bluuebunny/crossref_metadata_2025 huggingface dataset:
Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal
Authors: Kreipke R. E. , Birren S. J.
Published: September 2015

Key points

Cardiomyocytes withdraw from the cell cycle late in prenatal and early in postnatal life and subsequent heart growth occurs via cellular hypertrophy. The signals regulating this transition remain unknown.

Lesion of the neonatal sympathetic nervous system results in decreased heart size.

In vitro investigations show that sympathetic neurons delay cardiomyocyte maturation and cell cycle withdrawal.

Early sympathetic innervation may contribute to the determination of adult heart size by regulating the total number of cardiomyocytes via regulation of the proliferative/hypertrophic transition.

Early perturbations of sympathetic structure or function could have long‐term effects on adult heart function.

Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6‐hydroxydopamine (6‐OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β‐adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β‐adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.
Impact Isolation Through the Use of Compliant Interconnects for Microelectronic Packages
Authors: Chen Wei , Bhat Anirudh , Sitaraman Suresh K.
Published: September 2015

First-level and second-level compliant interconnect structures are being pursued in universities and industries to accommodate the differential displacement induced by the coefficient of thermal expansion mismatch between the die and the substrate or between the substrate and the board. The compliant interconnects mechanically decouple the die from the substrate or the substrate from the board, and thus reduce the thermally induced stresses in the assembly. This paper presents drop-test experimental and simulation data for scaled-up prototype of compliant interconnects. The simulations were based on Input-G method and performed using ANSYS® finite element software for varying drop heights. In parallel to the simulations, scaled-up compliant polymer interconnects sandwiched between a polymer die and a polymer substrate were fabricated using three-dimensional (3D) printing, and this fabrication provides a quick low-cost alternative to cleanroom fabrication. The prototype of the assembly was subjected to drop tests from varying drop heights. The response of the assembly during drop testing was captured using strain gauges and an accelerometer mounted on the prototype. The data from the experiments were compared with the predictions from the simulations. Based on such simulations, significant insight into the behavior of compliant interconnects under impact loading was obtained, which could be used for reliable design of compliant interconnect under impact loading. Both the experimental and simulation data reveal that the compliant interconnects are able to reduce the strains that transfer from substrate to die by one-order.
Characterizing Jetting in an Acoustic Fluidized Bed Using X-Ray Computed Tomography
Authors: Escudero David R. , Heindel Theodore J.
Published: September 2015

Understanding the jetting phenomena near the gas distributor plate in a fluidized bed is important to gas–solid mixing, heat and mass transfer, and erosion to any bed internals, which can all affect the performance of the bed. Moreover, acoustic vibration in a fluidized bed can be used to enhance the fluidization quality of the particulate matter and influence the jetting behavior. Characterizing the jetting structure using X-ray computed tomography (CT) in a three-dimensional (3D) fluidized bed, with and without acoustic intervention, is the focus of this study. A 10.2 cm ID fluidized bed filled with glass beads and ground walnut shell, with material densities of 2500 kg/m3 and 1440 kg/m3, respectively, and particle sizes ranging between 212 and 600 μm, is used in these experiments. X-ray CT imaging is used to determine local time-average gas holdup. From this information, qualitative and quantitative characteristics of the hydrodynamic structure of the multiphase flow system are determined. Local time-average gas holdup images of the fluidized bed under acoustic intervention at a high superficial gas velocity show that jets produced near the aeration plate merge with other jets at a higher axial position of the bed compared to the no acoustic condition. Acoustic fluidized beds also have a fewer number of active jets than the no acoustic fluidized bed, which allowed for a more homogeneous gas holdup region deep in the bed. Hence, the acoustic presence has a significant effect on the jetting phenomena near the aeration plate in a fluidized bed.
Experimental Investigation of Solid Particle Erosion in S-Bend
Authors: Mazumder Quamrul H. , Ahmed Kawshik , Zhao Siwen
Published: September 2015

Solid particle erosion is a micromechanical process that removes material from the surface. Erosion is a leading cause of failure in fluid handling equipment such as pumps and pipes. An investigation was conducted using an S-bend geometry with 12.7 mm inside diameter, r/D ratio of 1.5 with three different air velocities and two different particle sizes. This paper presents the preliminary results of an investigation to determine the location of erosion for a wide range of conditions. The experimental results showed the location of maximum erosion at 29–42 deg from the inlet at 45.72 m/s air velocity with 300 μm particle sizes.
Modeling Dilute Gas–Solid Flows Using a Polykinetic Moment Method Approach
Authors: Dunn Dennis M. , Squires Kyle D.
Published: September 2015

