DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.     

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.     

Adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) is a substrate for the nucleotide hydrolysis and RNA unwinding activities of eukaryotic translation initiation factor eIF4A

Whereas ATPgammaS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPgammaS is a good substrate for the RNA-stimulated nucleotide hydrolysis and RNA unwinding activities of eIF4A. In the presence of saturating single-stranded poly(U) RNA, eIF4A hydrolyzes ATPgammaS.Mg and ATP.Mg with similar steady-state parameters (KM(NTP.Mg) = 66 and 58 microM and kcat = 1.0 and 0.97 min(-1), respectively). ATPgammaS.Mg also supports catalysis of RNA unwinding within 10-fold of the rate supported by ATP.Mg. The identical steady-state rate parameters, in comparison with the expected difference in the intrinsic rate of hydrolysis for ATP and ATPgammaS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATPgammaS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.     

TreeSAAP: selection on amino acid properties using phylogenetic trees

The software program TreeSAAP measures the selective influences on 31 structural and biochemical amino acid properties during cladogenesis, and performs goodness-of-fit and categorical statistical tests.     

Golgin-84 is a rab1 binding partner involved in Golgi structure

Members of the golgin family of coiled-coil proteins have been implicated in the tethering of vesicles to Golgi membranes and cisternal membranes to each other. Many also bind to rab GTPases. Golgin-84 is a membrane-anchored golgin that we now show binds preferentially to the GTP form of the rab1 GTPase. It is also present throughout the Golgi stack by immuno-EM. Antibodies to golgin-84 inhibit stacking of cisternal membranes in a cell-free assay for Golgi reassembly, whereas the cytoplasmic domain of golgin-84 stimulates stacking and increases the length of re-assembled stacks. Transient expression of golgin-84 in NRK cells helps prevent the disassembly of the Golgi apparatus normally triggered by treatment with brefeldin A. Together these data suggest that golgin-84 is involved in generating and maintaining the architecture of the Golgi apparatus.     

Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

Infection of human urethral epithelium with Neisseria gonorrhoeae elicits an upregulation of host anti-apoptotic factors and protects cells from staurosporine-induced apoptosis

In order to better understand the host response to an infection with Neisseria gonorrhoeae, microarray technology was used to analyse the gene expression profile between uninfected and infected human urethral epithelium. The anti-apoptotic genes bfl-1, cox-2 and c-IAP-2 were identified to be upregulated approximately eight-, four- or twofold, respectively, following infection. Subsequent assays including RT-PCR, real time RT-PCR and RNase protection confirmed the increased expression of these apoptotic regulators, and identified that a fourth anti-apoptotic factor, mcl-1, is also upregulated. RT-PCR and RNase protection also showed that key pro-apoptotic factors including bax, bad and bak do not change in expression. Furthermore, our studies demonstrated that infection with the gonococcus partially protects urethral epithelium from apoptosis induced by the protein kinase inhibitor, staurosporine (STS). This work shows that following infection with Neisseria gonorrhoeae, several host anti-apoptotic factors are upregulated. In addition, a gonococcal infection protects host cells from subsequent STS-induced death. The regulation of host cell death by the gonococcus may represent a mechanism employed by this pathogen to survive and proliferate in host epithelium.     

Understanding the Hydraulics of Porous Pipes: Tradeoffs Between Water Uptake and Root Length Utilization

The water uptake region in roots is several hundred times longer than the root diameter. The distributed nature of the uptake zone requires that the hydraulic design of roots be understood by analogy to flow through a porous pipe. Here we present results of an analytical and experimental investigation that allowed an in-depth analysis of root hydraulic properties. Measurements on nodal maize roots confirm the nonlinear distribution of water uptake predicted by the porous pipe model. The major design parameter governing the distribution of water uptake along a porous pipe is the ratio between its axial and radial hydraulic resistance. However, total flow is proportional to the pipe's overall resistance. These results suggest the existence of a tradeoff between the effective utilization of root length and the total capacity for water uptake.     

Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential

Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6-1 cells (6-1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6-1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras-induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.