Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis.

The Escherichia coli lac operator has been placed on the 3' side of the promoter for the penicillinase gene of Bacillus licheniformis, creating a hybrid promoter controllable by the E. coli lac repressor. The E. coli lac repressor gene has been placed under the control of a promoter and ribosome-binding site that allows expression in Bacillus subtilis. When the penicillinase gene that contains the lac operator is expressed in B. subtilis on a plasmid that also produces the lac repressor, the expression of the penicillinase gene can be modulated by isopropyl beta-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. A similar system was constructed from a promoter of the B. subtilis phage SPO-1 and the leukocyte interferon A gene, which allowed the controlled expression of interferon in B. subtilis. These two examples show that a functional control system can be introduced into B. subtilis from E. coli.     

Design of molecular control mechanisms and the demand for gene expression.

Regulation by a repressor protein is the mechanism selected when, in the organism's natural environment, there is low demand for expression of the regulated structural genes. Regulation by an activator protein is selected when there is high demand for expression of the regulated structural genes. These general conclusions are useful in relating physiological function to underlying molecular determinants in a wide variety of systems that includes repressible biosynthetic pathways, inducible biosynthetic enzymes, inducible drug resistance, and prophage induction, as well as inducible catabolic pathways, for which a special case of this prediction previously was reported [Savageau, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 2453-2455].     

A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns.

A biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker joining biotin to the C-5 of the pyrimidine ring has been synthesized. This analog, Bio-SS-dUTP, is an efficient substrate for Escherichia coli DNA polymerase I. Bio-SS-dUTP supported DNA synthesis in a standard nick-translation reaction at 35%-40% the rate of an equal concentration of the normal nucleotide, TTP. DNA containing this analog was bound to an avidin-agarose affinity column and subsequently eluted after reduction of the disulfide bond by dithiothreitol. The ability to recover biotinylated DNA from an avidin affinity column under nondenaturing conditions should prove useful in the isolation of specific protein-DNA complexes. As a demonstration of this approach, Bio-SS-DNA was reconstituted with histones to form 11S monomer nucleosomes. Bio-SS-nucleosomes were shown to selectively bind to avidin-agarose. Ninety percent of the bound Bio-SS-nucleosomes were recovered from the affinity column by elution with buffer containing 50-500 mM dithiothreitol. The recovered nucleosomes were shown to be intact 11S particles as judged by velocity sedimentation in a sucrose gradient. This approach may prove to be generally useful in the isolation of protein-DNA complexes in a form suitable for further analysis of their native unperturbed structure.     

Covalent protein-DNA complexes at the 5'' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.     

Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion.

Mouse mammary epithelial cells (MMEC) secrete certain milk proteins only when cultured on floating collagen gels. We demonstrate here that modulation of milk proteins by substrata is manifested at several regulatory levels; (i) Cells cultured on floating collagen gels have 3- to 10-fold more casein mRNA than cells cultured on plastic or attached collagen gels. (ii) Cells on the latter two "flat" substrata, nevertheless, synthesize a significant amount of caseins, indicating that the remaining mRNA is functional. (iii) Cells on all substrata are inducible for casein mRNA and casein proteins by prolactin, but the extent of induction is greater on collagen than that on plastic--i.e., the substratum confers an altered degree of inducibility. (iv) Cells on all substrata synthesize casein proteins at rates proportional to the amount of casein mRNA, but the newly synthesized caseins in cells on plastic are degraded intracellularly, whereas those synthesized by cells on floating gels are secreted into the medium. (v) Cells on all substrata examined lose virtually all mRNA for whey acidic protein despite the fact that this mRNA is abundant in the mammary gland itself; we conclude that additional, as-yet-unknown, factors are necessary for synthesis and secretion of whey acidic protein in culture.