bluuebunny/crossref_metadata_2025 huggingface dataset:
Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor.
Authors: Yang X , Kang C M , Brody M S , Price C W
Published: June 2007

The general stress response of the bacterium Bacillus subtilis is governed by a signal transduction network that regulates activity of the sigma(B) transcription factor. We show that this network comprises two partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, which contribute to regulating sigma(B). Each module consists of a phosphatase (X or U), an antagonist protein (S or V), and a switch protein/kinase (T or W). In the downstream module, the W anti-sigma factor is the primary regulator of sigma(B) activity. If the V antagonist is phosphorylated, the W switch protein binds and inhibits sigma(B). If V is unphosphorylated, it complexes W, freeing sigma(B) to interact with RNA polymerase and promote transcription. The phosphorylation state of V is controlled by opposing kinase (W) and phosphatase (U) activities. The U phosphatase is regulated by the upstream module. The T switch protein directly binds U, stimulating phosphatase activity. The T-U interaction is governed by the phosphorylation state of the S antagonist, controlled by opposing kinase (T) and phosphatase (X) activities. This partner-switching mechanism provides a general regulatory strategy in which linked modules sense and integrate multiple signals by protein-protein interaction.
Vein is a novel component in the Drosophila epidermal growth factor receptor pathway with similarity to the neuregulins.
Authors: Schnepp B , Grumbling G , Donaldson T , Simcox A
Published: June 2007

The activation signal from tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR), is relayed via a highly conserved intracellular pathway involving Ras, Raf, and MAPK. In Drosophila, the EGFR and components of the intracellular pathway are broadly expressed, yet receptor activation evokes tissue-specific cell responses. Extracellular events that lead to receptor activation are one mechanism by which signaling is modulated. Here we show molecular and genetic evidence that Drosophila vein (vn) encodes a candidate EGFR ligand and that vn expression is spatially restricted. Consequently, vn may promote tissue-specific receptor activation. Unlike two other ligands, Gurken (Grk) and Spitz (Spi), which are transforming growth factor alpha-like proteins, Vn has both an immunoglobulin-like and an EGF-like domain. This combination of domains mirrors those in the vertebrate neuregulins that bind EGFR relatives.
Caenorhabditis elegans sex-determining protein FEM-2 is a protein phosphatase that promotes male development and interacts directly with FEM-3.
Authors: Chin-Sang I D , Spence A M
Published: June 2007

Male sexual development in the nematode Caenorhabditis elegans requires the genes fem-1, fem-2, and fem-3. The current model of sex determination portrays the FEM proteins as components of a novel signal transduction pathway, but the mechanisms involved in signaling through the pathway are not understood. We report the isolation of fem-2 cDNAs in a yeast two-hybrid screen for clones encoding proteins that interact with FEM-3. Association of FEM-3 and FEM-2 in two independent in vitro binding assays substantiates the interaction detected in the two-hybrid system. FEM-2 is related in sequence to protein serine/threonine phosphatases of Type 2C (PP2C). We demonstrate that FEM-2 exhibits magnesium-dependent casein phosphatase activity, typical of PP2C, in vitro. Point mutations that abolish the casein phosphatase activity of FEM-2 without affecting its FEM-3-binding activity reduce severely its ability to rescue male development in fem-2 mutant nematodes. These results suggest that protein phosphorylation regulates sex determination in C. elegans.
In vivo targeted mutagenesis of a regulatory element required for positioning the Hoxd-11 and Hoxd-10 expression boundaries.
Authors: Gérard M , Chen J Y , Gronemeyer H , Chambon P , Duboule D , Zákány J
Published: June 2007

Vertebrate Hox genes are required for the proper organization of structures along the rostrocaudal axis. Hoxd-11 is expressed in the posterior part of the embryo, up to the level of prevertebra 27, and its expression boundary is reproduced by a Hoxd-11/lacZ transgene. Expression of this transgene anterior to prevertebra 27 is prevented by the silencing activity of a cis-acting element, region IX. Using transgenic mice, we show that Hoxd-11 repression by region IX is necessary to position the sacrum properly. This silencing activity depends on phylogenetically conserved sequences able to bind in vitro retinoic acid receptors and COUP-TFs. ES cells were used to generate mice carrying a subtle mutation that abolishes binding of nuclear receptors to region IX. Mutant mice display an anterior shift of their lumbosacral transition inherited as a codominant trait. In mutant embryos, expression of both Hoxd-11 and Hoxd-10 mRNAs in the prevertebral column is anteriorized. These results illustrate the sharing, in cis, of a single regulatory element in order to establish the expression boundaries of two neighboring Hoxd genes.
Three sites of contact between the Bacillus subtilis transcription factor sigmaF and its antisigma factor SpoIIAB.
Authors: Decatur A L , Losick R
Published: June 2007

