bluuebunny/crossref_metadata_2025 huggingface dataset:
Recognition of pneumococcal isolates by antisera raised against PspA fragments from different clades
Authors: Darrieux Michelle , Moreno Adriana T. , Ferreira Daniela M. , Pimenta Fabiana C. , de Andrade Ana Lúcia S. S. , Lopes Alexandre P. Y. , Leite Luciana C. C. , Miyaji Eliane N.
Published: February 2008

Pneumococcal surface protein A (PspA) is an important vaccine candidate against pneumococcal infections, capable of inducing protection in different animal models. Based on its structural diversity, it has been suggested that a PspA-based vaccine should contain at least one fragment from each of the two major families (family 1, comprising clades 1 and 2, and family 2, comprising clades 3, 4 and 5) in order to elicit broad protection. This study analysed the recognition of a panel of 35 pneumococcal isolates bearing different PspAs by antisera raised against the N-terminal regions of PspA clades 1 to 5. The antiserum to PspA clade 4 was found to show the broadest cross-reactivity, being able to recognize pneumococcal strains containing PspAs of all clades in both families. The cross-reactivity of antibodies elicited against a PspA hybrid including the N-terminal region of clade 1 fused to a shorter and more divergent fragment (clade-defining region, or CDR) of clade 4 (PspA1–4) was also tested, and revealed a strong recognition of isolates containing clades 1, 4 and 5, and weaker reactions with clades 2 and 3. The analysis of serum reactivity against different PspA regions further revealed that the complete N-terminal region rather than just the CDR should be included in an anti-pneumococcal vaccine. A PspA-based vaccine is thus proposed to be composed of the whole N-terminal region of clades 1 and 4, which could also be expressed as a hybrid protein.
A newly identified DNA ligase of Saccharomyces cerevisiaeinvolved in RAD52-independent repair of DNA double-strand breaks
Authors: Schär Primo , Herrmann Gernot , Daly Graham , Lindahl Tomas
Published: February 2008

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4.Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of theLIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitizecdc9 or rad52 mutant cells. In contrast, lig4mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis.
Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2β
Authors: Moser Markus , Pscherer Armin , Roth Christina , Becker Jutta , Mücher Gabi , Zerres Klaus , Dixkens Christa , Weis Joachim , Guay-Woodford Lisa , Buettner Reinhard , Fässler Reinhard
Published: February 2008

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here thatAP-2β −/− mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2β-deficient mice. At the end of embryonic development expression of bcl-XL, bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2β gene was identified at chromosome 6p12–p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2β gene is located in close proximity to but distinct from the ARPKD gene.
Mutations in Drosophila DP and E2F distinguish G1–S progression from an associated transcriptional program
Authors: Royzman Irena , Whittaker Allyson J. , Orr-Weaver Terry L.
Published: February 2008

The E2F transcription factor, a heterodimer of E2F and DP subunits, is capable of driving the G1–S transition of the cell cycle. However, mice in which the E2F-1 gene had been disrupted developed tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The consequences of disrupting the DP genes have not been reported. We screened for mutations that disrupt G1–S transcription late inDrosophila embryogenesis and identified five mutations in thedDP gene. Although mutations in dDP or dE2Fnearly eliminate E2F-dependent G1–S transcription, S-phase still occurs. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and dE2F mutants the peaks of G1–S transcription of cyclin E are missing. Thus, greatly reduced levels of cyclin E transcript suffice for DNA replication until late in development. Both dDP anddE2F are necessary for viability, and mutations in the genes cause lethality at the late larval/pupal stage. The mutant phenotypes reveal that both genes promote progression of the cell cycle.
The response to extracytoplasmic stress in Escherichia coli is controlled by partially overlapping pathways
Authors: Connolly Lynn , Peñas Alejandro De Las , Alba Benjamin M. , Gross Carol A.
Published: February 2008

