Transcriptional activation by tetracyclines in mammalian cells.

A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.

Inducible gene expression systems for higher eukaryotic cells.

Of the wide variety of regulatory systems that have been developed to control gene expression in higher eukaryotes, those utilizing elements from prokaryotic systems presently achieve the highest specificity. In particular, systems based on the repressor/operator elements of an Escherichia coli tetracycline resistance operon appear broadly applicable. During the past year, the usefulness of these systems has been demonstrated not only at the cellular level, but also at the organismal level (i.e. in transgenic animals and plants).

Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein.

Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase.

Maltose transacetylase of Escherichia coli: a preliminary report.

Crude extracts of Escherichia coli K12 contain an enzyme that is able to transfer the acetyl group of acetyl-co-enzyme A to maltose (maltose transacetylase). Half maximal acetylation occurs at about 20 microM acetyl-coenzyme A. Half maximal concentration of maltose has not been determined precisely, but it is clear that it exceeds 10 mM. The appearance of maltose transacetylase is not induced by growth on maltose. Mutants carrying a defect in the malT gene, the positive regulator for the well known malA and malB regions, exhibit elevated levels of maltose transacetylase. The gene coding for the enzyme is not known that mutant analysis demonstrated that it is not part of malA, malB or malT. In addition, it is clear that the enzyme is not identical with thiogalactoside transacetylase, the gene product of the lacA gene. Besides maltose, maltodextrins and thiomaltose are substrates for maltose transacetylase.

Facilitated diffusion of p-nitrophenyl-alpha-D-maltohexaoside through the outer membrane of Escherichia coli. Characterization of LamB as a specific and saturable channel for maltooligosaccharides.

LamB, an outer membrane protein of Escherichia coli, is a component of the maltose-maltooligosaccharide transport system. We used p-nitrophenyl-alpha-D-maltohexaoside, a chromogenic analog of maltohexaose, and a periplasmic amylase that hydrolyzes this compound to study the LamB-mediated diffusion of p-nitrophenyl-alpha-D-maltohexaoside into the periplasm. Using this approach, we were able to characterize LamB in vivo as a saturable channel for maltooligosaccharides. Permeation through LamB follows Michaelis-Menten kinetics, with a Km of 0.13 mM and a Vmax of 3.3 nmol/min/10(9) cells. Previous studies suggested that maltose-binding protein increases the rate of maltooligosaccharide diffusion through LamB. We show here that, at least in strains that are unable to transport maltooligosaccharides into the cytoplasm, maltose-binding protein does not influence the rate of substrate diffusion. The periplasmic amylase had been previously described as being of the alpha-type. We have now purified this protein and analyzed its mode of action using chromogenic maltooligosaccharides of varying length. Analysis of the hydrolytic products revealed that the enzyme recognizes its substrate from the nonreducing end and preferentially liberates maltohexaose, in contrast to the behavior of classical alpha-amylases that are endohydrolases. Using p-nitrophenyl-alpha-D-maltohexaoside as a substrate, we determined a Km of 3 microM and a Vmax of 0.14 mumol/min/mg of protein.

Molecular characterization of the MalT-dependent periplasmic alpha-amylase of Escherichia coli encoded by malS.

malS, the gene encoding the periplasmic alpha-amylase, is under the regulatory control of the MalT protein, the gene activator of the Escherichia coli maltose system. We sequenced the DNA region encoding malS and its control elements. malS consists of an open reading frame of 2,028 base pairs encoding a protein of 676 amino acids with a deduced molecular weight of 75,664 including a typical amino-terminal signal sequence of 17 amino acids. The deduced amino acid sequence of malS was compared with that of other proteins. We found homologies to alpha-amylases and a cyclodextrin glucanotransferase but not to beta-amylases. In addition, MalS showed significant homology to another maltodextrin-utilizing and MalT-dependent enzyme of E. coli, maltodextrin glucosidase (MalZ) but not to amylomaltase (MalQ), the major maltodextrin-degrading enzyme. Conserved regions that have been proposed to constitute enzymatically active sites in alpha-amylases are present in MalS. Two of these sequences can also be found in the amino terminus of the lambda-receptor, a maltodextrin-specific channel in the outer membrane. The nucleotide sequence of the control region of malS revealed MalT binding sites correctly spaced for the start of the malS transcript. At the position 219 base pairs upstream of malS, we found a divergently transcribed gene, bax, which has been recognized previously. Downstream of malS, after a 513-base pair intergenic region, lies the convergently transcribed gene avtA, which codes for the alanine/valine transaminase.

A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells.

BACKGROUND: Tight control of gene activity has been achieved in cells and transgenic organisms using the Tet regulatory systems. Unregulated basal transcription can, however, be observed whenever integration of target genes driven by promoters responsive to tetracycline controlled transcriptional activators (tTA, rtTA) does not occur at suitable chromosomal sites. Moreover, in viral vectors containing both the tTA coding sequence and the regulated target gene, proximity of the enhancer element driving tTA/rtTA expression to the responsive unit will lead to elevated background levels. Similarly when tTA/rtTA responsive transcription units are in a non-integrated state as e.g., during transient expression, intrinsic residual transcription persists in their 'off' state, which can differ in intensity among different cell types. METHODS: To efficiently repress such background activities we generated tetracycline controlled transcriptional silencers (tTS) that bind promoters responsive for rtTA in absence of the effector doxycycline (Dox). Addition of Dox prevents binding of tTS thus relieving repression, promotes binding of rtTA and thereby switches the promoter from an actively repressed to an activated state. RESULTS: Of several tTS--fusions between a modified Tet repressor and transcriptional silencing domains--tTSKid was found to be most effective in reducing the activity of two target promoters. Ten to 200 fold repression is seen in transient expression whereas in stably transfected HeLa cells the regulatory range of the rtTA system was increased by three orders of magnitude. CONCLUSIONS: The new system appears particularly suited for the transfer of toxic genes into appropriate chromosomal sites as well as for tight regulation of genes carried by viral or episomal vectors.

A selective pathway for degradation of cytosolic proteins by lysosomes.

A lysosomal pathway of proteolysis is selective for cellular proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent cultured cells that are deprived of serum growth factors and in certain tissues of fasted animals. We have reconstituted this lysosomal degradation pathway in vitro. Transport into lysosomes requires a KFERQ-like sequence in the substrate protein and uptake and/or degradation is stimulated by ATP. A member of the heat shock 70 kDa protein family, the 73 kDa constitutive heat shock protein, binds to KFERQ-like peptide regions within proteins and, in some as yet unidentified manner, facilitates transfer of the proteins into lysosomes. Several possible mechanisms of selective protein transport into lysosomes are discussed.