bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.
Authors: Caras I W , Martin D W
Published: September 2015

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.
Genes that modify expression of major urinary proteins in mice.
Authors: Duncan R , Matthai R , Huppi K , Roderick T , Potter M
Published: September 2015

A survey of major urinary proteins (MUPs) from eight BALB/c mouse substrains by isoelectric focusing identified a common pattern with about 10 protein bands in males. One substrain, BALB/cJPt, differed in that it expressed two variant MUP patterns, designated 4.1lo and null. To find the chromosomal location of the gene which determines the 4.1lo phenotype, BALB/cJPt-MUP-4.1lo was crossed with a wild-derived Mus musculus domesticus inbred strain (CLA) that expresses the common BALB/c MUP pattern. The F1 phenotype revealed that the gene(s) controlling the MUP-4.1lo trait was recessive. A restriction fragment polymorphism between these strains found with a MUP cDNA probe allowed us to establish that a gene determining the MUP-4.1lo trait was not linked to the MUP structural genes on chromosome 4. Assays for other chromosomal marker loci revealed that a gene determining the MUP-4.1lo trait, designated Mupm-1, was closely linked to Myc-1 on chromosome 15. To determine the genetic basis of the null trait, BALB/cJPt-MUP-null mice were crossed with BALB/cJPt-MUP-4.1lo mice. A MUP restriction fragment polymorphism between these two lines was tightly linked to a gene or genes involved in determining the MUP-null phenotype. The two variant MUP phenotypes in BALB/cJ mice are determined by separate genes, one of which is located on chromosome 4 and the other on chromosome 15. The chromosomal location of Mupm-1 suggests that it produces a trans-acting factor which regulates MUP expression.
TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.
Authors: Gaber R F , Styles C A , Fink G R
Published: September 2015

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.
cis-acting translational effects of the 5' noncoding region of c-myc mRNA.
Authors: Parkin N , Darveau A , Nicholson R , Sonenberg N
Published: September 2015

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.
Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.
Authors: Conway G , Wooley J , Bibring T , LeStourgeon W M
Published: September 2015

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins A1 and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.
Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription.
Authors: Bogenhagen D F , Insdorf N F
Published: September 2015

The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA(Phe) and the displacement loop.
Template sequences required for transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters.
Authors: Bogenhagen D F , Romanelli M F
Published: September 2015

Previous work from our laboratory has shown that transcription of Xenopus laevis mitochondrial DNA initiates both in vivo and in vitro from bidirectional promoters located between the gene for tRNA(Phe) and the 5' termini of displacement loop DNA strands. A consensus sequence matching the octanucleotide ACGTTATA surrounds each transcription start site. In the present study, we used in vitro mutagenesis to define sequences required for specific transcription in vitro. First, cloned mitochondrial DNA templates generated by deletion mutagenesis were transcribed in vitro to define the limits of functional promoters. The bidirectional promoter located approximately 33 nucleotides upstream from the gene for tRNA(Phe), termed promoter 1, was studied in greatest detail. The results confirmed the hypothesis that the consensus octanucleotide sequence surrounding each start site is an essential promoter element. A duplex 18-base-pair oligonucleotide encoding the symmetrical promoter 1 region was synthesized and cloned in a plasmid vector. This synthetic oligonucleotide was sufficient to support bidirectional transcription. Point mutations within this oligonucleotide were used to identify critical residues within the consensus sequence.
Meiotic recombination between repeated transposable elements in Saccharomyces cerevisiae.
Authors: Kupiec M , Petes T D
Published: September 2015

We have measured the frequency of meiotic recombination between marked Ty elements in the Saccharomyces cerevisiae genome. These recombination events were usually nonreciprocal (gene conversions) and sometimes involved nonhomologous chromosomes. The frequency of ectopic gene conversion among Ty elements appeared lower than expected on the basis of previous studies of recombination between artificially constructed repeats. The conversion events involved either a subset of the total Ty elements in the genome or the conversion tract was restricted to a small region of the Ty element. In addition, the observed conversion events were very infrequently associated with reciprocal exchange.
The E1a gene of adenovirus type 2 reduces the metastatic potential of ras-transformed rat embryo cells.
Authors: Pozzatti R , McCormick M , Thompson M A , Khoury G
Published: September 2015