The activity of the alternate ς-factor ςE ofEscherichia coli is induced by several stressors that lead to the extracytoplasmic accumulation of misfolded or unfolded protein. The ςE regulon contains several genes, including that encoding the periplasmic protease DegP, whose products are thought to be required for maintaining the integrity of the cell envelope because cells lacking ςE are sensitive to elevated temperature and hydrophobic agents. Selection of multicopy suppressors of the temperature-sensitive phenotype of cells lacking ςErevealed that overexpression of the lipoprotein NlpE restored high temperature growth to these cells. Overexpression of NlpE has been shown previously to induce DegP synthesis by activating the Cpx two-component signal transduction pathway, and suppression of the temperature-sensitive phenotype by NlpE was found to be dependent on the Cpx proteins. In addition, a constitutively active form of the CpxA sensor/kinase also fully suppressed the temperature-sensitive defect of cells lacking ςE. DegP was found to be necessary, but not sufficient, for suppression. Activation of the Cpx pathway has also been shown to alleviate the toxicity of several LamB mutant proteins. Together, these results reveal the existence of two partially overlapping regulatory systems involved in the response to extracytoplasmic stress in E. coli.
Mice lacking the ski proto-oncogene have defects in neurulation, craniofacial patterning, and skeletal muscle development
Authors: Berk Michael , Desai Shailesh Y. , Heyman Hong Chen , Colmenares Clemencia
Published: February 2008

The c-ski proto-oncogene has been implicated in the control of cell growth and skeletal muscle differentiation. To determine its normal functions in vivo, we have disrupted the mouse c-ski gene. Our results show a novel role for ski in the morphogenesis of craniofacial structures and the central nervous system, and confirm its proposed function as a player in skeletal muscle development. Homozygous mutant mice show perinatal lethality resulting from exencephaly, a defect caused by failed closure of the cranial neural tube during neurulation. The timing of the neural tube defect in ski −/− embryos coincides with excessive apoptosis in the cranial neuroepithelium, as well as in the cranial mesenchyme. Homozygous ski mutants also exhibit a dramatic reduction in skeletal muscle mass, consistent with a defect in expansion of a myogenic precursor population. Nestin is an intermediate filament expressed in highly proliferative neuroepithelial stem cells and in myogenic precursors. Interestingly, we find decreased nestin expression in both the cranial neural tube and the somites ofski −/− embryos, compared with their normal littermates, but no reduction of nestin in the caudal neural tube. These results are consistent with a model in which skiactivities are required for the successful expansion of a subset of precursors in the neuroepithelial or skeletal muscle lineages.
The Pax2 homolog sparkling is required for development of cone and pigment cells in theDrosophila eye
Authors: Fu Weimin , Noll Markus
Published: February 2008

A new Drosophila Pax gene, sparkling(spa), implicated in eye development, was isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system and in cone, primary pigment, and bristle cells of larval and pupal eye discs. Inspapol mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. We suggest close functional analogies between Spa and Pax2 in the development of the insect and vertebrate eye.
The PAS domain confers target gene specificity of DrosophilabHLH/PAS proteins
Authors: Zelzer Elazar , Wappner Pablo , Shilo Ben-Zion
Published: February 2008

Trachealess (Trh) and Single-minded (Sim) are highly similarDrosophila bHLH/PAS transcription factors. They activate nonoverlapping target genes and induce diverse cell fates. A single Drosophila gene encoding a bHLH/PAS protein homologous to the vertebrate ARNT protein was isolated and may serve as a partner for both Trh and Sim. We show that Trh and Sim complexes recognize similar DNA-binding sites in the embryo. To examine the basis for their distinct target gene specificity, the activity of Trh–Sim chimeric proteins was monitored in embryos. Replacement of the Trh PAS domain by the analogous region of Sim was sufficient to convert it into a functional Sim protein. The PAS domain thus mediates all the features conferring specificity and the distinct recognition of target genes. The normal expression pattern of additional proteins essential for the activity of the Trh or Sim complexes can be inferred from the induction pattern of target genes and binding-site reporters, triggered by ubiquitous expression of Trh or Sim. We postulate that the capacity of bHLH/PAS heterodimers to associate, through the PAS domain, with additional distinct proteins that bind target-gene DNA, is essential to confer specificity.
The KH domain protein encoded by quaking functions as a dimer and is essential for notochord development inXenopus embryos
Authors: Zorn Aaron M. , Krieg Paul A.
Published: February 2008

