bluuebunny/crossref_metadata_2025 huggingface dataset:
First magenetic resonance imaging studies on aluminium maltolate‐treated aged New Zealand rabbits: an Alzheimer's animal model
Authors: Magisetty Obulesu , Dowlathabad Muralidhara Rao , Raichurkar Keshav P. , Mannar Shamasundar N.
Published: September 2015

Background

Alzheimer's disease is a devastative neurodegenerative disorder. To date, there has been no animal model that could unravel the complete disease pathology. Magnetic resonance imaging has played a pivotal role in the quantitative assessment of brain tissue atrophy for a few decades. In particular, temporal lobe atrophy and ventricular dilatation have been found to be sensitive in Alzheimer's disease.

Methods

The present study focused on the replication of these crucial pathological events to enable disease progression to be diagnosed at an early stage and stopped through the use of potential therapeutic strategies.

Result

The objective of this study was to show temporal lobe atrophy and ventricular dilatation in aluminium maltolate‐treated aged New Zealand rabbit, and our study was able to demonstrate this for the first time.

Conclusion

The present study makes this animal model a substantial one for further molecular level studies and opens up new targets for potential therapeutic strategies.
The role of external knowledge(s) in the introduction of product and process innovations
Authors: Antonelli Cristiano , Fassio Claudio
Published: September 2015

This paper investigates the heterogeneity of the sources of external knowledge and their differentiated effects on process and product innovations respectively. The results of the empirical investigations show that the upstream vertical sources of external knowledge from suppliers exert a strong and positive role on the introduction of process innovations, whereas horizontal and downstream vertical sources stemming respectively from competitors and customers have stronger effects on the introduction of product innovations. These results support the hypothesis that the matching between sources of external knowledge and types of innovation is necessary to implement successful innovation strategies. The study suggests that the strategic decisions of R&D managers in innovation practices should take in greater consideration the availability of the differentiated sources of external knowledge in the system in which firms are embedded.
Time allocations across collaborations of academic scientists and their impact on efforts to commercialize novel technologies: is more always better?
Authors: Libaers Dirk
Published: September 2015

We investigate how time spent in different collaborative research arrangements by academic scientists affect their propensity of involvement in the commercialization of novel, university‐originated technologies. Three common collaborative strategies used by academic scientists: (1) internal (within the research group or within the home university) research collaboration; (2) cosmopolitan research collaboration (with scientists in other US or foreign universities); or (3) university–industry research collaboration are assessed. Drawing on the concepts of cognitive and spatial distance, the empirical findings suggest that only one internal research collaboration strategy has a significant impact on the propensity of academic scientists to engage in the commercialization of novel technologies with a private firm; however, this relationship is inverse U shaped. More importantly, academic scientists adopting a university–industry collaboration strategy and spending more research time in such an arrangement have a significantly stronger propensity for being involved in technology commercialization with a private firm; however, this relationship is inverse U shaped as well. We discuss the managerial and policy implications of the findings.
TRPM2 protects against tissue damage following oxidative stress and ischaemia–reperfusion
Authors: Miller Barbara A. , Cheung Joseph Y.
Published: September 2015

TRPM channels are a subgroup of the transient receptor potential (TRP) channel superfamily whose members have important roles in cell proliferation and survival. TRPM2, the second subfamily member to be cloned, is expressed in many tissues including brain, heart, vasculature and haematopoietic cells. TRPM2 is activated by oxidative stress and several other extracellular signals including tumour necrosis factor α (TNF‐α) and amyloid β‐peptide, which increase production of ADP‐ribose (ADPR). ADPR binds to the TRPM2 C‐terminal NUDT9‐H domain, activating the channel. Early studies support the paradigm that TRPM2 activation induces cell death by sustained Ca2+ influx or by enhancing cytokine production, aggravating inflammation and tissue injury. However, more recent data show that for a number of physiological processes, TRPM2 is protective. TRPM2 protects lungs from endotoxin‐induced injury by reducing reactive oxygen species (ROS) production by phagocytes. It protects hearts from oxidative damage after ischaemia–reperfusion or hypoxia–reoxygenation by maintaining better mitochondrial bioenergetics and by decreasing ROS. Sustained Ca2+ entry through TRPM2 is required to maintain cellular bioenergetics and protect against hypoxia–reoxygenation injury. TRPM2 also protects neuroblastoma from moderate oxidative stress by decreasing ROS through increased levels of forkhead box transcription factor 3a (FOXO3a) and a downstream effector, superoxide dismutase 2. TRPM2 is important for tumour growth and cell survival through modulation of hypoxia‐inducible transcription factor expression, mitochondrial function and mitophagy. These findings in cardiac ischaemia and in neuroblastoma suggest that TRPM2 has a basic role in sustaining mitochondrial function and in cell survival that applies to a number of physiological systems and pathophysiological processes including ischaemia–reperfusion injury.

