Epigenetic marking systems confer stability of gene expression during mammalian development. Genome-wide epigenetic reprogramming occurs at stages when developmental potency of cells changes. At fertilization, the paternal genome exchanges protamines for histones, undergoes DNA demethylation, and acquires histone modifications, whereas the maternal genome appears epigenetically more static. During preimplantation development, there is passive DNA demethylation and further reorganization of histone modifications. In blastocysts, embryonic and extraembryonic lineages first show different epigenetic marks. This epigenetic reprogramming is likely to be needed for totipotency, correct initiation of embryonic gene expression, and early lineage development in the embryo. Comparative work demonstrates reprogramming in all mammalian species analysed, but the extent and timing varies, consistent with notable differences between species during preimplantation development. Parental imprinting marks originate in sperm and oocytes and are generally protected from this genome-wide reprogramming. Early primordial germ cells possess imprinting marks similar to those of somatic cells. However, rapid DNA demethylation after midgestation erases these parental imprints, in preparation for sex-specific de novo methylation during gametogenesis. Aberrant reprogramming of somatic epigenetic marks after somatic cell nuclear transfer leads to epigenetic defects in cloned embryos and stem cells. Links between epigenetic marking systems appear to be developmentally regulated contributing to plasticity. A number of activities that confer epigenetic marks are firmly established, while for those that remove marks, particularly methylation, some interesting candidates have emerged recently which need thorough testing in vivo. A mechanistic understanding of reprogramming will be crucial for medical applications of stem cell technology.

In this review, we look at the most recent studies of DNA elements that function over long genomic distances to regulate gene transcription and will discuss the mechanisms genes employ to overcome the positive and negative influences of their genomic neighbourhood in order to achieve accurate programmes of expression. Enhancer elements activate high levels of transcription of linked genes from distal locations. Recent technological advances have demonstrated chromatin loop interactions between enhancers and their target promoters. Moreover, there is increasing evidence that these dynamic interactions regulate the repositioning of genes to foci of active transcription within the nucleus. Enhancers have the potential to activate a number of neighbouring genes over a large chromosomal region, hence, their action must be restricted in order to prevent activation of non-target genes. This is achieved by specialized DNA sequences, termed enhancer blockers (or insulators), that interfere with an enhancer's ability to communicate with a target promoter when positioned between the two. Here, we summarize current models of enhancer blocking activity and discuss recent findings of how it can be dynamically regulated. It has become clear that enhancer blocking elements should not be considered only as structural elements on the periphery of gene loci, but as regulatory elements that are crucial to the outcome of gene expression. The transcription potential of a gene can also be susceptible to heterochromatic silencing originating from its chromatin environment. Insulator elements can act as barriers to the spread of heterochromatin. We discuss recent evidence supporting a number of non-exclusive mechanisms of barrier action, which mostly describe the modulation of chromatin structure or modification.

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of: 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating: a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.

Genomic imprinting results in parent-of-origin-dependent monoallelic expression of selected genes. Although their importance in development and physiology is recognized, few imprinted genes have been investigated for their effects on brain function. Gnas is a complex imprinted locus whose gene products are involved in early postnatal adaptations and neuroendocrine functions. Gnas encodes the stimulatory G-protein subunit Gsalpha and two other imprinted protein-coding transcripts. Of these, the Nesp transcript, expressed exclusively from the maternal allele, codes for neuroendocrine secretory protein 55 (Nesp55), a chromogranin-like polypeptide associated with the constitutive secretory pathway but with an unknown function. Nesp is expressed in restricted brain nuclei, suggesting an involvement in specific behaviors. We have generated a knockout of Nesp55 in mice. Nesp55-deficient mice develop normally, excluding a role of this protein in the severe postnatal effects associated with imprinting of the Gnas cluster. Behavioral analysis of adult Nesp55 mutants revealed, in three separate tasks, abnormal reactivity to novel environments independent of general locomotor activity and anxiety. This phenotype may be related to prominent Nesp55 expression in the noradrenergic locus coeruleus. These results indicate a role of maternally expressed Nesp55 in controlling exploratory behavior and are the first demonstration that imprinted genes affect such a fundamental behavior.

A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase gamma (PI3Kgamma) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function []. PI3Kgamma is currently thought to be composed of a p110gamma catalytic subunit and a single regulatory subunit, p101. The binding of p110gamma to p101 dramatically increases the activation of the complex by Gbetagamma subunits and, hence, is thought to be critical for the coupling of PI3Kgamma to G protein coupled receptors []. Here, we characterize a new regulatory subunit for PI3Kgamma. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110gamma specifically and with high affinity. Binding of p84 to p110gamma substantially increases the ability of Gbetagamma to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) production both in vitro and in vivo. However, the p84/p110gamma heterodimer is approximately 4-fold less sensitive to Gbetagammas than p101/p110gamma. Endogenous murine p84 expression is substantially reduced in the absence of p110gamma expression. We conclude that p110gamma has two potential regulatory subunits in vivo, p84 and p101.