The developmental regulatory protein sigmaF of Bacillus subtilis, a member of the sigma70-family of RNA polymerase sigma factors, is regulated negatively by the antisigma factor SpoIIAB, which binds to sigmaF to form an inactive complex. Complex formation between SpoIIAB, which contains an inferred adenosine nucleotide binding pocket, and sigmaF is stimulated strongly by the presence of ATP. Here we report that SpoIIAB contacts sigmaF at three widely spaced binding surfaces corresponding to conserved regions 2.1, 3.1, and 4.1 of sigma70-like sigma factors. This conclusion is based on binding studies between SpoIIAB and truncated portions of sigmaF, the isolation of mutants of sigmaF that were partially resistant to inhibition by SpoIIAB in vivo and were defective in binding to the antisigma factor in vitro, and the creation of alanine substitution mutants of regions 2.1, 3.1, or 4.1 of sigmaF that were impaired in complex formation. Because the interaction of SpoIIAB with all three binding surfaces was stimulated by ATP, we infer that ATP induces a conformational change in SpoIIAB that is needed for tight binding to sigmaF. Finally, we discuss the possibility that another antisigma factor, unrelated to SpoIIAB, may interact with its respective sigma factor in a similar topological pattern of widely spaced binding surfaces located in or near conserved regions 2.1, 3.1, and 4.1.
Mitotic regulation of TFIID: inhibition of activator-dependent transcription and changes in subcellular localization.
Authors: Segil N , Guermah M , Hoffmann A , Roeder R G , Heintz N
Published: June 2007

Mitosis in higher eukaryotes is accompanied by a general inhibition of transcription. To begin to understand the mechanisms underlying this inhibition we have examined the behavior of the general transcription factor TFIID during mitosis. Immunocytochemistry and subcellular fractionation studies indicate that the majority of TFIID is displaced from the disassembling prophase nucleus to the mitotic cytoplasm around the time of nuclear envelope breakdown. However, a subpopulation of TFIID remains associated tightly with the condensed mitotic chromosomes. Metabolic labeling of mitotic cells revealed that several subunits of TFIID undergo mitosis-specific phosphorylation, but in spite of these changes, the TFIID complex remains intact. Functional analysis of purified TFIID from mitotic cells shows that phosphorylated forms are unable to direct activator-dependent transcription, but that this activity is restored upon dephosphorylation. These results demonstrate that TFIID regulation by phosphorylation is likely to have an important role in mitotic inhibition of RNA polymerase II transcription. In addition, they suggest a mechanism for regulating gene expression through the selective disruption of polymerase II promoter structures during mitosis.
Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor.
Authors: Hammerschmidt M , Serbedzija G N , McMahon A P
Published: June 2007

According to a model based on embryological studies in amphibia, dorsoventral patterning is regulated by the antagonizing function of ventralizing bone morphogenetic proteins (BMPs) and dorsalizing signals generated by Spemann's organizer. Large-scale mutant screens in the zebrafish, Danio rerio, have led to the isolation of two classes of recessive lethal mutations affecting early dorsoventral pattern formation. dino mutant embryos are ventralized, whereas swirl mutants are dorsalized. We show that at early gastrula stages, dino and swirl mutants display an expanded or reduced Bmp4 expression, respectively. The dino and swirl mutant phenotypes both can be phenocopied and rescued by the modulation of BMP signaling in wild-type and mutant embryos. By suppressing BMP signaling in dino mutants, adult fertile dino -/- fish were generated. These findings, together with results from the analysis of dino-swirl double mutants, indicate that dino fulfills its dorsalizing activity via a suppression of swirl-dependent, BMP-like ventralizing activities. Finally, cell transplantation experiments show that dino is required on the dorsal side of early gastrula embryos and acts in a non-cell-autonomous fashion. Together, these results provide genetic evidence in support of a mechanism of early dorsoventral patterning that is conserved among vertebrate and invertebrate embryos.
CUS1, a suppressor of cold-sensitive U2 snRNA mutations, is a novel yeast splicing factor homologous to human SAP 145.
Authors: Wells S E , Neville M , Haynes M , Wang J , Igel H , Ares M
Published: June 2007

The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in Saccharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prp11 and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.
The Nanos gradient in Drosophila embryos is generated by translational regulation.
Authors: Dahanukar A , Wharton R P
Published: June 2007

Abdominal segmentation in the Drosophila embryo is governed by a gradient of Nanos (Nos) emanating from the posterior pole. This gradient is derived from translation of the nos mRNA that is localized in the pole plasm; in contrast, unlocalized nos mRNA is translationally repressed. Here we define the essential signals in the 3' untranslated region (UTR) of nos mRNA. Deletion of a 184-nucleotide translational control element (TCE) from the 3' UTR leads to the derepression of nos mRNA in the bulk cytoplasm and the development of lethal anterior defects. Furthermore, a minimal mRNA containing essentially only the TCE in its 3' UTR rescues nos- embryos to adulthood. The TCE is also sufficient to confer on maternal torso mRNA all three aspects of nos mRNA regulation: translational repression in the bulk cytoplasm, localization to the pole plasm, and translational activation at the posterior pole. These three phenomena are coupled intimately, as mutations in a pair of CUGGC pentamers within the TCE simultaneously abrogate all three regulatory events. This coupling suggests a model in which the polarized distribution of nos protein is generated primarily by translational control and that nos mRNA localization is a byproduct of this regulation, at least in part.
The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye.
Authors: Riesgo-Escovar J R , Jenni M , Fritz A , Hafen E
Published: June 2007