Modeling a dilute suspension of particles in a polykinetic Eulerian framework is described using the conditional quadrature method of moments (CQMOM). The particular regimes of interest are multiphase flows comprised of particles with diameters small compared to the smallest length scale of the turbulent carrier flow and particle material densities much larger than that of the fluid. These regimes correspond to moderate granular Knudsen number and large particle Stokes numbers in which interparticle collisions and/or particle trajectory crossing (PTC) can be significant. The probability density function (PDF) of the particle velocity space is discretized with a two-point quadrature, the minimum resolution required to capture PTC which is common to these flows. Both two-dimensional (2D) test cases (designed to assess numerical procedures) and a three-dimensional (3D) fully developed particle-laden turbulent channel flow were implemented for collisionless particles. The driving gas-phase carrier flow is computed using direct numerical simulation of the incompressible Navier–Stokes (N–S) equations and one-way coupled to the particle phase via the drag force. Visualizations and statistical descriptors demonstrate that CQMOM predicts physical features such as PTC, particle accumulation near the channel walls, and more uniform particle velocity profiles relative to the carrier flow. The improvements in modeling compared to monokinetic representations are highlighted.
Application of a Three-Dimensional Immersed Boundary Method for Free Convection From Single Spheres and Aggregates
Authors: Musong Samuel G. , Feng Zhi-Gang , Michaelides Efstathios E. , Mao Shaolin
Published: September 2015

A three-dimensional immersed boundary method (IBM) is applied for the solution of the thermal interactions between spherical particles in a viscous Newtonian fluid. At first, the free convection of an isolated isothermal sphere immersed in a viscous fluid is analyzed as a function of the Grashof number. A new correlation for the heat transfer rate from a single sphere is obtained, which is valid in the ranges 0.5 ≤ Pr ≤ 200 and 0 ≤ Gr ≤ 500. Second, the free convection heat transfer rate from pairs of spheres (bispheres) and from small spherical clusters immersed in air (Pr = 0.72) is investigated using this numerical technique. For bispheres, their orientation and the thermal plume interactions within a range of interparticle distances may cause the enhancement of the heat transfer rate above the values observed for two isolated spheres. For the simple triangular particle clusters, where the particles are in contact, it was observed that the average heat transfer rate per sphere decreases with the increased number of spheres in the cluster.
Ultrahigh-Temperature Continuous Reactors Based on Electrothermal Fluidized Bed Concept
Authors: Fedorov Sergiy S. , Singh Rohatgi Upendra , Barsukov Igor V. , Gubynskyi Mykhailo V. , Barsukov Michelle G. , Wells Brian S. , Livitan Mykola V. , Gogotsi Oleksiy G.
Published: September 2015

Authors introduce an ultrahigh-temperature (i.e., 2500–3000 °C) continuous fluidized bed furnace, in which the key operating variable is specific electrical resistance of the bed. A correlation has been established to predict the specific electrical resistance for the natural graphite-based precursors. Fluid dynamics models have been validated with the data from a fully functional prototype reactor. Data collected demonstrated that the difference between the calculated and measured values of specific resistance is approximately 25%; due to chaotic nature of electrothermal fluidized bed processes, this discrepancy was deemed acceptable. Optimizations proposed allow producing natural graphite-based end product with the purity level of 99.98 + wt. %C for battery markets.
Analysis of Turbulent Scalar Flux Models for a Discrete Hole Film Cooling Flow
Authors: Ling Julia , Ryan Kevin J. , Bodart Julien , Eaton John K.
Published: September 2015

Algebraic closures for the turbulent scalar fluxes were evaluated for a discrete hole film cooling geometry using the results from a high-fidelity large eddy simulation (LES). Several models for the turbulent scalar fluxes exist, including the widely used gradient diffusion hypothesis (GDH), the generalized GDH (GGDH), and the higher-order GDH (HOGGDH). By analyzing the results from the LES, it was possible to isolate the error due to these turbulent mixing models. Distributions of the turbulent diffusivity, turbulent viscosity, and turbulent Prandtl number were extracted from the LES results. It was shown that the turbulent Prandtl number varies significantly spatially, undermining the applicability of the Reynolds analogy for this flow. The LES velocity field and Reynolds stresses were fed into a Reynolds-averaged Navier–Stokes (RANS) solver to calculate the fluid temperature distribution. This analysis revealed in which regions of the flow various modeling assumptions were invalid and what effect those assumptions had on the predicted temperature distribution.
Lord Kelvin and Weaire–Phelan Foam Models: Heat Transfer and Pressure Drop
Authors: Cunsolo Salvatore , Iasiello Marcello , Oliviero Maria , Bianco Nicola , Chiu Wilson K. S. , Naso Vincenzo
Published: September 2015

