A neuronal isoform of <n>CPEB</n> regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in 
aplysia. Synapse-specific facilitation requires rapamycin-dependent local protein synthesis at the activated synapse. In Aplysia, rapamycin-dependent 
local protein synthesis serves two functions: (1) it provides a component of the mark at 
the activated synapse and thereby confers synapse specificity and (2) it stabilizes the synaptic growth 
associated with long-term facilitation. Here we report that a neuron-specific isoform of cytoplasmic polyadenylation element 
binding protein (<n>CPEB</n>) regulates this synaptic protein synthesis in an activity-dependent manner. Aplysia <n>CPEB</n> protein 
is upregulated locally at activated synapses, and it is needed not for the initiation but 
for the stable maintenance of long-term facilitation. We suggest that Aplysia <n>CPEB</n> is one of 
the stabilizing components of the synaptic mark. A neuronal isoform of the aplysia <n>CPEB</n> has 
prion-like properties. Prion proteins have the unusual capacity to fold into two functionally distinct conformations, 
one of which is self-perpetuating. When yeast prion proteins switch state, they produce heritable phenotypes. 
We report prion-like properties in a neuronal member of the <n>CPEB</n> family (cytoplasmic polyadenylation element 
binding protein), which regulates mRNA translation. Compared to other <n>CPEB</n> family members, the <n>neuronal protein</n> 
has an N-terminal extension that shares characteristics of yeast prion-determinants: a high glutamine content and 
predicted conformational flexibility. When fused to a reporter protein in yeast, this region confers upon 
it the epigenetic changes in state that characterize yeast prions. Full-length <n>CPEB</n> undergoes similar changes, 
but surprisingly it is the dominant, self-perpetuating prion-like form that has the greatest capacity to 
stimulate translation of <n>CPEB-regulated</n> mRNA. We hypothesize that conversion of <n>CPEB</n> to a prion-like state 
in stimulated synapses helps to maintain long-term synaptic changes associated with memory storage. Serotonylation of 
<n>small GTPases</n> is a signal transduction pathway that triggers platelet alpha-granule release. <n>Serotonin</n> is a 
neurotransmitter in the central nervous system. In the periphery, <n>serotonin</n> functions as a ubiquitous hormone 
involved in vasoconstriction and platelet function. <n>Serotonin</n> is synthesized independently in peripheral tissues and neurons 
by two different rate-limiting tryptophan <n>hydroxylase</n> (<n>TPH</n>) isoenzymes. Here, we show that mice selectively deficient 
in peripheral <n>TPH</n> and <n>serotonin</n> exhibit impaired hemostasis, resulting in a reduced risk of thrombosis 
and thromboembolism, although the ultrastructure of the platelets is not affected. While the aggregation of 
<n>serotonin-deficient</n> platelets in vitro is apparently normal, their adhesion in vivo is reduced due to 
a blunted secretion of adhesive alpha-granular proteins. In elucidating the mechanism further, we demonstrate that 
<n>serotonin</n> is transamidated to <n>small GTPases</n> by <n>transglutaminases</n> during activation and aggregation of platelets, rendering 
these <n>GTPases</n> constitutively active. Our data provides evidence for a receptor-independent signaling mechanism, termed herein 
as <n>"serotonylation,"</n> which leads to alpha-granule exocytosis from platelets. An intrinsic <n>adenylate kinase</n> activity regulates 
gating of the <n>ABC</n> transporter <n>CFTR</n>. Cystic fibrosis transmembrane conductance regulator (<n>CFTR</n>) is an anion 
channel in the ATP binding cassette (<n>ABC</n>) transporter family. Like other <n>ABC</n> transporters, it can 
hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that 
<n>CFTR</n> would require this reaction because anions flow passively through <n>CFTR</n>. Moreover, no other ion 
channel is known to require the large energy of ATP hydrolysis to gate. We found 
that <n>CFTR</n> also has <n>adenylate kinase</n> activity (ATP + AMP = ADP + ADP) that 
regulates gating. When functioning as an <n>adenylate kinase</n>, <n>CFTR</n> showed positive cooperativity for ATP suggesting 
its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two 
different enzymatic reactions, <n>ATPase</n> and <n>adenylate kinase</n>, that share a common ATP binding site in 
the second nucleotide binding domain. At physiologic nucleotide concentrations, <n>adenylate kinase</n> activity, rather than <n>ATPase</n> 
activity may control gating, and therefore involve little energy consumption. The C. elegans hook protein, 
<n>ZYG-12</n>, mediates the essential attachment between the centrosome and nucleus. The centrosome and nucleus are 
intimately associated in most animal cells, yet the significance of this interaction is unknown. Mutations 
in the <n>zyg-12</n> gene of Caenorhabditis elegans perturb the attachment of the centrosome to the 
nucleus, giving rise to aberrant spindles and ultimately, DNA segregation defects and lethality. These phenotypes 
indicate that the attachment is essential. <n>ZYG-12</n> is a member of the Hook family of 
cytoskeletal linker proteins and localizes to both the nuclear envelope (via <n>SUN-1</n>) and centrosomes. <n>ZYG-12</n> 
is able to bind the <n>dynein</n> subunit DLI-1 in a two-hybrid assay and is required 
for <n>dynein</n> localization to the nuclear envelope. Loss of <n>dynein</n> function causes a low percentage 
of defective centrosome/nuclei interactions in both Drosophila and Caenorhabditis elegans. We propose that <n>dynein</n> and 
<n>ZYG-12</n> move the centrosomes toward the nucleus, followed by a <n>ZYG-12/SUN-1-dependent</n> anchorage. Cell cycle regulated 
transport controlled by alterations in the nuclear pore complex. Eukaryotic cells have developed mechanisms for 
regulating the nuclear transport of macromolecules that control various cellular events including movement through defined 
stages of the cell cycle. In yeast cells, where the nuclear envelope remains intact throughout 
the cell cycle, these transport regulatory mechanisms must also function during mitosis. We have uncovered 
a mechanism for regulating transport that is controlled by <n>M phase</n> specific molecular rearrangements in 
the nuclear pore complex (<n>NPC</n>). These changes allow a transport inhibitory nucleoporin, <n>Nup53p</n>, to bind 
the karyopherin <n>Kap121p</n> specifically during mitosis, slowing its movement through the <n>NPC</n> and inducing cargo 
release. Yeast strains that possess defects in the function of <n>Kap121p</n> or the fidelity of 
the inhibitory pathway are delayed in mitosis. We propose that fluctuations in <n>Kap121p</n> transport mediated 
by the <n>NPC</n> contribute to controlling the subcellular distribution of molecules that direct progression through 
mitosis. Size selective recognition of siRNA by an RNA silencing suppressor. RNA silencing in plants 
likely exists as a defense mechanism against molecular parasites such as RNA viruses, retrotransposons, and 
transgenes. As a result, many plant viruses have adapted mechanisms to evade and suppress gene 
silencing. Tombusviruses express a 19 kDa protein (<n>p19</n>), which has been shown to suppress RNA 
silencing in vivo and bind silencing-generated and synthetic small interfering RNAs (siRNAs) in vitro. Here 
we report the 2.5 A crystal structure of <n>p19</n> from the Carnation Italian ringspot virus 
(CIRV) bound to a 21 nt siRNA and demonstrate in biochemical and in vivo assays 
that CIRV <n>p19</n> protein acts as a molecular caliper to specifically select siRNAs based on 
the length of the duplex region of the RNA. Prediction of mammalian microRNA targets. <n>MicroRNAs</n> 
(miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by <n>basepairing</n> to 
mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs 
are all unknown. Here we predict more than 400 regulatory target genes for the conserved 
vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA 
and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA 
controls supported a majority of these predictions, with the fraction of false positives estimated at 
31% for targets identified in human, mouse, and rat and 22% for targets identified in 
pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally 
using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched 
for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other 
functions. Plasmodium biology: genomic gleanings. The highly A+T rich genomes of human and rodent malarial 
parasites offer unprecedented glimpses of a lineage that is distinct from other model organisms. Plasmodium 
is distinguished by the presence of numerous low complexity inserts within globular domains of proteins. 
It displays several peculiarities in its transcription apparatus, and its DNA repair system appears to 
favor a certain innate level of mutability. Plasmodium possesses many cell surface molecules with "animal-like" 
adhesion modules. Potential genetic footprints of the ancestral eukaryotic algal precursor of the <n>apicoplast</n> are 
also detectable in its genome. The enemy at the gates. Ca2+ entry through <n>TRPM7 channels</n> 
and anoxic neuronal death. In brain ischemia, gating of postsynaptic glutamate receptors is thought to 
initiate Ca2+ overload leading to excitotoxic neuronal death. In this issue, <n>Aarts</n> and colleagues describe 
a novel mechanism, whereby gating of <n>TRPM7</n>, a Ca2+-permeable nonselective cation channel, mediates Ca2+ overload 
and demise of anoxic neurons. Memory, synaptic translation, and...prions? Two papers from Kausik Si, <n>Eric</n> 
Kandel, Susan Lindquist, and colleagues set forth a bold new idea for thinking about the 
mechanisms underlying the generation and maintenance of long-term memories (Si et al., <n>2003a</n>, <n>2003b</n> (this 
issue of Cell) ). Isomerization of the intersubunit disulphide-bond in <n>Env</n> controls retrovirus fusion. The 
membrane fusion activity of murine leukaemia virus <n>Env</n> is carried by the transmembrane (TM) and 
controlled by the peripheral (<n>SU</n>) subunit. We show here that all <n>Env</n> subunits of the 
virus form disulphide-linked <n>SU-TM</n> complexes that can be disrupted by treatment with <n>NP-40</n>, heat or 
urea, or by Ca(2+) depletion. Thiol mapping indicated that these conditions induced isomerization of the 
disulphide-bond by activating a thiol group in a Cys-X-X-Cys (<n>CXXC</n>) motif in <n>SU</n>. This resulted 
in dissociation of <n>SU</n> from the virus. The active thiol was hidden in uninduced virus 
but became accessible for alkylation by either Ca(2+) depletion or receptor binding. The alkylation inhibited 
isomerization, virus fusion and infection. DTT treatment of alkylated <n>Env</n> resulted in cleavage of the 
<n>SU-TM</n> disulphide-bond and rescue of virus fusion. Further studies showed that virus fusion was specifically 
inhibited by high and enhanced by low concentrations of Ca(2+). These results suggest that <n>Env</n> 
is stabilized by Ca(2+) and that receptor binding triggers a cascade of reactions involving Ca(2+) 
removal, <n>CXXC-thiol</n> exposure, <n>SU-TM</n> disulphide-bond isomerization and <n>SU</n> dissociation, which lead to fusion activation. Silencing 
of transgene transcription precedes methylation of promoter DNA and <n>histone H3</n> lysine 9. Transgenes stably 
integrated into cells or animals in many cases are silenced rapidly, probably under the influence 
of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by 
histone hypoacetylation, loss of methylation at H3 lysine 4, increase of <n>histone H3</n> lysine 9 
methylation as well as CpG DNA methylation at the promoter. However, the order and the 
timing of these modifications and their impact on transcription inactivation are less well understood. To 
determine the temporal order of these events, we examined a model system consisting of a 
transgenic cassette stably integrated in chicken erythroid cells. We found that <n>histone H3</n> and H4 
hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window 
of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that 
these histone modifications were the primary events in gene silencing. We show that the kinetics 
of silencing exclude <n>histone H3</n> K9 and promoter DNA methylation as the primary causative events 
in our transgene system. <n>Collagen</n> XVIII/endostatin is essential for vision and retinal pigment epithelial function. 
Age-related macular degeneration (<n>ARMD</n>) with abnormal deposit formation under the retinal pigment epithelium (<n>RPE</n>) is 
the major cause of blindness in the Western world. basal laminar deposits are found in 
early <n>ARMD</n> and are composed of excess basement membrane material produced by the <n>RPE</n>. Here, 
we demonstrate that mice lacking the basement membrane component <n>collagen</n> XVIII/endostatin have massive accumulation of 
sub-RPE deposits with striking similarities to basal laminar deposits, abnormal <n>RPE</n>, and age-dependent loss of 
vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a 
consequence of abnormal vitamin A metabolism in the <n>RPE</n>. In addition, aged mutant mice show 
photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our 
data demonstrate that <n>collagen</n> XVIII/endostatin is essential for <n>RPE</n> function, and suggest an important role 
of this <n>collagen</n> in Bruch's membrane. Consistent with such a role, the ultrastructural organization of 
<n>collagen</n> XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement 
membrane molecular networks. The SNARE <n>Ykt6</n> mediates protein palmitoylation during an early stage of homotypic 
vacuole fusion. The NSF homolog <n>Sec18</n> initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes 
during priming. <n>Sec18</n> is also required for palmitoylation of the fusion factor <n>Vac8</n>, although the 
acylation machinery has not been identified. Here we show that the SNARE <n>Ykt6</n> mediates <n>Vac8</n> 
palmitoylation and acts during a novel <n>subreaction</n> of vacuole fusion. This <n>subreaction</n> is controlled by 
a Sec17-independent function of <n>Sec18</n>. Our data indicate that <n>Ykt6</n> presents Pal-CoA via its N-terminal 
longin domain to <n>Vac8</n>, while transfer to <n>Vac8's</n> SH4 domain occurs spontaneously and not enzymatically. 
The conservation of <n>Ykt6</n> and its localization to several organelles suggest that its <n>acyltransferase</n> activity 
may also be required in other intracellular fusion events. Identification of a redox-regulated chaperone network. 
We have identified and reconstituted a multicomponent redox-chaperone network that appears to be designed to 
protect proteins against stress-induced unfolding and to refold proteins when conditions return to normal. The 
central player is <n>Hsp33</n>, a redox-regulated molecular chaperone. <n>Hsp33</n>, which is activated by disulfide bond 
formation and subsequent dimerization, works as an efficient chaperone holdase that binds to unfolding protein 
intermediates and maintains them in a folding competent conformation. Reduction of <n>Hsp33</n> is catalyzed by 
the <n>glutaredoxin</n> and <n>thioredoxin</n> systems in vivo, and leads to the formation of highly active, 
reduced <n>Hsp33</n> dimers. Reduction of <n>Hsp33</n> is necessary but not sufficient for substrate protein release. 