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Impact of Corrosion Test Container Material in Molten Fluorides
Authors: Olson Luke C. , Fuentes Roderick E. , Martinez-Rodriguez Michael J. , Ambrosek James W. , Sridharan Kumar , Anderson Mark H. , Garcia-Diaz Brenda L. , Gray Joshua , Allen Todd R.
Published: September 2015

The effects of crucible material choice on alloy corrosion rates in immersion tests in molten LiF–NaF–KF (46.5–11.5-42 mol. %) salt held at 850 °C for 500 hrs are described. Four crucible materials were studied. Molten salt exposures of Incoloy-800H in graphite, Ni, Incoloy-800H, and pyrolytic boron nitride (PyBN) crucibles all led to weight-loss in the Incoloy-800H coupons. Alloy weight loss was ∼30 times higher in the graphite and Ni crucibles in comparison to the Incoloy-800H and PyBN crucibles. It is hypothesized galvanic coupling between the alloy coupons and crucible materials contributed to the higher corrosion rates. Alloy salt immersion in graphite and Ni crucibles had similar weight-loss hypothesized to occur due to the rate limiting out diffusion of Cr in the alloys to the surface where it reacts with and dissolves into the molten salt, followed by the reduction of Cr from solution at the molten salt and graphite/Ni interfaces. Both the graphite and the Ni crucibles provided sinks for the Cr, in the formation of a Ni–Cr alloy in the case of the Ni crucible, and Cr carbide in the case of the graphite crucible.
Optical and Thermal Simulations of Photovoltaic Modules With and Without Sun Tracking System
Authors: Zarei Tahere , Abdolzadeh Morteza
Published: September 2015

The experimental method is extensively used to determine the temperature of a photovoltaic (PV) module at different hours of a day. In this method, the module temperature is measured using a temperature sensor mounted on the back of PV module. However, the experimental measurements have high cost and are not applicable everywhere. In this study, an optical–thermal model was used to predict all the PV module layer temperatures in two cases: tilted toward the south and fixed on a two-axis sun tracker. The impact of accurate consideration of the wind velocity and the ambient temperature on the PV module temperature was the main strength of the present simulation. This was carried out testing several correlations for prediction of convection heat transfer coefficient in the modeling process. The front and back layer temperatures as well as the silicon (Si) layer temperature of PV module were separately determined. To verify the results of the simulation, the temperatures of four PV modules measured in four different locations of the world, namely, China, Germany, Australia, and Brazil, were used. The results showed that the present study predicts the temperature of PV module more accurately compared to the previous studies. It was also shown that the average temperature errors between the measured and the predicted temperatures relative to the maximum module temperature were 2.19%, 2.3%, and 2.85%, for Australia, Brazil, and Germany, respectively.
Experimental Characterization Method of the Gas Diffusion Layers Compression Modulus for High Compressive Loads and Based on a Dynamic Mechanical Analysis
Authors: Faydi Younés , Lachat Remy , Lesage Philippe , Meyer Yann
Published: September 2015

In a proton exchange membrane fuel cell (PEMFC), gas diffusion layers (GDLs) play a major role in the overall system performances. This is the reason why many research investigations try to model and optimize the GDL physical properties. Currently, the major drawback of these models is to obtain representative GDL mechanical and physical input parameters under different excitations and, particularly, under dynamic excitations. In this paper, an experimental method using a dynamic mechanical analysis (DMA) is detailed to properly obtain the GDL Young's modulus in compression (or compression modulus) for high compressive loads under dynamic excitation. As an example, a very stiff GDL is characterized and analyzed. Only the first mechanical compression is considered. The GDL compression modulus is clearly nonlinear versus the compressive loads. The dynamic load amplitude has a strong effect on the GDL hysteretic behavior. However, the frequency value of the dynamic excitation seems to have no effect on the GDL compression modulus.
Thermal Conductivity of Self-Assembling Symmetric Block Copolymer Thin Films of Polystyrene-Block-Poly(methyl methacrylate)
Authors: George Matthew C. , Rodriguez Mark A. , Kent Michael S. , Brennecka Geoff L. , Hopkins Patrick E.
Published: September 2015

