bluuebunny/crossref_metadata_2025 huggingface dataset:
Does an extrinsic reward for R&D employees enhance innovation outcomes? Evidence from a Japanese innovation survey
Authors: Kanama Daisuke , Nishikawa Kohei
Published: October 2015

This paper examines the effects of extrinsic rewards for R&D employees on innovation outcomes based on evidence from a Japanese innovation survey. Theoretical and empirical studies present conflicting findings regarding the relationship between extrinsic rewards and innovation outcomes. This article seeks to shed light on the relationship between rewards and outcomes, as represented by the development of new products and services and their technological superiority and profitability. The analysis produced the following findings. First, companies that have introduced an evaluation system based on R&D performance are more likely to develop new products and services. The introduction of the evaluation system brings about success in product innovation with greater technological superiority. Second, monetary compensation has a negative impact on the development of new products and services and technological superiority. Third, these effects vary with the company size. Small‐ and medium‐sized companies achieve higher technological superiority with performance‐based evaluations. Large companies tend to adversely impact the development of new products and services and their technological superiority with monetary compensation.
Child Gender, Intergenerational Kinship and Parental Labor Market Outcomes
Authors: Wang Qing
Published: October 2015

This paper provides new evidence of the effects of child gender on parental labor market outcomes. Using data from the China Health and Nutrition Survey, I document a son premium on the intensive margin of parental labor supply in two‐parent families with one child. Parents with a newborn to a 6‐year‐old son have higher labor supply than parents with a daughter in the same age group. A further examination indicates that boys are likely to have better access to grandparent‐provided childcare than girls owing to grandson preference, and this allows parents with a preschool‐aged son to work more. The intensification of market work associated with having a son may affect economic outcomes over the lifecycle of parents through labor market attachment. This paper thus sheds light on the important distributional effects of family ties and culture on economic outcomes.
Rural–Urban Migration, Substitutability of Human Capital and City Productivity: Evidence from China
Authors: Yu Jixiang , Shen Kunrong , Liu Desu
Published: October 2015

Among the studies on the productivity effect of migration, the role of the substitutability of human capital between migrant workers and local workers has not received much attention. The elasticity of substitution is important for rural–urban migration in China, because there is a substantial difference in schooling quality between rural and urban regions and because there is imperfect competition in city labor markets. Using aggregate city‐level data from the 2010 China Population Census, we find that the positive effect of human capital on city productivity improves as the substitutability increases. Moreover, a grid‐search shows that the “best” estimate of the substitutability in China is between 2.1 and 2.5, far from being complete.
Outsourcing in Product Cycles
Authors: Chu Hsiao‐Lei
Published: October 2015

In this quality‐ladder product‐cycle model, a production process consists of core research and development (R&D), complementary R&D and manufacturing; and there exist northern imitation, southern innovation, and outsourcing of complementary R&D as well as manufacturing from the North to the South. It is found that a policy to protect intellectual property rights or to subsidize R&D may reduce the aggregate rate of innovation. In addition, not every globalized production strategy is innovation‐improving.
Democracy and Accession toGATT/WTO
Authors: Wong Ka‐fu , Yu Miaojie
Published: October 2015

In this paper we argue that the level of democracy of an applicant country affects the time it takes to gain General Agreement on Tariffs and Trade/World Trade Organization (GATT/WTO) accession. An extensive empirical search suggests that theGATT/WTOaccession duration is likely to be shorter for an applicant whose political regime is more democratic. Meanwhile, countries that initiated applications before 1995 took longer to accede toGATTthan those that initiated applications after 1995 to accede to theWTO. TheGATT/WTOaccession is shortened if the applicant country's economy is large. Such findings are robust to the choice of different econometric methods, data sets and model specifications.
The relationship between the Five‐Factor Model personality traits and peptic ulcer disease in a large population‐based adult sample
Authors: Realo Anu , Teras Andero , Kööts‐Ausmees Liisi , Esko Tõnu , Metspalu Andres , Allik Jüri
Published: October 2015