The cause of common organ-specific autoimmune diseases is poorly understood because of genetic and cellular complexity in humans and animals. Recent advances in the understanding of the mechanisms of the defects underlying autoimmune disease in autoimmune polyendocrinopathy syndrome type 1 and non-obese diabetic mice suggest that failures in central tolerance play a key role in predisposition towards organ-specific autoimmunity. The lessons from such rare monogenic autoimmune disorders and well-characterized polygenic traits demonstrate how subtle quantitative trait loci can result in large changes in the susceptibility to autoimmunity. These data allow us to propose a model relating efficiency of thymic deletion to T-cell tolerance and susceptibility to autoimmunity.

Phosphoinositide 3-kinase gamma (PI3Kgamma) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3Kgamma activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110gamma-/- mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110gamma-/- microvessels in vivo in response to tumor necrosis factor alpha (TNFalpha). This alteration in adhesion was further augmented by a deficiency in p110delta, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin-mediated adhesion in p110gamma-/-) mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-kappaB)-induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation.

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di- and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1beta) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HP1beta with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.

Lymphocytes are characterised by monoclonal expression of antigen receptors. This is achieved by silencing of one of two homologous antigen receptor alleles, a process known as allelic exclusion. This process is regulated both before and after V(D)J recombination, by a variety of mechanisms. These include nuclear localisation, changes in chromatin structure and histone modifications, non-coding sense and antisense RNA transcription, epigenetic alterations at the DNA level, feedback signalling from expressed alleles, locus contraction and decontraction, recruitment to heterochromatin. This review will focus on recent advances in the immunoglobulin heavy and kappa light chain loci. The current picture is of a complex, temporally ordered sequence of events, in which these loci share many contributory mechanisms, but clear and intriguing differences are emerging.

The BH3-only protein, Bim, exists as three splice variants (Bim(S), Bim(L), and Bim(EL)) of differing pro-apoptotic potency. Bim(EL), the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim(EL) splice variant that includes the major ERK1/2 phosphorylation site Ser(65). However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim(EL) (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80-97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim(EL) and also abolished ERK1/2-dependent phosphorylation of Bim(EL) in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim(EL) that may account for the distinct biological properties of this splice variant.

We have characterized three novel epsilon gamma delta beta-thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire beta-globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98-kb deletion extended 90-kb upstream of the epsilon gene to 8 kb upstream of the G gamma-gene, leaving the gamma,delta and beta-genes intact. The 439 kb deletion is the largest deletion reported so far to cause epsilon gamma delta beta-thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.

Myotonic dystrophy type 1 is an autosomal dominant disorder associated with the expansion of a CTG repeat in the 3' untranslated region (UTR) of the DMPK gene. Recent data suggest that pathogenesis is predominantly mediated by a gain of function of the mutant transcript. In patients, these expanded CUG repeat-containing transcripts are sequestered into ribonuclear foci that also contain the muscleblind-like proteins. To provide further insights into muscleblind function and the pathogenesis of myotonic dystrophy, we generated Drosophila incorporating CTG repeats in the 3'-UTR of a reporter gene. As in patients, expanded CUG repeats form discrete ribonuclear foci in Drosophila muscle cells that co-localize with muscleblind. Unexpectedly, however, foci are not observed in all cell types and muscleblind is neither necessary nor sufficient for their formation. The foci are dynamic transient structures with short half-lifes that do not co-localize with the proteasome, suggesting they are unlikely to contain mis-folded proteins. However, they do co-localize with non-A, the human orthologs of which are implicated in both RNA splicing and attachment of dsRNA to the nuclear matrix. Muscleblind is also revealed as having a previously unrecognized role in stabilizing CUG transcripts. Most interestingly, Drosophila expressing (CUG)162 repeats has no detectable pathological phenotype suggesting that in contrast to expanded polyglutamine-containing proteins, neither the expanded CUG repeat RNA nor the ribonuclear foci are directly toxic.

The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (WldS) mutant mice.

The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+ CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+ CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL-protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+ CD4+ T cells further up-regulate the IL-2R alpha chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+ CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+ CD4+ T cells is not a passive phenomenon but instead initiates--in conjunction with signals through the TCR--their differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo.

The slow Wallerian degeneration phenotype, Wld(S), which delays Wallerian degeneration and axon pathology for several weeks, has so far been studied only in mice. A rat model would have several advantages. First, rats model some human disorders better than mice. Second, the larger body size of rats facilitates more complex surgical manipulations. Third, rats provide a greater yield of tissue for primary culture and biochemical investigations. We generated transgenic Wld(S) rats expressing the Ube4b/Nmnat1 chimeric gene in the central and peripheral nervous system. As in Wld(S) mice, their axons survive up to 3 weeks after transection and remain functional for at least 1 week. Protection of axotomized nerve terminals is stronger than in mice, particularly in one line, where 95-100% of neuromuscular junctions remained intact and functional after 5 days. Furthermore, the loss of synaptic phenotype with age was much less in rats than in mice. Thus, the slow Wallerian degeneration phenotype can be transferred to another mammalian species and synapses may be more effectively preserved after axotomy in species with longer axons.