Mutations in the mouse indicate that quaking gene function is essential for both embryogenesis and for development of the nervous system. Recent isolation of the mouse quaking gene identified a putative RNA-binding protein containing a single KH domain. We have previously isolated the Xenopus homolog of quaking, Xqua, and shown that the sequence is highly conserved through evolution. Here, we report experimental data on the biochemical function of the quaking protein and its role during development. We demonstrate that the quaking protein expressed during early embryogenesis, pXqua357, can bind RNA in vitro, and we have mapped the regions of the protein that are essential for RNA binding. We present evidence that pXqua can form homodimers and that dimerization may be required for RNA binding. Oocyte injection experiments show that pXqua357 is located in both the nucleus and cytoplasm. In the Xenopus embryo, Xqua is first expressed during gastrulation in the organizer region and its derivative, the notochord. In later stage embryos, Xqua is expressed in a number of mesodermal and neural tissues. We demonstrate that disruption of normal Xqua function, by overexpression of a dominant inhibitory form of the protein, blocks notochord differentiation. Xqua function appears to be required for the accumulation of important mRNAs such as Xnot, Xbra, andgsc. These results indicate an essential role for the quaking RNA-binding protein during early vertebrate embryogenesis.
Translational repression by a transcriptional elongation factor
Authors: Wilson Helen R. , Kameyama Luis , Zhou Jian-guang , Guarneros Gabriel , Court Donald L.
Published: February 2008

One of the classical positive regulators of gene expression is bacteriophage λ N protein. N regulates the transcription of early phage genes by participating in the formation of a highly processive, terminator-resistant transcription complex and thereby stimulates the expression of genes lying downstream of transcriptional terminators. Also included in this antiterminating transcription complex are an RNA site (NUT) and host proteins (Nus). Here we demonstrate that N has an additional, hitherto unknown regulatory role, as a repressor of the translation of its own gene. N-dependent repression does not occur when NUT is deleted, demonstrating that N-mediated antitermination and translational repression both require the same cis-acting site in the RNA. In addition, we have identified one nut and several host mutations that eliminate antitermination and not translational repression, suggesting the independence of these two N-mediated mechanisms. Finally, the position of nutL with respect to the gene whose expression is repressed is important.
SKN-1 domain folding and basic region monomer stabilization upon DNA binding
Authors: Carroll Adam S. , Gilbert Dara E. , Liu Xiaoying , Cheung Jim W. , Michnowicz Jennifer E. , Wagner Gerhard , Ellenberger Tom E. , Blackwell T. Keith
Published: February 2008

The SKN-1 transcription factor specifies early embryonic cell fates in Caenorhabditis elegans. SKN-1 binds DNA at high affinity as a monomer, by means of a basic region like those of basic-leucine zipper (bZIP) proteins, which bind DNA only as dimers. We have investigated how the SKN-1 DNA-binding domain (the Skn domain) promotes stable binding of a basic region monomer to DNA. A flexible arm at the Skn domain amino terminus binds in the minor groove, but a support segment adjacent to the carboxy-terminal basic region can independently stabilize basic region–DNA binding. Off DNA, the basic region and arm are unfolded and, surprisingly, the support segment forms a molten globule of four α-helices. On binding DNA, the Skn domain adopts a tertiary structure in which the basic region helix extends directly from a support segment α-helix, which is required for binding. The remainder of the support segment anchors this uninterrupted helix on DNA, but leaves the basic region exposed in the major groove. This is similar to how the bZIP basic region extends from the leucine zipper, indicating that positioning and cooperative stability provided by helix extension are conserved mechanisms that promote binding of basic regions to DNA.
Binding specificity and in vivo targets of the EH domain, a novel protein–protein interaction module
Authors: Salcini Anna Elisabetta , Confalonieri Stefano , Doria Margherita , Santolini Elisa , Tassi Elena , Minenkova Olga , Cesareni Gianni , Pelicci Pier Giuseppe , Di Fiore Pier Paolo
Published: February 2008

EH is a recently identified protein–protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine–proline–phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene ofDrosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.
Dual pathways for induction of wingless expression by protein kinase A and Hedgehog inDrosophila embryos
Authors: Ohlmeyer Johanna Talavera , Kalderon Daniel
Published: February 2008

