Total current blockade in an ultracold dipolar quantum wire.

Cold-atom systems offer a great potential for the future design of new mesoscopic quantum systems with properties that are fundamentally different from semiconductor nanostructures. Here, we investigate the quantum-gas analogue of a quantum wire and find a new scenario for the quantum transport: Attractive interactions may lead to a complete suppression of current in the low-bias range, a total current blockade. We demonstrate this effect for the example of ultracold quantum gases with dipolar interactions.

Correlation-induced conductance suppression at level degeneracy in a quantum dot.

The large, level-dependent g factors in an InSb nanowire quantum dot allow for the occurrence of a variety of level crossings in the dot. While we observe the standard conductance enhancement in the Coulomb blockade region for aligned levels with different spins due to the Kondo effect, a vanishing of the conductance is found at the alignment of levels with equal spins. This conductance suppression appears as a canyon cutting through the web of direct tunneling lines and an enclosed Coulomb blockade region. In the center of the Coulomb blockade region, we observe the predicted correlation-induced resonance. Our findings are supported by numerical and analytical calculations.

Analysing the capacitance-voltage measurements of vertical wrapped-gated nanowires.

The capacitance of arrays of vertical wrapped-gate InAs nanowires is analysed. With the help of a Poisson-Schrödinger solver, information about the doping density can be obtained directly. Further features in the measured capacitance-voltage characteristics can be attributed to the presence of surface states as well as the coexistence of electrons and holes in the wire. For both scenarios, quantitative estimates are provided. It is furthermore shown that the difference between the actual capacitance and the geometrical limit is quite large, and depends strongly on the nanowire material.

E2F-1-induced p53-independent apoptosis in transgenic mice.

The E2F transcription factors are key targets for the retinoblastoma protein, pRB. By inactivation of E2Fs, pRB prevents progression to the S phase. To test proliferative functions of E2F, we generated transgenic mice expressing human E2F-1 and/or human DP-1. When the hydroxymethyl glutaryl coenzyme A reductase promoter was used to express DP-1, overexpression occurred in a variety of tissues and did not confer phenotypic changes. In contrast, expression of E2F-1 from the same promoter was obtained only in testicles, in which E2F-1 overexpression caused atrophy and sterility through a process involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered testicular atrophy.

A prospective nationwide study of Clostridium difficile-associated diarrhea in Sweden. The Swedish C. difficile Study Group.

Clostridium difficile-associated diarrhea (CDAD) is regarded as an emerging nosocomial infection. All patients positive for C. difficile in Sweden were recorded during 1995, including primary care patients. Those positive for toxin in feces were defined as CDAD cases. A total of 5,133 CDAD cases were recorded (58 per 100,000 inhabitants per year), as compared with 86 cases diagnosed in 1978 and 553 in 1983. CDAD was almost twice as prevalent as all (combined) diagnosed domestic cases of reportable bacterial and protozoal diarrhea. The age-specific incidence was little affected by gender but increased > 10-fold over the age range of 60-98 years. The differences in overall CDAD incidence were sixfold between counties and threefold between major hospitals. Among hospitalized patients the incidences were highest in geriatric/rehabilitation wards, followed by infectious diseases and internal medicine wards; 28% of all cases involved no recent hospitalization and were defined as community-acquired CDAD.

Tissue-specific posttranscriptional downregulation of expression of the S100A4(mts1) gene in transgenic animals.

The S100A4(mts1) is a gene associated with generation of metastatic disease. In order to analyze the consequences of alteration of the pattern of expression of the S100A4(mts1) gene we obtained strains of transgenic mice bearing the S100A4(mts1) gene under the control of a ubiquitous and constitutive 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) gene promoter. In transgenic animals the expression of the transgene RNA was detected in all organs, but only some of the organs showed elevated levels of the protein. Expression of the S100A4(Mts1) protein was downregulated in the organs that normally do not express the gene in the wild-type animal. The transgene RNA is detected in the polysomes indicating that it could be translated into the S100A4(Mts1) protein. The specificity of the S100A4(Mts1) protein expression is determined by a complex mechanism including regulation of translation and/or posttranslational degradation.

Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene.

Transgenic mice, carrying the mts1 gene, one of the genes involved in the acquisition of the metastatic phenotype, were generated. The mts1 gene was placed under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter leading to overexpression in the lactating mammary gland of transgenic animals. Animals bearing the transgene appear phenotypically normal. Animals of two transgenic lines (Tg463 and Tg507) were crossed with the GRS/A mice. The GRS/A strain is characterized by high incidence of mammary tumors which rarely metastasize. 40% of the tumor bearing hybrid GRS/A mts1 females were found to develop secondary tumors in the lungs. The Mts1 protein was detected in the transgene primary tumor cells as well as in the corresponding metastases. Nontransgenic littermates expressed the Mts1 protein only in the stromal cells surrounding the tumor but not in the tumor cells by itself. Taken together these observations indicate that overexpression of the mts1 gene in the mouse mammary carcinoma cells gives rise to more aggressive tumors which are able to metastasize.

