We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one ‘pseudo‐operator’ O2 or O3 decreases repression by wild‐type tetrameric Lac repressor approximately 2‐ to 3‐fold; absence of both ‘pseudo‐operators’ decreases repression greater than 50‐fold. O1 alone represses under these conditions only approximately 20‐fold. Dimeric active Lac repressor (iadi) represses the wild‐type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

No abstract

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 59-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient ''jumpstarting'' scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila. O NE of the main goals of modern genetics is to link the many thousands of genes identified through the sequencing of whole genomes of model organisms to gene function. The most powerful technique for this purpose so far has been transgenesis with mobile elements. This technique is a means to disrupt, overexpress, or misexpress single genes to identify expression patterns and also to characterize genetic pathways and their interactions. One of the main advantages of insertional mutagenesis over the classical method of chemical mutagenesis is the ease with which the targeted gene can be identified, since it carries an inserted tag.The P element was the first mobile element that enabled germ-line transformation of an insect species (Rubin and Spradling 1982). Since then, thousands of single P-element insertions causing lethality, semilethality, sterility, semisterility, and visible phenotypes have been created and analyzed in Drosophila (Cooley et al.

Gel‐filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel‐shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich‐complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5′‐ATGACGTCAT‐3′) at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4‐helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.

Exchanges in positions 1 and 2 of the putative recognition helix allow lac repressor to bind to ideal lac operator variants in which base pair 4 has been replaced. We show here that an Asn exchange in position 6 of the putative recognition helix of lac repressor abolishes lac repressor binding to ideal lac operator. This lac repressor variant, 7 6 however, binds to a variant of the ideal lac operator 5' TT-S 4 3 2 1 1 2 3456 7 TGAGCGCTCAAA 3' in which the original G-C of position 6 has been replaced by TEA. This result and our previous data confirm our suggestion that the N terminus of the recognition helix of lac repressor enters the major groove close to the center of symmetry of lac operator and that its C terminus leaves the major groove further away from the center of symmetry. The consequences of this model are discussed in regard to various phage and bacterial repressor operator systems.Twenty-seven years ago lac and A repressor became the paradigms of negative control (1). They were isolated at about the same time (2, 3), and soon after, it was shown that they both bound to their operator DNA specifically (4, 5). For a while it seemed that the lac system was easier to analyze. Overproducers of lac repressor led to the isolation of large amounts of this protein (6). This allowed protein sequence analysis of lac repressor (7). Genetic analysis identified the N-terminal domain of lac repressor as a small DNA and operator binding protrusion (8). The core of lac repressor was shown to be responsible for aggregation and inducer binding (9, 10). Active fusions between lac repressor and f3-galactosidase (11) led to the conclusion that although lac repressor is tetrameric a dimer suffices for lac operator recognition (12). lac operator (13, 14) and various lac operator mutants (15) were sequenced early and protection experiments outlined the active parts of lac operator (15).Yet, lac repressor did not yield suitable crystals for x-ray analysis. The x-ray structures of Acro protein (16), of 434 repressor-operator complex (17), of cap protein (18), and trp repressor (19) gave these systems a tremendous advantage. In particular, the A, P22, and 434 systems became well understood through the combined use of x-ray data and reverse genetics. An outstanding review of the phage repressor work can be found in the recent book of Ptashne (20).We have recently reported an analysis (21) that may bring lac repressor back into focus. We have set up a system that allows detection and selection of specificity changes in the lac repressor-operator complex. The system consists of two plasmids that can coexist in an Escherichia coli strain carrying a lac deletion. The plasmids have different sizes resistance genes and origins of replication. One carries a semisynthetic lacI gene. In its 5' end, which encodes the operator binding domain, short sequences can be replaced by short synthetic DNA double strands. The other plasmid contains a lac operon in which the lac operator has been deleted and replaced by a unique restric...

In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer–repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer–repressor stoichiometry in induction of the lac operon.

Transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific, economic, or medical interest. Minos, a member of the Tc1/mariner family of DNA transposons, exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates. Its use as a tool for transgenesis and genome analysis of rather different animal species is described.