The secreted Drosophila Hedgehog (Hh) protein induces transcription of specific genes by an unknown mechanism that requires the serpentine transmembrane protein Smoothened (Smo) and the transcription factor Cubitus interruptus (Ci). Protein kinase A (PKA) has been implicated in the mechanism of Hh signal transduction because it acts to repress Hh target genes in imaginal disc cells that express Ci. Changes in Ci protein levels, detected by an antibody that recognizes an epitope in the carboxy-terminal half of Ci, have been suggested to mediate the positive effects of Hh and the negative effects of PKA on Hh target gene expression in imaginal discs. Here we show that PKA inhibition, like Hh, leads to increased “carboxy-terminal” Ci staining and Hh target gene expression in embryos. In addition, we find that Hh and Smo can stimulate target gene expression at constant Ci levels and that increased PKA activity can induce ectopic Hh target gene expression in a manner that requires Smo and Ci activities but does not involve changes in Ci protein concentration. This suggests a branching pathway of Hh signal transduction downstream of Smo and that PKA exerts opposite effects on the two branches. Finally we show that Hh signaling in embryos does not depend on cAMP-dependent regulation of PKA activity.
The homeobox gene Distal-less induces ventral appendage development in Drosophila
Authors: Gorfinkiel Nicole , Morata Ginés , Guerrero Isabel
Published: February 2008

This study investigates the role of the homeobox geneDistal-less (Dll) in the development of the legs, antennae, and wings of Drosophila. Lack of Dll function causes a change in the identity of ventral appendage cells (legs and antennae) that often results in the loss of the appendage. Ectopic Dll expression in the proximal region of ventral appendages induces nonautonomous duplication of legs and antennae by the activation ofwingless and decapentaplegic. Ectopic Dllexpression in dorsal appendages produces transformation into corresponding ventral appendages; wings and halteres develop ectopic legs and the head–eye region develops ectopic antennae. In the wing, the exogenous Dll product induces this transformation by activating the endogenous Dll gene and repressing the wing determinant genevestigial. It is proposed that Dll induces the development of ventral appendages and also participates in a genetic address that specifies the identity of ventral appendages and discriminates the dorsal versus the ventral appendages in the adult. However, unlike other homeotic genes, Dll expression and function is not defined by a cell lineage border. Dll also performs a secondary and late function required for the normal patterning of the wing.
Oligodendrocyte precursor differentiation is perturbed in the absence of the cyclin-dependent kinase inhibitor p27Kip1
Authors: Casaccia-Bonnefil Patrizia , Tikoo Ravi , Kiyokawa Hiroaki , Friedrich Victor , Chao Moses V. , Koff Andrew
Published: February 2008

During development of the central nervous system, oligodendrocyte progenitor cells (O-2A) undergo an orderly pattern of cell proliferation and differentiation, culminating in the ability of oligodendrocytes to myelinate axons. Here we report that p27Kip1, a cyclin-dependent kinase inhibitor, is an important component of the decision of O-2A cells to withdraw from the cell cycle. In vitro, accumulation of p27 correlates with differentiation of oligodendrocytes. Furthermore, only a fraction of O-2A cells derived from p27-knockout mice differentiate successfully compared to controls. Inability to differentiate correlates with continued proliferation, suggesting that p27 is an important component of the machinery required for the G1/G0 transition in O-2A cells. In vivo, expansion of O-2A precursors before withdrawal, in part, leads to a greater number of oligodendrocytes. Together these data indicate a role for p27 during the decision to withdraw from the cell cycle in the oligodendrocyte lineage.
PARP is important for genomic stability but dispensable in apoptosis
Authors: Wang Zhao-Qi , Stingl Laura , Morrison Ciaran , Jantsch Michael , Los Marek , Schulze-Osthoff Klaus , Wagner Erwin F.
Published: February 2008

Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vivo is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells lacking this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor α, γ-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP−/− thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching andV(D)Jrecombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.
Intergenic transcription and transinduction of the human β-globin locus
Authors: Ashe Hilary L. , Monks Joan , Wijgerde Mark , Fraser Peter , Proudfoot Nick J.
Published: February 2008