We have previously demonstrated that second-passage rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. In contrast, rat embryo cells cotransformed with the ras oncogene and the adenovirus type 2 (Ad2) E1a gene are tumorigenic but either fail to metastasize or exhibit a very low metastatic potential. In this report, we demonstrate that transfection of the Ad2 E1a gene into four independent ras-transformed rat embryo cell lines results in a dramatic reduction in metastatic potential relative to that of the parental cell line. Transfection of cDNAs for the 12S and 13S E1a transcripts showed that the 12S cDNA was highly effective in reducing the metastatic potential of ras-transformed cell lines, while the 13S cDNA showed an effect in only one of the two cell lines tested. This effect is specific to the Ad2 E1a gene, since ras-transformed cell lines expressing the Ad12 E1a gene maintained their high metastatic potential. We hypothesize that the Ad2 E1a gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype.
Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta.
Authors: Coffey R J , Bascom C C , Sipes N J , Graves-Deal R , Weissman B E , Moses H L
Published: September 2015

Transforming growth factor beta (TGF beta) is a potent inhibitor of epithelial cell proliferation. A nontumorigenic epidermal growth factor (EGF)-dependent epithelial cell line, BALB/MK, is reversibly growth arrested by TGF beta. TGF beta will also abrogate EGF-stimulated mitogenesis of quiescent BALB/MK cells. Increased levels of calcium (greater than 1.0 mM) will induce differentiation in BALB/MK cells; in contrast, TGF beta-mediated growth inhibition does not result in induction of terminal differentiation. In the present study, the effects of TGF beta and calcium on growth factor-inducible gene expression were examined. TGF beta markedly decreased c-myc and KC gene expression in rapidly growing BALB/MK cells and reduced the EGF induction of c-myc and KC in a quiescent population of cells. TGF beta exerted its control over c-myc expression at a posttranscriptional level, and this inhibitory effect was dependent on protein synthesis. TGF beta had no effect on c-fos gene expression, whereas 1.5 mM calcium attenuated EGF-induced c-fos expression in quiescent cells. Expression of beta-actin, however, was slightly increased in both rapidly growing and EGF-restimulated quiescent BALB/MK cells treated with TGF beta. Thus, in this system, TGF beta selectively reduced expression of certain genes associated with cell proliferation (c-myc and KC), and at least part of the TGF beta effect was at a posttranscriptional level.
A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.
Authors: Shih C K , Wagner R , Feinstein S , Kanik-Ennulat C , Neff N
Published: September 2015

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.
Structure and expression of B-myc, a new member of the myc gene family.
Authors: Ingvarsson S , Asker C , Axelson H , Klein G , Sümegi J
Published: September 2015

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.
Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.
Authors: Barnhart K M , Kim C G , Banerji S S , Sheffery M
Published: September 2015

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.
Determinants that contribute to cytoplasmic stability of human c-fos and beta-globin mRNAs are located at several sites in each mRNA.
Authors: Kabnick K S , Housman D E
Published: September 2015

We have analyzed the contributions to cytoplasmic stability in an mRNA species with a very short half-life (human c-fos) and an mRNA species with a very long half-life (human beta-globin). When the human c-fos promoter was used to drive the expression of human c-fos, beta-globin, and chimeric DNAs between c-fos and beta-globin in transfected cells, a pulse of mRNA synthesis was obtained following induction of transcription by refeeding quiescent cells with medium containing 15% calf serum. The mRNA half-life was determined by using Northern (RNA) blot analysis of mRNAs prepared at various times following the pulse of transcription. Under these conditions human c-fos mRNA exhibited a half-life of 6.6 min and human beta-globin mRNA exhibited a half-life of 17.5 h. Replacement of the 3' end of the c-fos mRNA with the 3' end of the beta-globin mRNA increased the half-life of the resultant RNA from 6.6 to 34 min. The reciprocal chimera had a half-life of 34.6 min compared with the 17.5-h half-life of beta-globin mRNA. These results suggest that sequences which make a major contribution to mRNA stability reside in the 3' end of either or both molecules. A chimera in which the 5' untranslated region of globin was replaced by part of the 5' untranslated region of fos led to destabilization of the encoded mRNA. This construct produced an mRNA with a half-life of 6.8 h instead of the 17.5-h half-life of globin. This result suggests that additional determinants of stability reside in the 5' end of these mRNA molecules. Substitution of part of the 5' untranslated region of fos by the 5' untranslated region of beta-globin yielded an mRNA with stability similar to fos mRNA. These results suggest that interactions among sequences within each mRNA contribute to the stability of the respective molecules.
Herpes simplex virus induces the replication of foreign DNA.
Authors: Danovich R M , Frenkel N
Published: September 2015