Development and testing of improved suicide functions for biological containment of bacteria.

We have developed very efficient suicide functions for biological containment based on the lethal Escherichia coli relF gene. The suicide functions are placed in duplicate within a plasmid and arranged to prevent inactivation by deletion, recombination, and insertional inactivation. The efficiency of this concept was tested in a plasmid containment system that prevents transfer of plasmids to wild-type bacteria. Protection against plasmid transfer was assayed in test tubes and in rat intestine. Protection was efficient and refractory to inactivation by mutation and transposons. The efficiency of the suicide system was also tested in soil and seawater. We show that unprecedented suicide efficiency can be achieved in soil and seawater after suicide induction by IPTG (isopropyl-beta-D-thiogalactopyranoside). More than 7 orders of magnitude reduction in suicide bacteria was achieved.

Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts.

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serum free conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)2vitamin D3 significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 microM), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.

Development of efficient suicide mechanisms for biological containment of bacteria.

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.

Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon.

The parB region of plasmid R1 encodes two genes, hok and sok, which are required for the plasmid-stabilizing activity exerted by parB. The hok gene encodes a potent cell-killing factor, and it is regulated by the sok gene product such that cells losing a parB-carrying plasmid during cell division are rapidly killed. Coinciding with death of the host cell, a characteristic change in morphology is observed. Here we show that the killing factor encoded by the hok gene is a membrane-associated polypeptide of 52 amino acids. A gene located in the Escherichia coli relB operon, designated relF, is shown to be homologous to the hok gene. The relF gene codes for a polypeptide of 51 amino acids, which is 40% homologous to the hok gene product. Induced overexpression of the hok and relF gene products results in the same phenomena: loss of cell membrane potential, arrest of respiration, death of the host cell and change in cell morphology. The parB region and the relB genes were cloned into unstably inherited oriC minichromosomes. Whereas the parB region also conferred a high degree of genetic stability to an oriC minichromosome, the relB operon (with relF) did not; therefore the latter does not appear to 'stabilize' its replicon (the chromosome). The function of the relF gene is not known.

Association of RNA polymerase having increased Km for ATP and UTP with hyperexpression of the pyrB and pyrE genes of Salmonella typhimurium.

We investigated the transcription kinetics of RNA polymerase from an rpoBC mutant of Salmonella typhimurium which showed highly elevated, constitutive expression of the pyrB and pyrE genes as well as an increased cellular pool of UTP. When bacterial cultures containing an F' lac+ episome were induced for lac operon expression, the first active molecules of beta-galactosidase were formed with a delay of 73 +/- 3 s in rpo+ cells. The corresponding time was 104 to 125 s for cells carrying the rpoBC allele, indicating that this mutation causes a reduced RNA chain growth rate. In vitro the purified mutant RNA polymerase elongated transcripts of both T7 DNA and synthetic templates more slowly than the parental enzyme at a given concentration of nucleoside triphosphates. This defect was found to result from four- to sixfold-higher Km values for the saturation of the elongation site by ATP and UTP. The saturation kinetics of the RNA chain initiation step also seemed to be affected. The maximal elongation rate and Km for GTP and CTP were less influenced by the rpoBC mutation. Open complex formation at the promoters of T7 DNA and termination of the 7,100-nucleotide transcript showed no significant difference between the parental and mutant enzymes. Together with the phenotype of the rpoBC mutant, these results indicate that expression of pyrB and pyrE is regulated by the mRNA chain growth rate, which is controlled by the cellular UTP pool. The rate of gene expression is high when the saturation of RNA polymerase with UTP is low and vice versa.

Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene.

Escherichia coli relB mutants react to amino acid starvation by several abnormal responses, including accumulation of a translational inhibitor. We have isolated a relB-complementing plasmid from the Clarke and Carbon E. coli DNA library. From this plasmid we sequenced a 2140-bp segment which included the relB gene by the following two criteria: (i) it complements chromosomal relB mutations, (ii) the corresponding DNA segment cloned from chromosomal DNA of three relB mutants was defective in relB complementation. All three mutations fell within an open reading frame of 79 amino acids. A polypeptide of 9 kd compatible with this open reading frame was synthesized in maxicells and is in all probability the product of the relB gene. By nuclease S1 mapping we have determined the transcription start and stop of an 870 base transcript of the relB gene.

Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli.