The knowledge of thermal transport characteristics is of primary importance in the application of foams. The thermal characteristics of a foam heavily depend on its microstructure and, therefore, have to be investigated at a pore level. However, this analysis is a challenging task, because of the complex geometry of a foam. The use of foam models is a promising tool in their study. The Kelvin and the Weaire–Phelan foam models, among the most representative practical foam models, are used in this paper to numerically investigate heat transfer and pressure drop in metallic foams. They are developed in the “surface evolver” open source software. Mass, momentum, and energy equations, for air forced convection in open cell foams, are solved with a finite-element method, for different values of cell size and porosity. Heat transfer and pressure drop results are reported in terms of volumetric Nusselt number and Darcy–Weisbach friction factor, respectively. Finally, a comparison between the numerical predictions obtained with the two foam models is carried out, in order to evaluate the feasibility to substitute the more complex and computationally heavier Weaire–Phelan foam structure with the simpler Kelvin foam representation. Negligible differences between the two models are exhibited at high porosities.
Time Estimate for the Incipient Surface Melting of Regular-Shaped Metals Receiving Uniform Heat Flux Inside a Metal Melting Furnace
Authors: Marucho Marcelo D. , Campo Antonio , Ben Cheikh N.
Published: September 2015

This article addresses the continuous heating of regular-shaped metals (large plate, long cylinder, and sphere) at ambient temperature placed in a metal melting furnace. Under the assumption of temperature-independent thermophysical properties of the metal, the heat conduction problem entails to unsteady one-dimensional (1D) heat conduction with a boundary condition of uniform heat flux. Based on the exact, analytic spatiotemporal temperature distributions for the regular-shaped metals, the objective of this study is to construct simple predictive formulas so that engineers can estimate the incipient melting of these metals when heated continually. The time at which melting at the metal surface is initiated, tmelt, corresponds to setting the surface temperature, Tsur, equal to the melting temperature, Tmelt. The analysis will be done under the premises of two asymptotic solutions: one a “large-time” solution and the other a “short-time” solution. A collection of six formulas of simple form for predicting the melting time, tmelt, will be developed for those regular-shaped metals (large plate, long cylinder, and sphere).
Early infancy microbial and metabolic alterations affect risk of childhood asthma
Authors: Arrieta Marie-Claire , Stiemsma Leah T. , Dimitriu Pedro A. , Thorson Lisa , Russell Shannon , Yurist-Doutsch Sophie , Kuzeljevic Boris , Gold Matthew J. , Britton Heidi M. , Lefebvre Diana L. , Subbarao Padmaja , Mandhane Piush , Becker Allan , McNagny Kelly M. , Sears Malcolm R. , Kollmann Tobias ,   the CHILD Study Investigators, Mohn William W. , Turvey Stuart E. , Brett Finlay B.
Published: September 2015

Supplementing bacterial genera reduced in infants at high risk for asthma ameliorates lung inflammation in mice.
Novel insertion mutation in Caenorhabditis elegans.
Authors: Eide D J , Anderson P
Published: September 2015

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.
Linkage of the MBG locus to another functionally hemizygous gene locus (IDH2) on chromosome Z3 in Chinese hamster ovary cells.
Authors: Adair G M , Siciliano M J
Published: September 2015

To gain insight into the nature of hemizygosity in Chinese hamster ovary (CHO) cells and the mechanisms by which it has arisen, we are attempting to map and determine linkage relationships for as many hemizygous loci as possible. In this study, we have shown by segregation analysis of intraspecific somatic cell hybrids that the hemizygous gene locus associated with resistance to methylglyoxalbisguanyl hydrazone (MBG) in CHO cells is linked to the hemizygous IDH2 locus on chromosome Z3. Nine of the ten autosomal hemizygous gene loci that have been mapped to date in CHO cells are clustered on three chromosomes, with five such markers on chromosome 2, two on chromosome 8, and now two on the Z3 chromosome. With the mapping of MBG to the Z3 chromosome, selectable drug resistance markers are now available on eight different CHO chromosomes.
Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.
Authors: Wolf D , Harris N , Goldfinger N , Rotter V
Published: September 2015

