Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

The Bacillus subtilis spoOJ gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spoOJ-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for division. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria. The mechanism by which bacterial chromosomes are equipartitioned into daughter cells at division has remained obscure despite decades of study (Hiraga 1993;Wake and Errington 1995). There are several reports of abrupt movement of bacterial nucleoids (Sargent 1974;Hiraga et al. 1990;Begg and Donachie 1991), suggesting the existence of an active partitioning machinery equivalent to the mitotic apparatus of eukaryotes. However, there are some difficulties in interpreting these results and van Helvoort and Woldringh (1994)have shown convincingly that unperturbed nucleoids move apart gradually and continuously during cell growth (van Helvoort and Woldringh 1994). The tendency to assume that the mechanisms of chromosome segregation in bacteria are distinct from those of eukaryotes stems mainly from the absence of obvious structures such as the cytoskeleton and the mitotic spindle. However, this might be attributable in part to the difficulty in resolving such structures in ceils as small and tough as bacteria. Detection of mitotic-like activity is also hampered by the relatively unstructured state of the bacterial nucleoid and, particu-

Alzheimer's disease (AD) is a genetically complex and heterogeneous disorder. To date four genes have been established to either cause early-onset autosomal-dominant AD (APP, PSEN1, and PSEN2(1-4)) or to increase susceptibility for late-onset AD (APOE5). However, the heritability of late-onset AD is as high as 80%, (6) and much of the phenotypic variance remains unexplained to date. We performed a genome-wide association (GWA) analysis using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. We identified five SNPs showing either significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. One of these signals (p = 5.7 x 10(-14)) was elicited by SNP rs4420638 and probably reflects APOE-epsilon4, which maps 11 kb proximal (r2 = 0.78). The other four signals were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. Two of these SNPs showed significant association in the replication samples (combined p values 0.007 and 0.00002). The SNP (rs11159647, on chromosome 14q31) with the strongest association signal also showed evidence of association with the same allele in GWA data generated in an independent sample of approximately 1,400 AD cases and controls (p = 0.04). Although the precise identity of the underlying locus(i) remains elusive, our study provides compelling evidence for the existence of at least one previously undescribed AD gene that, like APOE-epsilon4, primarily acts as a modifier of onset age.

The spoOE locus ofBacillus subtilis codes for a negative regulator of sporulation that, when overproduced, represses sporulation and, if deleted, results in inappropriate timing of sporulation. The product of this locus, SpoOE, was purified and found to be a protein phosphatase, which specifically dephosphorylated the sporulation transcription factor SpoOA-P, converting it to an inactive form. SpoOE was not significantly active as a phosphatase on other components of the phosphorelay sinal-transduction pathway producing SpoOA-P. A mutant SpoOE protein that results in sporulation deficiency was purified and found to be hyperactive as a phosphatase. The SpoOE phosphatase may provide an additional control point for environmental, metabolic, or cell-cycle regulation of phosphate flow in the phosphorelay. These results reinforce the concept that the phosphorelay is subject to a host of positive and negative signals for sporulation that are recognized and interpreted as a signal integration circuit that has the role of regulating the cellular level of active phosphorylated SpoOA sporulation transcription factor.The initiation of sporulation in Bacillus subtilis is under control of the SpoOA transcription factor; this protein is a member of the response regulator class of two-component systems (1) and is inactive unless phosphorylated (2). Environmental conditions conducive to sporulation promote the phosphorylation of SpoOA through a complex signaltransduction pathway, the phosphorelay (3) (Fig. 1). Signal recognition by either of two kinases, KinA or KinB, is thought to activate autophosphorylation of the kinases, resulting in phosphorylation ofthe SpoOF protein. SpoOF-P is a substrate for the SpoOB phosphotransferase, which, in turn, phosphorylates SpoOA. SpoOA-P acts both as a repressor of certain vegetative genes and as an activator ofgenes required for the initiation of sporulation. Because it appears that the level of SpoOA-P in the cell is the factor determining the decision to either grow or sporulate (4), it is important to judiciously control this level to reflect the environmental and metabolic potential monitored by the cell. Regulation of the phosphorelay pathway may be accomplished by either transcriptional control of the cellular concentration of the key components of the phosphorelay, SpoOF and SpoOA, or by control of the phosphate flux through the pathway to SpoOA (5-7). Although transcriptional controls have been welldocumented, the activators of the kinases providing phosphate input into the pathway remain a mystery.The spoOE gene is believed to code for a negative regulator of sporulation because overproduction of its gene product inhibits sporulation and deletion of the gene results in increased pressure to sporulate (8). A spoOE deletion mutation has no effect on the transcriptional control of the kinA, spoOA, spoOF, or spoOB genes that code for the phosphorelay components, suggesting that the target for SpoOE negative regulation is not transcription but rather the flow, of phosphate in t...