We cloned and characterized the Drosophila homolog of mammalian Jun-N-terminal kinases (DJNK). We show that DJNK is encoded by basket (bsk). Like hemipterous (hep), which encodes the Drosophila JNK kinase, bsk is required in the embryo for dorsal closure, a process involving coordinate cell shape changes of ectodermal cells. Dorsal closure can also be blocked by dominant negative Drosophila cdc42, which has been shown to act upstream of JNKK in vertebrates. Therefore it appears that the JNK pathway is conserved and that it is involved in controlling cell morphogenesis in Drosophila. Although DJNK efficiently phosphorylates DJun in vitro, bsk function is not required for the specification of cell fate in the developing eye, a process that requires MAP kinase and DJun function.
The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway.
Authors: Hofmann F , Martelli F , Livingston D M , Wang Z
Published: June 2007

E2F-1 plays a crucial role in the regulation of cell-cycle progression at the G1-S transition. In keeping with the fact that, when overproduced, it is both an oncoprotein and a potent inducer of apoptosis, its transcriptional activity is subject to multiple controls. Among them are binding by the retinoblastoma gene product (pRb), activation by cdk3, and S-phase-dependent down-regulation of DNA-binding capacity by cyclin A-dependent kinase. Here we report that E2F-1 is actively degraded by the ubiquitin-proteasome pathway. Efficient degradation depends on the availability of selected E2F-1 sequences. Unphosphorylated pRb stabilized E2F-1, protecting it from in vivo degradation. pRb-mediated stabilization was not an indirect consequence of G1 arrest, but rather depended on the ability of pRb to interact physically with E2F-1. Thus, in addition to binding E2F-1 and transforming it into a transcriptional repressor, pRb has another function, protection of E2F-1 from efficient degradation during a period when pRb/E2F complex formation is essential to regulating the cell cycle. In addition, there may be a specific mechanism for limiting free E2F-1 levels, failure of which could compromise cell survival and/or homeostasis.
Discrimination between different E-box-binding proteins at an endogenous target gene of c-myc.
Authors: Desbarats L , Gaubatz S , Eilers M
Published: June 2007

c-myc plans a key role in regulating mammalian cell proliferation and apoptosis. The gene codes for a transcription factor, Myc, that belongs to the helix-loop-helix/leucine zipper (HLH/LZ) family of proteins. Myc heterodimerizes with a partner protein termed Max; the heterodimeric complex binds to CAC(G/A)TG (E-box) sequences and activates transcription from these sites. However, several other HLH/LZ proteins, including USF and TFE-3, bind to and trans-activate from the same element, yet have no documented effect on cell proliferation or apoptosis. Therefore, it is likely that mechanisms exist that discriminate between these proteins for activation of natural target genes of Myc. We now show that trans-activation from the E-box in the rat prothymosin-alpha intron enhancer is indeed specific for Myc, and identify both the distance from the start site of transcription and a second E-box element adjacent to that recognized by Myc as critical determinants of specificity. Surprisingly, transcription activation domains required for Myc to activate from this distal enhancer position differ from previously mapped domains and closely correlate with those domains essential for transformation. As observed in transformation assays, Myc and Max strongly synergize in activation from a distal enhancer position. Our data suggest that trans-activation from the prothymosin intron enhancer is a faithful reflection of the transforming properties of the Myc protein.
Transposase is the only nematode protein required for in vitro transposition of Tc1.
Authors: Vos J C , De Baere I , Plasterk R H
Published: June 2007

The Tc1 element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature. Here, we describe efficient and precise transposition of Tc1 in a cell-free system. Tc1 appears to jump by a cut-and-paste mechanism of transposition. The terminal 26 bp of the Tc1 terminal repeats together with the flanking TA sequence are sufficient for transposition. The target site choice in vitro is similar to that in vivo. Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tc1 transposase but also by recombinant transposase purified from Escherichia coli. The simple reaction requirements explain why horizontal spread of Tc1/mariner transposons can occur. They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.
Determinants of selectivity in Xer site-specific recombination.
Authors: Blakely G , Sherratt D
Published: June 2007

