The partial diploid lacl lacZlad lacZ produces pgalactosidase only in the presence of lactose because the lad gene is trans dominant

ß-Galactosidase

These mutations affected the production of both p-galactosi-dase and permease, because genes for both enzymes are in the same operon and are regulated coordinately.

Some of these mutations were constitutive, causing the lac enzymes to be produced all the time, whether lactose was present or not, and these mutations fell into two classes: regulator and operator. Jacob and Monod mapped one class to a site upstream of the structural genes; these mutations occurred in the regulator gene and were designated lacl~. The construction of partial diploids demonstrated that a lacl+ gene was dominant over a lacl~ gene; a single copy of lacl+ (genotype lacl+/lacl~) was sufficient to bring about normal regulation of enzyme production. Furthermore, lacl+ restored normal control to an operon even if the operon was located on a different DNA molecule, showing that lacl was able to act in trans. A partial diploid with genotype lacl+ lacZ~/lacI~ lacZ+ functioned normally, synthesizing p-galactosidase only when lactose was present (< Figure 16.9). In this strain, the lacI+ gene on the bacterial chromosome was functional, but the lacZ" gene was defec tive; on the plasmid, the lacZ" gene was defective, but the lacI+ gene was functional. The fact that a lacI+ gene could regulate a lacZ+ gene located on a different DNA molecule indicated to Jacob and Monod that the lacI+ gene product was able to diffuse to either the plasmid or the chromosome.

Some lacI mutations isolated by Jacob and Monod prevented transcription from taking place even in the presence of lactose and other inducers such as IPTG. These mutations were referred to as superrepressors (lacIs), because they produced repressors that could not be inactivated by an inducer. Recall that the repressor has two binding sites, one for the inducer and one for DNA. The lacIs mutations produced a repressor with an altered inducer-binding site, which made the inducer unable to bind to the repressor; consequently, the repressor was always able to attach to the operator site and prevent transcription of the lac genes. Superrepressor mutations were dominant over lacI+; partial diploids with genotype lacIs lacZ+/lacI+ lacZ+ were unable to synthesize either p-galactosidase or permease, whether or not lactose was present (< Figure 16.10).

Super-repressor Active repressor lac! s

Super-repressor Active repressor lacI+

Pi lacI+

The lacIs gene produces a superrepressor that does not bind lactose.

RNA " polymerase

Lactose

Inactive repressor

RNA polymerase

Cannot bind

Cannot bind lacO+ ~ operator

X lacZ+

lacP+

Cannot bind w

The lacIs gene is trans dominant: the superrepressor binds both operators and prevents transcription in the presence and absence of lactose.

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