Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.     

WNK1： analysis of protein kinase structure, downstream targets, and potential roles in hypertension

The WNK kinases are a recently discovered family of serine-threonine kinases that have been shown to play an essential role in the regulation of electrolyte homeostasis, lntronic deletions in the WNK1 gene resuk in its overexpression and lead to pseudohypoaldosteronism type Ⅱ, a disease with salt-sensitive hypertension and hyperkalemia. This review focuses on the recent evidence elucidating the structure of the kinase domain of WNK1 and functions of these kinases in normal and disease physiology. Their functions have implications for understanding the biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. The WNK kinases may be able to influence ion homeostasis through its effects on synaptotagmin function.     

Maize genetics: the treasure of the Sierra Madre

Massive morphological changes occurred during the domestication of maize from wild teosinte. Some of the most important shifts are due to altered expression patterns of major regulatory genes.     

High-resolution RH map of horse chromosome 22 reveals a putative ancestral vertebrate chromosome

High-resolution gene maps of individual equine chromosomes are essential to identify genes governing traits of economic importance in the horse. In pursuit of this goal we herein report the generation of a dense map of horse chromosome 22 (ECA22) comprising 83 markers, of which 52 represent specific genes and 31 are microsatellites. The map spans 831 cR over an estimated 64 Mb of physical length of the chromosome, thus providing markers at approximately 770 kb or 10 cR intervals. Overall, the resolution of the map is to date the densest in the horse and is the highest for any of the domesticated animal species for which annotated sequence data are not yet available. Comparative analysis showed that ECA22 shares remarkable conservation of gene order along the entire length of dog chromosome 24, something not yet found for an autosome in evolutionarily diverged species. Comparison with human, mouse, and rat homologues shows that ECA22 can be traced as two conserved linkage blocks, each related to individual arms of the human homologue-HSA20. Extending the comparison to the chicken genome showed that one of the ECA22 blocks that corresponds to HSA20q shares synteny conservation with chicken chromosome 20, suggesting the segment to be ancestral in mammals and birds.     

Use of pressurized liquid extraction (PLE)/gas chromatography-electron capture detection (GC-ECD) for the determination of biodegradation intermediates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in soils

A rapid, sensitive, and reproducible method was developed for quantitative determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its biodegradation intermediates, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soils. RDX, MNX, DNX, or TNX was extracted from soil by pressurized liquid extraction (PLE), followed by cleanup using florisil. Instrumental analysis was performed using gas chromatography with electron capture detection (GC-ECD), which was highly sensitive to the parent explosive and its metabolites. The method detection limits (MDLs) were 0.243, 0.095, 0.138, and 0.057 ng/g for RDX, MNX, DNX, and TNX, respectively. The method gave high recovery (98-102%), good precision (0.22-5.14%), and reproducibility, and proved to be suitable for real world sample analysis.     

Properties of WNK1 and implications for other family members

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast two-hybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a C-terminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.     

Genetics of species differences in the wild annual sunflowers, Helianthus annuus and H. petiolaris

Much of our knowledge of speciation genetics stems from quantitative trait locus (QTL) studies. However, interpretations of the size and distribution of QTL underlying species differences are complicated by differences in the way QTL magnitudes are estimated. Also, many studies fail to exploit information about QTL directions or to compare inter- and intraspecific QTL variation. Here, we comprehensively analyze an extensive QTL data set for an interspecific backcross between two wild annual sunflowers, Helianthus annuus and H. petiolaris, interpret different estimates of QTL magnitudes, identify trait groups that have diverged through selection, and compare inter- and intraspecific QTL magnitudes. Our results indicate that even minor QTL (in terms of backcross variance) may be surprisingly large compared to levels of standing variation in the parental species or phenotypic differences between them. Morphological traits, particularly flower morphology, were more strongly or consistently selected than life history or physiological traits. Also, intraspecific QTL were generally smaller than interspecific ones, consistent with the prediction that larger QTL are more likely to spread to fixation across a subdivided population. Our results inform the genetics of species differences in Helianthus and suggest an approach for the simultaneous mapping of inter- and intraspecific QTL.     

Structure of B-DNA with cations tethered in the major groove

Here, we describe the 1.6-A X-ray structure of the DDD (Dickerson-Drew dodecamer), which has been covalently modified by the tethering of four cationic charges. This modified version of the DDD, called here the DDD(4+), is composed of [d(CGCGAAXXCGCG)](2), where X is effectively a thymine residue linked at the 5 position to an n-propyl-amine. The structure was determined from crystals soaked with thallium(I), which has been broadly used as a mimic of K(+) in X-ray diffraction experiments aimed at determining positions of cations adjacent to nucleic acids. Three of the tethered cations are directed radially out from the DNA. The radially directed tethered cations do not appear to induce structural changes or to displace counterions. One of the tethered cations is directed in the 3' direction, toward a phosphate group near one end of the duplex. This tethered cation appears to interact electrostatically with the DNA. This interaction is accompanied by changes in helical parameters rise, roll, and twist and by a displacement of the backbone relative to a control oligonucleotide. In addition, these interactions appear to be associated with displacement of counterions from the major groove of the DNA.     

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.