Substrate dissociation from <n>Hsp33</n> is linked to the presence of the DnaK/DnaJ/GrpE foldase system, which 
alone, or in concert with the GroEL/GroES system, then supports the refolding of the substrate 
proteins. Upon substrate release, reduced <n>Hsp33</n> dimers dissociate into inactive monomers. This regulated substrate transfer 
ultimately links substrate release and <n>Hsp33</n> inactivation to the presence of available DnaK/DnaJ/GrpE, and, therefore, 
to the return of cells to non-stress conditions. <n>E2A</n> proteins enforce a proliferation checkpoint in 
developing thymocytes. <n>E2A</n> proteins regulate multiple stages of thymocyte development and suppress T-cell lymphoma. The 
activity of <n>E2A</n> proteins throughout thymocyte development is modulated by signals emanating from the pre-TCR 
and <n>TCR</n>. Here we demonstrate that <n>E2A</n> is required for the complete arrest in both 
differentiation and proliferation observed in thymocytes with defects in proteins that mediate pre-TCR signaling, including 
<n>LAT</n>, <n>Lck</n> and Fyn. We show that <n>E2A</n> proteins are required to prevent the accumulation 
of <n>TCRbeta</n> negative cells beyond the pre-TCR checkpoint. <n>E2A-deficient</n> thymocytes also exhibit abnormal cell-cycle progression 
prior to pre-TCR expression. Furthermore, we demonstrate that <n>E47</n> can act in concert with <n>Bcl-2</n> 
to induce cell-cycle arrest in vitro. These observations indicate that <n>E2A</n> proteins function during early 
thymocyte development to block cell-cycle progression prior to the expression of <n>TCRbeta</n>. In addition, these 
data provide further insight into how <n>deficiencies</n> in <n>E2A</n> lead to T lymphoma. Unravelling natural 
killer cell function: triggering and inhibitory human <n>NK receptors</n>. Natural killer (NK) cells represent a 
highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected 
cells. Their function is finely regulated by a series of inhibitory or <n>activating receptors</n>. The 
inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to 
discriminate between normal cells and cells that have lost the expression of MHC class I 
(e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, <n>NKp30</n>, <n>NKp44</n> 
and <n>NKG2D</n>. The NK-mediated lysis of tumor cells involves several such receptors, while killing of 
dendritic cells involves only <n>NKp30</n>. The target-cell ligands recognized by some receptors have been identified, 
but those to which major receptors bind are not yet known. Nevertheless, functional data suggest 
that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the 
ability of NK cells to lyse target cells requires both the lack of surface MHC 
class I molecules and the expression of appropriate ligands that trigger <n>NK receptors</n>. Blocking HIV-1 
infection via <n>CCR5</n> and <n>CXCR4</n> receptors by acting in trans on the <n>CCR2</n> chemokine receptor. 
The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to 
prevent HIV-1 infection. We generated <n>CCR2-01</n>, a <n>CCR2</n> receptor-specific monoclonal antibody that neither competes with 
the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of <n>monotropic</n> (R5) 
and <n>T-tropic</n> (X4) HIV-1 strains. This effect is explained by the ability of <n>CCR2-01</n> to 
induce oligomerization of <n>CCR2</n> with the <n>CCR5</n> or <n>CXCR4</n> viral coreceptors. HIV-1 infection through <n>CCR5</n> 
and <n>CXCR4</n> receptors can thus be prevented in the absence of steric hindrance or receptor 
downregulation by acting in trans on a receptor that is rarely used by the virus 
to infect cells. Structural bases for <n>CRMP</n> function in plexin-dependent semaphorin3A signaling. Collapsin response mediator 
proteins (<n>CRMPs</n>) are cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. <n>CRMP2</n> was previously 
shown to mediate the repulsive effect of <n>Sema3A</n> on axons and to participate in axonal 
specification. The X-ray crystal structure of <n>murine CRMP1</n> was determined at 2.1 A resolution and 
demonstrates that <n>CRMP1</n> is a bilobed 'lung-shaped' protein forming a tetrameric assembly. Structure-based mutagenesis of 
surface-exposed residues was employed to map functional domains. As a rapid assay for <n>CRMP</n>, we 
exploited a reconstituted <n>Sema3A</n> signaling system in COS-7 cells expressing the receptor components <n>Neuropilin1</n> and 
<n>PlexinA1</n> (<n>NP1/PlexA1</n>). In these cells, <n>CRMP</n> and <n>PlexA1</n> form a physical complex that is reduced 
in amount by <n>NP1</n> but enhanced by <n>Sema3A/NP1</n>. Furthermore, <n>CRMP</n> accelerates <n>Sema3A-induced</n> cell contraction. Alanine 
substitutions in one domain of <n>CRMP1</n> produce a constitutively active protein that causes <n>Sema3A-independent</n> COS-7 
contraction. This mutant <n>CRMP</n> mimics the DRG neurite outgrowth-inhibiting effects of <n>Sema3A</n> and reduces <n>Sema3A-induced</n> 
axonal repulsion. These data provide a structural view of <n>CRMP</n> function in Plex-dependent <n>Sema3A</n> signaling. 
Recruitment of <n>Cdc28</n> by <n>Whi3</n> restricts nuclear accumulation of the G1 cyclin-Cdk complex to late 
G1. The G1 <n>cyclin Cln3</n> is a key activator of cell-cycle entry in budding yeast. 
Here we show that <n>Whi3</n>, a negative G1 regulator of <n>Cln3</n>, interacts in vivo with 
the cyclin-dependent kinase <n>Cdc28</n> and regulates its localization in the cell. Efficient interaction with <n>Cdc28</n> 
depends on an N-terminal domain of <n>Whi3</n> that is also required for cytoplasmic localization of 
<n>Cdc28</n>, and for proper regulation of G1 length and filamentous growth. On the other hand, 
nuclear accumulation of <n>Cdc28</n> requires the nuclear localization signal of <n>Cln3</n>, which is also found 
in <n>Whi3</n> complexes. Both <n>Cln3</n> and <n>Cdc28</n> are mainly cytoplasmic during early G1, and become 
nuclear in late G1. However, <n>Whi3-deficient</n> cells show a distinct nuclear accumulation of <n>Cln3</n> and 
<n>Cdc28</n> already in early G1. We propose that <n>Whi3</n> constitutes a cytoplasmic retention device for 
<n>Cln3-Cdc28</n> complexes, thus defining a key G1 event in yeast cells. Identification of PKCzetaII: an 
endogenous inhibitor of cell polarity. A new member of the <n>atypical protein kinase C</n> (<n>aPKC</n>) 
family, designated PKCzetaII, is identified in this study. The gene contains no introns and is 
98% homologous with the cDNA encoding PKCzeta. The PKCzetaII coding region is frame-shifted with respect 
to the PKCzeta open reading frame, resulting in expression of an <n>aPKC</n> regulatory domain without 
associated kinase activity. PKCzetaII mRNA is detected in various mouse tissues and an immunoreactive 45 
kDa protein is present in epithelial cell cultures. PKCzetaII is shown to interact with the 
<n>Par6 protein</n> and functions in the development of cell polarity. HC11 epithelial cells express PKCzetaII 
and are maintained in a nondifferentiated state characterised by the absence of tight junctions and 
cell overgrowth. HC11 cells harbouring a PKCzetaII-specific <n>RNAi</n>, recruit <n>ZO-1</n> and other tight junction markers 
to cell-cell boundaries and adopt a monolayer phenotype in the presence of growth factors. The 
data demonstrate a regulatory role for PKCzetaII in the maintenance of cell transformation and the 
development of cell polarity. Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments. 
There is growing evidence that duplications have played a major role in eucaryotic genome evolution. 
Sequencing data revealed the presence of large duplicated regions in the genomes of many eucaryotic 
organisms, and comparative studies have suggested that duplication of large DNA segments has been a 
continuing process during evolution. However, little experimental data have been produced regarding this issue. Using 
a gene dosage assay for growth recovery in Saccharomyces cerevisiae, we demonstrate that a majority 
of the revertant strains (58%) resulted from the spontaneous duplication of large DNA segments, either 
intra- or <n>interchromosomally</n>, ranging from 41 to 655 kb in size. These events result in 
the concomitant duplication of dozens of genes and in some cases in the formation of 
chimeric open reading frames at the junction of the duplicated blocks. The types of sequences 
at the <n>breakpoints</n> as well as their superposition with the replication map suggest that spontaneous 
large segmental duplications result from replication accidents. Aneuploidization events or suppressor mutations that do not 
involve large-scale rearrangements accounted for the rest of the reversion events (in 26 and 16% 
of the strains, respectively). Modulation of <n>KSR</n> activity in Caenorhabditis elegans by Zn ions, <n>PAR-1 
kinase</n> and <n>PP2A</n> phosphatase. Vulval differentiation in Caenorhabditis elegans is controlled by a conserved signal 
transduction pathway mediated by <n>Ras</n> and a kinase cascade that includes <n>Raf</n>, <n>Mek</n> and <n>MAPK</n>. 
Activation of this cascade is positively regulated by a number of proteins such as <n>KSR</n> 
(kinase suppressor of <n>Ras</n>), SUR-8/SOC-2, SUR-6/PP2A-B and <n>CDF-1</n>. We describe the functional characterization of <n>sur-7</n> 
and several genes that regulate signaling downstream of <n>ras</n>. We identified <n>sur-7</n> by isolating a 
mutation that suppresses an activated <n>ras</n> allele, and showed that <n>SUR-7</n> is a divergent member 
of the cation diffusion facilitator family of heavy metal ion transporters that is probably localized 
to the endoplosmic recticulum membrane and regulates cellular Zn(2+) concentrations. Genetic double mutant analyses suggest 
that the <n>SUR-7-mediated</n> effect is not a general toxic response. Instead, Zn(2+) ions target a 
specific step of the pathway, probably regulation of the scaffolding protein <n>KSR</n>. Biochemical analysis in 
mammalian cells indicates that high Zn(2+) concentration causes a dramatic increase of <n>KSR</n> phosphorylation. Genetic 
analysis also indicates that <n>PP2A</n> phosphatase and <n>PAR-1</n> kinase act downstream of <n>Raf</n> to positively 
and negatively regulate <n>KSR</n> activity, respectively. Drosophila Cup is an <n>eIF4E-binding</n> protein that functions in 
<n>Smaug-mediated</n> translational repression. Translational regulation plays an essential role in development and often involves factors 
that interact with sequences in the 3' untranslated region (<n>UTR</n>) of specific mRNAs. For example, 
<n>Nanos</n> protein at the posterior of the Drosophila embryo directs posterior development, and this localization 
requires selective translation of posteriorly localized <n>nanos</n> mRNA. Spatial regulation of <n>nanos</n> translation requires <n>Smaug</n> 
protein bound to the <n>nanos</n> 3' <n>UTR</n>, which represses the translation of unlocalized <n>nanos</n> transcripts. 
While the function of 3' <n>UTR-bound</n> translational regulators is, in general, poorly understood, they presumably 
interact with the basic translation machinery. Here we demonstrate that <n>Smaug</n> interacts with the <n>Cup 
protein</n> and that Cup is an <n>eIF4E-binding</n> protein that blocks the binding of <n>eIF4G</n> to 
<n>eIF4E</n>. Cup mediates an indirect interaction between <n>Smaug</n> and <n>eIF4E</n>, and <n>Smaug</n> function in vivo 
requires Cup. Thus, <n>Smaug</n> represses translation via a Cup-dependent block in <n>eIF4G</n> recruitment. The multicoloured 
world of promoter recognition complexes. The expression pattern of regulated genes changes dynamically depending on 
the developmental stage and the differentiation state of the cell. Transcription factors regulate cellular events 
at the gene expression level by communicating signals to the general transcription machinery that forms 
a preinitiation complex (<n>PIC</n>) at class II core promoters. Recent data strongly suggest that PICs 
are composed of different sets of factors at distinct promoters, reflecting the spatiotemporal profile of 
gene expression in multicellular organisms. Thus, today it is important to ask the question: how 
universal are the promoter recognition factors? This review will focus on findings that support the 
new idea that core promoter recognition by distinct factors is an additional level of transcriptional 
regulation and that this step is developmentally regulated. <n>migS</n>, a cis-acting site that affects bipolar 
positioning of <n>oriC</n> on the Escherichia coli chromosome. During replication of the Escherichia coli chromosome, 
the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for 
bipolar migration is located in this region. To identify this cis-acting site, a series of 
mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 
kb subchromosome, was found to be defective in the bipolar positioning of <n>oriC</n>. The creation 
of deletion mutants allowed the identification of <n>migS</n>, a 25 bp sequence, as the cis-acting 
site for the bipolar positioning of <n>oriC</n>. When <n>migS</n> was located at the replication terminus, 
the chromosomal segment showed bipolar positioning. <n>migS</n> was able to rescue bipolar migration of plasmid 
DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the <n>migS</n> 
sequence on a plasmid in trans inhibited the bipolar positioning of <n>oriC</n>. Taken together, these 
findings indicate that <n>migS</n> plays a crucial role in the bipolar positioning of <n>oriC</n>. In 
addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and <n>migS</n> mutants 
suggests that bipolar positioning of the replicated <n>oriC</n> contributes to nucleoid organization. An episomal mammalian 
replicon: sequence-independent binding of the origin recognition complex. An extrachromosomally replicating plasmid was used to 
investigate the specificity by which the origin recognition complex (<n>ORC</n>) interacts with DNA sequences in 
mammalian cells in vivo. We first showed that the plasmid pEPI-1 replicates semiconservatively in a 
once-per-cell-cycle manner and is stably transmitted over many cell generations in culture without selection. Chromatin 
immunoprecipitations and quantitative polymerase chain reaction analysis revealed that, in G1-phase cells, <n>Orc1</n> and <n>Orc2</n>, 
as well as <n>Mcm3</n>, another component of the prereplication complex, are bound to multiple sites 
on the plasmid. These binding sites are functional because they show the S-phase-dependent dissociation of 
<n>Orc1</n> and <n>Mcm3</n> known to be characteristic for prereplication complexes in mammalian cells. In addition, 
we identified replicative nascent strands and showed that they correspond to many plasmid DNA regions. 
This work has implications for current models of replication origins in mammalian systems. It indicates 
that specific DNA sequences are not required for the chromatin binding of <n>ORC</n> in vivo. 
The conclusion is that epigenetic mechanisms determine the sites where mammalian DNA replication is initiated. 
A novel role for <n>XIAP</n> in copper homeostasis through regulation of <n>MURR1</n>. <n>XIAP</n> is a 
potent suppressor of apoptosis that directly inhibits specific members of the <n>caspase</n> family of cysteine 
<n>proteases</n>. Here we demonstrate a novel role for <n>XIAP</n> in the control of intracellular copper 
levels. <n>XIAP</n> was found to interact with <n>MURR1</n>, a factor recently implicated in copper homeostasis. 
<n>XIAP</n> binds to <n>MURR1</n> in a manner that is distinct from that utilized by <n>XIAP</n> 
to bind <n>caspases</n>, and consistent with this, <n>MURR1</n> did not affect the antiapoptotic properties of 
<n>XIAP</n>. However, cells and tissues derived from Xiap-deficient mice were found to contain reduced copper 
levels, while suppression of <n>MURR1</n> resulted in increased intracellular copper in cultured cells. Consistent with 
these opposing effects, <n>XIAP</n> was observed to negatively regulate <n>MURR1</n> protein levels by the formation 
of <n>K48</n> polyubiquitin chains on <n>MURR1</n> that promote its degradation. These findings represent the first 
described phenotypic alteration in Xiap-deficient mice and demonstrate that <n>XIAP</n> can function through <n>MURR1</n> to 
regulate copper homeostasis. <n>Olfactory receptor</n> antagonism between odorants. The detection of thousands of volatile odorants 
is mediated by several hundreds of different G protein-coupled olfactory receptors (ORs). The main strategy 
in encoding odorant identities is a combinatorial receptor code scheme in that different odorants are 
recognized by different sets of ORs. Despite increasing information on agonist-OR combinations, little is known 
about the antagonism of ORs in the mammalian olfactory system. Here we show that odorants 
inhibit odorant responses of <n>OR(s)</n>, evidence of antagonism between odorants at the receptor level. The 
antagonism was demonstrated in a heterologous OR-expression system and in single olfactory neurons that expressed 
a given OR, and was also visualized at the level of the olfactory epithelium. Dual 
functions of odorants as an agonist and an antagonist to ORs indicate a new aspect 
in the receptor code determination for odorant mixtures that often give rise to novel perceptual 
qualities that are not present in each component. The current study also provides insight into 
strategies to modulate perceived odorant quality. Regulated assembly of the Toll signaling complex drives Drosophila 
dorsoventral patterning. In Drosophila, the Toll pathway establishes the embryonic dorsoventral axis and triggers innate 
immune responses to infection. The transmembrane receptor Toll acts through three death domain-containing proteins, the 
kinase <n>Pelle</n> and the adapters Tube and <n>MyD88</n>, in signaling to downstream <n>NF-kappaB-like</n> transcription factors. 
