bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples
Authors: Ozkoc Soykan , Tuncay Sema , Delibas Songul Bayram , Akisu Ciler , Ozbek Zeynep , Durak Ismet , Walochnik Julia
Published: February 2008

In this study, two free-living amoebae strains, Acanthamoeba genotype T4 and Paravahlkampfia sp., which were isolated from keratitis cases are presented. While the Acanthamoeba strain was isolated as a single agent, the Paravahlkampfia strain was found together with herpes simplex virus. Neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. Amoebae were detected on non-nutrient agar covered with Escherichia coli. Based on PCR-amplified 18S rRNA-gene analysis the first isolate was identified as Acanthamoeba genotype T4 and the second as Paravahlkampfia sp. In thermotolerance tests, the maximum temperature at which trophozoites continued to divide was determined as 37 °C for this Acanthamoeba strain and 35 °C for the Paravahlkampfia strain. To the best of our knowledge, the Acanthamoeba strain described herein is the second molecularly identified Acanthamoeba strain in an Acanthamoeba keratitis patient in Turkey. However, the Paravahlkampfia isolate is believed to be the first strain that has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.
α2p controls donor preference during mating type interconversion in yeast by inactivating a recombinational enhancer of chromosome III
Authors: Szeto Lisa , Fafalios Maria K. , Zhong Hualin , Vershon Andrew K. , Broach James R.
Published: February 2008

Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at theMAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, α2p: Cells containing α2p select HMR, whereas those lacking α2p selectHML. As a repressor protein, α2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two α2p/Mcm1p-binding sites, DPS1 and DPS2,control donor selection. DPS1 and DPS2 are located ∼30 kb from the left arm of chromosome III, well removed fromHML, HMR, and MAT. Precise deletion of onlyDPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in α cells. Reciprocally, deletion of only the α2p binding segments in each of these two sites results in selection of the wrong donor loci in α cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of α2p, activates HML as donor. Binding of α2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML,resulting in default selection of HMR as donor. DPS1and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.
Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization
Authors: Qiu Yuhong , Pereira Fred A. , DeMayo Francesco J. , Lydon John P. , Tsai Sophia Y. , Tsai Ming-Jer
Published: February 2008

The COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. Multiple COUP-TF members have been cloned and they share a high degree of sequence homology between species as divergent as Drosophilaand humans, suggesting a conservation of function through evolution. The COUP-TFs are highly expressed in the developing nervous systems of several species examined, indicating their possible involvement in neuronal development and differentiation. In the mouse, there are two very homologous COUP-TF genes (I and II) and their expression patterns overlap extensively. To study the physiological function of mCOUP-TFI, a gene-targeting approach was undertaken. We report here that mCOUP-TFI null animals die perinataly. Mutant embryos display an altered morphogenesis of the ninth cranial ganglion and nerve. The aberrant formation of the ninth ganglion is most possibly attributable to extra cell death in the neuronal precursor cell population. In addition, at midgestation, aberrant nerve projection and arborization were oberved in several other regions of mutant embryos. These results indicate that mCOUP-TFI is required for proper fetal development and is essential for postnatal development. Furthermore, mCOUP-TFI possesses vital physiological functions that are distinct from mCOUP-TFII despite of their high degree of homology and extensive overlapping expression patterns.
eyelidantagonizes wingless signaling during Drosophiladevelopment and has homology to the Bright family of DNA-binding proteins
Authors: Treisman Jessica E. , Luk Alvin , Rubin Gerald M. , Heberlein Ulrike
Published: February 2008

In Drosophila, pattern formation at multiple stages of embryonic and imaginal development depends on the same intercellular signaling pathways. We have identified a novel gene, eyelid(eld), which is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, eld mutations have effects opposite to those caused by wingless (wg) mutations.eld encodes a widely expressed nuclear protein with a region homologous to a novel family of DNA-binding domains. Based on this homology and on the phenotypic analysis, we suggest that Eld could act as a transcription factor antagonistic to the Wg pathway.
An anteroposterior Dorsal gradient in theDrosophila embryo
Authors: Huang Audrey M. , Rusch Jannette , Levine Michael
Published: February 2008