We have identified novel nuclear transcripts in the human β-globin locus using nuclear run-on analysis in erythroid cell lines and in situ hybridization analysis of erythroid tissue. These transcripts extend across the LCR and intergenic regions but are undetectable in nonerythroid cells. Surprisingly, transient transfection of a β-globin gene (ε, γ, or β) induces transcription of the LCR and intergenic regions from the chromosomal β-globin locus in nonerythroid cell lines. The β-globin genes themselves, however, remain transcriptionally silent. Induction is dependent on transcription of the globin gene in the transfected plasmid but does not require protein expression. Using in situ hybridization analysis, we show that the plasmid colocalizes with the endogenous β-globin locus providing insight into the mechanism of transinduction.
Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast
Authors: Polymenis Michael , Schmidt Emmett V.
Published: February 2008

The eukaryotic cell cycle is driven by a cascade of cyclins and kinase partners including the G1 cyclin Cln3p in yeast. As the first step in this cascade, Cln3p is uniquely positioned to determine the critical growth-rate threshold for division. To analyze factors regulating CLN3 expression, we identified a short upstream open reading frame (uORF) in the 5′ leader of CLN3mRNA as a translational control element. This control element is critical for the growth-dependent regulation of Cln3p synthesis because it specifically represses CLN3 expression during conditions of diminished protein synthesis or slow growth. Inactivation of the uORF accelerates the completion of Start and entry into the cell cycle suggesting that translational regulation of CLN3 provides a mechanism coupling cell growth and division.
AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation
Authors: Fan Xinhao Cynthia , Myer Vic E. , Steitz Joan A.
Published: February 2008

AU-rich elements (AREs, usually containing repeated copies of AUUUA), when present in the 3′-untranslated regions (UTRs) of many mammalian mRNAs, confer instability on their host RNA molecules. The viral small nuclear RNA (snRNA) Herpesvirus saimiri U RNA 1 (HSUR 1) also contains an AUUUA-rich sequence. Here, we report that this ARE induces rapid degradation of HSUR 1 itself and of other snRNAs including HSUR 2 and cellular U1. Mutational analyses of the viral ARE establish that sequence requirements for mRNA and snRNA decay are the same, suggesting a similar mechanism. Moreover, the in vivo degradation activity of mutant AREs correlates with their in vitro binding to the HuR protein, implicated previously as a component of the mRNA degradation machinery. Our results suggest that ARE-mediated instability can be uncoupled from both ongoing translation and deadenylation of the target RNA.
Topoisomerase IV, not gyrase, decatenates products of site-specific recombination inEscherichia coli
Authors: Zechiedrich E. Lynn , Khodursky Arkady B. , Cozzarelli Nicholas R.
Published: February 2008

DNA replication and recombination generate intertwined DNA intermediates that must be decatenated for chromosome segregation to occur. We showed recently that topoisomerase IV (topo IV) is the only important decatenase of DNA replication intermediates in bacteria. Earlier results, however, indicated that DNA gyrase has the primary role in unlinking the catenated products of site-specific recombination. To address this discordance, we constructed a set of isogenic strains that enabled us to inhibit selectively with the quinolone norfloxacin topo IV, gyrase, both enzymes, or neither enzyme in vivo. We obtained identical results for the decatenation of the products of two different site-specific recombination enzymes, phage λ integrase and transposon Tn3 resolvase. Norfloxacin blocked decatenation in wild-type strains, but had no effect in strains with drug-resistance mutations in both gyrase and topo IV. When topo IV alone was inhibited, decatenation was almost completely blocked. If gyrase alone were inhibited, most of the catenanes were unlinked. We showed that topo IV is the primary decatenase in vivo and that this function is dependent on the level of DNA supercoiling. We conclude that the role of gyrase in decatenation is to introduce negative supercoils into DNA, which makes better substrates for topo IV. We also discovered that topo IV has an unexpectedly strong DNA relaxation activity that, together with gyrase and topo I, is able to set the supercoiling levels in Escherichia coli.
The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II
Authors: Cujec Thomas P. , Okamoto Hiroshi , Fujinaga Koh , Meyer Jon , Chamberlin Holly , Morgan David O. , Peterlin B. Matija
Published: February 2008