A remarkable property of some DNA-binding proteins that can interact with and pair distant DNA segments is that they mediate their biological function only when their binding sites are arranged in a specific configuration. Xer site-specific recombination at natural plasmid recombination sites (e.g., cer in ColE1) is preferentially intramolecular, converting dimers to monomers. In contrast, Xer recombination at the Escherichia coli chromosomal site dif can occur intermolecularly and intramolecularly. Recombination at both types of site requires the cooperative interactions of two related recombinases, XerC and XerD, with a 30-bp recombination core site. The dif core site is sufficient for recombination when XerC and XerD are present, whereas recombination at plasmid sites requires approximately 200 bp of adjacent accessory sequences and accessory proteins. These accessory factors ensure that recombination is intramolecular. Here we use a model system to show that selectivity for intramolecular recombination, and the consequent requirement for accessory factors, can arise by increasing the spacing between XerC- and XerD-binding sites from 6 to 8 bp. This reduces the affinity of the recombinases for the core site and changes the geometry of the recombinase/DNA complex. These changes are correlated with altered interactions of the recombinases with the core site and a reduced efficiency of XerC-mediated cleavage. We propose that the accessory sequences and proteins compensate for these changes and provide a nucleoprotein structure of fixed geometry that can only form and function effectively on circular molecules containing directly repeated sites.
CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein.
Authors: Timchenko N A , Wilde M , Nakanishi M , Smith J R , Darlington G J
Published: June 2007

C/EBPalpha has a role in growth arrest and differentiation of mouse preadipocytes. To study the mechanism of C/EBPalpha-induced growth arrest, we developed a cell line, HT1, that contained the human C/EBPalpha gene under Lac repressor control. IPTG-induced C/EBPalpha caused inhibition of cell proliferation and DNA synthesis as measured by colony growth assays, cell counting, and BrdU uptake. A number of proteins that are known to be involved in the regulation of the cell cycle, such as cyclin-dependent kinase (CDK)2 and CDK4, proliferating cell nuclear antigen (PCNA), p53, c-fos, and the CDK inhibitor p16 and p27 were investigated by Western analysis. No change in their expression was observed. However, the p21 (WAF-1/CIP-1/SDI-1) protein was significantly elevated in growth-arrested HT1 cells. Elevation of p21/SDI-1 mRNA (threefold) and activation of the p21/SDI-1 promoter by C/EBPalpha did not account for the 12- to 20-fold increase in p21/SDI-1 protein. Protein synthesis inhibition by cycloheximide (CHX) treatment indicated that the half-life of p21/SDI-1 in dividing HT1 cells was approximately 30 min. However, in C/EBPalpha growth-arrested cells, the level of the p21/SDI-1 did not change for > 80 min after CHX addition. Our studies demonstrate that C/EBPalpha activates p21/SDI-1 by increasing p21/SDI-1 gene expression and by post-translational stabilization of p21/SDI-1 protein. Furthermore, induction of p21/SDI-1 is responsible for the ability of C/EBPalpha to inhibit proliferation because transcription of antisense p21/SDI-1 mRNA eliminated growth inhibition by C/EBPalpha.
Tissue-specific inactivation of p53 tumor suppression in the mouse.
Authors: Bowman T , Symonds H , Gu L , Yin C , Oren M , Van Dyke T
Published: June 2007

The p53 gene is the most frequent target of structural and functional genetic mutations in human cancer. Thus, considerable effort has been devoted to mapping the functional domains of p53 with regard to their impact on tumorigenesis in vivo. Studies have shown that the carboxy-terminal domain of p53 is sufficient for transformation in vitro. To determine whether a transdominant-negative p53 protein could be used to elicit a tissue-specific p53-null effect in vivo, we tested whether a carboxy-terminal p53 fragment (amino acids 302-390) could abolish p53-dependent apoptosis in an established tumor progression model. We showed previously that loss of p53-dependent apoptosis accelerates brain tumorigenesis in a transgenic mouse model. Here, we show that the same effect can be elicited by expressing a dominant-negative p53 protein tissue specifically in the presence of wild-type p53. Transgenic mice in which pRb function has been disrupted and that coexpress a p53 carboxy-terminal dominant-negative fragment (p53DD) develop aggressive brain tumors mimicking genetic loss of p53 in this model. Inactivation of endogenous p53, which we show to be complexed with p53DD, results in a reduction in apoptosis and acceleration of tumorigenesis. These studies establish a mechanism for tissue-specific knock out of p53 function in vivo.
Differential effects of cdk2 and cdk3 on the control of pRb and E2F function during G1 exit.
Authors: Hofmann F , Livingston D M
Published: June 2007