The current study examined the relationship between the Five‐Factor Model personality traits and physician‐confirmed peptic ulcer disease (PUD) diagnosis in a large population‐based adult sample, controlling for the relevant behavioral and sociodemographic factors. Personality traits were assessed by participants themselves and by knowledgeable informants using the NEO Personality Inventory‐3 (NEO PI‐3). When controlling for age, sex, education, and cigarette smoking, only one of the five NEO PI‐3 domain scales – higher Neuroticism – and two facet scales – lower A1: Trust and higher C1: Competence – made a small, yet significant contribution (p < 0.01) to predicting PUD in logistic regression analyses. In the light of these relatively modest associations, our findings imply that it is certain behavior (such as smoking) and sociodemographic variables (such as age, gender, and education) rather than personality traits that are associated with the diagnosis of PUD at a particular point in time. Further prospective studies with a longitudinal design and multiple assessments would be needed to fully understand if the FFM personality traits serve as risk factors for the development of PUD.
Subset Selection in Linear Regression using Sequentially Normalized Least Squares: Asymptotic Theory
Authors: Määttä Jussi , Schmidt Daniel F. , Roos Teemu
Published: October 2015

This article examines the recently proposed sequentially normalized least squares criterion for the linear regression subset selection problem. A simplified formula for computation of the criterion is presented, and an expression for its asymptotic form is derived without the assumption of normally distributed errors. Asymptotic consistency is proved in two senses: (i) in the usual sense, where the sample size tends to infinity, and (ii) in a non‐standard sense, where the sample size is fixed and the noise variance tends to zero.
Feeling the Hands of Time: Intersections of Time and Emotion
Authors: Mullaney Jamie L. , Shope Janet Hinson
Published: October 2015

While the fields of the sociology of time and the sociology of emotion have grown exponentially, missing from the literature is explicit theorizing on the intersections of emotion and time. In this review article, we begin by examining literature that explores both how time (e.g. temporal control, perceived amount) influences emotional responses and how emotions (e.g. emotional intensity, valence) impact perceptions of time. Although such research occurs in a variety of disciplines, much of it fails to look at individuals' active manipulation of time and emotion to produce desired experiences. After discussing the established concepts of emotion work (Hochschild 1979) and time work (Flaherty 2003), we illustrate how two newer concepts – “temporal emotion work” (Lois 2010, 2012) and “emoting time” (Mullaney and Shope 2012) – serve as starting points for thinking about how individuals actively use time in emotion work and emotions in time work. Although much of the thinking to date occurs within the scholarly literature on work and family, we call for more intentional and systematic theorizing about the relationship between time and emotion.
All Our Stuff: Families and Consumption
Authors: Yodanis Carrie , Schramm Michelle , Czekajlo Caroline
Published: October 2015

In this article, we provide a different lens through which to understand how we evaluate family relationships. There is general consensus that family relationships, whether between adults or between parents and children, should be rooted in love and caring. But how do we evaluate love and care? We review of literature that shows that consumption and commodities are key criteria through which we determine the quantity and quality of the love and care in family relationships.
Bestiality: An Overview and Analytic Discussion
Authors: Navarro John C. , Tewksbury Richard
Published: October 2015

Individuals who experience sexual and emotional attractions to animals are widely considered deviant in American society. Such a condition is frequently referred to as bestiality and zoophilia, which in fact describe two separate types of sexual behaviors. A multitude of studies have uncovered a range of prevalence rates across different demographic groups, which may vary due to the terminology and the type of data collection methods utilized. Dogs and horses are consistently chosen as the preferred animal to engage in sexual activities by humans, while male animals have been presented as the heavily favored sex of animal. It is questionable whether one's sexual orientation corresponds with the preference of the sex of the animal. Those engaged in human–animal sexual contacts vary in their reasoning behind their sexual behavior with animals, which are generally environmentally or belief based, and reinforce certain myths. Since the development of the Internet not only has it promoted an enhanced research perspective of the sexual behaviors of bestialists and zoophiles, but they have also found an environment populated by like‐minded individuals with similar sexual behaviors/interests.
Expanding Boundaries of Whiteness? A Look at the Marital Patterns of Part‐White Multiracial Groups
Authors: Miyawaki Michael H.
Published: October 2015