The human immunodeficiency virus encodes the transcriptional transactivator Tat, which binds to the transactivation response (TAR) RNA stem–loop in the viral long terminal repeat (LTR) and increases rates of elongation rather than initiation of transcription by RNA polymerase II (Pol II). In this study, we demonstrate that Tat binds directly to the cyclin-dependent kinase 7 (CDK7), which leads to productive interactions between Tat and the CDK-activating kinase (CAK) complex and between Tat and TFIIH. Tat activates the phosphorylation of the carboxy-terminal domain (CTD) of Pol II by CAK in vitro. The ability of CAK to phosphorylate the CTD can be inhibited specifically by a CDK7 pseudosubstrate peptide that also inhibits transcriptional activation by Tat in vitro and in vivo. We conclude that the phosphorylation of the CTD by CAK is essential for Tat transactivation. Our data identify a cellular protein that interacts with the activation domain of Tat, demonstrate that this interaction is critical for the function of Tat, and provide a mechanism by which Tat increases the processivity of Pol II.
Inhibition ofCaenorhabditis elegansvulval induction bygap-1and bylet-23receptor tyrosine kinase
Authors: Hajnal Alex , Whitfield Charles W. , Kim Stuart K.
Published: February 2008

During induction of theCaenorhabditis eleganshermaphrodite vulva, a signal from the anchor cell activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase signaling pathway in the vulval precursor cells. We have characterized two mechanisms that limit the extent of vulval induction. First, we found thatgap-1may directly inhibit the LET-60 Ras signaling pathway. We identified thegap-1gene in a genetic screen for inhibitors of vulval induction.gap-1is predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the signaling activity of LET-60 Ras. A loss-of-function mutation ingap-1suppresses the vulvaless phenotype of mutations in thelet-60 rassignaling pathway, but agap-1single mutant does not exhibit excess vulval induction. Second, we found thatlet-23 EGFRprevents vulval induction in a cell-nonautonomous manner, in addition to its cell-autonomous role in activating thelet-60 ras/mpk-1signaling pathway. Using genetic mosaic analysis, we show thatlet-23activity in the vulval precursor cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive signal to distal vulval precursor cells.
Alternative 3′-end processing of U5 snRNA by RNase III
Authors: Chanfreau Guillaume , Elela Sherif Abou , Ares Manuel , Guthrie Christine
Published: February 2008

The cellular components required to form the 3′ ends of small nuclear RNAs are unknown. U5 snRNA fromSaccharomyces cerevisiaeis found in two forms that differ in length at their 3′ ends (U5L and U5S). When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5. The two forms of U5 are produced in vitro by alternative 3′-end processing. A temperature-sensitive mutation in theRNT1gene encoding RNase III blocks accumulation of U5L in vivo. In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III. These results identify RNase III as atrans-acting factor involved in 3′-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.
Normal human chromosomes have long G-rich telomeric overhangs at one end
Authors: Wright Woodring E. , Tesmer Valerie M. , Huffman Kenneth E. , Levene Stephen D. , Shay Jerry W.
Published: February 2008

Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 ± 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3′ overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.
The yeast nucleoporin Rip1p contributes to multiple export pathways with no essential role for its FG-repeat region
Authors: Stutz Françoise , Kantor Julia , Zhang Dong , McCarthy Terri , Neville Megan , Rosbash Michael
Published: February 2008

The FG-repeat domain of the yeast Rip1 protein (Rip1p) was identified initially as a possible target for the nuclear export signal (NES) of the HIV-1 Rev protein in a yeast two-hybrid assay. Rip1p is inessential, associated with nuclear pore complexes, and structurally related to the FG-nucleoporin family of pore proteins. It contributes to HIV-1 Rev-mediated RNA export and is also important for the export of heat shock RNAs at 42°C. We show here that Rip1p is essential for the export of heat shock RNAs, and this function is fulfilled by the unique carboxyl terminus of Rip1p with no substantial contribution from the FG-repeat region. Genetic interactions between Rip1p and the RNA export mediator Gle1p are described, which support a role of the carboxyl terminus of Rip1p in poly(A)+RNA export. Finally, this domain of Rip1p also contributes to Rev-mediated RNA export. The data suggest that Rip1p promotes the nuclear export of different classes of substrates by contributing to optimal pore function.
c-Myc promotes differentiation of human epidermal stem cells
Authors: Gandarillas Alberto , Watt Fiona M.
Published: February 2008