The cyclin-dependent kinases cdk2 and cdk3 are required for the G1-S transition in mammalian cells. Here we show that G1 arrest induced by the corresponding dominant-negative mutants of these enzymes, cdk2dn or cdk3dn, is resistant to the action of SV40 T antigen (T). In the presence of cdk2dn, T released active E2F from negative control by pRb and its related family members (pocket proteins) but failed to induce S-phase. Therefore, among other targets, cdk2 also phosphorylates nonpocket protein substrates in promoting S-phase entry, and T does not mimic all cdk2 functions. In the presence of cdk3dn, however, T failed to induce cell cycle progression or stimulate E2F-dependent transcription activity. Dominant-negative cdk3 inhibited E2F-1, E2F-2, and, less significantly, E2F-3, but not E2F-4 transcription activity. The inhibition occurred in a pRb-independent manner and did not affect the DNA-binding capacity of the transcription factor. Cdk3 bound specifically to E2F-1/DP-1 complexes in vivo, most likely through DP-1. Thus, cdk3 function contributes to the activation of E2F-1, E2F-2, and partially E2F-3 and, thereby, participates in the process of S-phase entry.
Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes.
Authors: Tucker K L , Beard C , Dausmann J , Jackson-Grusby L , Laird P W , Lei H , Li E , Jaenisch R
Published: June 2007

Embryonic stem (ES) cells homozygous for a disruption of the DNA (cytosine-5)-methyltransferase gene (Dnmt) proliferate normally with their DNA highly demethylated but die upon differentiation. Expression of the wild-type Dnmt cDNA in mutant male ES cells caused an increase in methylation of bulk DNA and of the Xist and Igf2 genes to normal levels, but did not restore the methylation of the imprinted genes H19 and Igf2r. These cells differentiated normally in vitro and contributed substantially to adult chimeras. While the Xist gene was not expressed in the remethylated male ES cells, no restoration of the normal expression profile was seen for H19, Igf2r, or Igf2. This indicates that ES cells can faithfully reestablish normal methylation and expression patterns of nonimprinted genes but lack the ability to restore those of imprinted genes. Full restoration of monoallelic methylation and expression was imposed on H19, Igf2, and Igf2r upon germ-line transmission. These results are consistent with the presence of distinct de novo DNA methyltransferase activities during oogenesis and spermatogenesis, which specifically recognize imprinted genes but are absent in the postimplantation embryo and in ES cells.
TANK, a co-inducer with TRAF2 of TNF- and CD 40L-mediated NF-kappaB activation.
Authors: Cheng G , Baltimore D
Published: June 2007

We describe a new signal mediator of NF-kappaB activation, TANK, that acts in a pathway common to two surface receptors CD40 and TNFR II. TRAF family members interact directly with these receptors. Using the yeast two-hybrid system, TANK was identified as an intracellular protein without previous homologs that interacts with all three known TRAF family members. In cotransfection experiments, TANK and TRAF2 activate NF-kappaB synergistically, requiring both the amino-terminal portion of TANK and the ring finger domain of TRAF2. TANK has a negatively acting carboxyl terminus and is constitutively inactive, but TRAF2 binding overcomes the internal inhibitory influence. We propose that ligand binding to CD40 or TNFR II leads to the formation of a TRAF2/TANK complex, mediating NF-kappaB activation.
Pollux, a novel Drosophila adhesion molecule, belongs to a family of proteins expressed in plants, yeast, nematodes, and man.
Authors: Zhang S D , Kassis J , Olde B , Mellerick D M , Odenwald W F
Published: June 2007

Adhesion molecules have pivotal roles in cellular processes critical to the development and maintenance of multicellular organisms. Here we describe a new member of the adhesive repertoire encoded by the Drosophila pollux (plx) gene. Marked by a novel 74-amino-acid domain, Plx belongs to a highly conserved family with members in plants, yeast, nematodes, and man, including the human oncoprotein TRE17. Essential for viability, plx mutant analysis indicates that larval death is attributable to asphyxiation brought on by fluid-congested tracheal tubes. Ultrastructural examination of mutant tracheae reveals defects in cell-extracellular matrix contacts. During embryogenesis, Plx uniformly covers the apical surface of cellular blastoderm cells. It is later found regionally concentrated along subsets of central nervous system axon pathways and on the apical surface of the trachea's tubular epithelium. Cell attachment assays demonstrate that Plx can serve as a ligand for cell surface integrins. Plx also contains a motor neuron-selective adhesive site, multiple proteoglycan-binding motifs, and a leucine zipper: all suggest possible associations with additional components of the adhesion complex.
The alternative splicing factor PSI regulates P-element third intron splicing in vivo.
Authors: Adams M D , Tarng R S , Rio D C
Published: June 2007

Splicing of the Drosophila P-element third intron (IVS3) is inhibited in somatic cells, restricting transposase expression to the germ line. Somatic inhibition of IVS3 splicing involves the assembly of a multiprotein complex on a regulatory sequence in the IVS3 5' exon. The P-element somatic inhibitor protein (PSI) is a component of this ribonucleoprotein complex and is required for inhibition of IVS3 splicing in vitro. The soma-specific expression pattern of PSI suggests that its low abundance in the germ line allows IVS3 splicing. We demonstrate that ectopic expression of PSI in the female germ line is sufficient to repress splicing of an IVS3 reporter transgene. We also show that IVS3 splicing is activated in somatic embryonic cells in the presence of an antisense PSI ribozyme. These results support the model that PSI is a tissue-specific regulator of IVS3 splicing in vivo.
SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.
Authors: Strahl-Bolsinger S , Hecht A , Luo K , Grunstein M
Published: June 2007

Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.
Oncogene-dependent apoptosis in extracts from drug-resistant cells.
Authors: Fearnhead H O , McCurrach M E , O'Neill J , Zhang K , Lowe S W , Lazebnik Y A
Published: June 2007

Many genotoxic agents kill tumor cells by inducing apoptosis; hence, mutations that suppress apoptosis produce resistance to chemotherapy. Although directly activating the apoptotic machinery may bypass these mutations, how to achieve this activation in cancer cells selectively is not clear. In this study, we show that the drug-resistant 293 cell line is unable to activate components of the apoptotic machinery-the ICE-like proteases (caspases)-following treatment with an anticancer drug. Remarkably, extracts from untreated cells spontaneously activate caspases and induce apoptosis in a cell-free system, indicating that drug-resistant cells have not only the apoptotic machinery but also its activator. Comparing extracts from cells with defined genetic differences, we show that this activator is generated by the adenovirus E1A oncogene and is absent from normal cells. We provide preliminary characterization of this oncogene generated activity (OGA) and show that partially purified OGA activates caspases when added to extracts from untransformed cells. We suggest that agents that link OGA to caspases in cells would kill tumor cells otherwise resistant to conventional cancer therapy. As this killing relies on an activity generated by an oncogene, the effect of these agents should be selective for transformed cells.
A novel Mcm1-dependent element in the SWI4, CLN3, CDC6, and CDC47 promoters activates M/G1-specific transcription.
Authors: McInerny C J , Partridge J F , Mikesell G E , Creemer D P , Breeden L L
Published: June 2007

We have identified a novel promoter element that confers M/G1-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI4 promoter, but it is also present in the promoter of a G1 cyclin CLN3, as well as in the promoters of three DNA replication genes: CDC6, CDC47, and CDC46. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcm1 binding site to which Mcm1 binds in vitro, and an Mcm1-VP16 fusion, which places a constitutive activator on Mcm1-binding sites in vivo, can deregulate ECB-containing promoters. Mcm1 is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication.
Three human RNA polymerase III-specific subunits form a subcomplex with a selective function in specific transcription initiation.
Authors: Wang Z , Roeder R G
Published: June 2007

Transcription by RNA polymerase III involves recruitment of the polymerase by template-bound accessory factors, followed by initiation, elongation, and termination steps. An immunopurification approach has been used to demonstrate that human RNA Pol III is composed of 16 subunits, some of which are apparently modified in HeLa cells. Partial denaturing conditions and sucrose gradient sedimentation at high salt result in the dissociation of a subcomplex that includes hRPC32, hRPC39, and hRPC62. Cognate cDNAs were isolated and shown to encode three subunits that are specific to RNA Pol III and homologous to three yeast subunits. The human RNA Pol III core lacking the subcomplex functions in transcription elongation and termination following nonspecific initiation on a tailed template, but fails to show promoter-dependent transcription initiation in conjunction with accessory factors. The capability for specific transcription initiation can be restored either by the natural subcomplex or by a stable subcomplex composed of recombinant hRPC32, hRPC39, and hRPC62 polypeptides. One component (hRPC39) of this subcomplex interacts physically with both hTBP and hTFIIIB90, two subunits of human RNA Pol III transcription initiation factor IIIB. These data strongly suggest that the hRPC32-hRPC39-hRPC62 subcomplex directs RNA Pol III binding to the TFIIIB-DNA complex via the interactions between TFIIIB and hRPC39.
Cyclin E-CDK2 is a regulator of p27Kip1.
Authors: Sheaff R J , Groudine M , Gordon M , Roberts J M , Clurman B E
Published: June 2007

CDK inhibitors are thought to prevent cell proliferation by negatively regulating cyclin-CDK complexes. We propose that the opposite is also true, that cyclin-CDK complexes in mammmalian cells can promote cell cycle progression by directly down-regulating CDK inhibitors. We show that expression of cyclin E-CDK2 in murine fibroblasts causes phosphorylation of the CDK inhibitor p27Kip1 on T187, and that cyclin E-CDK2 can directly phosphorylate p27 T187 in vitro. We further show that cyclin E-CDK2-dependent phosphorylation of p27 results in elimination of p27 from the cell, allowing cells to transit from G1 to S phase. Moreover, mutation of T187 in p27 to alanine creates a p27 protein that causes a G1 block resistant to cyclin E and whose level of expression is not modulated by cyclin E. A kinetic analysis of the interaction between p27 and cyclin E-CDK2 explains how p27 can be regulated by the same enzyme it targets for inhibition. We show that p27 interacts with cyclin E-CDK2 in at least two distinct ways: one resulting in p27 phosphorylation and release, the other in tight binding and cyclin E-CDK2 inhibition. The binding of ATP to the CDK governs which state predominates. At low ATP (< 50 microM) p27 is primarily a CDK inhibitor, but at ATP concentrations approaching physiological levels (> 1 mM) p27 is more likely to be a substrate. Thus, we have identified p27 as a biologically relevant cyclin E-CDK2 substrate, demonstrated the physiological consequences of p27 phosphorylation, and developed a kinetic model to explain how p27 can be both an inhibitor and a substrate of cyclin E-CDK2.
An activating mutation in a Caenorhabditis elegans Gs protein induces neural degeneration.
Authors: Korswagen H C , Park J H , Ohshima Y , Plasterk R H
Published: June 2007