Using a boundary perspective (Alba and Nee ), I examine the marital behavior of three self‐identified multiracial groups: black/whites, American Indian/whites, Asian/whites. With a focus on marriage with whites, I assess whether the boundaries of whiteness are expanding to include these part‐white multiracial groups. Marrying whites at a large scale may signify that part‐white multiracial Americans are in the process of being accepted as “white.” At the same time, due to differences in the racial identity experiences of multiracial groups, marital patterns may differ by racial combination. Based on analysis of 2008–2012 American Community Survey data, I find that the majority of all three groups are married to whites, suggesting that most members in these groups are on the path to whiteness. On the other hand, multinomial logistic regression analysis demonstrates that American Indian/whites and Asian/whites are more likely than black/whites to have a white spouse, relative to spouses of another race/ethnicity. Moreover, separate regression analyses by multiracial group reveal gender differences in their likelihood of marrying whites for black/whites and Asian/whites. These results indicate racial stratification in the marriage market among part‐white multiracial Americans, with further stratification by gender for some groups.
Mode I Fracture Toughness of CNT‐Reinforced PMMA
Authors: Simhi T. , Banks‐Sills L. , Fourman V. , Shlayer A.
Published: October 2015

The influence of carbon nanotube (CNT) concentration on the fracture toughness of poly(methyl methacrylate) (PMMA) was examined on single‐edge V‐notched‐beam (SEVNB) specimens. Six groups of SEVNB specimens containing 0.5, 1, 2, 4, 8.5 wt% of CNTs and neat PMMA as a reference were tested. First, a notch was introduced into the specimens by a specially made disk whose edge is V‐shaped with a 30∘ angle and a 30 μm tip width. As suggested by an American Society for Testing and Materials Standard for polymers, induction of a natural crack was attempted, without success. Therefore, fracture toughness values were determined with the ‘sharp’ machined notch by means of a calibration formula. These were compared to values obtained using a stress concentration factor and found to differ by less than 3%. The latter calculation takes into account the geometry of the notch. Results showed a decrease in the fracture toughness values with an increase in the CNT concentration. For specimens in which a natural crack was attempted, referred to as a razor‐cut notch, a significant increase in the apparent fracture toughness was observed, as a result of the induced damage.
Human Norovirus prevalence in Africa: a review of studies from 1990 to 2013
Authors: Kabue Jean Pierre , Meader Emma , Hunter Paul R. , Potgieter Natasha
Published: October 2015

Objectives

To assess the contribution of Human Norovirus to diarrhoeal diseases in Africa.

Methods

We conducted a systematic review of the PubMed and EMBASE databases for published articles of Human Norovirus in Africa between 1990 and 2013. Data were extracted from selected studies and analysed.

Results

A total of 208 eligible studies were identified, of which 55 (from 19 countries) met the inclusion criteria. Many cases were of sporadic gastroenteritis (70.9%) in children (82%), 65.4% of which were seen in an outpatient setting. Over half (59.4%) of the affected children were under 5 years of age. The pooled prevalence rate of Human NoV was 11% (95% CI 8–14%), and the meta‐analysis indicated significant heterogeneity between the studies. However, the conditional negative binomial regression could not clearly find the factors affecting the Human NoV prevalence rates reported. A close relationship was found between Human Norovirus strains from environmental and clinical samples.