The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that regulate exit from the stem cell compartment, we constitutively expressed c-Myc in primary human keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell types, activation of c-Myc in keratinocytes caused a progressive reduction in growth rate, without inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched for stem or transit amplifying cells on the basis of β1 integrin expression and by use of this method to fractionate cells prior to c-Myc activation, we found that c-Myc acted selectively on stem cells, driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis reconstituted on a dermal equivalent led to premature execution of the differentiation program. The transcriptional regulatory domain of c-Myc was required for these effects because a deletion within that domain acted as a dominant-negative mutation. Our results reveal a novel biological role for c-Myc and provide new insights into the mechanism regulating epidermal stem cell fate.
end-1 encodes an apparent GATA factor that specifies the endoderm precursor in Caenorhabditis elegans embryos
Authors: Zhu Jiangwen , Hill Russell J. , Heid Paul J. , Fukuyama Masamitsu , Sugimoto Asako , Priess James R. , Rothman Joel H.
Published: February 2008

The endoderm in the nematode Caenorhabditis elegans is clonally derived from the E founder cell. We identified a single genomic region (the endoderm-determining region, or EDR) that is required for the production of the entire C. elegans endoderm. In embryos lacking the EDR, the E cell gives rise to ectoderm and mesoderm instead of endoderm and appears to adopt the fate of its cousin, the C founder cell. end-1, a gene from the EDR, restores endoderm production in EDR deficiency homozygotes.end-1 transcripts are first detectable specifically in the E cell, consistent with a direct role for end-1 in endoderm development. The END-1 protein is an apparent zinc finger-containing GATA transcription factor. As GATA factors have been implicated in endoderm development in other animals, our findings suggest that endoderm may be specified by molecularly conserved mechanisms in triploblastic animals. We propose that end-1, the first zygotic gene known to be involved in the specification of germ layer and founder cell identity in C. elegans, may link maternal genes that regulate the establishment of the endoderm to downstream genes responsible for endoderm differentiation.
A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron
Authors: Matsuura Manabu , Saldanha Roland , Ma Hongwen , Wank Herbert , Yang Jian , Mohr Georg , Cavanagh Stacey , Dunny Gary M. , Belfort Marlene , Lambowitz Alan M.
Published: February 2008

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli.The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA inE. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression inE. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.
The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis
Authors: Kuo Chay T. , Veselits Margaret L. , Barton Kevin P. , Lu Min Min , Clendenin Cynthia , Leiden Jeffrey M.
Published: February 2008

The transcriptional programs that regulate blood vessel formation are largely unknown. In this paper, we examine the role of the zinc finger transcription factor LKLF in murine blood vessel morphogenesis and homeostasis. By in situ hybridization and immunohistochemistry, we show that LKLF is expressed as early as embryonic day 9.5 (E9.5) in vascular endothelial cells throughout the developing mouse embryo. To better understand the function of LKLF, we used homologous recombination in embryonic stem (ES) cells to generate LKLF-deficient (LKLF−/−) mice. Both angiogenesis and vasculogenesis were normal in the LKLF−/−mice. However, LKLF−/− embryos died between E12.5 and E14.5 from severe intra-embryonic and intra-amniotic hemorrhaging. This bleeding disorder was associated with specific defects in blood vessel morphology. Umbilical veins and arteries in the LKLF−/− embryos displayed an abnormally thin tunica media and aneurysmal dilatation before rupturing into the amniotic cavity. Similarly, vascular smooth muscle cells in the aortae from the LKLF−/− animals displayed a cuboidal morphology and failed to organize into a compact tunica media. Consistent with these findings, electron microscopic analyses demonstrated endothelial cell necrosis, significant reductions in the number of vessel-wall pericytes and differentiating smooth muscle cells, and decreased deposition of extracellular matrix in the LKLF−/− vessels. Despite these defects, in situ hybridization demonstrated normal expression of platelet-derived growth factor B, Tie1, Tie2, transforming growth factor β, and heparin-binding epidermal growth factor in the vasculature of the LKLF−/− embryos. Therefore, LKLF defines a novel transcriptional pathway in which endothelial cells regulate the assembly of the vascular tunica media and concomitant vessel wall stabilization during mammalian embryogenesis.
TheXenopus Brachyurypromoter is activated by FGF and low concentrations ofactivinandsuppressed by high concentrationsof activin and by paired-type homeodomain proteins
Authors: Latinkic Brancko V. , Umbhauer Muriel , Neal Kathy A. , Lerchner Walter , Smith James C. , Cunliffe Vincent
Published: February 2008