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.
Insertional activation of a promoterless thymidine kinase gene.
Authors: Hiller S , Hengstler M , Kunze M , Knippers R
Published: September 2015

A plasmid carrying a promoterless herpes simplex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk-) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, we first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. We found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, two CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient for full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.
Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.
Authors: Kane S E , Troen B R , Gal S , Ueda K , Pastan I , Gottesman M M
Published: September 2015

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.
Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.
Authors: Gould K L , Hunter T
Published: September 2015

We have shown previously that pp60c-src is a substrate for protein kinase C in vivo and that the target of protein kinase C phosphorylation in mammalian pp60c-src is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2 alpha) lead to the phosphorylation of pp60c-src at serine 12. In addition to stimulating serine 12 phosphorylation in pp60c-src, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.
Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.
Authors: Kozarsky K F , Call S M , Dower S K , Krieger M
Published: September 2015

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.
Transcriptional and posttranscriptional modulation of myeloid colony-stimulating factor expression by tumor necrosis factor and other agents.
Authors: Koeffler H P , Gasson J , Tobler A
Published: September 2015

Granulocyte (G) and granulocyte-macrophage (GM) colony-stimulating factors (CSF) are necessary for proliferation and differentiation of myeloid hematopoietic cells. Fibroblasts stimulated by tumor necrosis factor alpha (TNF alpha) and several other agents are a rich source of these CSF. The GM-CSF synthesized by these cells had the same molecular weight and glycosylation pattern as that produced by activated T lymphocytes, as shown by [35S]methionine labeling studies. Northern (RNA) blot analysis showed that the fibroblasts had trace levels of G- and GM-CSF mRNA. Both G- and GM-CSF mRNA concentrations coordinately increased after exposure of the cells to TNF alpha (greater than or equal to 5 ng/ml), 12-O-tetradecanoylphorbol 13-acetate (TPA) (greater than or equal to 5 x 10(-10) M), or cycloheximide (20 micrograms/ml). Both TNF alpha and TPA increased levels of G- and GM-CSF mRNA in the absence of new protein synthesis. Transcriptional run-on studies demonstrated that fibroblasts constitutively transcribed GM-CSF, and transcription was enhanced 3.0-fold by TNF alpha and 2.5-fold by TPA and was unchanged by cycloheximide. The stability of G- and GM-CSF transcripts was determined after exposure of the cells to actinomycin D; the half-lives of G- and GM-CSF mRNA in unstimulated cells were less than 0.25 h and were increased 2- to 16-fold in cells cultured with TNF, TPA, or cycloheximide. In summary, both transcriptional and posttranscriptional signals acted coordinately to modulate the levels of G- and GM-CSF mRNAs in fibroblasts.
Purification and characterization of human mitochondrial transcription factor 1.
Authors: Fisher R P , Clayton D A
Published: September 2015

We purified to near homogeneity a transcription factor from human KB cell mitochondria. This factor, designated mitochondrial transcription factor 1 (mtTF1), is required for the in vitro recognition of both major promoters of human mitochondrial DNA by the homologous mitochondrial RNA polymerase. Furthermore, it has been shown to bind upstream regulatory elements of the two major promoters. After separation from RNA polymerase by phosphocellulose chromatography, mtTF1 was chromatographed on a MonoQ anion-exchange fast-performance liquid chromatography column. Analysis of mtTF1-containing fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single major polypeptide with an Mr of approximately 25,000. Centrifugation in analytical glycerol gradients indicated a sedimentation coefficient of approximately 2.5 S, consistent with a monomeric 25-kilodalton protein. Finally, when the 25-kilodalton polypeptide was excised from a stained sodium dodecyl sulfate-polyacrylamide gel and allowed to renature, it regained DNA-binding and transcriptional stimulatory activities at both promoters. Although mtTF1 is the only mitochondrial DNA-binding transcription factor to be purified and characterized, its properties, such as a high affinity for random DNA and a weak specificity for one of its target sequences, may typify this class of regulatory proteins.
Heat shock protection against cold stress of Drosophila melanogaster.
Authors: Burton V , Mitchell H K , Young P , Petersen N S
Published: September 2015