Here, we delineate the critical events in the earliest stages of Toll signaling. Mutational studies 
based on structural modeling reveal that the direct interaction of the bivalent Tube death domain 
with <n>MyD88</n> is critical for signaling in vivo. The complex of <n>MyD88</n> and Tube forms 
prior to signaling and is localized to the embryonic plasma membrane by <n>MyD88</n>. Upon Toll 
homodimerization, this complex is rapidly recruited to Toll. Binding of <n>Pelle</n> to the <n>MyD88-Tube</n> complex 
promotes <n>Pelle</n> activation, leading to degradation of the IkappaB-like inhibitor, Cactus. Together, these experiments convert 
a linear picture of gene function into a dynamic mechanistic and structural understanding of signaling 
complex assembly and function. Identification of a family of animal sphingomyelin synthases. <n>Sphingomyelin</n> (<n>SM</n>) is 
a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from 
phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by <n>SM</n> 
synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and 
ceramide as anti- and proapoptotic stimuli, respectively. <n>SM</n> synthesis occurs in the lumen of the 
Golgi as well as on the cell surface. As no gene for <n>SM</n> synthase has 
been cloned so far, it is unclear whether different enzymes are present at these locations. 
Using a functional cloning strategy in yeast, we identified a novel family of integral membrane 
proteins exhibiting all enzymatic features previously attributed to animal <n>SM</n> synthase. Strikingly, human, mouse and 
Caenorhabditis elegans genomes each contain at least two different <n>SM synthase</n> (<n>SMS</n>) genes. Whereas human 
<n>SMS1</n> is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these 
findings open up important new avenues for studying sphingolipid function in animals. Roles of <n>SWI/SNF</n> 
and HATs throughout the dynamic transcription of a yeast glucose-repressible gene. Eucaryotic gene expression requires 
chromatin-remodeling activities. We show by time-course studies that transcriptional induction of the yeast glucose-regulated <n>SUC2</n> 
gene is rapid and shows a striking biphasic pattern, the first phase of which is 
partly mediated by the general stress transcription factors <n>Msn2p/Msn4p</n>. The <n>SWI/SNF</n> ATP-dependent chromatin-remodeling complex associates 
with the promoter in a similar biphasic manner and is essential for both phases of 
transcription. Two different <n>histone acetyltransferases</n>, <n>Gcn5p</n> and <n>Esa1p</n>, enhance the binding of <n>SWI/SNF</n> to the 
promoter during early transcription and are required for optimal <n>SUC2</n> induction. <n>Gcn5p</n> is recruited to 
<n>SUC2</n> simultaneously with <n>SWI/SNF</n>, whereas <n>Esa1p</n> associates constitutively with the promoter. This study reveals an 
unusual transcription pattern of a metabolic gene and suggests a novel strategy by which distinct 
chromatin remodelers cooperate for the dynamic activation of transcription. <n>Nbp2</n> targets the <n>Ptc1-type</n> 2C Ser/Thr 
phosphatase to the HOG <n>MAPK</n> pathway. The yeast high osmolarity glycerol (HOG) pathway signals via 
the Pbs2 <n>MEK</n> and the <n>Hog1</n> <n>MAPK</n>, whose activity requires phosphorylation of Thr and Tyr 
in the activation loop. The <n>Ptc1-type</n> 2C Ser/Thr phosphatase (<n>PP2C</n>) inactivates <n>Hog1</n> by dephosphorylating phospho-Thr, 
while the <n>Ptp2</n> and <n>Ptp3</n> protein tyrosine <n>phosphatases</n> dephosphorylate phospho-Tyr. In this work, we show 
that the SH3 domain-containing protein <n>Nbp2</n> negatively regulates <n>Hog1</n> by recruiting <n>Ptc1</n> to the Pbs2-Hog1 
complex. Consistent with this role, <n>NBP2</n> acted as a negative regulator similar to <n>PTC1</n> in 
phenotypic assays. Biochemical analysis showed that <n>Nbp2</n>, like <n>Ptc1</n>, was required to inactivate <n>Hog1</n> during 
adaptation. As predicted for an adapter, deletion of <n>NBP2</n> disrupted <n>Ptc1-Pbs2</n> complex formation. Furthermore, <n>Nbp2</n> 
contained separate binding sites for <n>Ptc1</n> and Pbs2: the novel N-terminal domain bound <n>Ptc1</n>, while 
the SH3 domain bound Pbs2. In addition, the Pbs2 scaffold bound the <n>Nbp2</n> SH3 via 
a Pro-rich motif distinct from that which binds the SH3 domain of the positive regulator 
<n>Sho1</n>. Thus, <n>Nbp2</n> recruits <n>Ptc1</n> to Pbs2, a scaffold for both negative and positive regulators. 
Regulation of <n>InsP(3) receptor</n> activity by neuronal Ca(2+)-binding proteins. Inositol 1,4,5-trisphosphate receptors (<n>InsP(3)Rs</n>) were recently 
demonstrated to be activated independently of <n>InsP(3)</n> by a family of <n>calmodulin</n> (<n>CaM</n>) -like neuronal 
Ca(2+)-binding proteins (<n>CaBPs</n>). We investigated the interaction of both naturally occurring long and short <n>CaBP1</n> 
isoforms with <n>InsP(3)Rs</n>, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, 
including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and 
(45)Ca(2+) flux from permeabilised COS cells, we demonstrated that <n>CaBPs</n> decrease the sensitivity of <n>InsP(3)-induced</n> 
Ca(2+) release (<n>IICR</n>). In addition, we found a Ca(2+)-independent interaction between <n>CaBP1</n> and the NH(2)-terminal 
159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased <n>InsP(3)</n> binding 
to the receptor reminiscent of that observed for <n>CaM</n>. Unlike <n>CaM</n>, however, <n>CaBPs</n> do not 
inhibit ryanodine receptors, have a higher affinity for <n>InsP(3)Rs</n> and more potently inhibited <n>IICR</n>. We 
also show that phosphorylation of <n>CaBP1</n> at a casein kinase 2 consensus site regulates its 
inhibition of <n>IICR</n>. Our data suggest that <n>CaBPs</n> are endogenous regulators of <n>InsP(3)Rs</n> tuning the 
sensitivity of cells to <n>InsP(3)</n>. Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane 
conductance regulator. Cystic fibrosis transmembrane conductance regulator (<n>CFTR</n>) is an <n>ATP-binding cassette</n> (<n>ABC</n>) transporter that 
functions as a chloride channel. Nucleotide-binding domain 1 (<n>NBD1</n>), one of two <n>ABC</n> domains in 
<n>CFTR</n>, also contains sites for the predominant <n>CF-causing</n> mutation and, potentially, for regulatory phosphorylation. We 
have determined crystal structures for mouse <n>NBD1</n> in unliganded, <n>ADP-</n> and ATP-bound states, with and 
without phosphorylation. This <n>NBD1</n> differs from typical <n>ABC</n> domains in having added regulatory segments, a 
foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated <n>NBD1</n> has undetectable <n>ATPase</n> activity 
and its structure is essentially the same independent of ligand state. Phe508, which is commonly 
deleted in <n>CF</n>, is exposed at a putative <n>NBD1-transmembrane</n> interface. Our results are consistent with 
a <n>CFTR</n> mechanism, whereby channel gating occurs through ATP binding in an <n>NBD1-NBD2</n> nucleotide sandwich 
that forms upon displacement of <n>NBD1</n> regulatory segments. What <n>kinesin</n> does at roadblocks: the coordination 
mechanism for molecular walking. Competing models for the coordination of <n>processive</n> stepping in <n>kinesin</n> can 
be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an 
irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of 
<n>kinesin</n> monomers or dimers with and without the T93N roadblock using microflash photolysis combined with 
stopped flow. Control nucleotide-induced monomer (<n>rK340</n>) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) 
for ADP. Control ADP-induced dimer (<n>rK430</n>) unbinding was 18.6 s(-1). Added 20 mM Pi slowed 
both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers 
is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than 
two-fold slower than the stepping rate of unimpeded <n>rK430</n> dimers (50-70 s(-1)), indicating that during 
walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of 
trail head detachment. As we discuss, this implies a coordination model having very fast (2000 
s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the 
lead head. Structural basis of ligand recognition by <n>PABC</n>, a highly specific peptide-binding domain found 
in <n>poly(A)-binding protein</n> and a HECT <n>ubiquitin</n> ligase. The C-terminal domain of <n>poly(A)-binding protein</n> (<n>PABC</n>) 
is a peptide-binding domain found in poly(A)-binding proteins (<n>PABPs</n>) and a HECT (homologous to <n>E6-AP</n> 
C-terminus) family E3 <n>ubiquitin</n> ligase. In protein synthesis, the <n>PABC</n> domain of <n>PABP</n> functions to 
recruit several translation factors possessing the <n>PABP-interacting</n> motif 2 (<n>PAM2</n>) to the mRNA poly(A) tail. 
We have determined the solution structure of the human <n>PABC</n> domain in complex with two 
peptides from <n>PABP-interacting</n> <n>protein-1</n> (<n>Paip1</n>) and <n>Paip2</n>. The structures show a novel mode of peptide 
recognition, in which the peptide binds as a pair of <n>beta-turns</n> with extensive hydrophobic, electrostatic 
and aromatic stacking interactions. Mutagenesis of <n>PABC</n> and peptide residues was used to identify key 
protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and <n>GST</n> pull-down assays. The 
results provide insight into the specificity of <n>PABC</n> in mediating <n>PABP-protein</n> interactions. Estrogen receptor-alpha directs 
ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Transcriptional activation of 
a gene involves an orchestrated recruitment of components of the basal transcription machinery and intermediate 
factors, concomitant with an alteration in local chromatin structure generated by posttranslational modifications of <n>histone 
tails</n> and nucleosome remodeling. We provide here a comprehensive picture of events resulting in transcriptional 
activation of a gene, through evaluating the <n>estrogen receptor-alpha</n> (<n>NR3A1</n>) target <n>pS2</n> gene promoter in 
MCF-7 cells. This description integrates chromatin remodeling with a kinetic evaluation of cyclical networks of 
association of 46 transcription factors with the promoter, as determined by chromatin immunoprecipitation assays. We 
define the concept of a "transcriptional clock" that directs and achieves the sequential and combinatorial 
assembly of a transcriptionally productive complex on a promoter. Furthermore, the unanticipated findings of key 
roles for <n>histone deacetylases</n> and nucleosome-remodeling complexes in limiting transcription implies that transcriptional activation is 
a cyclical process that requires both activating and repressive epigenetic processes. Ribosome loading onto the 
mRNA cap is driven by conformational coupling between <n>eIF4G</n> and <n>eIF4E</n>. The <n>eukaryotic initiation factor 
4G</n> (<n>eIF4G</n>) is the core of a multicomponent switch controlling gene expression at the level 
of translation initiation. It interacts with the small ribosomal subunit interacting protein, <n>eIF3</n>, and the 
<n>eIF4E/cap-mRNA</n> complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe 
the solution structure of the complex between yeast <n>eIF4E/cap</n> and <n>eIF4G</n> (393-490). Binding triggers a 
coupled folding transition of <n>eIF4G</n> (393-490) and the <n>eIF4E</n> N terminus resulting in a molecular 
bracelet whereby <n>eIF4G</n> (393-490) forms a right-handed helical ring that wraps around the N terminus 
of <n>eIF4E</n>. Cofolding allosterically enhances association of <n>eIF4E</n> with the cap and is required for 
maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, <n>eIF4E</n>, 
and <n>eIF4G</n> exists as a stable mRNP that may facilitate multiple rounds of <n>ribosomal loading</n> 
during translation initiation, a key determinant in the overall rate of protein synthesis. The deacetylase 
<n>HDAC6</n> regulates <n>aggresome</n> formation and cell viability in response to <n>misfolded</n> protein stress. The efficient 
clearance of cytotoxic <n>misfolded</n> protein aggregates is critical for cell survival. <n>Misfolded</n> protein aggregates are 
transported and removed from the cytoplasm by <n>dynein</n> motors via the microtubule network to a 
novel organelle termed the <n>aggresome</n> where they are processed. However, the means by which <n>dynein</n> 
motors recognize <n>misfolded</n> protein cargo, and the cellular factors that regulate <n>aggresome</n> formation, remain unknown. 
We have discovered that <n>HDAC6</n>, a microtubule-associated deacetylase, is a component of the <n>aggresome</n>. We 
demonstrate that <n>HDAC6</n> has the capacity to bind both polyubiquitinated <n>misfolded</n> proteins and <n>dynein</n> motors, 
thereby acting to recruit <n>misfolded</n> protein cargo to <n>dynein</n> motors for transport to <n>aggresomes</n>. Indeed, 
cells deficient in <n>HDAC6</n> fail to clear <n>misfolded</n> protein aggregates from the cytoplasm, cannot form 
<n>aggresomes</n> properly, and are hypersensitive to the accumulation of <n>misfolded</n> proteins. These findings identify <n>HDAC6</n> 
as a crucial player in the cellular management of <n>misfolded</n> protein-induced stress. <n>O-GlcNAc</n> modification is 
an endogenous inhibitor of the proteasome. The <n>ubiquitin</n> proteasome system classically selects its substrates for 
degradation by tagging them with <n>ubiquitin</n>. Here, we describe another means of controlling proteasome function 
in a global manner. The 26S proteasome can be inhibited by modification with the enzyme, 
<n>O-GlcNAc</n> <n>transferase</n> (<n>OGT</n>). This reversible modification of the proteasome inhibits the proteolysis of the transcription 
factor <n>Sp1</n> and a hydrophobic peptide through inhibition of the <n>ATPase</n> activity of 26S <n>proteasomes</n>. 