The thermal conductivities of both disordered and self-assembled symmetric polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) copolymer films were measured using time-domain thermoreflectance (TDTR). The variation in out-of-plane thermal conductivity with changing block copolymer thickness is similar to that of PMMA polymer brushes and thick spun-cast films. The results suggest that the interfaces between the PS and PMMA, and reorganization of the PS and PMMA chains around these interfaces, do not significantly affect the thermal transport in these PS-b-PMMA films. However, for thin PS-b-PMMA films, the thermal boundary resistances at the sample interfaces limit the thermal transport.
Technical Note: MRI only prostate radiotherapy planning using the statistical decomposition algorithm
Authors: Siversson Carl , Nordström Fredrik , Nilsson Terese , Nyholm Tufve , Jonsson Joakim , Gunnlaugsson Adalsteinn , Olsson Lars E.
Published: September 2015

Purpose:

In order to enable a magnetic resonance imaging (MRI) only workflow in radiotherapy treatment planning, methods are required for generating Hounsfield unit (HU) maps (i.e., synthetic computed tomography, sCT) for dose calculations, directly from MRI. The Statistical Decomposition Algorithm (SDA) is a method for automatically generating sCT images from a single MR image volume, based on automatic tissue classification in combination with a model trained using a multimodal template material. This study compares dose calculations between sCT generated by the SDA and conventional CT in the male pelvic region.

Methods:

The study comprised ten prostate cancer patients, for whom a 3D T2 weighted MRI and a conventional planning CT were acquired. For each patient, sCT images were generated from the acquired MRI using the SDA. In order to decouple the effect of variations in patient geometry between imaging modalities from the effect of uncertainties in the SDA, the conventional CT was nonrigidly registered to the MRI to assure that their geometries were well aligned. For each patient, a volumetric modulated arc therapy plan was created for the registered CT (rCT) and recalculated for both the sCT and the conventional CT. The results were evaluated using several methods, including mean average error (MAE), a set of dose‐volume histogram parameters, and a restrictive gamma criterion (2% local dose/1 mm).

Results:

The MAE within the body contour was 36.5 ± 4.1 (1 s.d.) HU between sCT and rCT. Average mean absorbed dose difference to target was 0.0% ± 0.2% (1 s.d.) between sCT and rCT, whereas it was −0.3% ± 0.3% (1 s.d.) between CT and rCT. The average gamma pass rate was 99.9% for sCT vs rCT, whereas it was 90.3% for CT vs rCT.

Conclusions:

The SDA enables a highly accurate MRI only workflow in prostate radiotherapy planning. The dosimetric uncertainties originating from the SDA appear negligible and are notably lower than the uncertainties introduced by variations in patient geometry between imaging sessions.
Characterization of Vertical Accelerations Experienced by Older People Attending an Aerobics Class Designed to Produce High Impacts
Authors: Hannam Kimberly , Deere Kevin , Worrall Sue , Hartley April , Tobias Jon H.
Published: September 2015

The purpose of this study was to establish the feasibility of using an aerobics class to produce potentially bone protective vertical impacts of ≥ 4g in older adults and to determine whether impacts can be predicted by physical function. Participants recruited from older adult exercise classes completed an SF-12 questionnaire, short physical performance battery, and an aerobics class with seven different components, performed at low and high intensity. Maximum g and jerk values were identified for each activity. Forty-one participants (mean 69 years) were included. Mean maximal values approached or exceeded the 4g threshold for four of the seven exercises. In multivariate analyses, age (−0.53; −0.77, −0.28) (standardized beta coefficient; 95% CI) and 4-m walk time (−0.39; −0.63, −0.16) were inversely related to maximum g. Aerobics classes can be used to produce relatively high vertical accelerations in older individuals, although the outcome is strongly dependent on age and physical function.
Endogenous retroviruses in ALS: A reawakening?
Authors: Brown Robert H. , Al-Chalabi Ammar
Published: September 2015

New data incriminate the human endogenous retrovirus HERV-K in the pathogenesis of ALS and document that the HERV-K envelope protein can be neurotoxic (Li
et al
., this issue).
The heavy toll of influenza
Authors: Bouvier Nicole M.
Published: September 2015

