Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.     

The DNA-dependent protein kinase catalytic subunit is phosphorylated in vivo on threonine 3950, a highly conserved amino acid in the protein kinase domain

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.     

The Role of Climate in Settlement and Landscape Change in the North Atlantic Islands: An Assessment of Cumulative Deviations in High-Resolution Proxy Climate Records

In order to assess possible contributions of climate change to the human ecology of the North Atlantic islands we evaluate the utility of cumulative deviations from the mean, calculated for the Greenland ice core storm frequency proxy (GISP2 Na⁺) and sea ice proxy (GISP2 chloride excess). Our aim is to identify episodes of unpredictable change in the context of long-term trends of cultural and environmental development. Key changes are identified in the proxy climate records in 975 and 980 ad, 1025 and 1040 ad, 1180 ad, 1425 and 1450 ad, and 1520 and 1525 ad. Some of these changes are consistent with those inferred from new studies of the palaeoecological record of the Faroes. This indicates that the cumulative deviation measure could give greatest prominence to the most important climate changes affecting landscapes and settlement (such as the changes of 1425 and 1450 ad and their immediate aftermath), rather than extreme events, such as great single storms.     

Why Folkbiology Matters: Resource Conflict Despite Shared Goals and Knowledge

There is a continuing controversy over Native American fishing and hunting rights. We show that Native American (Menominee) and European American fish experts have a common knowledge base and share values and attitudes associated with fishing practices (though organized around different ethical principles). Nonetheless, perceived group differences are dramatic (especially European American perceptions of Native Americans). Cultural differences in models of nature and associated inference processes appear to mediate these stereotypes and may hold the key to reducing intergroup conflict over resources.

Dynamic use of multiple parameter sets in sequence alignment

The level of conservation between two homologous sequences often varies among sequence regions; functionally important domains are more conserved than the remaining regions. Thus, multiple parameter sets should be used in alignment of homologous sequences with a stringent parameter set for highly conserved regions and a moderate parameter set for weakly conserved regions. We describe an alignment algorithm to allow dynamic use of multiple parameter sets with different levels of stringency in computation of an optimal alignment of two sequences. The algorithm dynamically considers various candidate alignments, partitions each candidate alignment into sections, and determines the most appropriate set of parameter values for each section of the alignment. The algorithm and its local alignment version are implemented in a computer program named GAP4. The local alignment algorithm in GAP4, that in its predecessor GAP3, and an ordinary local alignment program SIM were evaluated on 257716 pairs of homologous sequences from 100 protein families. On 168475 of the 257716 pairs (a rate of 65.4%), alignments from GAP4 were more statistically significant than alignments from GAP3 and SIM.     

Molecular phylogeny, biogeography, and systematics of Dicerandra (Lamiaceae), a genus endemic to the southeastern United States

Dicerandra, an endemic mint of the southeastern United States, comprises nine species, all of which are threatened or endangered and restricted to sandhill vegetation and a mosaic of scrub habitats. Molecular phylogenetic analyses of Dicerandra based on data from the nuclear and plastid genomes for all 13 taxa of the genus, identified two strongly supported clades, corresponding to the four annual and to the five perennial species of Dicerandra. However, the nuclear and plastid trees were incongruent in their placement of two perennial taxa, D. cornutissima and D. immaculata var. savannarum, perhaps due to ancient hybridization or to lineage sorting. Based on these analyses, the widespread D. linearifolia is not monophyletic, with populations of D. linearifolia var. linearifolia falling into either western or eastern clades. The western clade, comprising populations of D. linearifolia var. linearifolia and var. robustior, occurs in an area drained by rivers flowing toward the Gulf of Mexico, whereas the eastern clade, comprising populations of D. linearifolia var. linearifolia, D. densiflora, D. odoratissima, and D. radfordiana (i.e., all the annual species), occupies a region drained by rivers flowing to the Atlantic Ocean. Although this pattern of genetic differentiation between populations from these two river drainages has been documented in several animal species, it has not previously been reported for plants. A revised subgeneric classification is presented to reflect the annual and perennial clades.     

Utility of multiple chemical techniques in archaeological residential mobility studies: case studies from Tiwanaku- and Chiribaya-affiliated sites in the Andes

In the south central Andes, archaeologists have long debated the extent of Tiwanaku colonization during the Middle Horizon (AD 500-1000). We tested the hypotheses regarding the nature of Tiwanaku influence using strontium isotope, trace element concentration, and oxygen isotope data from archaeological human tooth enamel and bone from Tiwanaku- and Chiribaya-affiliated sites in the south central Andes. Strontium isotope analysis of 25 individuals buried at the Tiwanaku-affiliated Moquegua Valley site of Chen Chen demonstrates that it was likely a Tiwanaku colony. In contrast, no immigrants from the Lake Titicaca Basin were present in 27 individuals analyzed from the San Pedro de Atacama cemeteries of Coyo Oriental, Coyo-3, and Solcor-3; it is likely that these sites represent economic and religious alliances, but not colonies. However, strontium isotope analysis alone cannot distinguish movement between the Tiwanaku- and Chiribaya-affiliated sites in the Moquegua and Ilo Valleys of southern Peru. Analyzing oxygen isotope and trace element concentration data and comparing it with strontium isotope data from the same individuals provides a more detailed picture of residential mobility in the Tiwanaku and Chiribaya polities. In addition to monitoring diagenetic contamination, trace element concentration data identified movement during adulthood for certain individuals. However, these data could not distinguish movement between the Moquegua and Ilo Valleys. While oxygen isotope data could clearly distinguish the high-altitude sites from others, more data is needed to characterize the local oxygen isotope ratios of these regions. These data demonstrate the potential for archaeological reconstruction of residential mobility through multiple lines of evidence.     

Flooded flatheads: evidence of increased growth in Mississippi River <Emphasis Type="Italic">Pylodictis olivaris</Emphasis> (Pisces: Ictaluridae) following the Great Midwest Flood of 1993

Both the Mississippi and Missouri rivers experienced major flooding in the spring and summer of 1993, inundating much of their floodplains for long periods, and allowing fish access to vast but previously inaccessible benthic foraging areas. In response to the latter, we hypothesized that flathead catfish (Pylodictis olivaris) would exhibit altered post-flooding growth as compared to like-aged fish whose lives were flood-free. To test this hypothesis, we obtained the left pectoral fin spines from hoop-netted Mississippi River flathead catfish collected in the vicinity of Crystal City, Missouri, USA, and conducted an individual-based back-calculated growth comparison of individuals captured pre-flood (1991) and post-flood (1996). For like-aged cohorts, our back-calculated length-at-age data clearly show that the post-flooding growth in 1996-collected fish was superior to that of the 1991-collected fish that had experienced less exceptional flow regimes. This outcome suggests that increased forage or/and foraging opportunities can be a consequence of flooding, and that flathead catfish are able to capitalize on this. The natural flooding regime of big rivers may thus be an important contributor to the growth and success of certain fish species; consequently, modifying the river’s natural hydrograph and channel morphology may be detrimental. Handling editor: J. A. Cambray An erratum to this article is available at .     

The T4 RI antiholin has an N-terminal signal anchor release domain that targets it for degradation by DegP

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a degP host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.