The <n>Rpt2</n> <n>ATPase</n> in the mammalian proteasome 19S cap is modified by <n>O-GlcNAc</n> in vitro 
and in vivo and as its modification increases, proteasome function decreases. This mechanism may couple 
<n>proteasomes</n> to the general metabolic state of the cell. The <n>O-GlcNAc</n> modification of <n>proteasomes</n> may 
allow the organism to respond to its metabolic needs by controlling the availability of amino 
acids and regulatory proteins. The bacterial cytoskeleton: an intermediate filament-like function in cell shape. Various 
cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly 
understood. Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape 
in higher organisms. No such filaments have been found in prokaryotes. Here, we describe a 
bacterial equivalent to IF proteins, named <n>crescentin</n>, whose cytoskeletal function is required for the <n>vibrioid</n> 
and helical shapes of Caulobacter crescentus. Without <n>crescentin</n>, the cells adopt a straight-rod morphology. <n>Crescentin</n> 
has characteristic features of IF proteins including the ability to assemble into filaments in vitro 
without energy or cofactor requirements. In vivo, <n>crescentin</n> forms a helical structure that colocalizes with 
the inner cell curvatures beneath the cytoplasmic membrane. We propose that IF-like filaments of <n>crescentin</n> 
assemble into a helical structure, which by applying its geometry to the cell, generates a 
<n>vibrioid</n> or helical cell shape depending on the length of the cell. Drosophila <n>PAR-1</n> and 
14-3-3 inhibit <n>Bazooka/PAR-3</n> to establish complementary cortical domains in polarized cells. <n>PAR-1</n> <n>kinases</n> are required 
for polarity in diverse cell types, such as epithelial cells, where they localize laterally. <n>PAR-1</n> 
activity is believed to be transduced by binding of 14-3-3 proteins to its phosphorylated substrates, 
but the relevant targets are unknown. We show that <n>PAR-1</n> phosphorylates <n>Bazooka/PAR-3</n> on two conserved 
serines to generate 14-3-3 binding sites. This inhibits formation of the Bazooka/PAR-6/aPKC complex by blocking 
Bazooka oligomerization and binding to <n>aPKC</n>. In epithelia, this complex localizes apically and defines the 
apical membrane, whereas Bazooka lacking <n>PAR-1</n> phosphorylation/14-3-3 binding sites forms ectopic lateral complexes. Lateral exclusion 
by <n>PAR-1/14-3-3</n> cooperates with apical anchoring by <n>Crumbs/Stardust</n> to restrict Bazooka localization, and loss of 
both pathways disrupts epithelial polarity. <n>PAR-1</n> also excludes Bazooka from the posterior of the oocyte, 
and disruption of this regulation causes anterior-posterior polarity defects. Thus, antagonism of Bazooka by <n>PAR-1/14-3-3</n> 
may represent a general mechanism for establishing complementary cortical domains in polarized cells. <n>EIN3-dependent</n> regulation 
of plant ethylene hormone signaling by two arabidopsis F box proteins: <n>EBF1</n> and <n>EBF2</n>. The 
plant hormone ethylene regulates a wide range of developmental processes and the response of plants 
to stress and pathogens. Genetic studies in Arabidopsis led to a partial elucidation of the 
mechanisms of ethylene action. Ethylene signal transduction initiates with ethylene binding at a family of 
<n>ethylene receptors</n> and terminates in a transcription cascade involving the <n>EIN3/EIL</n> and ERF families of 
plant-specific transcription factors. Here, we identify two Arabidopsis F box proteins called <n>EBF1</n> and <n>EBF2</n> 
that interact physically with <n>EIN3/EIL</n> transcription factors. <n>EBF1</n> overexpression results in plants insensitive to ethylene. 
In contrast, plants carrying the <n>ebf1</n> and <n>ebf2</n> mutations display a constitutive ethylene response and 
accumulate the <n>EIN3</n> protein in the absence of the hormone. Our work places <n>EBF1</n> and 
<n>EBF2</n> within the genetic framework of the ethylene-response pathway and supports a model in which 
ethylene action depends on <n>EIN3</n> protein stabilization. Plant responses to ethylene gas are mediated by 
SCF(EBF1/EBF2)-dependent proteolysis of <n>EIN3</n> transcription factor. Plants use ethylene gas as a signal to regulate 
myriad developmental processes and stress responses. The Arabidopsis <n>EIN3</n> protein is a key transcription factor 
mediating ethylene-regulated gene expression and morphological responses. Here, we report that <n>EIN3</n> protein levels rapidly 
increase in response to ethylene and this response requires several ethylene-signaling pathway components including the 
<n>ethylene receptors</n> (<n>ETR1</n> and <n>EIN4</n>), <n>CTR1</n>, <n>EIN2</n>, <n>EIN5</n>, and <n>EIN6</n>. In the absence of ethylene, 
<n>EIN3</n> is quickly degraded through a <n>ubiquitin/proteasome</n> pathway mediated by two F box proteins, <n>EBF1</n> 
and <n>EBF2</n>. Plants containing mutations in either gene show enhanced ethylene response by stabilizing <n>EIN3</n>, 
whereas <n>efb1</n> efb2 double mutants show constitutive ethylene phenotypes. Plants overexpressing either F box gene 
display ethylene insensitivity and destabilization of <n>EIN3</n> protein. These results reveal that a <n>ubiquitin/proteasome</n> pathway 
negatively regulates ethylene responses by targeting <n>EIN3</n> for degradation, and pinpoint <n>EIN3</n> regulation as the 
key step in the response to ethylene. A central role of the BK potassium channel 
in behavioral responses to ethanol in C. elegans. The activities of many neuronal proteins are 
modulated by ethanol, but the fundamental mechanisms underlying behavioral effects of ethanol remain unclear. To 
identify mechanisms responsible for intoxication, we screened for Caenorhabditis elegans mutants with altered behavioral responses 
to ethanol. We found that <n>slo-1</n> mutants, which were previously recognized as having slightly uncoordinated 
movement, are highly resistant to ethanol in two behavioral assays. Numerous loss-of-function <n>slo-1</n> alleles emerged 
from our screens, indicating that <n>slo-1</n> has a central role in ethanol responses. <n>slo-1</n> encodes 
the BK potassium channel. Electrophysiological analysis shows that ethanol activates the channel in vivo, which 
would inhibit neuronal activity. Moreover, behaviors of <n>slo-1</n> gain-of-function mutants resemble those of ethanol-intoxicated animals. 
These results demonstrate that selective activation of <n>BK channels</n> is responsible for acute intoxicating effects 
of ethanol in C. elegans. <n>BK channel</n> activation may explain a variety of behavioral responses 
to ethanol in invertebrate and vertebrate systems. The secret life of <n>ACE2</n> as a receptor 
for the SARS virus. The membrane-associated carboxypeptidase angiotensin-converting enzyme 2 (<n>ACE2</n>) is an essential regulator 
of heart function. Now, Li at al. identify and characterize an unexpected second function of 
<n>ACE2</n> as a partner of the SARS-CoV <n>spike glycoprotein</n> in mediating virus entry and cell 
fusion. Coupled folding during translation initiation. The structure of the eukaryotic initiation factor <n>eIF4E</n> bound 
to a cognate domain of <n>eIF4G</n> and <n>m(7)GDP</n> in this issue of Cell shows that 
these factors undergo coupled folding to form a stable complex with high cap binding activity 
that promotes efficient ribosomal attachment to mRNA during translation initiation. Another cytoskeleton in the closet. 
Many eukaryotic cells contain up to three families of cytoskeletal proteins that are responsible for 
the spatial organization of the cell. Although the prokaryotic origins of the <n>actin</n> and <n>tubulin</n> 
families have now been established, the origin of the third was unknown. In this issue 
of Cell, provide evidence that the third family, comprising the intermediate filaments, also has origins 
in bacteria and is responsible for producing curved cells. SCF-mediated proteolysis and negative regulation in 
ethylene signaling. Ethylene is an important hormonal regulator of plant growth that acts by regulating 
gene expression. In this issue of Cell, <n>Guo</n> and <n>Ecker</n>, and Potuschak et al., show 
that ethylene increases the abundance of the transcription factor <n>EIN3</n>, an activator of ethylene-inducible genes, 
by relieving its SCF-mediated destruction. The crucial role of <n>caspase-9</n> in the disease progression of 
a transgenic <n>ALS</n> mouse model. Mutant copper/zinc superoxide dismutase (<n>SOD1</n>) -overexpressing transgenic mice, a mouse 
model for familial amyotrophic lateral sclerosis (<n>ALS</n>), provides an excellent resource for developing novel therapies 
for <n>ALS</n>. Several observations suggest that mitochondria-dependent apoptotic signaling, including <n>caspase-9</n> activation, may play an 
important role in mutant <n>SOD1-related</n> neurodegeneration. To elucidate the role of <n>caspase-9</n> in <n>ALS</n>, we 
examined the effects of an inhibitor of X chromosome-linked inhibitor of apoptosis (<n>XIAP</n>), a mammalian 
inhibitor of <n>caspase-3</n>, -7 and -9, and <n>p35</n>, a baculoviral broad <n>caspase</n> inhibitor that does 
not inhibit <n>caspase-9</n>. When expressed in spinal motor neurons of mutant <n>SOD1</n> mice using transgenic 
techniques, <n>XIAP</n> attenuated disease progression without delaying onset. In contrast, <n>p35</n> delayed onset without slowing 
disease progression. Moreover, <n>caspase-9</n> was activated in spinal motor neurons of human <n>ALS</n> subjects. These 
data strongly suggest that <n>caspase-9</n> plays a crucial role in disease progression of <n>ALS</n> and 
constitutes a promising therapeutic target. Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only <n>Bcl-2</n> 
family member <n>Bim</n>. Osteoclasts (<n>OCs</n>) undergo rapid apoptosis without trophic factors, such as macrophage colony-stimulating 
factor (<n>M-CSF</n>). Their apoptosis was associated with a rapid and sustained increase in the pro-apoptotic 
BH3-only <n>Bcl-2</n> family member <n>Bim</n>. This was caused by the reduced ubiquitylation and proteasomal degradation 
of <n>Bim</n> that is mediated by <n>c-Cbl</n>. Although the number of <n>OCs</n> was increased in 
the skeletal tissues of bim-/- mice, the mice exhibited mild osteosclerosis due to reduced bone 
resorption. <n>OCs</n> differentiated from bone marrow cells of bim-/- animals showed a marked prolongation of 
survival in the absence of <n>M-CSF</n>, compared with <n>bim+/+</n> <n>OCs</n>, but the bone-resorbing activity of 
bim-/- <n>OCs</n> was significantly reduced. Overexpression of a degradation-resistant lysine-free <n>Bim</n> mutant in bim-/- cells 
abrogated the anti-apoptotic effect of <n>M-CSF</n>, while wild-type <n>Bim</n> did not. These results demonstrate that 
ubiquitylation-dependent regulation of <n>Bim</n> levels is critical for controlling apoptosis and activation of <n>OCs</n>. <n>IAP-antagonists</n> 
exhibit non-redundant modes of action through differential <n>DIAP1</n> binding. The Drosophila inhibitor of apoptosis protein 
<n>DIAP1</n> ensures cell viability by directly inhibiting <n>caspases</n>. In cells destined to die this <n>IAP-mediated</n> 
inhibition of <n>caspases</n> is overcome by <n>IAP-antagonists</n>. Genetic evidence indicates that <n>IAP-antagonists</n> are non-equivalent and 
function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of 
action of Reaper, Grim, Hid and <n>Jafrac2</n>. We find that these <n>IAP-antagonists</n> display differential and 
selective binding to specific <n>DIAP1</n> BIR domains. Consistently, we show that each <n>DIAP1</n> BIR region 
associates with distinct <n>caspases</n>. The differential <n>DIAP1</n> BIR interaction seen both between initiator and effector 
<n>caspases</n> and within <n>IAP-antagonist</n> family members suggests that different <n>IAP-antagonists</n> inhibit distinct <n>caspases</n> from interacting 
with <n>DIAP1</n>. Surprisingly, we also find that the <n>caspase-binding</n> residues of <n>XIAP</n> predicted to be 
strictly conserved in <n>caspase-binding</n> <n>IAPs</n>, are absent in <n>DIAP1</n>. In contrast to <n>XIAP</n>, residues C-terminal 
to the <n>DIAP1</n> BIR1 domain are indispensable for <n>caspase</n> association. Our studies on <n>DIAP1</n> and 
<n>caspases</n> expose significant differences between <n>DIAP1</n> and <n>XIAP</n> suggesting that <n>DIAP1</n> and <n>XIAP</n> inhibit <n>caspases</n> 
in different ways. Visualizing telomere dynamics in living mammalian cells using <n>PNA</n> probes. Chromosome ends 
are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG 
and associated proteins. These so-called <n>telomeric</n> complexes are suggested to play an important role in 
establishing a functional nuclear chromatin organization. Using peptide nucleic acid (<n>PNA</n>) probes, we studied the 
dynamic behavior of <n>telomeric</n> DNA repeats in living human osteosarcoma U2OS cells. A fluorescent cy3-labeled 
<n>PNA</n> probe was introduced in living cells by glass bead loading and was shown to 
specifically associate with <n>telomeric</n> DNA shortly afterwards. Telomere dynamics were imaged for several hours using 
digital fluorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in 
a human cell nucleus showed significant directional movements. Also, a subfraction of telomeres were shown 
to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic 
structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (<n>PML</n>) bodies in a dynamic 
manner. Mouse <n>Rev1 protein</n> interacts with multiple DNA <n>polymerases</n> involved in translesion DNA synthesis. Polkappa 
and <n>Rev1</n> are members of the Y family of DNA <n>polymerases</n> involved in tolerance to 
DNA damage by replicative bypass (translesion DNA synthesis (<n>TLS</n>) ). We demonstrate that mouse <n>Rev1</n> 
protein physically associates with Polkappa. We show too that <n>Rev1</n> interacts independently with <n>Rev7</n> (a 
subunit of a <n>TLS</n> polymerase, Polzeta) and with two other Y-family <n>polymerases</n>, <n>Poliota</n> and <n>Poleta</n>. 
Mouse Polkappa, <n>Rev7</n>, <n>Poliota</n> and <n>Poleta</n> each bind to the same approximately 100 amino acid 
C-terminal region of <n>Rev1</n>. Furthermore, <n>Rev7</n> competes directly with Polkappa for binding to the <n>Rev1</n> 
C-terminus. Notwith standing the physical interaction between <n>Rev1</n> and Polkappa, the DNA polymerase activity of 
each measured by primer extension in vitro is unaffected by the complex, either when extending 
normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer 
termini. Our observations suggest that <n>Rev1</n> plays a role(s) in mediating protein-protein interactions among DNA 
<n>polymerases</n> required for <n>TLS</n>. The precise function(s) of these interactions during <n>TLS</n> remains to be 
determined. The <n>Mre11</n> complex is required for <n>ATM</n> activation and the G(2)/M checkpoint. The maintenance 
of genome integrity requires a rapid and specific response to many types of DNA damage. 
The conserved and related PI3-like protein <n>kinases</n>, ataxia-telangiectasia mutated (<n>ATM</n>) and <n>ATM-Rad3-related</n> (<n>ATR</n>), orchestrate signal 
transduction pathways in response to genomic insults, such as DNA double-strand breaks (<n>DSBs</n>). It is 
unclear which proteins recognize <n>DSBs</n> and activate these pathways, but the <n>Mre11/Rad50/NBS1</n> complex has been 
suggested to act as a damage sensor. Here we show that infection with an adenovirus 
lacking the E4 region also induces a cellular DNA damage response, with activation of <n>ATM</n> 
and <n>ATR</n>. Wild-type virus blocks this signaling through degradation of the <n>Mre11</n> complex by the 
viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the <n>Mre11</n> complex is required 
for both <n>ATM</n> activation and the <n>ATM-dependent</n> G(2)/M checkpoint in response to <n>DSBs</n>. These results 
demonstrate that the <n>Mre11</n> complex can function as a damage sensor upstream of <n>ATM/ATR</n> signaling 
in mammalian cells. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. The anaphase-promoting complex 
(APC) or <n>cyclosome</n> is a <n>ubiquitin</n> ligase that initiates anaphase and mitotic exit. APC activation 
is thought to depend on APC phosphorylation and <n>Cdc20</n> binding. We have identified 43 phospho-sites 
on APC of which at least 34 are mitosis specific. Of these, 32 sites are 
clustered in parts of <n>Apc1</n> and the tetratricopeptide repeat (<n>TPR</n>) subunits <n>Cdc27</n>, <n>Cdc16</n>, <n>Cdc23</n> and 
<n>Apc7</n>. In vitro, at least 15 of the mitotic phospho-sites can be generated by <n>cyclin-dependent 
kinase 1</n> (<n>Cdk1</n>), and 3 by <n>Polo-like kinase 1</n> (<n>Plk1</n>). APC phosphorylation by <n>Cdk1</n>, but 
not by <n>Plk1</n>, is sufficient for increased <n>Cdc20</n> binding and APC activation. Immunofluorescence microscopy using 
phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of <n>cyclin B1</n>. 