The lung cells of obese individuals may be more susceptible to severe influenza infection.
Predicting drug resistance before it occurs
Authors: Behjati Sam
Published: September 2015

Koch and colleagues define possible resistance mechanisms to a small molecule inhibitor before it is tested in cancer patients.
A Phase-Variable Surface Layer from the Gut Symbiont Bacteroides thetaiotaomicron
Authors: Taketani Mao , Donia Mohamed S. , Jacobson Amy N. , Lambris John D. , Fischbach Michael A.
Published: September 2015

The capsule from
Bacteroides
, a common gut symbiont, has long been a model system for studying the molecular mechanisms of host-symbiont interactions. The
Bacteroides
capsule is thought to consist of an array of phase-variable polysaccharides that give rise to subpopulations with distinct cell surface structures. Here, we report the serendipitous discovery of a previously unknown surface structure in
Bacteroides thetaiotaomicron
: a surface layer composed of a protein of unknown function, BT1927. BT1927, which is expressed in a phase-variable manner by ~1:1,000 cells in a wild-type culture, forms a hexagonally tessellated surface layer. The BT1927-expressing subpopulation is profoundly resistant to complement-mediated killing, due in part to the BT1927-mediated blockade of C3b deposition. Our results show that the
Bacteroides
surface structure is capable of a far greater degree of structural variation than previously known, and they suggest that structural variation within a
Bacteroides
species is important for productive gut colonization.

IMPORTANCE
Many bacterial species elaborate a capsule, a structure that resides outside the cell wall and mediates microbe-microbe and microbe-host interactions. Species of
Bacteroides
, the most abundant genus in the human gut, produce a capsule that consists of an array of polysaccharides, some of which are known to mediate interactions with the host immune system. Here, we report the discovery of a previously unknown surface structure in
Bacteroides thetaiotaomicron
. We show that this protein-based structure is expressed by a subset of cells in a population and protects
Bacteroides
from killing by complement, a component of the innate immune system. This novel surface layer protein is conserved across many species of the genus
Bacteroides
, suggesting an important role in colonization and host immune modulation.
Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion
Authors: Tenor Jennifer L. , Oehlers Stefan H. , Yang Jialu L. , Tobin David M. , Perfect John R.
Published: September 2015

The human fungal pathogen
Cryptococcus neoformans
is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of
Cryptococcus
with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of
Cryptococcus
mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that
C. neoformans
is able to penetrate the zebrafish brain following intravenous infection. By studying a
C. neoformans
FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-
C. neoformans
platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents.

IMPORTANCE
Cryptococcus neoformans
is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of
C. neoformans
infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.
Selective transfer of individual human chromosomes to recipient cells.
Authors: Saxon P J , Srivatsan E S , Leipzig G V , Sameshima J H , Stanbridge E J
Published: September 2015

Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.
Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli.
Authors: Fonzi W A , Sypherd P S
Published: September 2015

Diploid cells of Saccharomyces cerevisiae homozygous for the spe1A mutation, which eliminates ornithine decarboxylase activity, were found to sporulate at a greatly reduced frequency in the absence of polyamines. Plasmids which complement the spe1A mutation were isolated by their ability to restore sporulation competence to these cells. Three distinct plasmids were isolated. Each plasmid insert overlapped the same 8.0-kilobase region, and each plasmid restored ornithine decarboxylase activity to spe1A mutants. These plasmids also conferred ornithine decarboxylase activity to Escherichia coli EWH319 from which the ornithine decarboxylase gene is deleted. The plasmid-encoded activity expressed in E. coli resembled S. cerevisiae ornithine decarboxylase in its kinetic characteristics, indicating that the yeast ornithine decarboxylase gene was cloned. Southern blot analysis suggested that ornithine decarboxylase is a single-copy gene in S. cerevisiae. A single 2.1-kilobase transcript was demonstrated by Northern blot analysis.
A domain of methylation change at the albumin locus in rat hepatoma cell variants.
Authors: Orlofsky A , Chasin L A
Published: September 2015

A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites.
Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells.
Authors: Elliott E M , Okayama H , Sarangi F , Henderson G , Ling V
Published: September 2015

Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.
Concerted evolution of dispersed Neurospora crassa 5S RNA genes: pattern of sequence conservation between allelic and nonallelic genes.
Authors: Morzycka-Wroblewska E , Selker E U , Stevens J N , Metzenberg R L
Published: September 2015