The mesoderm ofXenopus laevisarises through an inductive interaction in which signals from the vegetal hemisphere of the embryo act on overlying equatorial cells. One candidate for an endogenous mesoderm-inducing factor is activin, a member of the TGFβ superfamily. Activin is of particular interest because it induces different mesodermal cell types in a concentration-dependent manner, suggesting that it acts as a morphogen. These concentration-dependent effects are exemplified by the response ofXbra,expression of which is induced in ectodermal tissue by low concentrations of activin but not by high concentrations.Xbratherefore offers an excellent paradigm for studying the way in which a morphogen gradient is interpreted in vertebrate embryos. In this paper we examine the trancriptional regulation ofXbra2,a pseudoallele ofXbrathat shows an identical response to activin. Our results indicate that 381 bp 5′ of theXbra2transcription start site are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. We present evidence that the suppression ofXbraexpression at high concentrations of activin is mediated by paired-type homeobox genes such asgoosecoid, Mix.1,andXotx2.
Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants
Authors: Liang Chun , Stillman Bruce
Published: February 2008

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.
Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1
Authors: Saka Yasushi , Esashi Fumiko , Matsusaka Takahiro , Mochida Satoru , Yanagida Mitsuhiro
Published: February 2008

Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes ofcut5andcrb2mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.
Budding yeast centromere composition and assembly as revealed by in vivo cross-linking
Authors: Meluh Pamela B. , Koshland Douglas
Published: February 2008

The centromere–kinetochore complex is a specialized chromatin structure that mediates bipolar attachment of replicated chromosomes to the mitotic spindle, thereby ensuring proper sister chromatid separation during anaphase. The manner in which this important multimeric structure is specified and assembled within chromatin is unknown. Using in vivo cross-linking followed by immunoprecipitation, we show that the Mif2 protein of the budding yeast Saccharomyces cerevisiae, previously implicated in centromere function by genetic criteria, resides specifically at centromeric loci in vivo. This provides definitive evidence for structural conservation between yeast and mammalian centromeres, as Mif2p shares homology with CENP-C, a mammalian centromere protein. Ndc10p and Cbf1p, previously implicated in centromere function by genetic and in vitro biochemical assays, were also found to interact with centromeric DNA in vivo. By examining Mif2p, Ndc10p, and Cbf1p association with centromeric DNA derivatives, we demonstrate the existence of centromeric subcomplexes that may correspond to assembly intermediates. Based on these observations, we provide a simple model for centromere assembly. Finally, given the sensitivity of this technique, its application to other sequence-specific protein–DNA complexes within the cell, such as origins of replication and enhancer–promoter regions, could be of significant value.
A role for CKII phosphorylation of the Cactus PEST domain in dorsoventral patterning of the Drosophila embryo
Authors: Liu Zhi-Ping , Galindo Rene L. , Wasserman Steven A.
Published: February 2008

Regulated proteolysis of Cactus, the cytoplasmic inhibitor of the Rel-related transcription factor Dorsal, is an essential step in patterning of the Drosophila embryo. Signal-induced Cactus degradation frees Dorsal for nuclear translocation on the ventral and lateral sides of the embryo, establishing zones of gene expression along the dorsoventral axis. Cactus stability is regulated by amino-terminal serine residues necessary for signal responsiveness, as well as by a carboxy-terminal PEST domain. We have identifiedDrosophila casein kinase II (CKII) as a Cactus kinase and shown that CKII specifically phosphorylates a set of serine residues within the Cactus PEST domain. These serines are phosphorylated in vivo and are required for wild-type Cactus activity. Conversion of these serines to alanine or glutamic acid residues differentially affects the levels and activity of Cactus in embryos, but does not inhibit the binding of Cactus to Dorsal. Taken together, these data indicate that wild-type axis formation requires CKII-catalyzed phosphorylation of the Cactus PEST domain.