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. We investigate the consequences of expressing a constitutively activated form of the Toll receptor, Toll10b, in anterior regions of the early embryo using the bicoid3′ UTR. Localized Toll10b products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo. This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo.
A role for FGF-6 in skeletal muscle regeneration
Authors: Floss Thomas , Arnold Hans-Henning , Braun Thomas
Published: February 2008

Fibroblast growth factor-6 (FGF-6) belongs to a family of cytokines that control cell proliferation, cell differentiation, and morphogenetic events. Individual FGFs are either expressed widely or in a restricted pattern during embryonic, fetal, and adult life. FGF-6 exhibits a restricted expression profile predominantly in the myogenic lineage. Important functions in wound healing and tissue regeneration have been proposed for various FGFs in the past, although data from knockout mice have not supported this view. We have inactivated the FGF-6 gene in mice to investigate the role of FGF-6 in skeletal muscle development and regeneration. Wild-type mice up-regulate FGF-6 after skeletal muscle injuries and completely restore experimentally damaged skeletal muscle. In contrast, FGF-6(−/−) mutant mice show a severe regeneration defect with fibrosis and myotube degeneration. The number of MyoD- and Myogenin-expressing activated satellite cells after injury were significantly reduced in mutants. This reduction was not caused by a reduced pool of quiescent satellite cells but presumably by a lack of activation or proliferation. Interbreeding of FGF-6(−/−) mutants with mdx mice leads to striking dystrophic changes in skeletal muscles of double homozygous mice characterized by myotube degeneration, the presence of large amounts of mononuclear cells, and deposition of collagen. RNA analysis revealed an up-regulation of MyoD mRNA in mdx but not in FGF-6(−/−)/mdx double mutant mice. We conclude that FGF-6 is a critical component of the muscle regeneration machinery in mammals, possibly by stimulating or activating satellite cells.
Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium
Authors: Ginsburg Gail T. , Kimmel Alan R.
Published: February 2008

Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4− cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by another cAMP receptor, CAR1 and/or CAR3. Additional data indicate that these CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by an ancient mechanism that is shared by the Wnt/Fz circuits for dorsoventral patterning during early Xenopus development and establishing Drosophila segment polarity.
Drosophila P-element transposase is a novel site-specific endonuclease
Authors: Beall Eileen L. , Rio Donald C.
Published: February 2008

We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5′ and 3′ P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3′ cleavage site is at the end of the P-element, whereas the 5′ cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo.
An essential role for C/EBPβ in female reproduction
Authors: Sterneck Esta , Tessarollo Lino , Johnson Peter F.
Published: February 2008

A large number of intercellular signaling molecules have been identified that orchestrate female reproductive physiology. However, with the exception of steroid hormone receptors, little information exists about the transcriptional regulators that mediate cellular responses to these signals. The transcription factor C/EBPβ (CCAAT/enhancer-bindingprotein β) is expressed in ovaries and testes, as well as many other tissues of adult mice. Here we show that mice carrying a targeted deletion of the C/EBPβ gene exhibit reproductive defects. Although these animals develop normally and males are fertile, adult females are sterile. Transplantation of normal ovaries into mutant females restored fertility, thus localizing the primary reproductive defect to the ovary proper. In normal ovaries, C/EBPβ mRNA is specifically induced by luteinizing hormone (LH/hCG) in the granulosa layer of preovulatory antral follicles. C/EBPβ-deficient ovaries lack corpora lutea and fail to down-regulate expression of the prostaglandin endoperoxidase synthase 2 and P450 aromatase genes in response to gonadotropins. These findings demonstrate that C/EBPβ is essential for periovulatory granulosa cell differentiation in response to LH. C/EBPβ is thus established as a critical downstream target of G-protein-coupled LH receptor signaling and one of the first transcription factors, other than steroid hormone receptors, known to be required for ovarian follicle development in vivo.
Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage
Authors: Zou Hongyan , Wieser Rotraud , Massagué Joan , Niswander Lee
Published: February 2008