Heat shock protein synthesis can be induced during recovery from cold treatment of Drosophila melanogaster larvae. Survival of larvae after a cold treatment is dramatically improved by a mild heat shock just before the cold shock. The conditions which induce tolerance to cold are similar to those which confer tolerance to heat.
p21-ras effector domain mutants constructed by "cassette" mutagenesis.
Authors: Stone J C , Vass W C , Willumsen B M , Lowy D R
Published: September 2015

A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could not be altered, even conservatively, without a loss of function (codons 32 and 35); one which retained detectable biologic activity with conservative changes but which lost function with more drastic substitutions (codons 36 and 40); and one which retained function even with a nonconservative substitution (codon 39).
Transpositional competence and transcription of endogenous Ty elements in Saccharomyces cerevisiae: implications for regulation of transposition.
Authors: Curcio M J , Sanders N J , Garfinkel D J
Published: September 2015

Transposition of Ty elements in the yeast Saccharomyces cerevisiae occurs through an RNA intermediate. Although Ty RNA accounts for 5 to 10% of the total polyadenylated RNA in a haploid cell, the transposition frequency is only 10(-7) to 10(-8) per gene. To determine whether Ty elements native to the yeast genome are transpositionally competent, two elements were fused to the GAL1 promoter and tested for their ability to transpose. These native elements, Ty1-588 and Ty2-117, transposed at high levels when the GAL1 promoter was induced. Three Ty's identified as spontaneous transpositions in specific target genes were also tested. Of these three, Ty2-917 and the previously characterized element Ty1-H3 were shown to be transpositionally competent. The third element, Ty1-H1, was transposition defective. In addition, we marked the chromosomal copy of Ty1-588 with the NEO gene and demonstrated that Ty1-588NEO was actively transcribed in yeast cells. Ty1-588NEO transcription was regulated by the SPT3 and MAT loci in the same manner as that observed for Ty's collectively. These results indicate that the yeast genome contains functional Ty elements. The presence of a transpositionally competent, actively transcribed element suggests that regulation of Ty transposition occurs at a posttranscriptional level.
Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.
Authors: Kondo K , Hodgkin J , Waterston R H
Published: September 2015

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.
Transcription of HLA class II genes in the absence of B-cell-specific octamer-binding factor.
Authors: Stimac E , Lyons S , Pious D
Published: September 2015

HLA-DR and other human class II histocompatibility genes are expressed by Epstein-Barr virus-transformed B-lymphocyte cell lines but not by most T-cell leukemia lines. We determined by transcriptional run-on experiments that regulation of class II expression in these cells is at the level of gene transcription; nuclei isolated from B-cell lines actively transcribe class II mRNA, whereas nuclei from non-class II-expressing T-cell lines and from the class II transactive factor-deficient B-cell mutant 6.1.6 do not. In searching for DNA-binding proteins which might regulate transcription, we found both a ubiquitous (B1) and a B-cell-specific (B2) factor which bind to the octamer sequence ATTTGCAT 52 base pairs 5' of the cap site in the DR alpha gene. We examined the relationship of these factors to DR alpha transcription. HUT-78, a T-cell line which expresses class II mRNA constitutively, contains only the ubiquitous B1 octamer-binding factor also found in non-class II-expressing T-cell leukemias. Human fibroblast, HeLa, and melanoma cell lines similarly contain only the ubiquitous factor, even when these cells are induced to express class II mRNA by treatment with gamma interferon. Both B1 and B2 binding factors are present in the B-cell mutant 6.1.6, which nevertheless fails to transcribe class II mRNA. Although we have not ruled out the requirement of B-cell-specific octamer-binding factor B2 for class II expression in B cells, it is clear that in other cells substantial DR alpha transcription occurs in the absence of this factor.
Key features of heat shock regulatory elements.
Authors: Amin J , Ananthan J , Voellmy R
Published: September 2015