About 100 genes coding for 5S RNA in Neurospora crassa are dispersed throughout the genome (Selker et al., Cell 24:815-818, 1981; R. L. Metzenberg, J. N. Stevens, E. U. Selker, and E. Morzycka-Wroblewska, manuscript in preparation). The majority of them correspond to the most abundant species (alpha) of 5S RNA found in the cell. Gene conversion, gene transposition, or both may be responsible for the maintenance of sequence homogeneity (concerted evolution) of alpha-type 5S genes. To explore these possibilities, we isolated and characterized separate 5S regions from two distantly related laboratory strains of N. crassa. Restriction and sequence analyses revealed no differences in molecular location of allelic 5S genes between the two strains. However, the DNA sequences around the 5S genes are ca. 10% divergent. We concluded that transposition is not frequent enough to account for the concerted evolution of N. crassa alpha-5S genes. In contrast to sequence divergence in the flanking regions between the two strains, the 5S transcribed regions are identical (with one exception), suggesting that these genes are being corrected. We have found that flanking sequences of various N. crassa 5S genes within each strain are largely different. Thus, if the correction mechanism is based on gene conversion, it is limited to the transcribed regions of the genes. However, we did find a short region of consensus including the sequence TATA located 25 to 30 nucleotides preceding the position of transcription initiation. This region may be involved in the transcription of N. crassa 5S genes.
Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.
Authors: Folger K R , Thomas K , Capecchi M R
Published: September 2015

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.
Family of developmentally regulated, maternally expressed Drosophila RNA species detected by a v-myc probe.
Authors: Madhavan K , Bilodeau-Wentworth D , Wadsworth S C
Published: September 2015

Drosophila melanogaster genomic sequences that hybridize with v-myc have been reported (B.-Z. Shilo and R. A. Weinberg, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792, 1981). We have detected Drosophila RNA sequences that also hybridize with v-myc. In an attempt to characterize these RNA sequences, we used v-myc hybridization probes to isolate Drosophila genomic segments. None of the Drosophila genomic or cDNA clones that we have isolated hybridize with the 3' exon of v-myc. Preliminary nucleotide sequence analyses have revealed sufficient homology to account for the observed hybridization between v-myc and the Drosophila clones but have failed to detect significant amino acid sequence homology. Thus is seems unlikely that the mRNA sequences or the genomic sequences that we have isolated by hybridization with v-myc represent homologs of the vertebrate myc gene. Despite the lack of structural homology between the cloned Drosophila sequences and v-myc, we have investigated the pattern of expression of those RNA species that hybridize with v-myc. Polyadenylic acid-containing transcripts of 2.7, 2.2, and 1.7 kilobases (kb) in embryos, pupae, adults, and Kc cells and an additional 1.4-kb transcript in adults were complementary to the Drosophila genomic clones and to v-myc. The 1.7- and 2.2-kb transcripts were localized on polyribosomes in Kc cells. The 1.7- and 2.2-kb transcripts were present after 45 min, 2 h, and 4 h of embryonic development, but by 16 h of development their levels had decreased by more than sixfold. During metamorphosis, two peaks of expression of the 1.7- and 2.2-kb transcripts were observed, at 6 and 72 h postpupariation. The 1.4-kb RNA species was first detected at 72 h postpupariation. In adults, the 1.7- and 2.2-kb transcripts were detected only in ovaries in females, whereas the 1.4-kb transcript was present in female nonovarian RNA and in males. These results suggest that the transcripts in early embryos are of maternal origin.
Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents.
Authors: Ruby S W , Szostak J W
Published: September 2015

When exposed to DNA-damaging agents, the yeast Saccharomyces cerevisiae induces the expression of at least six specific genes. We have previously identified one damage inducible (DIN) gene as a gene fusion (din-lacZ fusion) whose expression increases in response to DNA-damaging treatments. We describe here the identification of five additional DIN genes as din-lacZ fusions and the responses of all six DIN genes to DNA-damaging agents. Northern blot analyses of the transcripts of two of the DIN genes show that their levels increase after exposure to DNA-damaging agents. Five of the din-lacZ fusions are induced in S. cerevisiae cells exposed to UV light, gamma rays, methotrexate, or alkylating agents. One of the din-lacZ fusions is induced by either UV or methotrexate but not by the other agents. This finding suggests that there are sets of DIN genes that are regulated differently.
The Caenorhabditis elegans vitellogenin gene family includes a gene encoding a distantly related protein.
Authors: Spieth J , Blumenthal T
Published: September 2015