In prometaphase phospho-APC accumulates on centrosomes where <n>cyclin B</n> ubiquitination is initiated, appears throughout the 
cytosol and disappears during mitotic exit. <n>Plk1</n> depletion neither prevents APC phosphorylation nor <n>cyclin A</n> 
destruction in vivo. These observations imply that APC activation is initiated by <n>Cdk1</n> already in 
the nuclei of late prophase cells. <n>Nsl1p</n> is essential for the establishment of bipolarity and 
the localization of the Dam-Duo complex. We identified a physical complex consisting of <n>Mtw1p</n>, an 
established kinetochore protein, with <n>Nnf1p</n>, <n>Nsl1p</n> and <n>Dsn1p</n> and have demonstrated that <n>Nnf1p</n>, <n>Nsl1p</n> and 
<n>Dsn1p</n> localize to the Saccharomyces cerevisiae kinetochore. When challenged prior to metaphase, the temperature-sensitive mutants 
nsl1-16 and <n>nsl1-42</n> as well as <n>Nsl1p-depleted</n> cells failed to establish a bipolar spindle-kinetochore interaction 
and executed monopolar segregation of sister chromatids. In contrast, an nsl1-16 defect could not be 
evoked after the establishment of bipolarity. The observed phenotype is characteristic of that of mutants 
with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex. However 
nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the <n>ipl1-321</n> mutant does. 
Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting 
this to be the cause for the failure of nsl1 cells to establish bipolarity. Moreover 
the analysis of <n>Nsl1p-depleted</n> cells indicated that <n>Nsl1p</n> is required for the spindle checkpoint and 
kinetochore integrity. Yeast <n>Nop15p</n> is an RNA-binding protein required for pre-rRNA processing and cytokinesis. <n>Nop15p</n> 
is an essential protein that contains an RNA recognition motif (<n>RRM</n>) and localizes to the 
nucleoplasm and nucleolus. Cells depleted of <n>Nop15p</n> failed to synthesize the 25S and <n>5.8S</n> rRNA 
components of the 60S ribosomal subunit, and exonucleolytic 5' processing of <n>5.8S</n> rRNA was strongly 
inhibited. Pre-rRNAs co-precipitated with tagged <n>Nop15p</n> confirmed its association with early pre-60S particles and <n>Nop15p</n> 
bound a pre-rRNA transcript in vitro. <n>Nop15p-depleted</n> cells show an unusually abrupt growth arrest prior 
to substantial depletion of ribosomal subunits. Following cell synchronization in mitosis, <n>Nop15p-depleted</n> cells undergo nuclear 
division with wild-type kinetics, activate the mitotic exit network and disassemble their mitotic spindle. However, 
they uniformly arrest at cytokinesis and fail to assemble a contractile <n>actin</n> ring at the 
bud neck. In dividing wild-type cells, segregation of nucleolar proteins to the daughter nuclei occurs 
after separation of the nucleoplasm. In these late mitotic cells, <n>Nop15p</n> was partially delocalized from 
the nucleolus to the nucleoplasm, consistent with a specific function in cell division in addition 
to its role in ribosome synthesis. A La protein requirement for efficient pre-tRNA folding. The 
La protein protects the 3' ends of many nascent small RNAs from <n>exonucleases</n>. Here we 
report that La is required for efficient folding of certain pre-tRNAs. A mutation in pre-tRNA(Arg)(CCG) 
causes yeast cells to be cold-sensitive and to require the La protein <n>Lhp1p</n> for efficient 
growth. When the mutant cells are grown at low temperature, or when <n>Lhp1p</n> is depleted, 
mature <n>tRNA(Arg)(CCG)</n> is not efficiently aminoacylated. The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to 
<n>misfold</n> into an alternative helix in vitro. Intragenic suppressor mutations that disrupt the <n>misfolded</n> helix 
or strengthen the correct helix alleviate the requirement for <n>Lhp1p</n>, providing evidence that the anticodon 
stem misfolds in vivo. Chemical and enzymatic footprinting experiments suggest a model in which <n>Lhp1p</n> 
stabilizes the correctly folded stem. <n>Lhp1p</n> is also required for efficient aminoacylation of two wild-type 
tRNAs when yeast are grown at low temperature. These experiments reveal that pre-tRNAs can require 
protein assistance for efficient folding in vivo. Regulation of HIV-1 gene expression by histone acetylation 
and factor recruitment at the LTR promoter. In HIV-1 infected cells, the LTR promoter, once 
organized into chromatin, is transcriptionally inactive in the absence of stimulation. To examine the chromosomal 
events involved in transcriptional activation, we analyzed histone acetylation and factor recruitment at contiguous LTR 
regions by a quantitative chromatin immunoprecipitation assay. In chronically infected cells treated with a phorbol 
ester, we found that acetylation of both histones H3 and H4 occurs at discrete nucleosomal 
regions before the onset of viral mRNA transcription. Concomitantly, we observed the recruitment of known 
cellular acetyl-transferases to the promoter, including <n>CBP</n>, <n>P/CAF</n> and <n>GCN5</n>, as well as that of 
the <n>p65</n> subunit of <n>NF-kappaB</n>. The specific contribution of the viral <n>Tat</n> transactivator was assayed 
in cells harboring the sole LTR. We again observed nucleosomal acetylation and the recruitment of 
specific co-factors to the viral LTR upon activation by either recombinant <n>Tat</n> or a phorbol 
ester. Strikingly, <n>P/CAF</n> was found associated with the promoter only in response to <n>Tat</n>. Taken 
together, these results contribute to the elucidation of the molecular events underlying HIV-1 transcriptional activation. 
Critical loss of <n>CBP/p300</n> histone acetylase activity by <n>caspase-6</n> during neurodegeneration. By altering chromatin structure, 
<n>histone acetyltransferases</n> (HATs) act as transcriptional regulators. We observed in a model of primary neurons 
that histone acetylation levels decreased at the onset of apoptosis. The <n>CREB-binding protein</n> (<n>CBP</n>) is 
a HAT of particular interest because it also acts as a co-activator controlling, among others, 
CREB-dependent transcriptional activity. It has been demonstrated that CREB exerts neuroprotective functions, but the fate 
of <n>CBP</n> during neuronal apoptosis remained unclear till now. This work provided evidence that <n>CBP</n> 
is specifically targeted by <n>caspases</n> and <n>calpains</n> at the onset of neuronal apoptosis, and <n>CBP</n> 
was futher identified as a new <n>caspase-6</n> substrate. This ultimately impinged on the <n>CBP/p300</n> HAT 
activity that decreased with time during apoptosis entry, whereas total cellular HAT activity remained unchanged. 
Interestingly, <n>CBP</n> loss and histone deacetylation were observed in two different pathological contexts: amyloid precursor 
protein-dependent signaling and amyotrophic lateral sclerosis model mice, indicating that these modifications are likely to 
contribute to neurodegenerative diseases. In terms of function, we demonstrated that fine-tuning of <n>CBP</n> HAT 
activity is necessary to ensure neuroprotection. Transcriptional activation of known and novel apoptotic pathways by 
<n>Nur77</n> orphan <n>steroid receptor</n>. <n>Nur77</n> is a nuclear orphan <n>steroid receptor</n> that has been implicated 
in negative selection. Expression of <n>Nur77</n> in thymocytes and cell lines leads to apoptosis through 
a mechanism that remains unclear. In some cell lines, <n>Nur77</n> was reported to act through 
a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show 
here that <n>Nur77-mediated</n> apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot 
be rescued by <n>Bcl-2</n>. Microarray analysis shows that <n>Nur77</n> induces many genes, including two novel 
genes (<n>NDG1</n>, <n>NDG2</n>) and known apoptotic genes FasL and <n>TRAIL</n>. Characterization of <n>NDG1</n> and <n>NDG2</n> 
indicates that <n>NDG1</n> initiates a novel apoptotic pathway in a <n>Bcl-2-independent</n> manner. Thus <n>Nur77-mediated</n> apoptosis 
in T cells involves <n>Bcl-2</n> independent transcriptional activation of several known and novel apoptotic pathways. 
Cooperation between the <n>GATA</n> and <n>RUNX</n> factors Serpent and Lozenge during Drosophila hematopoiesis. Members of 
the <n>GATA</n> and <n>RUNX</n> families of genes appear to have conserved functions during hematopoiesis from 
Drosophila to mammals. In Drosophila, the <n>GATA</n> factor Serpent (<n>Srp</n>) is required in blood cell 
progenitors for the formation of the two populations of blood cells (plasmatocytes and crystal cells), 
while the <n>RUNX factor Lozenge</n> (<n>Lz</n>) is specifically required for crystal cell development. Here we 
investigate the function and the mechanisms of action of <n>Lz</n> during hematopoiesis. Our results indicate 
that <n>Lz</n> can trigger crystal cell development. Interestingly, we show that <n>Lz</n> function is strictly 
dependent on the presence of functional <n>Srp</n> and that <n>Srp</n> and <n>Lz</n> cooperate to induce 
crystal cell differentiation in vivo. Furthermore, we show that <n>Srp</n> and <n>Lz</n> directly interact in 
vitro and that this interaction is conserved between Drosophila and mammals. Moreover, both <n>Srp</n> and 
mouse GATA1 synergize with mouse <n>RUNX1</n> to activate transcription. We propose that interaction and cooperation 
between <n>GATA</n> and <n>RUNX</n> factors may play an important role in regulating blood cell formation 
from Drosophila to mammals. Growth inhibition by the mammalian SWI-SNF subunit <n>Brm</n> is regulated by 
acetylation. In mammalian cells, the SWI-SNF chromatin-remodeling complex is a regulator of cell proliferation, and 
overexpression of the catalytic subunit <n>Brm</n> interferes with cell cycle progression. Here, we show that 
treatment with <n>histone deacetylase</n> (<n>HDAC</n>) inhibitors reduces the inhibitory effect of <n>Brm</n> on the growth 
of mouse fibroblasts. This observation led to the identification of two carboxy-terminal acetylation sites in 
the <n>Brm protein</n>. Mutation of these sites into non-acetylatable sequences increased both the growth-inhibitory and 
the transcriptional activities of <n>Brm</n>. We also show that culture in the presence of <n>HDAC</n> 
inhibitors facilitates the isolation of clones overexpressing <n>Brm</n>. Removal of the <n>HDAC</n> inhibitors from the 
growth medium of these clones leads to downregulation of <n>cyclin D1</n>. This downregulation is absent 
in cell transformed by oncogenic <n>ras</n>. A novel docking site on Mediator is critical for 
activation by <n>VP16</n> in mammalian cells. <n>ARC92/ACID1</n> was identified as a novel specific target of 
the herpes simplex transactivator <n>VP16</n> using an affinity purification procedure. Characterization of the protein revealed 
tight interactions with human Mediator mediated through a von Willebrand type A domain. <n>ARC92/ACID1</n> further 
contains a novel activator-interacting domain (<n>ACID</n>), which it shares with at least one other human 
gene termed <n>PTOV1/ACID2</n>. The structure of <n>ARC92/ACID1</n> is of ancient origin but is conserved in 
mammals and in selected higher eukaryotes. A subpopulation of Mediator is associated with <n>ARC92/ACID1</n>, which 
is specifically required for <n>VP16</n> activation both in vitro and in mammalian cells, but is 
dispensable for other activators such as <n>SP1</n>. Despite many known targets of <n>VP16</n>, <n>ARC92/ACID1</n> appears 
to impose a critical control on transcription activation by <n>VP16</n> in mammalian cells. Discontinuous movement 
and conformational change during pausing and termination by T7 RNA polymerase. Time-resolved characterization of T7 
RNA polymerase pausing and terminating at a class II termination site has been carried out 
using site-specifically tethered chemical nucleases. The data indicate that <n>T7RNAP</n> normally moves uniformly down the 
template as a rigid body. However, at the class II site this movement is interrupted, 
and the leading edge of the polymerase moves further along the DNA than the trailing 
edge. This discontinuous movement may persist until it can no longer be accommodated by conformational 
changes in the elongation complex, at which point the polymerase can either pause or terminate. 
Termination, but not pausing, is abrogated by introduction of a disulfide bond between the polymerase 
fingers and thumb subdomains. The introduced cysteines disrupt a thumb-fingers salt-bridge and, under reducing conditions, 
this mutant enzyme shows reduced processivity coincident with extension of the RNA to 5 nt. 
These observations suggest that termination requires that the thumb and fingers subdomains move apart, in 
a reversal of a conformational change important for initially forming a stable transcription complex. A 
novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy. We describe 
a novel signaling mechanism mediated by the <n>G-protein-coupled receptor</n> (<n>GPCR</n>) angiotensin II (<n>Ang II</n>) type 
2 receptor (<n>AT(2)</n>). Yeast two-hybrid studies and affinity column binding assay show that the isolated 
<n>AT(2)</n> C-terminus binds to the transcription factor promyelocytic zinc finger protein (<n>PLZF</n>). Cellular studies employing 
confocal microscopy show that <n>Ang II</n> stimulation induces cytosolic <n>PLZF</n> to co-localize with <n>AT(2)</n> at 
the plasma membrane, then drives <n>AT(2)</n> and <n>PLZF</n> to internalize. <n>PLZF</n> slowly emerges in the 
nucleus whereas <n>AT(2)</n> accumulates in the perinuclear region. Nuclear <n>PLZF</n> binds to a consensus sequence 
of the phosphatidylinositol-3 kinase <n>p85alpha</n> subunit (<n>p85alpha</n> <n>PI3K</n>) gene. <n>AT(2)</n> enhances expression of <n>p85alpha</n> <n>PI3K</n> 
followed by enhanced p70(S6) kinase, essential to protein synthesis. An inactive mutant of <n>PLZF</n> abolishes 
this effect. <n>PLZF</n> is expressed robustly in the heart in contrast to many other tissues. 
This cardiac selective pathway involving <n>AT(2)</n>, <n>PLZF</n> and <n>p85alpha</n> <n>PI3K</n> may explain the absence of 
a cardiac hypertrophic response in <n>AT(2)</n> gene-deleted mice. <n>Arkadia</n> amplifies <n>TGF-beta</n> superfamily signalling through degradation 
of <n>Smad7</n>. <n>Arkadia</n> was originally identified as a protein that enhances signalling activity of Nodal 
and induces mammalian nodes during early embryogenesis; however, the mechanisms by which <n>Arkadia</n> affects <n>transforming 
growth factor-beta</n> (<n>TGF-beta</n>) superfamily signalling have not been determined. Here we show that <n>Arkadia</n> is 
widely expressed in mammalian tissues, and that it enhances both <n>TGF-beta</n> and bone morphogenetic protein 
(<n>BMP</n>) signalling. <n>Arkadia</n> physically interacts with inhibitory <n>Smad</n>, <n>Smad7</n>, and induces its poly-ubiquitination and degradation. 