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.
Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.
Authors: Kelly R , Kelly L J , Ennis H L
Published: September 2015

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.
Transcription unit of the rabbit beta 1 globin gene.
Authors: Rohrbaugh M L , Johnson J E , James M D , Hardison R C
Published: September 2015

We have hybridized pulse-labeled nuclear transcripts to cloned DNA fragments from the rabbit beta-like globin genes to determine the developmental timing, extent, and asymmetry of their transcription. The fetal-adult gene beta 1 was transcribed in fetal liver but not embryonic nuclei, whereas genes beta 3 and beta 4, which encode embryonic globin polypeptides, were transcribed only in embryonic nuclei. This shows that the switch from embryonic to fetal-adult globin production in rabbits is accomplished primarily by differential transcription of the beta-like globin genes. Gene beta 1 was subdivided into M13 subclones and tested for hybridization to nascent RNA. The nucleotide sequence of the 3' flanking region of gene beta 1 was also determined for 2,447 base pairs past the polyadenylation [poly(A)] site. No transcripts were found 5' to the cap site, but asymmetric transcription of gene beta 1 proceeded at a high level through the gene and past the poly(A) addition site for 603 nucleotides. The level of transcription declined after this, gradually dropping through the next 568 nucleotides. No polymerases were found on a fragment that begins 1,707 nucleotides past the poly(A) site; this fragment was part of a segment of repetitive DNA. These data show that the transcription unit of gene beta 1 begins at or near the cap nucleotide and extends at least 1,171 but no more than 1,706 nucleotides past the poly(A) addition site. The DNA segment that precedes the region of declining transcription contained an inverted repeat and encoded a short RNA transcribed by RNA polymerase II from the strand opposite the beta 1 transcript. These two features may function to attenuate the transcription of gene beta 1. An inverted repeat and a potential polymerase II transcription unit were also found in the homologous segment 3' to the human beta-globin gene. A short DNA segment close to the 3' end of the beta 1 transcription unit was transcribed more actively than the surrounding DNA, and it contained sequences that match the consensus internal control region for RNA polymerase III. This DNA segment may contain a separate polymerase III transcription unit. A member of the D repeat family located 3' to gene beta 1 was not transcribed in its entirety coordinately with beta 1.
RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation.
Authors: Naumovski L , Chu G , Berg P , Friedberg E C
Published: September 2015

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.
Structural requirements of adenovirus VAI RNA for its translation enhancement function.
Authors: Bhat R A , Domer P H , Thimmappaya B
Published: September 2015

Recently, by genetic and biochemical approaches, it has been shown that adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. To understand the nucleotide sequences and the domains of the VAI RNA that are responsible for the role of VAI RNA in enhancement of translation, a mutational analysis of the VAI gene was undertaken. Deletion, substitution, and insertion mutations covering most of the nucleotide sequences of VAI RNA were introduced into the VAI gene at the plasmid level. These mutant genes were then reintroduced into the virus, and growth properties of the mutant viruses were studied. The majority of the mutants retained normal or nearly normal levels of biological function. Mutations in the region between +43 and +53 and between +107 and the 3' end of the gene resulted in a considerable loss of activity. These mutants, however, grew significantly better than did an adenovirus type 5 mutant lacking both functional VAI and VAII genes, indicating that they retain a portion of their activity. Because no one mutation was able to completely abolish the function, we suggest that the VAI RNA may have multiple functional sites for its translation modulation function. These multiple sites may be short oligonucleotide sequences that may interact with cellular or viral components or both during translation.
The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.
Authors: Amin J , Mestril R , Lawson R , Klapper H , Voellmy R
Published: September 2015

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.
Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase.
Authors: Peterson T A , Prakash L , Prakash S , Osley M A , Reed S I
Published: September 2015

We have cloned CDC9, the structural gene for Saccharomyces cerevisiae DNA ligase, and investigated its transcriptional regulation both as a function of cell cycle stage and after UV irradiation. The steady-state level of DNA ligase mRNA increases at least fourfold in late G1, after the completion of start but before S phase. This high level of CDC9 mRNA then decays with an apparent half-life of ca. 20 min and remains at a low basal level throughout the rest of the cell cycle. The accumulation of CDC9 mRNA in late G1 is dependent upon the completion of start but not the CDC7 and CDC8 functions. Exposure of cells to UV light elicits an eightfold increase in DNA ligase mRNA levels.
Increased secretion of type beta transforming growth factor accompanies viral transformation of cells.
Authors: Anzano M A , Roberts A B , De Larco J E , Wakefield L M , Assoian R K , Roche N S , Smith J M , Lazarus J E , Sporn M B
Published: September 2015