The promoters of heat shock protein genes are among the best-studied inducible eucaryotic promoters. Regions responsible for heat regulation have been identified previously by deletion experiments with several different heat shock genes. In this paper the critical importance of two novel features of heat shock regulatory elements was investigated. First, the elements were modular and, as a consequence, displayed a characteristic 5-nucleotide periodicity produced by multiple GAA blocks that were arranged in alternating orientations and at 2-nucleotide intervals. Functional heat shock regulatory elements appeared to include three or more of these blocks. Second, the nucleotides at the two positions immediately upstream from GAA segments played an important role in defining the competence of regulatory elements.
Purification and characterization of TTFI, a factor that mediates termination of mouse ribosomal DNA transcription.
Authors: Bartsch I , Schoneberg C , Grummt I
Published: September 2015

Termination of rRNA gene transcription is dependent on an 18-base-pair sequence motif, AGGTCGAC CAG AT TA NTCCG (the Sal box), which is present several times in the spacer region downstream of the 3' end of the pre-rRNA coding region. We report here the purification to molecular homogeneity of a nuclear factor which specifically interacts with the Sal box element. Addition of the isolated protein to S-100 extracts which contain low levels of the Sal box-binding protein and are therefore termination incompetent restores terminating activity, indicating that this protein is a polymerase I-specific transcription termination factor. The purified protein (termed TTFI) has a molecular weight of approximately 105,000 on sodium dodecyl sulfate-polyacrylamide gels. Mild proteolysis generates a relatively protease-resistant core which still specifically recognizes its target sequence. However, the termination activity has been lost, suggesting that the interaction with the DNA and the interaction with the transcription apparatus reside in different protein domains.
Differential expression of a tropomyosin isoform in low- and high-metastatic Lewis lung carcinoma cells.
Authors: Takenaga K , Nakamura Y , Sakiyama S
Published: September 2015

Two-dimensional electrophoretograms of newly synthesized polypeptides from low-metastatic (P29) and high-metastatic (D6) Lewis lung carcinoma cells were compared. The results showed that the synthesis of tropomyosin 2 (TM2) was significantly less in D6 cells than in P29 cells. Furthermore, suppression of TM2 synthesis was induced in P29 cells during incubation in medium containing dimethyl sulfoxide or butyric acid, which induced the metastatic phenotype of P29 cells. These results suggest that the suppression of TM2 synthesis is linked to the metastatic potential of Lewis lung carcinoma cells.
Gallstone is correlated with an increased risk of idiopathic sudden sensorineural hearing loss: a retrospective cohort study
Authors: Chiu Chong-Chi , Lee Kuan-Ji , Weng Shih-Feng , Yang Yuan-Ming , Lin Yung-Song
Published: September 2015

Objectives

This study aims to test the hypothesis that gallstone disease (GSD) is a risk factor for the development of idiopathic sudden sensorineural hearing loss (ISSNHL). Research has shown risks of cardiovascular and cerebrovascular events in patients with GSD; however, well-conducted English studies on the association between GSD and the development of ISSNHL are lacking.

Design and setting

Retrospective cohort study using the Taiwan Longitudinal Health Insurance Database.

Participants

We compared 26 449 patients diagnosed with GSD between 1 January 2001 and 31 December 2007, with 52 898 age-matched, gender-matched and comorbidities propensity scores-matched controls.

Outcome measured

We followed each patient until the end of 2011 and evaluated the incidence of ISSNHL for at least 4 years after the initial GSD diagnosis.

Results

The incidence of ISSNHL was 1.42 times higher in the GS cohort than in the non-GS cohort (9.27 vs 6.52/10 000 person-years). Using Cox proportional hazard regressions, the adjusted HR was 1.44 (95% CI 1.19 to 1.74). In the cohort of patients with GSD who needed a cholecystectomy, 37 patients suffered from ISSNHL. Among those patients, 31 (83.7%) patients sustained ISSNHL before cholecystectomy and 6 (16.2%) patients sustained ISSNHL after cholecystectomy.