The bone morphogenetic proteins (BMPs), TGFβ superfamily members, play diverse roles in embryogenesis, but how the BMPs exert their action is unclear and how different BMP receptors (BMPRs) contribute to this process is not known. Here we demonstrate that the two type I BMPRs, BMPR-IA and BMPR-IB, regulate distinct processes during chick limb development. BmpR-IB expression in the embryonic limb prefigures the future cartilage primordium, and its activity is necessary for the initial steps of chondrogenesis. During later chondrogenesis, BmpR-IA is specifically expressed in prehypertrophic chondrocytes. BMPR-IA regulates chondrocyte differentiation, serving as a downstream mediator of Indian Hedgehog (IHH) function in both a local signaling loop and a longer-range relay system to PTHrP. BMPR-IB also regulates apoptosis: Expression of activated BMPR-IB results in increased cell death, and we showed previously that dominant-negative BMPR-IB inhibits apoptosis. Our studies indicate that in TGFβ signaling systems, different type I receptor isoforms are dedicated to specific functions during embryogenesis.
GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation inSaccharomyces cerevisiae
Authors: Huang Han-kuei , Yoon Heejeong , Hannig Ernest M. , Donahue Thomas F.
Published: February 2008

We have isolated and characterized two suppressor genes,SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus inSaccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the γ subunit of the eIF-2 complex and contains a mutation in the G2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2γ complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the β subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 ⋅ GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.
A tetramer of the Flp recombinase silences the trimers within it during resolution of a Holliday junction substrate
Authors: Lee Jehee , Jayaram Makkuni
Published: February 2008

Recombination catalyzed by the Flp site-specific recombinase involves breakage and joining of four DNA strands between two target substrates. The reaction is carried out in two steps of pairwise strand exchanges by a DNA–protein assembly in which four Flp monomers act cooperatively to execute strand cleavage and joining. Two models for recombination have been proposed. In the trimer model, the two active sites required for each step are assembled from three Flp monomers. In the tetramer (or dimer of asymmetric dimers) model, the two active sites are assembled from four Flp monomers, two monomers each contributing one active site. Experiments in which the two models challenge each other reveal that, within the Flp tetramer arranged on a Holliday junction, the two active sites required for its resolution are derived from all four, rather than three, Flp monomers. Thus, the relative protein subunit configuration of the tetramer silences the trimers within it by excluding them from assembling a functional active site pair.
Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3′-end formation in yeast
Authors: Kessler Marco M. , Henry Michael F. , Shen Elisa , Zhao Jing , Gross Stefan , Silver Pamela A. , Moore Claire L.
Published: February 2008

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3′-end processing and mRNA export from the nucleus.
A novel histidine kinase inhibitor regulating development inBacillus subtilis
Authors: Wang Ling , Grau Roberto , Perego Marta , Hoch James A.
Published: February 2008

Kinase A is the sensor histidine kinase responsible for processing postexponential phase information and providing phosphate input to the phosphorelay that activates developmental transcription via phosphorylated Spo0A. A protein inhibitor, KipI, of kinase A was discovered encoded in an operon of genes of unknown function but regulated by the availability of fixed nitrogen. KipI is a potent inhibitor of the autophosphorylation reaction of kinase A but does not inhibit phosphate transfer to the Spo0F response regulator once kinase A is phosphorylated. KipI is an inhibitor of the catalytic domain of kinase A affecting the ATP/ADP reactions and not the phosphotransferase functions of this domain. The inhibitory activity of KipI is counteracted by the product of another gene in the operon, KipA. This protein may bind to KipI, preventing its function as an inhibitor of kinase A. KipI may be the first representative of a new class of signal transduction inhibitors that function by direct interaction with the catalytic domain of histidine kinases to counteract signals influencing the “sensor” domain of such kinases. This inhibitor represents yet another way by which the phosphorelay signal transduction system is affected by negative regulators under the control of metabolic, environmental, or cell cycle influences antithetical to the initiation of developmental transcription.
TAFII250-dependent transcription of cyclin A is directed by ATF activator proteins
Authors: Wang Edith H. , Zou Sharleen , Tjian Robert
Published: February 2008