In contrast to <n>Smurf1</n>, which interacts with <n>TGF-beta receptor</n> complexes through <n>Smad7</n> and degrades them, 
<n>Arkadia</n> fails to associate with <n>TGF-beta</n> receptors. In contrast to <n>Smad7</n>, expression of <n>Arkadia</n> is 
down-regulated by <n>TGF-beta</n>. Silencing of the <n>Arkadia</n> gene resulted in repression of transcriptional activities induced 
by <n>TGF-beta</n> and <n>BMP</n>, and accumulation of the <n>Smad7</n> protein. <n>Arkadia</n> may thus play an 
important role as an amplifier of <n>TGF-beta</n> superfamily signalling under both physiological and pathological conditions. 
Ribosome binding to the <n>Oxa1</n> complex facilitates co-translational protein insertion in mitochondria. The <n>Oxa1</n> translocase 
of the mitochondrial inner membrane facilitates the insertion of both <n>mitochondrially</n> and nuclear-encoded proteins from 
the matrix into the inner membrane. Most <n>mitochondrially</n> encoded proteins are hydrophobic membrane proteins which 
are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism 
of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of 
<n>Oxa1</n> forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion 
of this <n>Oxa1</n> domain strongly diminished the efficiency of membrane insertion of subunit 2 of 
<n>cytochrome oxidase</n>, a <n>mitochondrially</n> encoded substrate of the <n>Oxa1</n> translocase. This suggests that co-translational membrane 
insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with 
the membrane-bound translocase. Yeast <n>Oxa1</n> interacts with mitochondrial ribosomes: the importance of the C-terminal region 
of <n>Oxa1</n>. The yeast mitochondrial <n>Oxa1 protein</n> is a member of the conserved <n>Oxa1/YidC/Alb3</n> protein 
family involved in the membrane insertion of proteins. <n>Oxa1</n> mediates the insertion of proteins (<n>nuclearly</n> 
and <n>mitochondrially</n> encoded) into the inner membrane. The <n>mitochondrially</n> encoded substrates interact directly with <n>Oxa1</n> 
during their synthesis as nascent chains and in a manner that is supported by the 
associated ribosome. We have investigated if the <n>Oxa1</n> complex interacts with the mitochondrial ribosome. Evidence 
to support a physical association between <n>Oxa1</n> and the large ribosomal subunit is presented. Our 
data indicate that the matrix-exposed C-terminal region of <n>Oxa1</n> plays an important role supporting the 
ribosomal-Oxa1 interaction. Truncation of this C-terminal segment compromises the ability of <n>Oxa1</n> to support insertion 
of substrate proteins into the inner membrane. <n>Oxa1</n> can be cross-linked to <n>Mrp20</n>, a component 
of the large ribosomal subunit. <n>Mrp20</n> is homologous to <n>L23</n>, a subunit located next to 
the peptide exit tunnel of the ribosome. We propose that the interaction of <n>Oxa1</n> with 
the ribosome serves to enhance a coupling of translation and membrane insertion events. The lysosomal 
trafficking of sphingolipid activator proteins (SAPs) is mediated by <n>sortilin</n>. Most soluble lysosomal proteins bind 
the <n>mannose 6-phosphate receptor</n> (<n>M6P-R</n>) to be sorted to the lysosomes. However, the lysosomes of 
I-cell disease (<n>ICD</n>) patients, a condition resulting from a mutation in the <n>phosphotransferase</n> that adds 
mannose 6-phosphate to <n>hydrolases</n>, have near normal levels of several lysosomal proteins, including the sphingolipid 
activator proteins (SAPs), <n>G(M2)AP</n> and <n>prosaposin</n>. We tested the hypothesis that SAPs are targeted to 
the lysosomal compartment via the <n>sortilin receptor</n>. To test this hypothesis, a dominant-negative construct of 
<n>sortilin</n> and a <n>sortilin</n> small interfering RNA (siRNA) were introduced into COS-7 cells. Our results 
showed that both the truncated <n>sortilin</n> and the <n>sortilin</n> siRNA block the traffic of <n>G(M2)AP</n> 
and <n>prosaposin</n> to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated 
that <n>G(M2)AP</n> and <n>prosaposin</n> are interactive partners of <n>sortilin</n>. Furthermore, a dominant-negative mutant GGA prevented 
the trafficking of <n>prosaposin</n> and <n>G(M2)AP</n> to lysosomes. In conclusion, our results show that the 
trafficking of SAPs is dependent on <n>sortilin</n>, demonstrating a novel lysosomal trafficking. Protein kinase A 
regulates <n>AKAP250</n> (<n>gravin</n>) scaffold binding to the beta(2)-adrenergic receptor. A-kinase-anchoring <n>protein 250</n> (<n>AKAP250</n>; <n>gravin</n>) acts 
as a scaffold that binds <n>protein kinase A</n> (<n>PKA</n>), <n>protein kinase C</n> and protein <n>phosphatases</n>, 
associating reversibly with the beta(2)-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation 
of the receptor-scaffold association was revealed through mutagenesis and biochemical analyses. The <n>AKAP</n> domain found 
in other members of this superfamily is essential for the <n>scaffold-receptor</n> interactions. <n>Gravin</n> constructs lacking 
the <n>AKAP</n> domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate 
agonist-stimulated phosphorylation of <n>gravin</n> and enhanced <n>gravin-receptor</n> association. Analysis of the <n>AKAP</n> domain revealed two 
canonical <n>PKA</n> sites phosphorylated in response to elevated <n>cAMP</n>, blocked by <n>PKA</n> inhibitor, and essential 
for <n>scaffold-receptor</n> association and for resensitization of the receptor. The <n>AKAP</n> appears to provide the 
catalytic <n>PKA</n> activity responsible for phosphorylation of the scaffold in response to agonist activation of 
the receptor as well as for the association of the scaffold with the receptor, a 
step critical to receptor resensitization. Integrated analysis of protein composition, tissue diversity, and gene regulation 
in mouse mitochondria. Mitochondria are tailored to meet the metabolic and signaling needs of each 
cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse 
brain, heart, kidney, and liver and combined the results with existing gene annotations to produce 
a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. 
The protein expression data were largely concordant with large-scale surveys of RNA abundance and both 
measures indicate tissue-specific differences in organelle composition. RNA expression profiles across tissues revealed networks of 
mitochondrial genes that share functional and regulatory mechanisms. We also determined a larger "neighborhood" of 
genes whose expression is closely correlated to the mitochondrial genes. The combined analysis identifies specific 
genes of biological interest, such as candidates for mtDNA repair enzymes, offers new insights into 
the biogenesis and ancestry of mammalian mitochondria, and provides a framework for understanding the organelle's 
contribution to human disease. Neural tissue in ascidian embryos is induced by <n>FGF9/16/20</n>, acting via 
a combination of maternal <n>GATA</n> and <n>Ets</n> transcription factors. In chordates, formation of neural tissue 
from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in 
vertebrates. Here, using the early neural marker Otx as an entry point, we dissected the 
neural induction pathway in the simple embryos of Ciona intestinalis. We first isolated the regulatory 
element driving Otx expression in the prospective neural tissue, showed that this element directly responds 
to <n>FGF</n> signaling and that <n>FGF9/16/20</n> acts as an endogenous neural inducer. Binding site analysis 
and gene loss of function established that <n>FGF9/16/20</n> induces neural tissue in the ectoderm via 
a synergy between two maternal response factors. <n>Ets1/2</n> mediates general <n>FGF</n> responsiveness, while the restricted 
activity of <n>GATAa</n> targets the neural program to the ectoderm. Thus, our study identifies an 
endogenous <n>FGF</n> neural inducer and its early downstream gene cascade. It also reveals a role 
for <n>GATA</n> factors in <n>FGF</n> signaling. Churchill, a zinc finger transcriptional activator, regulates the transition 
between gastrulation and neurulation. Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the 
neural plate is established within the epiblast-both events require <n>FGF</n> signaling. We describe a zinc 
finger transcriptional activator, Churchill (<n>ChCh</n>), which acts as a switch between different roles of <n>FGF</n>. 
<n>FGF</n> induces <n>ChCh</n> slowly; this activates <n>Smad-interacting-protein-1</n> (<n>Sip1</n>), which blocks further induction of the mesoderm 
markers <n>brachyury</n> and <n>Tbx6L</n> by <n>FGF</n>. <n>ChCh</n> is first expressed as cells stop migrating through 
the primitive streak, and we show that it regulates cell ingression. We propose a simple 
mechanism by which <n>FGF</n> sensitizes cells to <n>BMP</n> signals. These results reveal that neural induction 
requires cessation of mesoderm formation at the midline in addition to the decision between epidermis 
and neural plate. Local, efflux-dependent <n>auxin</n> gradients as a common module for plant organ formation. 
Plants, compared to animals, exhibit an amazing adaptability and plasticity in their development. This is 
largely dependent on the ability of plants to form new organs, such as lateral roots, 
leaves, and flowers during postembryonic development. Organ primordia develop from founder cell populations into organs 
by coordinated cell division and differentiation. Here, we show that organ formation in Arabidopsis involves 
dynamic gradients of the signaling molecule <n>auxin</n> with maxima at the primordia tips. These gradients 
are mediated by cellular efflux requiring asymmetrically localized PIN proteins, which represent a functionally redundant 
network for <n>auxin</n> distribution in both aerial and underground organs. <n>PIN1</n> polar localization undergoes a 
dynamic rearrangement, which correlates with establishment of <n>auxin</n> gradients and primordium development. Our results suggest 
that PIN-dependent, local <n>auxin</n> gradients represent a common module for formation of all plant organs, 
regardless of their mature morphology or developmental origin. <n>TSC2</n> mediates cellular energy response to control 
cell growth and survival. Mutations in either the <n>TSC1</n> or <n>TSC2</n> tumor suppressor gene are 
responsible for Tuberous Sclerosis Complex. The gene products of <n>TSC1</n> and <n>TSC2</n> form a functional 
complex and inhibit the phosphorylation of <n>S6K</n> and 4EBP1, two key regulators of translation. Here, 
we describe that <n>TSC2</n> is regulated by cellular energy levels and plays an essential role 
in the cellular energy response pathway. Under energy starvation conditions, the <n>AMP-activated protein kinase</n> (<n>AMPK</n>) 
phosphorylates <n>TSC2</n> and enhances its activity. Phosphorylation of <n>TSC2</n> by <n>AMPK</n> is required for translation 
regulation and cell size control in response to energy deprivation. Furthermore, <n>TSC2</n> and its phosphorylation 
by <n>AMPK</n> protect cells from energy deprivation-induced apoptosis. These observations demonstrate a model where <n>TSC2</n> 
functions as a key player in regulation of the common mTOR pathway of protein synthesis, 
cell growth, and viability in response to cellular energy levels. Sequential modification of <n>NEMO/IKKgamma</n> by 
<n>SUMO-1</n> and <n>ubiquitin</n> mediates <n>NF-kappaB</n> activation by genotoxic stress. The transcription factor <n>NF-kappaB</n> is critical 
for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to 
<n>NF-kappaB</n> signaling pathways is <n>NEMO/IKKgamma</n>, the regulatory subunit of the cytoplasmic <n>IkappaB kinase</n> (<n>IKK</n>) complex. 
While <n>NF-kappaB</n> activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the 
mechanism by which nuclear DNA damage modulates <n>NEMO</n> to activate cytoplasmic <n>IKK</n> remains unknown. Here, 
we show that genotoxic stress causes nuclear localization of <n>IKK-unbound</n> <n>NEMO</n> via site-specific <n>SUMO-1</n> attachment. 
Surprisingly, this sumoylation step is <n>ATM-independent</n>, but nuclear localization allows subsequent <n>ATM-dependent</n> ubiquitylation of <n>NEMO</n> 
to ultimately activate <n>IKK</n> in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, 
<n>SUMO-1</n> modification and <n>ATM</n> activation, which work in concert to sequentially cause nuclear targeting and 
ubiquitylation of free <n>NEMO</n> to permit the <n>NF-kappaB</n> survival pathway. These <n>SUMO</n> and <n>ubiquitin</n> modification 
pathways may serve as anticancer drug targets. The hyperpolarization-activated <n>HCN1</n> channel is important for motor 
learning and neuronal integration by cerebellar Purkinje cells. In contrast to our increasingly detailed understanding 
of how synaptic plasticity provides a cellular substrate for learning and memory, it is less 
clear how a neuron's voltage-gated <n>ion channels</n> interact with plastic changes in synaptic strength to 
influence behavior. We find, using generalized and regional knockout mice, that deletion of the <n>HCN1</n> 
channel causes profound motor learning and memory deficits in swimming and <n>rotarod</n> tasks. In cerebellar 
Purkinje cells, which are a key component of the cerebellar circuit for learning of correctly 
timed movements, <n>HCN1</n> mediates an inward current that stabilizes the integrative properties of Purkinje cells 
and ensures that their input-output function is independent of the previous history of their activity. 
We suggest that this nonsynaptic integrative function of <n>HCN1</n> is required for accurate decoding of 
input patterns and thereby enables synaptic plasticity to appropriately influence the performance of motor activity. 
A <n>Rad53</n> kinase-dependent surveillance mechanism that regulates <n>histone protein</n> levels in S. cerevisiae. <n>Rad53</n> and 
<n>Mec1</n> are protein <n>kinases</n> required for DNA replication and recovery from DNA damage in Saccharomyces 
cerevisiae. Here, we show that <n>rad53</n>, but not <n>mec1</n> mutants, are extremely sensitive to <n>histone 
overexpression</n>, as <n>Rad53</n> is required for degradation of excess histones. Consequently, excess histones accumulate in 
<n>rad53</n> mutants, resulting in slow growth, DNA damage sensitivity, and chromosome loss phenotypes that are 
significantly suppressed by a reduction in <n>histone gene dosage</n>. <n>Rad53</n> monitors excess histones by associating 
with them in a dynamic complex that is modulated by its kinase activity. Our results 
argue that <n>Rad53</n> contributes to genome stability independently of <n>Mec1</n> by preventing the damaging effects 
of excess histones both during normal cell cycle progression and in response to DNA damage. 
<n>EMSY</n> links the <n>BRCA2</n> pathway to sporadic breast and ovarian cancer. The <n>BRCA2</n> gene is 
mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair 
and transcriptional regulation. Here we identify a protein, <n>EMSY</n>, which binds <n>BRCA2</n> within a region 
(<n>exon 3</n>) deleted in cancer. <n>EMSY</n> is capable of silencing the activation potential of <n>BRCA2</n> 
<n>exon 3</n>, associates with chromatin regulators <n>HP1beta</n> and <n>BS69</n>, and localizes to sites of repair 
following DNA damage. <n>EMSY</n> maps to chromosome <n>11q13.5</n>, a region known to be involved in 
breast and ovarian cancer. We show that the <n>EMSY</n> gene is amplified almost exclusively in 
sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, <n>EMSY</n> amplification is associated 
with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic 
value. The remarkable clinical overlap between sporadic <n>EMSY</n> amplification and familial <n>BRCA2</n> deletion implicates a 
<n>BRCA2</n> pathway in sporadic breast and ovarian cancer. Endocytosis and signaling: a relationship under development. 