Conclusion

Unreported sporadic gastroenteritis cases of Human Norovirus are common in Africa. Most are community‐associated infections. Possible environmental transmission routes have been documented. Combined environmental and clinical studies are required for targeted actions to control transmission of Human Norovirus in Africa. Systematic surveillance of Human Norovirus is needed to measure the burden of Norovirus‐induced gastroenteritis in Africa and support any requirements for vaccine development.
Nitrogen isotope analysis of NaNO3 and KNO3 by nano secondary ion mass spectrometry using the 15N16O2−/14N16O2− ratio
Authors: Li Kexue , Sinha Baerbel , Hoppe Peter
Published: October 2015

The authors demonstrate that the Cameca NanoSIMS 50 ion probe is capable of measuring species specific stable nitrogen isotope ratios accurately on bulk sodium nitrate (NaNO3) and potassium nitrate (KNO3) standards deposited on gold substrate by using a Cs+ primary ion beam and the secondary molecular ion ratio 15N16O2−/14N16O2−. The typical precision in a given session is ±1.3‰ and the accuracy for long term measurements on the in-house NaNO3 standard is ±1.9‰ for a raster size of 5 × 5 μm2. The difference in the matrix specific instrument mass fractionation between NaNO3 and KNO3 is 7.1 ± 0.9‰. The results shown in this paper indicate that single micrometer sized nitrate particles can be measured accurately for N isotopic composition. This method can be used to conduct laboratory studies to better understand the isotope fractionation during reactions of NO on sea salt and dust surfaces.
Canadian High School Coaches’ Experiences, Insights, and Perceived Roles With Sport-Related Concussions
Authors: Caron Jeffrey G. , Bloom Gordon A. , Bennie Andrew
Published: October 2015

There is a need to improve concussion education and prevention efforts for youth athletes and those responsible for their care. The purpose of this study was to understand Canadian high school coaches’ insights and perceptions of concussions. Using a case study design, eight high school coaches were interviewed and the data were analysed using a hierarchical content analysis. Findings indicated that participants primarily acquired information about concussions through their own experiences as athletes and parents, and from reports in the sports media. The coaches’ felt their role with concussions was to teach athletes safety techniques during practices and competitions and to encourage them to accurately report their concussion symptoms. In addition, participants forwarded a number of recommendations to improve the dissemination of information to coaches. Results from this study will add to a limited body of concussion research with youth sport coaches. Participants’ insights provide researchers and clinicians with information about coaches’ perceived role with sport-related concussions.
IFT-A for Wnt signaling
Authors: Gough Nancy R.
Published: October 2015

The intraflagellar transport complex A (IFT-A) functions in the cytosol to promote Wnt–β-catenin signaling.
Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection
Authors: Detalle Laurent , Stohr Thomas , Palomo Concepción , Piedra Pedro A. , Gilbert Brian E. , Mas Vicente , Millar Andrena , Power Ultan F. , Stortelers Catelijne , Allosery Koen , Melero José A. , Depla Erik
Published: October 2015

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated
in vitro
neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.
Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase
Authors: Melody Kevin , McBeth Sarah , Kline Christopher , Kashuba Angela D. M. , Mellors John W. , Ambrose Zandrea
Published: October 2015

Preexposure prophylaxis (PrEP) using antiretroviral drugs is effective in reducing the risk of human immunodeficiency virus type 1 (HIV-1) infection, but adherence to the PrEP regimen is needed. To improve adherence, a long-acting injectable formulation of the nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV LA) has been developed. However, there are concerns that PrEP may select for drug-resistant mutations during preexisting or breakthrough infections, which could promote the spread of drug resistance and limit options for antiretroviral therapy. To address this concern, we administered RPV LA to macaques infected with simian immunodeficiency virus containing HIV-1 RT (RT-SHIV). Peak plasma RPV levels were equivalent to those reported in human trials and waned over time after dosing. RPV LA resulted in a 2-log decrease in plasma viremia, and the therapeutic effect was maintained for 15 weeks, until plasma drug concentrations dropped below 25 ng/ml. RT mutations E138G and E138Q were detected in single clones from plasma virus in separate animals only at one time point, and no resistance mutations were detected in viral RNA isolated from tissues. Wild-type and E138Q RT-SHIV displayed similar RPV susceptibilitiesin vitro, whereas E138G conferred 2-fold resistance to RPV. Overall, selection of RPV-resistant variants was rare in an RT-SHIV macaque model despite prolonged exposure to slowly decreasing RPV concentrations following injection of RPV LA.
CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of
pnp
Transcripts That Have Been Previously Processed by RNase III and PNPase
Authors: Park Hongmarn , Yakhnin Helen , Connolly Michael , Romeo Tony , Babitzke Paul
Published: October 2015

Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of
Escherichia coli
is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5′ untranslated leader or initial coding sequence of target transcripts.
pnp
mRNA, encoding the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a
pnp
autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5′ fragment, leads to
pnp
repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in
pnp
leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the
pnp
Shine-Dalgarno sequence such that bound CsrA causes strong repression of
pnp
translation. CsrA-mediated translational repression also leads to a small increase in the
pnp
mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation.

IMPORTANCE
CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of
pnp
by binding to two sites in the
pnp
leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the
pnp
ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link
pnp
expression to conditions affecting CsrA activity.
Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.
Authors: Qureshi S A , Alexandropoulos K , Joseph C K , Spangler R , Foster D A
Published: October 2015

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.
Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.
Authors: Milner J , Medcalf E A , Cook A C
Published: October 2015

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.
Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart.
Authors: Bacher N , Zisman Y , Berent E , Livneh E
Published: October 2015

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.
In vivo genomic footprint of a yeast centromere.
Authors: Densmore L , Payne W E , Fitzgerald-Hayes M
Published: October 2015

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.
The Xenopus B1 factor is closely related to the mammalian activator USF and is implicated in the developmental regulation of TFIIIA gene expression.
Authors: Kaulen H , Pognonec P , Gregor P D , Roeder R G
Published: October 2015

The Xenopus laevis TFIIIA promoter contains a motif that has been implicated in promoter activation in late-stage oocytes and contains the sequence (-269) CACGTG (-264). A cDNA encoding a protein (B1) that binds to this element has been cloned from X. laevis and Xenopus borealis ovarian cDNA libraries. We show that this protein is a member of the helix-loop-helix family of regulatory proteins and contains 80% sequence identity with the human adenovirus major late transcription factor (MLTF or USF). A survey of B1 protein expression during oogenesis and embryogenesis revealed both oocyte-specific and somatic cell-specific B1 protein-DNA complexes. Immunological data, RNA blot analysis, and proteolytic clipping band shift assays indicated that these complexes most likely represent altered forms of a single B1 polypeptide. Implications for TFIIIA gene regulation during development are discussed.
Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control.
Authors: Abastado J P , Miller P F , Jackson B M , Hinnebusch A G
Published: October 2015

GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcd1 mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORF1 from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcd1 cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORF1 fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORF1 translation, causing them all to reinitiate at uORF4 rather than at GCN4.
Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP.
Authors: Iyer S V , Davis D L , Seal S N , Burch J B
Published: October 2015

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.
Progesterone enhances target gene transcription by receptor free of heat shock proteins hsp90, hsp56, and hsp70.
Authors: Bagchi M K , Tsai S Y , Tsai M J , O'Malley B W
Published: October 2015

Steroid receptors regulate transcription of target genes in vivo and in vitro in a steroid hormone-dependent manner. Unoccupied progesterone receptor exists in the low-salt homogenates of target cells as a functionally inactive 8 to 10S complex with several nonreceptor components such as two molecules of 90-kDa heat shock protein (hsp90), a 70-kDa heat shock protein (hsp70), and a 56-kDa heat shock protein (hsp56). Ligand-induced dissociation of receptor-associated proteins such as hsp90 has been proposed as the mechanism of receptor activation. Nevertheless, it has not been established whether, beyond release of heat shock proteins, the steroidal ligand plays a role in modulating receptor activity. To examine whether the release of these nonreceptor proteins from receptor complex results in a constitutively active receptor, we isolated an unliganded receptor form essentially free of hsp90, hsp70, and hsp56. Using a recently developed steroid hormone-responsive cell-free transcription system, we demonstrate for the first time that the dissociation of heat shock proteins is not sufficient to generate a functionally active receptor. This purified receptor still requires hormone for high-affinity binding to a progesterone response element and for efficient transcriptional activation of a target gene. When an antiprogestin, Ru486, is bound to the receptor, it fails to promote efficient transcription. We propose that in the cell, in addition to the release of receptor-associated inhibitory proteins, a distinct hormone-mediated activation event must precede efficient gene activation.
The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.
Authors: Brown J A , Holmes S G , Smith M M
Published: October 2015