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

A large cluster of imprinted genes is located on the mouse distal chromosome 7. This cluster is well conserved in humans and its dysregulation results in the overgrowth- and tumour-associated Beckwith-Wiedemann syndrome. Two imprinting centres (IC1 and IC2) controlling different sets of genes have been identified in the cluster, raising the hypothesis that the cluster is divided into two functionally independent domains. However, the mechanisms by which imprinting of genes in the IC2 domain (e.g. Cdkn1c and Kcnq1) is regulated have not been well defined, and recent evidence indicates that distantly located cis-acting elements are required for IC2 imprinting. We show that the maternal germ-line methylation at IC2 and the imprinted expression of five genes of the IC2 domain are correctly reproduced on an 800 kb YAC transgene when transferred outside of their normal chromosomal context. These results, together with previous transgenic studies, locate key imprinting control elements within a 400 kb region centromeric of IC2 and demonstrate that each of the two domains of the cluster contains the cis-acting elements required for the imprinting control of its own genes. Finally, maternal, but not paternal, transmission of the transgene results in fetal growth restriction, suggesting that during evolution the acquisition of imprinting may have been facilitated by the opposite effects of the two domains on embryo growth.

Xenotransplantation offers one way to circumvent the widening gap between the demand for and supply of human organs for transplantation, and the pig is widely regarded as the donor animal most likely to prove appropriate. Most attention has focused on the adaptive immune response to xenogeneic tissue. However, there is optimism that it may soon be possible to overcome that hurdle. In this paper, we consider the possibility of the direct recognition of xenogeneic tissue by neutrophils.

Mammalian embryonic stem (ES) cells can either self-renew or generate progenitor cells that have a more restricted developmental potential. This provides an important model system to ask how pluripotency, cell commitment and differentiation are regulated at the level of chromatin-based changes that distinguish stem cells from their differentiated progeny. Here we show that the differentiation of ES cells to neural progenitors results in dynamic changes in the epigenetic status of multiple genes that encode transcription factors critical for early embryonic development or lineage specification. In particular, we demonstrate that DNA replication at a subset of neural-associated genes including Pax3, Pax6, Irx3, Nkx2.9 and Mash1 is advanced upon neural induction, consistent with increased locus accessibility. Conversely, many ES-associated genes including Oct4, Nanog, Utf1, Foxd3, Cripto and Rex1 that replicate early in ES cells switch their replication timing to later in S-phase in response to differentiation. Detailed analysis of the Rex1 locus reveals that delayed replication extends to a 2.8 Mb region surrounding the gene and is associated with substantial reductions in the level of histone H3K9 and H4 acetylation at the promoter. These results show that loss of pluripotency (and lineage choice) is associated with extensive and predictable changes in the replication timing of key regulator genes.

In concert with the TCR, CD28 promotes T cell survival by regulating the expression of the antiapoptotic protein Bcl-x(L). The mechanism by which CD28 mediates the induction of Bcl-x(L) remains unknown. We show that although signaling through the TCR is sufficient to stimulate transcription of Bcl-x(L) mRNA, CD28, by activating PI3K and mammalian target of rapamycin, provides a critical signal that regulates the translation of Bcl-x(L) transcripts. We observe that CD28 induced 4E-binding protein-1 phosphorylation, an inhibitor of the translational machinery, and that CD28 costimulation directly augmented the translation of a Bcl-x(L) 5'-untranslated region reporter construct. Lastly, costimulation by CD28 shifted the distribution of Bcl-x(L) mRNA transcripts from the pretranslation complex to the translationally active polyribosomes. These results demonstrate that CD28 relieves the translational inhibition of Bcl-x(L) in a PI3K/mammalian target of rapamycin-dependent manner.

Phosphoinositide 3-Kinase (PI3K) gamma is a lipid kinase that is regulated by G-protein-coupled receptors. It plays a crucial role in inflammatory and allergic processes. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits. The regulatory p101 subunit of PI3Kgamma binds to Gbetagamma and, thereby, recruits the catalytic p110gamma subunit to the plasma membrane. Despite its crucial role in the activation of PI3Kgamma, the structural organization of p101 is still largely elusive. Employing fluorescence resonance energy transfer measurements, coimmunoprecipitation and colocalization studies with p101 deletion mutants, we show here that distinct regions within the p101 primary structure are responsible for interaction with p110gamma and Gbetagamma. The p110gamma binding site is confined to the N terminus, whereas binding to Gbetagamma is mediated by a C-terminal domain of p101. These domains appear to be highly conserved among various species ranging from Xenopus to men. In addition to establishing a domain structure for p101, our results point to the existence of a previously unknown, p101-related regulatory subunit for PI3Kgamma.

We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis. A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With Smoldyn, we constructed a three-dimensional model of an E. coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.