A specific mutation in TAFII250, the largest subunit of the transcription factor TFIID, disrupts cell growth control in the temperature-sensitive mutant hamster cell line ts13. Transcription from the cyclin A and D1 but not the c-fos and myc promoters is also dramatically reduced in ts13 cells at the nonpermissive temperature. These findings provide an intriguing link between TAF-mediated transcriptional regulation and cell cycle progression. Here we report the mapping of an enhancer element in the cyclin A promoter (TSRE) that responds to mutations in TAFII250. An analysis of chimeric promoter constructs reveals that the cyclin A TSRE can confer TAFII250 dependence to the core promoter of c-fos. In addition, reciprocal hybrid promoter constructs suggest that TAFII250 also contributes to the transcriptional properties of the cyclin A core promoter. We have purified and identified cellular activators that specifically bind to the TSRE and mediate transcription in a TAFII250-dependent manner. By micropeptide sequencing, we determined that TSRE-binding proteins include members of the activating transcription factor (ATF) family. These results suggest that the ts13 mutation of TAFII250 has compromised the ability of TFIID to mediate activation of transcription by specific enhancer factors such as ATF, as well as to perform certain core promoter functions. These defects in TAFII250 apparently result in the down-regulation of key molecules, such as cyclin A, which may be responsible for the ts13 cell cycle arrest phenotype.
The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coliRNA polymerase without abolishing its catalytic activities
Authors: Hung Siu Chun , Gottesman Max E.
Published: February 2008

Bacteriophage HK022 Nun protein blocks transcription elongation byEscherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3′-OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex.
Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila
Authors: Morcillo Patrick , Rosen Christina , Baylies Mary K. , Dorsett Dale
Published: February 2008

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by theDrosophila suppressor of Hairy-wingprotein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in thecutgene also appears to act interchromosomally to antagonize enhancer–promoter interactions on the homologous chromosome when activity of theChipgene is reduced. This implicatesChipin enhancer–promoter communication. We clonedChipand find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 andXenopusXldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, andChipinteracts genetically withapterous,showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair–rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer–promoter interactions.
Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p
Authors: Saavedra Claudio A. , Hammell Christopher M. , Heath Catherine V. , Cole Charles N.
Published: February 2008

We reported previously that heat or ethanol shock inSaccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.
Conversion of Dorsal from an activator to a repressor by the global corepressor Groucho
Authors: Dubnicoff Todd , Valentine Scott A. , Chen Guoqing , Shi Tao , Lengyel Judith A. , Paroush Ze’ev , Courey Albert J.
Published: February 2008

The Dorsal morphogen acts as both an activator and a repressor of transcription in the Drosophila embryo to regulate the expression of dorsal/ventral patterning genes. Circumstantial evidence has suggested that Dorsal is an intrinsic activator and that additional factors (corepressors) convert it into a repressor. These corepressors, however, have previously eluded definitive identification. We show here, via the analysis of embryos lacking the maternally encoded Groucho corepressor and via protein-binding assays, that recruitment of Groucho to the template by protein:protein interactions is required for the conversion of Dorsal from an activator to a repressor. Groucho is therefore a critical component of the dorsal/ventral patterning system.
TheArabidopsis HY5gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl
Authors: Oyama Tokitaka , Shimura Yoshiro , Okada Kiyotaka
Published: February 2008

Plant developmental processes are controlled by both endogenous programs and environmental stimuli. As a photomorphogenetic mutant,hy5ofArabidopsishas been isolated and characterized. Our detailed characterization has revealed that the mutant is deficient in a variety of stimulus responses, including gravitropic response and waving growth of roots, as well as light-dependent hypocotyl elongation. In the roots and hypocotyl, thehy5mutation also affects greening and specific cell proliferation such as lateral root formation and secondary thickening. Those phenotypes indicate that theHY5gene is responsible for the regulation of fundamental developmental processes of the plant cell: cell elongation, cell proliferation, and chloroplast development. Molecular cloning of theHY5gene using a T-DNA-tagged mutant has revealed that the gene encodes a protein with a bZIP motif, one of the motifs found in transcriptional regulators. Nuclear localization of the HY5 protein strongly suggests that theHY5gene modulates the signal transduction pathways under the HY5-related development by controlling expression of genes downstream of these pathways.
Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 andCLN2 transcription occur in response to DNA damage inSaccharomyces cerevisiae
Authors: Sidorova Julia M. , Breeden Linda L.
Published: February 2008

Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage. We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also aMEC1-, RAD53-, and RAD9-dependent delay in G1. This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to α-factor, and have lowCLN1 and CLN2 transcript levels for a longer time than untreated cells. We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels. The initial drop in CLN transcript levels in MMS is notRAD53 dependent, but the kinetics of reaccumulation ofCLN messages as cells recover from the damage is faster inrad53-11 cells than in wild type cells. This is not an indirect effect of faster progression through G1, because CLNtranscripts reaccumulate faster in rad53-11 mutants arrested in G1 as well. In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6. This indicates that both Rad53 and Swi6 are negative regulators ofCLN expression after DNA damage. Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro. On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription.
Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast
Authors: Henchoz Sandra , Chi Yong , Catarin Barbara , Herskowitz Ira , Deshaies Raymond J. , Peter Matthias
Published: February 2008

Cyclin-dependent kinase inhibitors (CKIs) play key roles in controlling the eukaryotic cell cycle by coordinating cell proliferation and differentiation. Understanding the roles of CKIs requires knowledge of how they are regulated both through the cell cycle and in response to extracellular signals. Here we show that the yeast CKI, Far1p, is controlled by ubiquitin-dependent proteolysis. Wild-type Far1p was stable only in the G1 phase of the cell cycle. Biochemical and genetic evidence indicate that its degradation required the components of the G1–S ubiquitination system, Cdc34p, Cdc4p, Cdc53p, and Skp1p. We isolated a mutant form of Far1p (Far1p-22) that was able to induce cell cycle arrest in the absence of α-factor. Cells that overexpress Far1-22p arrested in G1 as large unbudded cells with low Cdc28p–Clnp kinase activity. Wild-type Far1p, but not Far1-22p, was readily ubiquitinated in vitro in aCDC34- and CDC4-dependent manner. Far1-22p harbors a single amino acid change, from serine to proline at residue 87, which alters phosphorylation by Cdc28p–Cln2p in vitro. Our results show that Far1p is regulated by ubiquitin-mediated proteolysis and suggest that phosphorylation of Far1p by the Cdc28p–Clnp kinase is part of the recognition signal for ubiquitination.
u-shaped encodes a zinc finger protein that regulates the proneural genesachaete and scute during the formation of bristles in Drosophila
Authors: Cubadda Yolande , Heitzler Pascal , Ray Robert P. , Bourouis Marc , Ramain Philippe , Gelbart William , Simpson Pat , Haenlin Marc
Published: February 2008

The pattern of the large sensory bristles on the notum ofDrosophila arises as a consequence of the expression of theachaete and scute genes. The gene u-shapedencodes a novel zinc finger that acts as a transregulator ofachaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression ofachaete and scute. In contrast, overexpression ofu-shaped causes a loss of achaete–scute expression and consequently a loss of dorsal bristles. The effects on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The effects ofu-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute.A study of the sites at which the dorsocentral bristles arise in mosaicu-shaped nota, suggests that the levels of the u-shapedprotein are crucial for the precise positioning of the precursors of these bristles.
Transcriptional activity of Pannier is regulated negatively by heterodimerization of the GATA DNA-binding domain with a cofactor encoded by the u-shaped gene of Drosophila
Authors: Haenlin Marc , Cubadda Yolande , Blondeau Francois , Heitzler Pascal , Lutz Yves , Simpson Pat , Ramain Philippe
Published: February 2008

The genes pannier (pnr) and u-shaped(ush) are required for the regulation of achaete-scuteduring establishment of the bristle pattern in Drosophila. pnrencodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. We show here that both wild-type Pannier and the dominant mutant form activate transcription from the heterologous α globin promoter when transfected into chicken embryonic fibroblasts. Furthermore, Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr and when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. These interactions have been investigated in vivo by overexpression of the mutant and wild-type proteins. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development.
Human telomerase contains evolutionarily conserved catalytic and structural subunits
Authors: Harrington Lea , Zhou Wen , McPhail Timothy , Oulton Rena , Yeung David S.K. , Mar Vernon , Bass Michael B. , Robinson Murray O.
Published: February 2008

We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits.
Disruption of downstream chromatin directed by a transcriptional activator
Authors: Brown Steven A. , Kingston Robert E.
Published: February 2008