The ability to internalize macromolecules by endocytosis is a property of all eukaryotic cells. Frontline 
research on endocytosis has been presented in a successful series of biannual meetings in Europe. 
This year's meeting on "Membrane Dynamics in Endocytosis" was held September 13-18 in Acquafredda di 
Maratea, on the coast of southern Italy. Four key questions were addressed: What are the 
molecular mechanisms of <n>endocytic</n> membrane trafficking? How does endocytosis modulate receptor signaling and vice versa? 
What is the importance of endocytosis during development? How do <n>endocytic</n> organelles contribute to immunity 
or susceptibility to pathogens? Brain or brawn: how <n>FGF</n> signaling gives us both. How does 
<n>FGF</n> (<n>fibroblast growth factor</n>) signaling induce both neural and mesodermal cell fates in the early 
embryo? Two papers address this fundamental question in this issue of Cell. Bertrand et al. 
show in the ascidian that a <n>GATA</n> factor determines the neural response of animal cells 
to <n>FGF</n> signaling, while in the chick, Sheng et al. demonstrate that the slow induction 
by <n>FGF</n> of a new transcription factor (Churchill) in the neural plate in turn induces 
expression of <n>Sip1</n> (<n>Smad</n> interacting <n>protein-1</n>), which inhibits mesodermal genes and sensitizes cells to later 
neural inducing factors. <n>Rad53</n>: a controller ensuring the fine-tuning of histone levels. Checkpoint proteins are 
activated in response to genotoxic insults or replication stress to maintain genome integrity. Their function 
is believed to depend largely on the detection of the DNA damage or defects occurring 
during replication fork progression. The <n>BRCA2-EMSY</n> connection: implications for breast and ovarian tumorigenesis. In this 
issue, Hughes-Davies et al. describe a novel gene product, <n>EMSY</n>, which suppresses the transactivational activity 
of <n>BRCA2</n>. <n>EMSY</n> is located within an amplicon in sporadic breast and ovarian cancers, suggesting 
that its overexpression may mimic the effects of <n>BRCA2</n> inactivation. The implications for <n>BRCA2</n> function 
are discussed. Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is 
genetic information transferred between organelles via RNA? We describe an open reading frame (ORF) with 
high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all 
the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 
39% T in the third codon position. It is located between a sequence fragment from 
the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 
of the <n>NADH dehydrogenase</n>. The nuclear derived sequence consists of 528 nucleotides from the small 
ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains 
part of the <n>ribosomal protein S4</n> and the complete tRNA(Ser). The observation that only transcribed 
sequences have been found i more than one subcellular compartment in higher plants suggests that 
interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and 
genomic integration. Double and triple mutant combinations of bithorax complex of Drosophila. We have constructed 
double and triple mutant combinations for the <n>Ubx</n>, <n>abd-A</n> and <n>Abd-B</n> genes of the bithorax 
complex and have examined the homeotic transformations they produce in the larval and adult patterns. 
Embryos hemizygous for the triple combination exhibit a metameric pattern consisting of <n>parasegments</n> 5-12 being 
transformed into parasegment 4. In addition, parasegment 13 develops like a mixture of <n>parasegment 3</n> 
and 4, and <n>parasegment 14</n> is abnormal. The same phenotype is displayed by embryos homozygous 
for <n>DfP9</n>, lacking all the <n>BX-C</n> DNA, 300 kb. This result strongly supports the notion 
that the <n>BX-C</n> contains only three genes which account for all the developmental functions of 
the complex. The phenotypes of the different double combinations also support the same view; the 
<n>Ubx</n> abd-a comthoracic and several abdominal functions. The <n>abd-A</n> <n>Abd-B</n> combination exhibits the same phenotype 
of <n>DpP10</n> <n>DfP9</n>, lacking all the abdominal functions except those specific for A1. Our results 
also indicate that each <n>BX-C</n> gene becomes active autonomously regardless of the presence or functional 
state of the other <n>BX-C</n> genes. The instability of the <n>TE-like</n> mutation Dp(2:2)GYL of Drosophila 
melanogaster is intimately associated with the hobo element. We have characterized molecularly several derivatives of 
the <n>TE-like</n> element Dp(2:2)GYL of Drosophila melanogaster. This highly unstable mutation occurred in a dysgenic 
cross involving the 23.5 MRF chromosome, and represents an inverted insertional duplication of approximately 130 
polytene bands of the paternal 2L, at 50AB of the right arm of the maternal 
2R. The instability of this mutation is characterized by deletion of some of duplicated material, 
by the induction of rearrangements in its vicinity and by the transposition of parts of 
the original element. We have found that the mobile element hobo is present at, or 
<n>very near</n>, the <n>breakpoints</n> of all GYL derivatives analysed, demonstrating that hobo is not only 
active in dysgenic crosses, but also that it can promote genetic instability reminiscent of transposable 
elements (<n>TE</n>). Sps-3 transcript levels are determined by multiple remote sequence elements. The region from 
1.4 to 2.7 kb upstream of Drosophila melanogaster gene <n>Sgs-3</n> is responsible for a 10-fold 
increase in <n>Sgs-3</n> transcript levels in the third instar larval salivary gland. This region includes 
the related <n>Sgs-7</n> gene from the 68C glue gene cluster as well as 400 bp 
of its 5' sequences. We show that two elements are involved, each contributing a modest 
3-fold effect. One of these includes <n>Sgs-7</n> transcribed sequences some 2.3 kb upstream of <n>Sgs-3</n>, 
although <n>Sgs-7</n> transcription is not involved. Although important for the overall levels of <n>Sgs-3</n> expression, 
they are clearly not strong, viral-like enhancer elements. We propose that many position effects observed 
in P element transformation studies are the consequence of insertion in the vicinity of similar 
elements dispersed throughout the genome and having modest effects on transcript levels. The AAA-ATPase <n>Cdc48/p97</n> 
regulates spindle disassembly at the end of mitosis. Spindle disassembly at the end of mitosis 
is a complex and poorly understood process. Here, we report that the AAA-ATPase <n>Cdc48/p97</n> and 
its adapters Ufd1-Npl4, which have a well-established role in membrane functions, also regulate spindle disassembly 
by modulating microtubule dynamics and bundling at the end of mitosis. In the absence of 
<n>p97-Ufd1-Npl4</n> function, microtubules in Xenopus egg extracts remain as monopolar spindles attached to condensed chromosomes 
after Cdc2 kinase activity has returned to the interphase level. Consequently, interphase microtubule arrays and 
nuclei are not established. Genetic analyses of <n>Cdc48</n>, the yeast homolog of <n>p97</n>, reveal that 
<n>Cdc48</n> is also required for disassembly of mitotic spindles after execution of the mitotic exit 
pathway. Furthermore, <n>Cdc48/p97-Ufd1-Npl4</n> directly binds to spindle assembly factors and regulates their interaction with microtubules 
at the end of mitosis. Therefore, <n>Cdc48/p97-Ufd1-Npl4</n> is an essential chaperone that regulates transformation of 
the microtubule structure as cells reenter interphase. <n>ACF7</n>. An essential integrator of microtubule dynamics. <n>ACF7</n> 
is a member of the <n>spectraplakin</n> family of cytoskeletal crosslinking proteins possessing <n>actin</n> and microtubule 
binding domains. Here, we show that <n>ACF7</n> is an essential integrator of MT-actin dynamics. In 
endodermal cells, <n>ACF7</n> binds along microtubules but concentrates at their distal ends and at cell 
borders when polarized. In ACF7's absence, microtubules still bind <n>EB1</n> and <n>CLIP170</n>, but they no 
longer grow along polarized <n>actin</n> bundles, nor do they pause and tether to <n>actin-rich</n> cortical 
sites. The consequences are less stable, long microtubules with skewed cytoplasmic trajectories and altered dynamic 
instability. In response to wounding, <n>ACF7</n> null cultures activate polarizing signals, but fail to maintain 
them and coordinate migration. Rescue of these defects requires ACF7's <n>actin</n> and microtubule binding domains. 
Thus, <n>spectraplakins</n> are important for controlling microtubule dynamics and reinforcing links between microtubules and polarized 
<n>F-actin</n>, so that cellular polarization and coordinated cell movements can be sustained. Temporal regulation of 
salmonella virulence effector function by proteasome-dependent protein degradation. Salmonella enterica invasion of host cells requires 
the reversible activation of the Rho-family <n>GTPases</n> <n>Cdc42</n> and <n>Rac1</n> by the bacterially encoded GEF 
<n>SopE</n> and the GAP <n>SptP</n>, which exert their function at different times during infection and 
are delivered into host cells by a type 3 secretion system. We found that <n>SopE</n> 
and <n>SptP</n> are delivered in equivalent amounts early during infection. However, <n>SopE</n> is rapidly degraded 
through a proteosome-mediated pathway, while <n>SptP</n> exhibits much slower degradation kinetics. The half-lives of these 
effector proteins are determined by their secretion and translocation domains. Chimeric protein analysis indicated that 
delivery of <n>SptP</n> into host cells by the <n>SopE</n> secretion and translocation domain drastically shortened 
its half-life. Conversely, delivery of <n>SopE</n> by the <n>SptP</n> secretion and translocation signals significantly increased 
its half-life, resulting in persistent <n>actin</n> cytoskeleton rearrangements. This regulatory mechanism constitutes a remarkable example 
of a pathogen's adaptation to modulate cellular functions. Reverse transcriptase of <n>Moloney</n> murine leukemia virus 
binds to <n>eukaryotic release factor 1</n> to modulate suppression of translational termination. The pol (for 
polymerase) gene of the murine leukemia viruses (<n>MuLVs</n>) is expressed in the form of a 
large <n>Gag-Pol</n> precursor protein by the suppression of translational termination, or enhanced readthrough, of a 
UAG stop codon at the end of gag. A search for cellular proteins that interact 
with the reverse transcriptase of <n>Moloney</n> MuLV resulted in the identification of <n>eRF1</n>, the eukaryotic 
translation release factor 1. The proteins bound strongly in vitro, and the overexpression of <n>eRF1</n> 
resulted in the <n>RT-dependent</n> incorporation of the protein into assembling virion particles. The overexpression of 
<n>RT</n> in trans enhanced the translational readthrough of a reporter construct containing the <n>Gag-Pol</n> boundary 
region. Noninteracting mutants of <n>RT</n> failed to synthesize adequate levels of <n>Gag-Pol</n> and could not 
replicate. These results suggest that <n>RT</n> enhances suppression of termination and that the interaction of 
<n>RT</n> with <n>eRF1</n> is required for an appropriate level of translational readthrough. Neutrophil <n>elastase</n> cleaves 
<n>PML-RARalpha</n> and is important for the development of acute promyelocytic leukemia in mice. The fusion 
protein <n>PML-RARalpha</n>, generated by the t(15;17)(q22;q11.2) translocation associated with acute promyelocytic leukemia (APL), initiates APL 
when expressed in the early myeloid compartment of transgenic mice. <n>PML-RARalpha</n> is cleaved in several 
positions by a neutral serine protease in a human myeloid cell line; purification revealed that 
the protease is neutrophil <n>elastase</n> (<n>NE</n>). Immunofluorescence localization studies suggested that the cleavage of <n>PML-RARalpha</n> 
must occur within the cell, and perhaps, within the nucleus. The functional importance of <n>NE</n> 
for APL development was assessed in <n>NE</n> deficient mice. Greater than 90% of bone marrow 
<n>PML-RARalpha</n> cleaving activity was lost in the absence of <n>NE</n>, and <n>NE</n> (but not <n>Cathepsin 
G</n>) deficient animals were protected from APL development. Primary mouse and human APL cells also 
contain <n>NE-dependent</n> <n>PML-RARalpha</n> cleaving activity. Since <n>NE</n> is maximally produced in promyelocytes, this protease may 
play a role in APL pathogenesis by facilitating the leukemogenic potential of <n>PML-RARalpha</n>. <n>Taspase1</n>: a 
threonine aspartase required for cleavage of <n>MLL</n> and proper <n>HOX</n> gene expression. The Mixed-Lineage Leukemia 
gene (<n>MLL/HRX/ALL1</n>) encodes a large nuclear protein homologous to Drosophila trithorax that is required for 
the maintenance of <n>HOX</n> gene expression. <n>MLL</n> is cleaved at two conserved sites generating N320 
and C180 fragments, which heterodimerize to stabilize the complex and confer its subnuclear destination. Here, 
we purify and clone the protease responsible for cleaving <n>MLL</n>. We entitle it <n>Taspase1</n> as 
it initiates a class of endopeptidases that utilize an N-terminal threonine as the active site 
nucleophile to proteolyze polypeptide substrates following aspartate. <n>Taspase1</n> proenzyme is intramolecularly proteolyzed generating an active 
28 kDa alpha/22 kDa beta heterodimer. <n>RNAi-mediated</n> knockdown of <n>Taspase1</n> results in the appearance of 
unprocessed <n>MLL</n> and the loss of proper <n>HOX</n> gene expression. <n>Taspase1</n> coevolved with <n>MLL/trithorax</n> as 
Arthropoda and Chordata emerged from Metazoa suggesting that <n>Taspase1</n> originated to regulate complex segmental body 
plans in higher organisms. <n>BMP</n> induction of Id proteins suppresses differentiation and sustains embryonic stem 
cell self-renewal in collaboration with <n>STAT3</n>. The cytokine leukemia inhibitory factor (<n>LIF</n>) drives self-renewal of 
mouse embryonic stem (ES) cells by activating the transcription factor <n>STAT3</n>. In serum-free cultures, however, 
<n>LIF</n> is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone 
morphogenetic proteins (<n>BMPs</n>) act in combination with <n>LIF</n> to sustain self-renewal and preserve multilineage differentiation, 
chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and 
derived de novo without serum or feeders using <n>LIF</n> plus <n>BMP</n>. The critical contribution of 
<n>BMP</n> is to induce expression of Id genes via the <n>Smad</n> pathway. Forced expression of 
Id liberates ES cells from <n>BMP</n> or serum dependence and allows self-renewal in <n>LIF</n> alone. 
Upon <n>LIF</n> withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. 