Heterotrimeric guanine nucleotide-binding proteins (G proteins) act as signal-transducing molecules that connect serpentine-transmembrane receptors to a variety of intracellular effectors. We characterized a Caenorhabditis elegans G(s) gene, gsa-1, which encodes a G(s) alpha-subunit (G alpha(s)) that is expressed throughout the nervous system and in muscle cells. gsa-1 is an essential gene; a loss-of-function mutation in gsa-1 results in lethality at the first stage of larval development. Partial (mosaic) loss of G alpha(s) expression or overexpression of the protein results in reciprocal defects in movement and egg-laying, suggesting a role for G alpha(s) in the regulation of these behaviors. Expression of a constitutively active form of G alpha(s) from an inducible promotor results in hypercontraction of body-wall muscle cells and vacuolization and degeneration of neurons within hours of induction. Neurons that are susceptible to the degeneration induced by activated G alpha(s) are predominantly motoneurons located within the ventral nerve cord. Phenotypic analysis shows that the induced neural degeneration is not the result of programmed cell death but is probably caused by the activation of ion channels. A genetic suppressor of activated G alpha(s) was isolated that identifies a putative downstream target of G(s) signaling.
The UNC-14 protein required for axonal elongation and guidance in Caenorhabditis elegans interacts with the serine/threonine kinase UNC-51.
Authors: Ogura K , Shirakawa M , Barnes T M , Hekimi S , Ohshima Y
Published: June 2007

Certain unc mutants in the nematode Caenorhabditis elegans, such as unc-14 and unc-51, show abnormal axonal elongation and axonal structures. We cloned the unc-51 gene previously and predicted that it encodes a novel serine/threonine protein kinase. In this study, we precisely localized the activity to rescue an unc-14 mutation. Also, we identified four cDNA clones encoded by the unc-14 rescuing region, in screens for proteins that bind to UNC-51 using a yeast two-hybrid system. A mutation site in the cDNA was identified for each of the six unc-14 mutants, establishing that the unc-14 gene was cloned. The unc-14 gene encodes a novel protein of 665 amino acids, and is coexpressed with the unc-51 gene in the cell bodies and axons of almost all neurons including DD/VD and hermaphrodite-specific neurons. Another clone recovered in the two-hybrid screen encodes a carboxy-terminal region of UNC-51. Analysis using the yeast two-hybrid system suggested that a central region of UNC-14 bound to a carboxy-terminal region of UNC-51, and that the UNC-51 carboxy-terminal region oligomerized. In in vitro binding studies using recombinant fusion proteins, UNC-14 interacted with UNC-51 directly. We propose that UNC-51 protein kinase acts as an oligomer, and that UNC-14 is a regulator of UNC-51, in axonal elongation and guidance.
Mutation in Sos1 dominantly enhances a weak allele of the EGFR, demonstrating a requirement for Sos1 in EGFR signaling and development.
Authors: Wang D Z , Hammond V E , Abud H E , Bertoncello I , McAvoy J W , Bowtell D D
Published: June 2007

We have investigated the role of the mammalian Son of sevenless 1 (Sos1) protein in growth factor signaling in vivo by generating mice and cell lines that lacked the Sos1 protein. Homozygous null embryos were smaller than normal, died mid-gestation with cardiovascular and yolk sac defects, and their fibroblasts showed reduced mitogen-activated protein kinase activation in response to epidermal growth factor (EGF). An intercross of mice mutant for Sos1 and the EGF receptor (EGFR) demonstrated that a heterozygous mutation in Sos1 dominantly enhanced the phenotype of a weak allele of the EGFR allele (wa-2). These animals had distinctive eye defects that closely resembled those seen in mice that were null for the EGFR or its ligand, TGF alpha. Our findings provide the first demonstration of a functional requirement for Sos1 in growth factor signaling in vivo. They also show that the genetic test of enhancement of weak receptor allele by heterozygous mutation in one component represents a powerful tool for analyzing the ras pathway in mammals.
Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins.
Authors: Perona R , Montaner S , Saniger L , Sánchez-Pérez I , Bravo R , Lacal J C
Published: June 2007