While the nematode Caenorhabditis elegans is more primitive than most egg-laying organisms, it's vitellogenins, or yolk protein precursors, appear to be more complex. C. elegans oocytes accumulate two major classes of yolk proteins. The first consists of two polypeptides with an Mr of about 170,000 (yp170A and yp170B) encoded by a family of five closely related genes called vit-1 through vit-5. The second class consists of two smaller proteins with Mr values of 115,000 (yp115) and 88,000 (yp88) which are cut from a single precursor. Here we report the cloning and analysis of a single-copy gene (vit-6) that encodes this precursor. The lengths of the gene and its mRNA are about 5 X 10(3) base pairs. Like vit-1 through vit-5, vit-6 is expressed exclusively in adult hermaphrodites. Comparison of portions of the coding sequence indicates that vit-6 is distantly related to the vit-1 through vit-5 gene family. Thus, even though the two classes of yolk proteins are antigenically and physically distinct, they are encoded by a single highly diverged gene family.
Inhibition of DNA-dependent transcription by the leader RNA of vesicular stomatitis virus: role of specific nucleotide sequences and cell protein binding.
Authors: Grinnell B W , Wagner R R
Published: September 2015

The leader RNA transcript of vesicular stomatitis virus inhibits transcription of the adenovirus major late promoter and virus-associated genes in a soluble HeLa cell transcription system. We examined the specific nucleotide sequence involved and the potential role of leader-protein interactions in this inhibition of RNA polymerase II- and III-directed transcription. Using synthetic oligodeoxynucleotides homologous to regions of the leader RNA molecule, we extend our previous results (B.W. Grinnell and R.R. Wagner, Cell 36:533-543, 1984) that suggest a role for the AU-rich region of the leader RNA or the homologous AT region of a cloned cDNA leader in the inhibition of DNA-dependent transcription. Our results indicate that a short nucleotide sequence (AUUAUUA) or its deoxynucleotide homolog (ATTATTA) appears to be the minimal requirement for the leader RNA to inhibit transcription by both RNA polymerases, but sequences flanking both sides of this region increase the inhibitory activity. Nucleotide changes in the homologous AT-rich region drastically decrease the transcriptional inhibitory activity. Leader RNAs from wild-type virus, but not from a 5'-defective interfering particle, form a ribonuclease-resistant, protease-sensitive ribonucleoprotein complex in the soluble HeLa cell extract. Several lines of evidence suggest that the leader RNA specifically interacts with a 65,000-dalton (65K) cellular protein. In a fractionated cell extract, only those fractions containing this 65K protein could reverse the inhibition of DNA-dependent RNA synthesis by the plus-strand vesicular stomatitis virus leader RNA or by homologous DNA. In studies with synthetic oligodeoxynucleotides homologous to leader RNA sequences, only those oligonucleotides containing the inhibitory sequence were able to bind to a gradient fraction containing the 65K protein.
Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line.
Authors: Jenh C H , Geyer P K , Johnson L F
Published: September 2015

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that over produces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20- to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)- RNA species.
Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport.
Authors: Babiss L E , Ginsberg H S , Darnell J E
Published: September 2015

Late in adenovirus infection, large amounts of viral mRNA accumulate while cell mRNA transport and translation decrease. Viruses deleted in the E1B region of type 5 adenovirus do not produce the same outcome: (i) viral mRNA synthesis by the mutants is normal, delivery to the cytoplasm is 50 to 75% of normal, but steady-state levels of viral mRNA are decreased 10-fold; (ii) cell mRNA synthesis and transport continue normally in the mutant virus-infected cell; and (iii) translation of preexisting cell mRNA which is disrupted in wild-type infection remains normal in mutant-virus-infected cells. Thus E1B proteins are required for accumulation of virus mRNA and for induction of the failure of host cell mRNA transport and translation. If a single function is involved, by inference the transport and some aspect of translation of mRNAs could be linked.
A polymorphic, prespore-specific cell surface glycoprotein is present in the extracellular matrix of Dictyostelium discoideum.
Authors: Grant W N , Welker D L , Williams K L
Published: September 2015