Cells transformed by Harvey or Moloney sarcoma virus secrete at least 40 times as much type beta transforming growth factor as their respective untransformed control cells. The transformed cells bind only 20 to 50% as much type beta transforming growth factor as the control cells, suggesting that transformation causes down-regulation of the type beta transforming growth factor receptor.
Blockage of tropoelastin secretion by monensin represses tropoelastin synthesis at a pretranslational level in rat smooth muscle cells.
Authors: Frisch S M , Davidson J M , Werb Z
Published: September 2015

The blockage of protein secretion in the R22 cultured rat aortic smooth muscle cell strain with monensin repressed tropoelastin gene expression at the mRNA level by ca. 50-fold as measured by biosynthetic pulse-labeling, in vitro translation, and hybridization with a tropoelastin genomic DNA probe. These results suggest that tropoelastin gene expression is autoregulated, and they represent the first reported effect of monensin on gene expression.
Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.
Authors: Antler A M , Greenberg M E , Edelman G M , Hanafusa H
Published: September 2015

The level of phosphotyrosine in vinculin was determined in chicken embryo fibroblasts transformed by various strains of avian sarcoma virus. As previously reported (Sefton et al., Cell 24:165-174, 1981), vinculin was phosphorylated at tyrosine residues in most cultures examined, but the level varied greatly and no detectable change was found in cultures infected with Fujinami sarcoma virus or UR2 sarcoma virus. Regardless of the level of vinculin phosphorylation, the number of organized microfilament bundles was found to be decreased in all transformed cells. These results strongly suggest that tyrosine phosphorylation of vinculin is not an obligatory step in cell transformation by this class of oncogenes, nor is it correlated with the associated cytoskeletal disarray.
Stable transcription complex on a class III gene in a minichromosome.
Authors: Lassar A B , Hamer D H , Roeder R G
Published: September 2015

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.
Method of mapping DNA replication origins.
Authors: Spotila L D , Huberman J A
Published: September 2015

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.
Elimination of micronuclear specific DNA sequences early in anlagen development.
Authors: Brunk C F , Conover R K
Published: September 2015

After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop. During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen. A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated. DNAs from anlagen of various developmental stages were examined by Southern analysis. It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen. The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.
Characterization of yeast strains with conditionally expressed variants of ribosomal protein genes tcm1 and cyh2.
Authors: Fried H M , Nam H G , Loechel S , Teem J
Published: September 2015

We placed a regulatory sequence derived from the GAL10 locus of Saccharomyces cerevisiae at various distances from the start sites of transcription of two yeast ribosomal protein genes, tcm1 and cyh2. The hybrid ribosomal protein genes were transcribed at wild-type levels in the presence of galactose. In the absence of galactose, the hybrid genes were transcribed either at a reduced level or essentially not at all. Yeast cells which transcribe the ribosomal protein genes at a reduced rate continued to grow, suggesting that enhanced translation of the ribosomal protein mRNA may permit an adequate rate of synthesis of the corresponding protein. Consistent with this suggestion is the finding that preexisting mRNA decayed at a reduced rate when transcription was halted abruptly by removal of galactose. Yeast cells unable to transcribe tcm1 or cyh2 without galactose did not grow. These conditional lethal strains demonstrate that the ribosomal proteins encoded by tcm1 and cyh2 are essential; furthermore, these strains are potentially useful for isolating mutations in the tcm1 and cyh2 proteins affecting their transport, assembly, or function.
Cell-specific expression of secreted versus membrane forms of immunoglobulin gamma 2b mRNA involves selective use of alternate polyadenylation sites.
Authors: Milcarek C , Hall B
Published: September 2015

Immunoglobulin heavy chain genes encode at least two forms of mRNA, secretory- and membrane-specific. In less mature B cells and tumors arising from them, lymphomas, the membrane form of the protein and mRNA are in high abundance, while in more mature stages, plasma cells, and myeloma tumor cells, the secreted forms of protein and mRNA predominate. In myeloma cells producing approximately 8:1 ratios of secretory- to membrane-encoding forms of gamma-heavy chain mRNA, we observed equimolar transcription of the secretory- and membrane-encoding exons of the gene. In isolated nuclei from 4T001 (gamma 2b) and K23 (gamma 2a) myeloma cells, the secretory-encoding mRNA polyadenylation site was used at least three times as often as the membrane-encoding mRNA polyadenylation site. In the A20 (gamma 2a) lymphoma, which produces equal amounts of mature secretory- and membrane-encoding heavy chain mRNAs, results of experiments with isolated nuclei showed that the membrane mRNA polyadenylation site was used about two times as often as the secretory mRNA polyadenylation site. Selective use of alternate polyadenylation and cleavage sites, therefore, can modulate the production of the two mRNAs from a single gene during B cell differentiation.
Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae.
Authors: Sarokin L , Carlson M
Published: September 2015

