Underproduction of sigma 70 mimics a stringent response. A proteome approach

When Escherichia coli cells enter stationary phase due to carbon starvation the synthesis of ribosomal proteins is rapidly repressed. In a DeltarelA DeltaspoT mutant, defective in the production of the alarmone guanosine tetraphosphate (ppGpp), this regulation of the levels of the protein synthesizing system is abolished. Using a proteomic approach we demonstrate that the production of the vast majority of detected E. coli proteins are decontrolled during carbon starvation in the DeltarelA DeltaspoT strain and that the starved cells behave as if they were growing exponentially. In addition we show that the inhibition of ribosome synthesis by the stringent response can be qualitatively mimicked by artificially lowering the levels of the housekeeping sigma factor, sigma(70). In other words, genes encoding the protein-synthesizing system are especially sensitive to reduced availability of sigma(70) programmed RNA polymerase. This effect is not dependent on ppGpp since lowering the levels of sigma(70) gives a similar but less pronounced effect in a ppGpp(0) strain. The data is discussed in view of the models advocating for a passive control of gene expression during stringency based on alterations in RNA polymerase availability.     

Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14-1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3-5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes-VK16, VK18, VK23, VK24, VK37, and VK47--detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen-RN1,VK24-VK47-VK23-VK16,FRAXA-++ +VK21A-VK18-IDS-VK37-U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA.     

Sperm deliver a new second messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.     

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Extending the family of UNCG-like tetraloop motifs: NMR structure of a CACG tetraloop from coxsackievirus B3

Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA-RNA and RNA-protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on three most frequently occurring families of tetraloop sequences, namely, the cUNCGg, the cGNRAg, and the gCUUGc sequences. Our knowledge surely is not exhaustive, and efforts are still being made to gain a better understanding. Here we report the NMR structure of a uCACGg tetraloop that occurs naturally within the cloverleaf RNA structure of the 5'-UTR of coxsackievirus B3. This tetraloop is the major determinant for interaction between the cloverleaf RNA and viral 3C protease, which is an essential part of a ribonucleoprotein complex that plays a critical role in the regulation of viral translation and replication. Our structure shows that the CACG tetraloop is closed by a wobble U.G base pair. The structure of the CACG tetraloop is stabilized by extensive base stacking and hydrogen bonding interactions strikingly similar to those previously reported for the cUUCGg tetraloop. Identification of these hallmark structural features strongly supports the existence of an extended YNCG tetraloop family. The U.G base pair closing the stem and the A residue in the loop introduce some small structural and themodynamic distinctions from the canonical cUUCGg tetraloop that may be important for recognition by the viral 3C protease.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

Temperature impact on the midsummer decline of Daphnia galeata: an analysis of long-term data from the biomanipulated Bautzen Reservoir (Germany)

1. The influence of water temperature on occurrence and duration of a midsummer decline (MSD) of Daphnia galeata was studied in the biomanipulated Bautzen Reservoir in Germany. The proportion of piscivores in the fish community of the reservoir has been enhanced experimentally since 1981. As a consequence, Daphnia galeata has dominated the zooplankton. Over 18 years of study (1981–1998), a long‐lasting MSD (longer than 30 days) occurred in 7 years, whereas a short MSD (shorter than 30 days) was observed in 6 years. During the remaining 5 years, an MSD was not observed.
2. Two hypotheses were examined to explain the observed patterns. First, we postulated that high water temperature during winter and early spring (January–April) leads to an MSD after an early and high spring peak of daphnids. On the other hand, low temperature during winter and early spring should not cause an MSD owing to a slower increase of the population, resulting in a later peak of daphnids. Second, we hypothesized that the mean water temperature during early summer (May and June) influences the occurrence of an MSD (by controlling young‐of‐the‐year (YOY) fish predation on daphnids).
3. The water temperature during winter and early spring explains 83%, and the early summer water temperature 55%, of interannual variation in the occurrence of an MSD.
4. The interannual variation in duration of an MSD was neither explained by temperature during winter and early spring nor by early summer temperature alone, but in 14 of the 18 years (78%) by a combination of both.
5. We conclude that water temperature during winter and early spring had a strong impact on Daphnia mortality by influencing height and timing of the spring peak which, in turn, influenced the extent of overexploitation of their food resources. By contrast, the water temperature during early summer probably influenced the mortality of daphnids caused by predation of YOY fish. The relative timing of both sources of mortality, which depends on the temperature regime during the first 6 months of the year, is the key process in controlling the occurrence and duration of an MSD. A long‐lasting MSD, therefore, is likely in Bautzen Reservoir only if temperatures are high during winter and early spring, as well as during early summer.
6. As a consequence of climate warming, recent climate records reveal warming during winter, spring and early summer in middle Europe, rather than an increase in mean annual temperatures. If our findings and conclusions are related to this regional and temporal pattern of climate warming, an increasing frequency of years with a long‐lasting MSD and, consequently, a decreasing efficiency of biomanipulation can be predicted.     

Effects of Cell-Bound Microcystins on Survival and Feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.     

Helianthus maximiliani and species fine‐scale spatial pattern affect diversity interactions in reconstructed tallgrass prairies

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Influence of fire and mechanical sagebrush reduction treatments on restoration seedings in Utah,United States

Overabundance of woody plants in semiarid ecosystems can degrade understory herbaceous vegetation and often requires shrub reduction and seeding to recover ecosystem services. We used meta‐analysis techniques to assess the effects of fire and mechanical shrub reduction over two post‐treatment timeframes (1–4 and 5–10 years) on changes in cover and frequency of 15 seeded species at 63 restoration sites with high potential for recovery. Compared to mechanical treatments, fire resulted in greater increases in seeded species. Native shrubs did not increase, and forbs generally declined over time; however, large increases in perennial grasses were observed, suggesting that seeding efforts contributed to enhanced understory herbaceous conditions. We found greater increases in a few non‐native species than native species across all treatments, suggesting the possibility that interference among seeded species may have influenced results of this regional assessment. Differences among treatments and species were likely driven by seedbed conditions, which should be carefully considered in restoration planning. Site characteristics also dictated seeded species responses: while forbs showed greater increases in cover over the long term at higher elevation sites considered to be more resilient to disturbance, surprisingly, shrubs and grasses had greater increases in cover and frequency at lower elevation sites where resilience is typically much lower. Further research is needed to understand the causes of forb mortality over time, and to decipher how greater increases of non‐native relative to native seeded species will influence species diversity and successional trajectories of restoration sites.