred before gal.