The SUC2 gene produces two differently regulated mRNAs that encode two forms of invertase. The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose (carbon catabolite) repression, and the 1.8-kilobase mRNA encoding intracellular invertase is synthesized constitutively. Previous work has shown that the 5' noncoding region between -650 and -418 is required for derepression of secreted invertase in response to glucose deprivation. We show here that this upstream region can confer glucose-repressible expression to a heterologous gene, a LEU2-lacZ gene fusion, that is not normally regulated by glucose repression. This expression was found to respond appropriately to mutations in trans-acting genes that affect regulation of SUC2 expression. Mutations in the SNF1 through SNF6 loci reduced derepression of beta-galactosidase, and a mutation at the SSN6 locus caused constitutive expression. These findings indicate that the SUC2 upstream region mediates the regulatory effects of these genes and suggest that regulation occurs at the level of transcription. In addition, the upstream region was partially active in the inverted orientation.
Regulation of the transcript for a lysosomal protein: evidence for a gene program modified by platelet-derived growth factor.
Authors: Frick K K , Doherty P J , Gottesman M M , Scher C D
Published: September 2015

Platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize MEP, a lysosomal protein. This enhanced synthesis appears to be largely regulated by the PDGF-modulated accumulation of MEP mRNA, a 1.8-kilobase species. The increase in the MEP transcript, which is dependent on the PDGF concentration, begins 3 to 4 h after PDGF addition and is maximal at 12 h. The accumulation of the MEP transcript is growth-factor specific: PDGF and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, an agent which acts like PDGF, induce MEP RNA accumulation, whereas epidermal growth factor, somatomedin C, insulin, and whole plasma do not. A spontaneously transformed BALB/c-3T3 cell line (ST2-3T3), which does not require PDGF for growth, optimally expresses MEP RNA in the absence of PDGF. The PDGF-modulated increase in MEP RNA is unlike PDGF-modulated c-myc and c-fos RNA accumulation because it is blocked by cycloheximide, suggesting a requirement for de novo protein synthesis. It appears that PDGF modulates a program of gene expression with the accumulation of some transcripts, typified by MEP, being dependent upon the translation of others.
Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.
Authors: Singh K , Saragosti S , Botchan M
Published: September 2015

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.
Liver-specific RNA metabolism in hepatoma cells: variations in transcription rates and mRNA levels.
Authors: Clayton D F , Weiss M , Darnell J E
Published: September 2015

The transcription rate and abundance of several liver-specific mRNAs as well as mRNAs common to many cell types were compared in a series of rodent hepatoma cell lines, normal liver cells, and primary hepatocyte cultures. The rat hepatoma cell line, Fao, which displays a liver-specific phenotype, contained eight of eight liver-specific mRNAs examined. However, the transcription rates of most liver-specific mRNAs were found to be low (1 to 30%) compared with normal liver in this and other differentiated cell lines. This low rate is similar to the transcription rates of liver-specific mRNA sequences measured in primary cultures of hepatocytes. Several variant cell lines that had lost differentiated traits contained few or none of the liver-specific mRNAs; clonal descendents which had regained differentiated function regained the tissue-specific mRNAs as a group, but at various concentrations. Because all of the changes observed in mRNA levels were not accompanied by parallel changes in transcription of the same sequences, differential posttranscriptional stabilization of the liver-specific mRNAs must also occur in the different cell lines. These results qualify the utility of cultured cell lines in the study of tissue-specific transcriptional control, but raise the possibility that posttranscriptional mechanisms act in cooperation with transcriptional controls to bring the level of tissue-specific mRNAs closer to those found in liver cells.
Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.
Authors: Cartwright C A , Hutchinson M A , Eckhart W
Published: September 2015

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.
X chromosome reactivation in mouse embryonal carcinoma cells.
Authors: Paterno G D , Adra C N , McBurney M W
Published: September 2015

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.
In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.
Authors: Hanley-Bowdoin L , Orozco E M , Chua N H
Published: September 2015

The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.