The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.
Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I.
Authors: Wooden S K , Li L J , Navarro D , Qadri I , Pereira L , Lee A S
Published: October 2015

The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) protein that can form stable associations with a variety of proteins retained in the ER because of underglycosylation or other conformational changes. In this study, we provide evidence at the transcriptional level that a conformationally abnormal protein, an altered herpes simplex virus type 1 envelope protein that is retained in the ER of a mammalian cell line, transactivates the grp78 promoter. In contrast, the normal viral envelope glycoprotein does not elevate grp78 promoter activity. Using a series of 5' deletions, linker-scanning, and internal deletion mutations spanning a 100-bp region from -179 to -80, we correlate the cis-acting regulatory elements mediating the activation of grp78 by malfolded proteins, glycosylation block, and the calcium ionophore A23187. We show that they all act through the same control elements, suggesting that they share a common signal. We report here that the highly conserved grp element, while important for basal level and induced grp78 expression, is functionally redundant. The single most important element, by linker-scanning analysis, is a 10-bp region that contains a CCAAT motif. It alone is not sufficient for promoter activity, but a 40-bp region (-129 to -90) that contains this motif is essential for mediating basal level and stress inducibility of the grp78 promoter. We show that the transcription factor CTF/NF-I is able to transactivate the grp78 promoter through interaction with this CCAAT motif.
CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus.
Authors: Rowley A , Singer R A , Johnston G C
Published: October 2015

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.
nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.
Authors: Yuan G F , Fu Y H , Marzluf G A
Published: October 2015

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.
A novel protein factor is required for use of distal alternative 5' splice sites in vitro.
Authors: Harper J E , Manley J L
Published: October 2015

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.
Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor).
Authors: Mondino A , Giordano S , Comoglio P M
Published: October 2015

The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (p190 alpha beta) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an alpha chain of 50 kDa and a beta chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (pr170). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (p190NC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) p190NC is sensitive to mild trypsin digestion of the cell surface, generating alpha and beta chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. p190NC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect.
Evidence for torsional stress in transcriptionally activated chromatin.
Authors: Leonard M W , Patient R K
Published: October 2015

The existence of torsional stress in eukaryotic chromatin has been controversial. To determine whether it could be detected, we probed the structure of an alternating AT tract. These sequences adopt cruciform geometry when the DNA helix is torsionally strained by negative supercoiling. The single-strand-specific nuclease P1 was used to determine the structure of an alternating AT sequence upstream of the Xenopus beta-globin gene when assembled into chromatin in microinjected Xenopus oocytes. The pattern of cleavage by P1 nuclease strongly suggests that the DNA in this chromatin template is under torsional stress. The cruciform was detected specifically in the most fully reconstituted templates at later stages of chromatin assembly, suggesting that negative supercoiling is associated with chromatin maturation. Furthermore, the number of torsionally strained templates increased dramatically at the time when transcription of assembled chromatin templates began. Transcription itself has been shown to induce supercoiling, but the requisite negative supercoiling for cruciform extrusion by (AT)n in oocytes was not generated in this way since the characteristic P1 cutting pattern was retained even when RNA polymerase elongation was blocked with alpha-amanitin. Thus, torsional stress is associated with transcriptional activation of chromatin templates in the absence of ongoing transcription.