Promoter–proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the humanhsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. We demonstrate that activation of hsp70leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor α-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to α-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. We suggest that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation.
Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture
Authors: Andrä Kerstin , Lassmann Hans , Bittner Reginald , Shorny Sigrid , Fässler Reinhard , Propst Friedrich , Wiche Gerhard
Published: February 2008

Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin’s function in a living organism, we have disrupted its gene in mice. Plectin (−/−) mice died 2–3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (−/−) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (−/−) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation.
Mediation of NGF signaling by post-translational inhibition of HES-1, a basic helix–loop–helix repressor of neuronal differentiation
Authors: Ström Anders , Castella Paul , Rockwood Julia , Wagner John , Caudy Michael
Published: February 2008

The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process are not known. Here we show that the bHLH transcriptional repressor HES-1 is a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling.
Localization of the modified base J in telomericVSGgene expression sites ofTrypanosoma brucei
Authors: van Leeuwen Fred , Wijsman Eric R. , Kieft Rudo , van der Marel Gijs A. , van Boom Jacques H. , Borst Piet
Published: February 2008

African trypanosomes such asTrypanosoma bruceiundergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing thevariant surface glycoprotein(VSG) gene expressed. The transcribedVSGgene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change theVSGgene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously thatVSGgenes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base β-d-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomericVSGexpression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomericVSGgenes in silenced expression sites and not in actively transcribed telomericVSGgenes. J was absent from inactive chromosome-internalVSGgenes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of theVSGgene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.
Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophila eye
Authors: Domínguez María , Hafen Ernst
Published: February 2008

Patterning of the compound eye begins at the posterior edge of the eye imaginal disc and progresses anteriorly toward the disc margin. The advancing front of ommatidial differentiation is marked by the morphogenetic furrow (MF). Here we show by clonal analysis that Hedgehog (Hh), secreted from two distinct populations of cells has two distinct functions: It was well documented that Hh expression in the differentiating photoreceptor cells drives the morphogenetic furrow. Now we show that, in addition, Hh, secreted from cells at the posterior disc margin, is absolutely required for the initiation of patterning and predisposes ommatidial precursor cells to enter ommatidial assembly later. These two functions of Hh in eye patterning are similar to the biphasic requirement for Sonic Hh in patterning of the ventral neural tube in vertebrates. We show further that Hh induces ommatidial development in the absence of its secondary signals Wingless (Wg) and Dpp and that the primary function of Dpp in MF initiation is the repression of wg, which prevents ommatidial differentiation. Our results show that the regulatory relationships between Hh, Dpp, and Wg in the eye are similar to those found in other imaginal discs such as the leg disc despite obvious differences in their modes of development.
5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II
Authors: McCracken Susan , Fong Nova , Rosonina Emanuel , Yankulov Krassimir , Brothers Greg , Siderovski David , Hessel Andrew , Foster Stephen , Program Amgen EST , Shuman Stewart , Bentley David L.
Published: February 2008

We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5′ capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5′ ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation.
Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway
Authors: Caelles Carme , González-Sancho José M. , Muñoz Alberto
Published: February 2008

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Here we present evidence that hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73 and, consequently, AP-1 activation, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite to interact with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents.
The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability
Authors: Chávez Sebastián , Aguilera Andrés
Published: February 2008

The yeast HPR1 gene plays an important role in genome stability, as indicated by the observation that hpr1 mutants have high frequencies of DNA repeat recombination and chromosome loss. Here we report that HPR1 is required for transcriptional elongation. Transcription driven from constitutive and regulated yeast promoters cannot elongate through the bacterial lacZ coding region in hpr1Δ cells, but progresses efficiently through other sequences such as yeast PHO5. We show that HPR1 is not required for transcription activation and that the previously reported effects of hpr1Δon the activation of different promoters is a consequence of the incapacity of hpr1Δ cells to elongate transcription through lacZ, used as reporter. Transcriptional defects are also observed in yeast DNA sequences ofhpr1Δ cells in the presence of the transcription elongation inhibitor 6-azauracil. In all cases, the blockage of transcription elongation inhpr1Δ is associated with both the high frequency of deletions and the increase in plasmid instability that we report here. Therefore, in addition to the identification of a new element involved in transcriptional elongation, our work provides evidence for a new source of genomic instability.