We have constructed a series of plasmid vectors (pBAD vectors) containing the P BAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/ repression can be 1,200-fold, compared with 50-fold for P TAC -based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the P BAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.In bacterial physiology studies, it is often useful to express a cloned gene from an inducible promoter and assess the effect of the expression or depletion of the gene product in mutants lacking the chromosomal gene. In these situations, it is highly desirable to use a system that can be efficiently shut off. This is particularly the case when the phenotype caused by the absence of a protein can be obscured by leakiness from a partially repressed promoter or when even low levels of a protein are detrimental to the cell. Also, it would be desirable to modulate the expression system to achieve synthesis levels similar to those of the wild-type gene. However, the available repertoire of Escherichia coli expression systems usually produce high levels of the corresponding cloned gene product (4,13,18,45,48) and in many cases still produce substantial levels of synthesis in uninduced or repressed conditions (4,13,15,16,48,49). These systems include controllable expression vectors based on the strong inducible promoters P LAC (48), P TAC (13), P TRC (4), P L and P R (18), and P T7 (45). Some are better repressed than others, but induction of expression requires changes of temperature (18, 45) and produces very high levels of protein, resulting in conditions detrimental to cell growth and viability (17, 45).We have been studying the function of several essential genes that encode membrane proteins involved in cell division of E. coli (7a, 22). To analyze their role, we have sought to deplete cells of the proteins and then examine the phenotype of cells so depleted under conditions that would minimize alterations in cell physiology. For these purposes, we wished to use a plasmid that would satisfy the following two conditions: (i) the synthesis of the proteins should be shut off rapidly and efficiently without changes of temperature, and (ii) expression before depletion should not produce very high levels of protein, which itself may give a phe...

We report on results of an all-sky search for high-energy neutrino events interacting within the IceCube neutrino detector conducted between May 2010 and May 2012. The search follows up on the previous detection of two PeV neutrino events, with improved sensitivity and extended energy coverage down to about 30 TeV. Twenty-six additional events were observed, substantially more than expected from atmospheric backgrounds. Combined, both searches reject a purely atmospheric origin for the 28 events at the 4σ level. These 28 events, which include the highest energy neutrinos ever observed, have flavors, directions, and energies inconsistent with those expected from the atmospheric muon and neutrino backgrounds. These properties are, however, consistent with generic predictions for an additional component of extraterrestrial origin.

A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G 2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the doublestrand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G 2 , these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.Many temperature-sensitive mutations that cause cells to arrest at specific stages in the cell cycle have been isolated in the yeast Saccharomyces cerevisiae (49). Mutations in four genes, CDC2, -9, -13, and -17, result in arrest in late S or G 2 phase. Cells defective for these genes probably arrest because of defects in the completion of DNA replication. CDC2, -9, and -17 are all known to encode components of the DNA replication machinery: CDC2 and CDC17 code for DNA polymerases (5, 13), whereas CDC9 codes for DNA ligase (36). Furthermore, mutations in all four genes induce high levels of mitotic recombination at the restrictive temperature, a result consistent with the presence of DNA lesions that remain after incomplete DNA replication (25). RAD9 is part of a system of genes that detects the presence of X-ray-induced DNA damage and stops the cell cycle in G 2 to allow time for repair before the cell enters mitosis (64). The RAD9 checkpoint may also recognize spontaneous damage and/or incomplete DNA replication, since rad9 deletion mutants experience an elevated level of chromosome loss (65). In a rad9 mutant background, cells with mutations in any one of these four CDC genes fail to arrest in G 2 ; instead they proceed into the next cell cycle. This result has been interpreted to mean that mutations in all four genes leave damage after DNA replication and that this damage produces a G 2 arrest mediated by the RAD9 pathway (66). In a RAD9 background, these mutants retain viability for a few hours at the restrictive temperature because mitosis in the presence of DNA damage is prevented; however, in a rad9 background, all four mutant strains die rapidly.Unlike those of CDC2, CDC9, and CDC17, the function of CDC13 is not yet understood. The sequence, reported here, for the CDC13 gene does not suggest a biochemical function. The DNA lesions left in cdc13 mutants display two properties in a rad9 background that distinguish them from the lesions left in cdc2, cdc9, and cdc17 mutants. First, the maxi...

We report on the observation of two neutrino-induced events which have an estimated deposited energy in the IceCube detector of 1.04±0.16 and 1.14±0.17 PeV, respectively, the highest neutrino energies observed so far. These events are consistent with fully contained particle showers induced by neutral-current ν(e,μ,τ) (ν(e,μ,τ)) or charged-current ν(e) (ν(e)) interactions within the IceCube detector. The events were discovered in a search for ultrahigh energy neutrinos using data corresponding to 615.9 days effective live time. The expected number of atmospheric background is 0.082±0.004(stat)(-0.057)(+0.041)(syst). The probability of observing two or more candidate events under the atmospheric background-only hypothesis is 2.9×10(-3) (2.8σ) taking into account the uncertainty on the expected number of background events. These two events could be a first indication of an astrophysical neutrino flux; the moderate significance, however, does not permit a definitive conclusion at this time.

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