The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor kappaB (NF-kappaB) by a mechanism that involves phosphorylation of Ikappa Balpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-kappaB by TNFalpha depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-kappaB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-kappaB-dependent transcription.
The subcellular locations of p15(Ink4b) and p27(Kip1) coordinate their inhibitory interactions with cdk4 and cdk2.
Authors: Reynisdóttir I , Massagué J
Published: June 2007

In dividing cells, p27(Kip1) is predominantly bound to cyclin D-cdk4 without inhibiting this kinase. Upon being induced by TGF-beta or with a conditional expression system in lung epithelial cells, p15(Ink4b) binds to and inhibits the cyclin D-dependent kinases, prevents p27 binding to these cdk complexes, and promotes p27 binding and inhibition of cyclin-cdk2. In vitro, however, p15 prevents p27 binding only if it has access to cyclin D-cdk4 first. We present evidence that the different subcellular location of p15 and p27 ensures the prior access of p15 to cdk4. In the cell, p15 is localized mostly in the cytoplasm, whereas p27 is nuclear. p15 prevails over p27 or a p27 construct consisting of the cdk inhibitory domain tagged with a nuclear localization signal. However, when p15 and p27 are forced to reside in the same subcellular location, either the cytoplasm or the nucleus, p15 no longer prevents p27 from binding to cdk4. These properties allow p15 and p27 to coordinately inhibit cdk4 and cdk2.
Stn1, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdc13.
Authors: Grandin N , Reed S I , Charbonneau M
Published: June 2007

We have isolated STN1, an essential Saccharomyces cerevisiae gene, as a suppressor of the cdc13-1 mutation. A synthetic lethal interaction between a temperature-sensitive mutant allele of STN1, stn1-13, and cdc13-1 was observed. Stn1 and Cdc13 proteins displayed a physical interaction by two-hybrid analysis. As shown previously for cdc13-1, stn1-13 cells at the restrictive temperature accumulate single-stranded DNA in subtelomeric regions of the chromosomes, but to a lesser extent than cdc13-1 cells. In addition, both Cdc13 and Stn1 were found to be involved in the regulation of telomere length, mutations in STN1 or CDC13 conferring an increase in telomere size. Loss of Stn1 function activated the RAD9 and MEC3 G2/M checkpoints, therefore confirming that DNA damage is generated. We propose that Stn1 functions in telomere metabolism during late S phase in cooperation with Cdc13.
Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro.
Authors: Prescott J , Blackburn E H
Published: June 2007

The ribonucleoprotein enzyme telomerase adds telomeric DNA to chromosomal ends. In most eukaryotes the telomeric repeat units are repeated precisely, consistent with the action of a telomerase that faithfully copies its RNA template. In contrast, Saccharomyces cerevisiae telomeric repeats are degenerate, suggesting that its telomerase has unusual mechanistic properties. We mutated the S. cerevisiae telomerase RNA (TLC1) with a series of 3-base (GUG) substitutions in and next to the 17-nucleotide templating domain. All mutant telomerases were active in TLC1/tlc1 diploids and synthesized patterns of mixed wild-type and mutant telomeric repeats into telomeric DNA, consistent with nonprocessive action. Telomerase isolated from cells containing each mutated tlc1 allele by itself had altered reaction properties in vitro. One mutant template enzyme, 476GUG, was active in vivo and in vitro in the presence of wild-type TLC1 RNA but lacked detectable activity in its absence. Haploid tlc1-476GUG cells containing only this mutant tlc1 allele underwent senescence. Other tlc1 template region mutations allowed maintenance of shortened telomeres in vivo but altered specific enzymatic properties of telomerase in vitro, including induction of primer-template slippage (472GUG) or alteration of the 5' boundary of the template (467GUG). These data demonstrate that telomerase RNA bases influence enzyme activity profoundly, suggesting that their roles are not confined to serving simply as the template for this specialized reverse transcriptase.
The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme.
Authors: Anderson D G , Kowalczykowski S C
Published: June 2007

Homologous recombination in Escherichia coli is stimulated at DNA sequences known as chi sites. Stimulation requires the multifunctional RecBCD enzyme, which is both a helicase and a 3' --> 5' exonuclease. Upon recognition of a properly oriented chi site, the 3' --> 5' exonuclease activity is attenuated. Here we show that in addition to attenuation of the 3' --> 5' exonuclease activity, recognition of chi by the RecBCD enzyme also up-regulates a nuclease activity of the opposite polarity, resulting in an enzyme that now preferentially degrades 5' --> 3'. These results demonstrate that chi is a unique regulatory element that converts the antirecombinogenic form of the RecBCD enzyme into a recombinogenic form by causing two distinct enzymatic changes: attenuation of the 3' --> 5' nuclease activity, and up-regulation of the 5' --> 3' nuclease activity. The consequence of chi recognition is the production of a recombination intermediate possessing a 3'-ssDNA overhang terminating at the chi sequence. This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs during the initation of homologous recombination in other pathways in E. coli, and in other organisms such as the yeast Saccharomyces cerevisiae.