We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response 
to <n>LIF/STAT3</n>. <n>Homothorax</n> switches function of Drosophila photoreceptors from color to polarized light sensors. Different 
classes of photoreceptors (<n>PRs</n>) allow animals to perceive various types of visual information. In the 
Drosophila eye, the outer <n>PRs</n> of each ommatidium are involved in motion detection while the 
inner <n>PRs</n> mediate color vision. In addition, flies use a specialized class of <n>inner PRs</n> 
in the "dorsal rim area" of the eye (<n>DRA</n>) to detect the e-vector of polarized 
light, allowing them to exploit skylight polarization for orientation. We show that <n>homothorax</n> is both 
necessary and sufficient for <n>inner PRs</n> to adopt the polarization-sensitive <n>DRA</n> fate instead of the 
color-sensitive default state. <n>Homothorax</n> increases rhabdomere size and uncouples R7-R8 communication to allow both cells 
to express the same opsin rather than different ones as required for color vision. <n>Homothorax</n> 
expression is induced by the iroquois complex and the wingless (<n>wg</n>) pathway. However, crucial <n>wg</n> 
pathway components are not required, suggesting that additional signals are involved. The receptors for mammalian 
sweet and <n>umami</n> taste. Sweet and <n>umami</n> (the taste of monosodium glutamate) are the main 
attractive taste modalities in humans. <n>T1Rs</n> are candidate mammalian taste receptors that combine to assemble 
two heteromeric <n>G-protein-coupled receptor</n> complexes: <n>T1R1+3</n>, an <n>umami</n> sensor, and <n>T1R2+3</n>, a sweet receptor. We 
now report the behavioral and physiological characterization of <n>T1R1</n>, <n>T1R2</n>, and <n>T1R3</n> knockout mice. We 
demonstrate that sweet and <n>umami</n> taste are strictly dependent on <n>T1R-receptors</n>, and show that selective 
elimination of T1R-subunits differentially abolishes detection and perception of these two taste modalities. To examine 
the basis of sweet tastant recognition and coding, we engineered animals expressing either the human 
<n>T1R2-receptor</n> (<n>hT1R2</n>), or a modified <n>opioid-receptor</n> (<n>RASSL</n>) in sweet cells. Expression of <n>hT1R2</n> in mice 
generates animals with humanized sweet taste preferences, while expression of <n>RASSL</n> drives strong attraction to 
a synthetic opiate, demonstrating that sweet cells trigger dedicated behavioral outputs, but their tastant selectivity 
is determined by the nature of the receptors. <n>HtrA2/Omi</n>, a Sheep in Wolf's Clothing. Mammalian 
mitochondrial <n>HtrA2/Omi</n> was originally described as an apoptosis inducer, but rather than having extra cells, 
mice with mutant <n>HtrA2/Omi</n> suffer from a neurodegenerative disease due to progressive mitochondrial damage. This 
suggests that instead of promoting cell death by antagonizing inhibitor of apoptosis (<n>IAP</n>) proteins, the 
primary function of <n>HtrA2/Omi</n> is to handle <n>misfolded</n> proteins in the mitochondria. MoMLV Reverse Transcriptase 
Regulates Its Own Expression. A precise ratio of Gag:Gag-Pol expression is required for assembly of 
infectious retroviral virions. In this issue of Cell, Orlova et al. show that MoMLV reverse 
transcriptase binds the translation release factor <n>eRF1</n>, and that this interaction promotes translation readthrough to 
make <n>Gag-Pol</n>. Proteolytic processing in development and leukemogenesis. There are now numerous examples in the 
hematopoietic system of genes that are critical for normal hematopoietic development, but when mutated, rearranged, 
or overexpressed, contribute to leukemogenesis. Two papers in this issue of Cell provide a fascinating 
twist on this paradigm, and suggest that proteolytic processing of certain of these genes plays 
an important role both in development and in leukemogenesis. These findings also suggest the possibility 
that <n>proteases</n> may be therapeutic targets in leukemia. Embryonic stem cell self-renewal, analyzed. Maintaining the 
pluripotency of mouse ES cells requires both <n>LIF</n> (leukemia inhibitory factor) and unknown factors in 
serum. The paper from Ying et al. in this issue of Cell shows that <n>BMP</n> 
(bone morphogenetic protein) can replace serum in this capacity, defining molecular requirements for ES cell 
self-renewal. Competitive processivity-clamp usage by DNA <n>polymerases</n> during DNA replication and repair. Protein clamps are 
ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that 
interact with a large variety of proteins. In this report we identify residues that are 
required for binding of the <n>beta-clamp</n> to DNA polymerase III of Escherichia coli, a polymerase 
of the <n>Pol C</n> family. We show that the alpha polymerase subunit of DNA polymerase 
III interacts with the <n>beta-clamp</n> via its extreme seven C-terminal residues, some of which are 
conserved. Moreover, interaction of Pol III with the clamp takes place at the same site 
as that of the delta-subunit of the clamp loader, providing the basis for a switch 
between the clamp loading machinery and the polymerase itself. Escherichia coli DNA <n>polymerases</n> I, II, 
<n>IV</n> and V (<n>UmuC</n>) interact with beta at the same site. Given the limited amounts 
of clamps in the cell, these results suggest that clamp binding may be competitive and 
regulated, and that the different <n>polymerases</n> may use the same clamp sequentially during replication and 
repair. Decatenation of DNA circles by <n>FtsK-dependent</n> Xer site-specific recombination. DNA replication results in interlinked 
(catenated) sister duplex molecules as a consequence of the intertwined helices that comprise duplex DNA. 
DNA topoisomerases play key roles in decatenation. We demonstrate a novel, efficient and directional decatenation 
process in vitro, which uses the combination of the Escherichia coli XerCD site-specific recombination system 
and a protein, <n>FtsK</n>, which facilitates simple synapsis of <n>dif</n> recombination sites during its translocation 
along DNA. We propose that the <n>FtsK-XerCD</n> recombination machinery, which converts chromosomal dimers to monomers, 
may also function in vivo in removing the final catenation links remaining upon completion of 
DNA replication. <n>YaeL</n> proteolysis of <n>RseA</n> is controlled by the PDZ domain of <n>YaeL</n> and 
a Gln-rich region of <n>RseA</n>. <n>sigmaE</n> is an alternative sigma factor involved in a pathway 
of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, <n>sigmaE</n> activity is down-regulated 
by the membrane-bound anti-sigmaE protein, <n>RseA</n>. Extracytoplasmic stress signals induce degradation of <n>RseA</n> by two 
successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by <n>YaeL-mediated</n> second proteolysis 
at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first 
cleavage (site-1 cleavage). Here, we show that <n>YaeL</n> variants with the periplasmic PDZ domain deleted 
or mutated allows unregulated cleavage of <n>RseA</n> and consequent <n>sigmaE</n> activation. It was also found 
that a glutamine-rich region in the periplasmic domain of <n>RseA</n> was required for the avoidance 
of the <n>YaeL-mediated</n> proteolysis in the absence of site-1 cleavage. These results indicate that multiple 
negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine 
the strict dependence of the site-2 proteolysis on the site-1 cleavage. <n>Tab2</n> is a novel 
conserved RNA binding protein required for translation of the chloroplast <n>psaB</n> mRNA. The chloroplast <n>psaB</n> 
mRNA encodes one of the reaction centre polypeptides of <n>photosystem I</n>. Protein pulse-labelling profiles indicate 
that the mutant strain of Chlamydomonas reinhardtii, <n>F14</n>, affected at the nuclear locus <n>TAB2</n>, is 
deficient in the translation of <n>psaB</n> mRNA and thus deficient in <n>photosystem I</n> activity. Genetic 
studies reveal that the target site for <n>Tab2</n> is situated within the <n>psaB</n> 5'UTR. We 
have used genomic complementation to isolate the nuclear <n>Tab2</n> gene. The deduced amino acid sequence 
of <n>Tab2</n> (358 residues) displays 31-46% sequence identity with several orthologues found only in eukaryotic 
and prokaryotic organisms performing oxygenic photosynthesis. Directed mutagenesis indicates the importance of a highly conserved 
C-terminal tripeptide in <n>Tab2</n> for normal <n>psaB</n> translation. The <n>Tab2</n> protein is localized in the 
chloroplast stroma where it is associated with a high molecular mass protein complex containing the 
<n>psaB</n> mRNA. Gel mobility shift assays reveal a direct and specific interaction between <n>Tab2</n> and 
the <n>psaB</n> 5'UTR. We propose that <n>Tab2</n> plays a key role in the initial steps 
of <n>PsaB</n> translation and <n>photosystem I</n> assembly. <n>MDM2</n> promotes <n>p21waf1/cip1</n> proteasomal turnover independently of ubiquitylation. 
The <n>CDK</n> inhibitor <n>p21waf1/cip1</n> is degraded by a <n>ubiquitin-independent</n> proteolytic pathway. Here, we show that 
<n>MDM2</n> mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization 
signal (NLS) -deleted, <n>MDM2</n> decreased <n>p21waf1/cip1</n> levels without ubiquitylating this protein and affecting its mRNA 
level in <n>p53(-/-)</n> cells. This decrease was reversed by the proteasome inhibitors MG132 and <n>lactacystin</n>, 
by <n>p19(arf)</n>, and by small interfering RNA (siRNA) against <n>MDM2</n>. <n>p21waf1/cip1</n> bound to <n>MDM2</n> in 
vitro and in cells. The <n>p21waf1/cip1-binding-defective</n> mutant of <n>MDM2</n> was unable to degrade <n>p21waf1/cip1</n>. <n>MDM2</n> 
shortened the half-life of both exogenous and endogenous <n>p21waf1/cip1</n> by 50% and led to the 
degradation of its lysine-free mutant. Consequently, <n>MDM2</n> suppressed <n>p21waf1/cip1-induced</n> cell growth arrest of human <n>p53(-/-)</n> 
and <n>p53(-/-)/Rb(-/-)cells</n>. These results demonstrate that <n>MDM2</n> directly inhibits <n>p21waf1/cip1</n> function by reducing <n>p21waf1/cip1</n> stability 
in a <n>ubiquitin-independent</n> fashion. The <n>PTB</n> interacting protein <n>raver1</n> regulates <n>alpha-tropomyosin</n> alternative splicing. Regulated switching 
of the mutually exclusive exons 2 and 3 of <n>alpha-tropomyosin</n> (TM) involves repression of <n>exon 
3</n> in smooth muscle cells. Polypyrimidine tract-binding protein (<n>PTB</n>) is necessary but not sufficient for 
regulation of TM splicing. <n>Raver1</n> was identified in two-hybrid screens by its interactions with the 
cytoskeletal proteins <n>actinin</n> and <n>vinculin</n>, and was also found to interact with <n>PTB</n>. Consistent with 
these interactions <n>raver1</n> can be localized in either the nucleus or cytoplasm. Here we show 
that <n>raver1</n> is able to promote the smooth muscle-specific alternative splicing of TM by enhancing 
<n>PTB-mediated</n> repression of <n>exon 3</n>. This activity of <n>raver1</n> is dependent upon characterized <n>PTB-binding</n> regulatory 
elements and upon a region of <n>raver1</n> necessary for interaction with <n>PTB</n>. Heterologous recruitment of 
<n>raver1</n>, or just its C-terminus, induced very high levels of <n>exon 3</n> skipping, bypassing the 
usual need for <n>PTB</n> binding sites downstream of <n>exon 3</n>. This suggests a novel mechanism 
for <n>PTB-mediated</n> splicing repression involving recruitment of <n>raver1</n> as a potent splicing co-repressor. <n>FinO</n> is 
an RNA chaperone that facilitates sense-antisense RNA interactions. The protein <n>FinO</n> represses F-plasmid conjugative transfer 
by facilitating interactions between the mRNA of the major F-plasmid transcriptional activator, <n>TraJ</n>, and an 
antisense RNA, <n>FinP</n>. <n>FinO</n> is known to bind stem-loop structures in both <n>FinP</n> and <n>traJ</n> 
RNAs; however, the mechanism by which <n>FinO</n> facilitates sense-antisense pairing is poorly understood. Here we 
show that <n>FinO</n> acts as an RNA chaperone to promote strand exchange and duplexing between 
minimal RNA targets derived from <n>FinP</n>. This strongly suggests that <n>FinO</n> may function to destabilize 
internal secondary structures within <n>FinP</n> and <n>traJ</n> RNAs that would otherwise act as a kinetic 
trap to sense-antisense pairing. The energy for <n>FinO-catalyzed</n> base-pair destabilization does not arise from ATP 
hydrolysis but appears to be supplied directly from <n>FinO</n> RNA binding free energy. An analysis 
of the activities of mutants that are specifically deficient in strand exchange but not RNA-binding 
activity demonstrates that strand exchange is essential to the ability of <n>FinO</n> to mediate sense-antisense 
RNA recognition, and that this function also plays a role in repression of conjugation in 
vivo. Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer. Methyl-CpG binding 
proteins (<n>MBDs</n>) mediate <n>histone deacetylase-dependent</n> transcriptional silencing at methylated CpG islands. Using chromatin immunoprecitation (<n>ChIP</n>) 
we have found that gene-specific profiles of <n>MBDs</n> exist for hypermethylated promoters of breast cancer 
cells, whilst a common pattern of histone modifications is shared. This unique distribution of <n>MBDs</n> 
is also characterized in chromosomes by comparative genomic hybridization of immunoprecipitated DNA and immunolocalization. Most 
importantly, we demonstrate that <n>MBD</n> association to methylated DNA serves to identify novel targets of 
epigenetic inactivation in human cancer. We combined the <n>ChIP</n> assay of <n>MBDs</n> with a CpG 
island microarray (<n>ChIP</n> on chip). The scenario revealed shows that, while many genes are regulated 
by multiple <n>MBDs</n>, others are associated with a single <n>MBD</n>. These target genes displayed methylation- 
associated transcriptional silencing in breast cancer cells and primary <n>tumours</n>. The candidates include the homeobox 
gene PAX6, the prolactin hormone receptor, and dipeptidylpeptidase <n>IV</n> among others. Our results support an 
essential role for <n>MBDs</n> in gene silencing and, when combined with genomic strategies, their potential 
to 'catch' new hypermethylated genes in cancer. Transcript cleavage factors <n>GreA</n> and <n>GreB</n> act as 
transient catalytic components of RNA polymerase. Prokaryotic transcription elongation factors <n>GreA</n> and <n>GreB</n> stimulate intrinsic 
<n>nucleolytic</n> activity of RNA polymerase (<n>RNAP</n>). The proposed biological role of <n>Gre-induced</n> RNA hydrolysis includes 
transcription proofreading, suppression of transcriptional pausing and arrest, and facilitation of <n>RNAP</n> transition from transcription 
initiation to transcription elongation. Using an array of biochemical and molecular genetic methods, we mapped 
the interaction interface between <n>Gre</n> and <n>RNAP</n> and identified the key residues in <n>Gre</n> responsible 
for induction of <n>nucleolytic</n> activity in <n>RNAP</n>. We propose a structural model in which the 
C-terminal globular domain of <n>Gre</n> binds near the opening of the <n>RNAP</n> secondary channel, the 
N-terminal coiled-coil domain (<n>NTD</n>) protrudes inside the <n>RNAP channel</n>, and the tip of the <n>NTD</n> 
is brought to the immediate vicinity of <n>RNAP</n> catalytic center. Two conserved acidic residues D41 
and <n>E44</n> located at the tip of the <n>NTD</n> assist <n>RNAP</n> by coordinating the Mg2+ 
ion and water molecule required for catalysis of RNA hydrolysis. If so, <n>Gre</n> would be 
the first transcription factor known to directly participate in the catalytic act of <n>RNAP</n>. <n>NRSF</n> 
regulates the fetal cardiac gene program and maintains normal cardiac structure and function. Reactivation of 
the fetal cardiac gene program is a characteristic feature of hypertrophied and failing hearts that 
correlates with impaired cardiac function and poor prognosis. However, the mechanism governing the reversible expression 
of fetal cardiac genes remains unresolved. Here we show that neuron-restrictive silencer factor (<n>NRSF</n>), a 
transcriptional repressor, selectively regulates expression of multiple fetal cardiac genes, including those for atria