Polymorphisms of a major developmentally regulated prespore-specific protein (PsA) in Dictyostelium discoideum slugs are described. These polymorphisms allowed discrimination between PsA (found on the cell surface and in the extracellular matrix) and a similar extracellular but nonpolymorphic protein, ShA. The two proteins were also distinguished by their differing reactivities with a range of monoclonal antibodies and by their sensitivity to release from the sheath with cellulase. The results are discussed in terms of the molecular and genetic relationships between the cell surface and the extracellular matrix during development.
P-element-induced control mutations at the r gene of Drosophila melanogaster.
Authors: Tsubota S , Ashburner M , Schedl P
Published: September 2015

The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non-P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression.
Alternative excision products originating from a single integration of polyomavirus DNA.
Authors: Huberdeau D , Sylla B S , Herring-Gillam E , Bourgaux-Ramoisy D , Bourgaux P
Published: September 2015

The Cyp cell line consists of mouse cells transformed by a thermosensitive polyomavirus (Py) genome and routinely propagated at 39 degrees C. Cyp cells are readily induced to synthesize free Py DNA by being transferred to 33 degrees C. In one subclone (C12/a1/S48, or S48) of this line, such induction resulted in the intracellular accumulation of three discrete species of cyclic DNA, i.e., genomic Py DNA, RmI, and RmII. RmI and RmII are Py-mouse chimeras, each of which contains a distinct set of sequences originating from the site of integration. Conceivably, genomic Py DNA, RmI, and RmII could persist at 39 degrees C as free replicating plasmids or originate from distinct populations of cells in S48 cultures. The data indicated that all three species arise at 33 degrees C from a genetically homogeneous cell population in which neither RmI nor RmII replicates at 39 degrees C. Examination of the sequence at the viral-cellular junction unique to RmII indicated that this chimera is excised from the host chromosome through a recombination event involving a complex viral sequence and a simple cellular sequence. Therefore, RmII provides another example of precise recombination occurring between nonhomologous sequences in a mammalian cell, as already observed for RmI (B. S. Sylla, D. Huberdeau, D. Bourgaux-Ramoisy, and P. Bourgaux, Cell 37:661-667, 1984).
A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs.
Authors: Lorenz L J , Richter J D
Published: September 2015

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.
Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes.
Authors: Ng S Y , Gunning P , Eddy R , Ponte P , Leavitt J , Shows T , Kedes L
Published: September 2015

We have assigned six members of the human beta-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned beta-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome. Using intervening sequence probes, we showed that the functional gene is single copy and that all of the other beta-actin related sequences are recently generated in evolution and are probably processed pseudogenes. The entire nucleotide sequence of the functional gene has been determined and is identical to cDNA clones in the coding and 5' untranslated regions. We have previously reported that the 3' untranslated region is well conserved between humans and rats (Ponte et al., Nucleic Acids Res. 12:1687-1696, 1984). Now we report that four additional noncoding regions are evolutionarily conserved, including segments of the 5' flanking region, 5' untranslated region, and, surprisingly, intervening sequences I and III. These conserved sequences, especially those found in the introns, suggest a role for internal sequences in the regulation of beta-actin gene expression.
Processing of p60v-src to its myristylated membrane-bound form.
Authors: Garber E A , Cross F R , Hanafusa H
Published: September 2015

p60src of wild-type Rous sarcoma virus is myristylated at its N-terminal glycine residue. We have shown previously that this myristylation is necessary for p60src membrane association and for cell transformation by using src mutants with alterations within the N-terminal 30 kilodaltons of p60src. In this study we analyzed the process of p60src myristylation in wild type- and mutant-infected cells. All myristylated src proteins examined lack the initiator methionine, but two mutant src proteins lacking the initiator methionine are not myristylated, indicating that removal of the initiator methionine and myristylation are not obligatorily coupled. Analysis of the kinetics of myristylation and the association of p60src with cellular proteins p50 and p90 indicated that myristylation occurs before p60src becomes membrane associated and that transient association with p50 and p90 occurs regardless of myristylation. Myristylation is required for stable association of p60src with the plasma membrane but is not sufficient for membrane association. A mutant with an src deletion of amino acids 169 through 264 has an src protein that is myristylated but not membrane bound, remaining stably associated with p50 and p90. This mutant is transformation defective. Several N-terminal deletion mutants possessing tyrosine kinase activity have myristylated and membrane-bound src proteins but are not fully active in cell transformation, suggesting that additional N-terminal functional domains exist.
N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.
Authors: Cross F R , Garber E A , Hanafusa H
Published: September 2015

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.