A 273 bp DNA fragment containing the attenuator of the pyrBI operon was inserted into a synthetic cloning site early in the lacZ gene on a plasmid. By this operation the first few codons of lacZ were joined through a linker to the last 39 codons of the open reading frame for the putative pyrB leader peptide. In addition a gene fusion encoding a hybrid protein with beta-galactosidase activity was formed between the pyrB start and the rest of lacZ. This gene fusion is expressed from the lac promoter and the transcript is subject to facultative termination at the pyrBI attenuator. Different variants of the lacZ start were used that either contained a stop codon or directed the translation toward the attenuator in any of the alternative reading frames. The following results were obtained. No significant read-through of transcription over the pyrB attenuator was seen when the leader translation ended 49 nucleotide residues, or more, upstream of the attenuator symmetry, but a UTP-modulated attenuation was established if the leader translation was allowed to proceed across the attenuator as for the putative leader peptide or in a frame-shifted version. The regulation, however, was not as great as for the native pyrB gene. This is probably because the substitution of the normal start of the leader peptide by the start of lacZ alters the coupling between transcription and translation and thereby the attenuation frequency. It cannot, however, be ruled out that the pyrBI operon is regulated at the promoters in addition to the control by attenuation.

Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription.

The pyrE gene of Escherichia coli is part of an operon where it is preceded by an unknown gene (orfE) that ends 8 bp before the start of the symmetry of the UTP-modulated pyrE attenuator. On a plasmid we have inserted this attenuator region in a synthetic cloning site early in lacZ. The resulting structure contains the lac promoter-operator, the first few codons of lacZ, 42 bp of DNA from the orfE end, the pyrE attenuator, and an in-frame fusion pyrE-lacZ+. The synthetic cloning sites have been used to vary the length and reading frame of the translation that begins at the lacZ start and proceeds towards the attenuator. The effects of these variations on pyrE attenuation were determined by monitoring the synthesis of beta-galactosidase from the pyrE-lacZ hybrid gene in cells grown with either low or high pools of UTP. Thus, a very low level of pyrE expression was observed, regardless of UTP pool size, when the translation from the lacZ start ended 31 or 62 nucleotide residues upstream to the pyrE attenuator symmetry, but a proper UTP controlled attenuation could be established if this translation ended only 8 bp before the symmetry region of the attenuator (as the native orfE gene) or 10 bp after this structure. However, a single 'leader peptide' read from only frequently used codons gave a high level of pyrE expression both at high and low UTP pools. These observations indicate that the coupling between transcription and translation determines the degree of mRNA chain terminations at the pyrE attenuator.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of the dut gene of Escherichia coli. I. Cloning in thermoinducible plasmids.

We have constructed thermoinducible plasmids carrying the gene (dut) for the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli. A 9.4-kb BamHI restriction enzyme fragment carrying the dut gene was inserted into the runaway-replication plasmid pKN402A (Uhlin et al., Gene 6 (1979) 91-106). Strains carrying such plasmids increased their dUTPase activity considerably. In minimal medium a 200-fold increase was demonstrated. A smaller (1.5-kb) SacI-BamHI fragment from the dut region was also cloned into pKN402A. The dUTPase production in dut mutant strains carrying this plasmid (pKK141) was only at about wild-type level after temperature shift. To test the hypothesis that the SacI cleavage used affects a control region for the dut gene, we recloned the dut fragment by transferring it from pKK141 into pHUB2 (Bernard et al., Gene 5 (1979) 59-76), a plasmid carrying the phage lambda pL promoter. A 3.6-kb EcoRI-BamHI fragment from pKK141, including the 1.5-kb SacI-BamHI segment from the dut region, was inserted downstream from the pL promoter. When this plasmid was present in a strain containing a thermosensitive lambda repressor gene, thermoinduction of dUTPase was negligible, apparently due to the presence of some termination signals between pL and dut. Therefore, we removed a 1.9-kb EcoRI-SacI fragment from the region between pL and the dut gene and replaced it with a 0.22-kb EcoRI-SacI fragment, obtained from the b2 region of lambda. Strains carrying such a shortened dut-pHUB2 derivative and a temperature-sensitive lambda repressor overproduced dUTPase very dramatically after heat induction. The final level reached was 300-400 times the wild-type level, corresponding to 10% of the total soluble protein. The information obtained, together with analysis of plasmid-directed polypeptide products described by Lundberg et al. (Gene 22 (1983) 127-131) shows that the SacI site is indeed on the promoter-proximal side of the dut gene.

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products.

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.

Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12.

The nucleotide sequence of the dUTPase structural gene, dut, of Escherichia coli has been determined. The DNA sequence predicts a polypeptide chain of 150 amino acid residues (mol. wt. 16 006) corresponding in size and composition to the purified dUTPase subunit. In a tentative promoter region preceding the dut gene, the -35 and -10 regions are separated by a SacI (SstI) site. Cloning of the dut gene utilization this SacI site was previously shown to reduce dut expression dramatically. The nucleotide sequence also contains a 210-codon open reading frame 106 bp downstream of dut and co-directional with dut. Previous protein synthesis experiments using dut plasmids allocated the gene of a polypeptide of mol. wt. 23 500 to this DNA region. The open reading frame thus may correspond to a protein of unknown function co-transcribed with the dut gene.