Conclusions

A diagnosis of GSD may be an independent risk for ISSNHL. This finding suggests that an underlying vascular and inflammatory mechanism may contribute to the development of ISSNHL. Physicians may want to counsel patients with GSD to seek medical attention if they have hearing impairments, because patients may be at an increased risk of developing ISSNHL.
The importance of tissue handling of surgically removed breast cancer for an accurate assessment of the Ki-67 index
Authors: Arima Nobuyuki , Nishimura Reiki , Osako Tomofumi , Nishiyama Yasuyuki , Fujisue Mamiko , Okumura Yasuhiro , Nakano Masahiro , Tashima Rumiko , Toyozumi Yasuo
Published: September 2015

Aim

Insufficient attention for the Ki-67 immunohistochemistry has been given to the importance of tissue handling for surgical breast cancer specimens. We sought to investigate the effect of fixation status on the Ki-67.

Methods

We examined the effect of fixative, time to and duration of fixation using surgical specimens, and finally, compared the paired Ki-67 index in the tumour between core needle and surgical specimen.

Results

The Ki-67 was significantly higher when 10% neutral buffered formalin was used (p=0.0276). Insufficient fixation caused a drastic reduction in the Ki-67 index (p=0.0177), but not significant in oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Sixteen hours delayed time to fixation also caused a reduction of the Ki-67 (p=0.0284), but not significant in ER. Prolonged fixation significantly led to a gradual reduction in the Ki-67 in a time-dependent manner, but not in both ER and HER2. Finally, cutting the tumour before fixation improved fixation status and consequently caused an increased level of the Ki-67 index (p=0.0181), which resulted in a strong correlation of the Ki-67 between core needle and surgical specimen (r=0.8595).

Conclusions

Tissue handling of surgical specimen is critical for assessing the Ki-67 compared with ER and HER2. We should pay more attention to tissue fixation status for the standard assessment of the Ki-67 index.
Study of nonleptonic B(s)∗→ M1M2 (M = D,Ds,π,K) weak decays with factorization approach
Authors: Chang Qin , Li Pan-Pan , Hu Xiao-Hui , Han Lin
Published: September 2015

Motivated by the experiments of heavy flavor physics at running LHC and upgrading SuperKEKB/Belle-II in the future, the nonleptonic [Formula: see text] [Formula: see text] weak decays are studied in this paper. The amplitudes are calculated with factorization approach, and the transition form factors [Formula: see text] are evaluated within BSW model. With the reasonable approximation [Formula: see text], our predictions of branching fractions are presented. Numerically, the CKM-favored tree-dominated [Formula: see text] and [Formula: see text] decays have the largest branching fractions of the order [Formula: see text], and hence will be firstly observed by forthcoming Belle-II experiment. However, most of the other decay modes have the branching fractions [Formula: see text] and thus are hardly to be observed soon. Besides, for the possible detectable [Formula: see text] decays with branching fractions [Formula: see text], some useful ratios, such as [Formula: see text], etc. are presented and discussed in detail.
Massive scalar Casimir interaction beyond proximity force approximation
Authors: Teo L. P.
Published: September 2015

Since massive scalar field plays an important role in theoretical physics, we consider the interaction between a sphere and a plate due to the vacuum fluctuation of a massive scalar field. We consider combinations of Dirichlet and Neumann boundary conditions. There is a simple prescription to obtain the functional formulas for the Casimir interaction energies, known as TGTG formula, for the massive interactions from the massless interactions. From the TGTG formulas, we discuss how to compute the small separation asymptotic expansions of the Casimir interaction energies up to the next-to-leading order terms. Unlike the massless case, the results could not be expressed as simple algebraic expressions, but instead could only be expressed as infinite sums over some integrals. Nonetheless, it is easy to show that one can obtain the massless limits which agree with previously established results. We also show that the leading terms agree with that derive using proximity force approximation. The dependence of the leading order terms and the next-to-leading order terms on the mass of the scalar field is studied both numerically and analytically. In particular, we derive the small mass asymptotic expansions of these terms. Surprisingly, the small mass asymptotic expansions are quite complicated as they contain terms that are of odd powers in mass as well as logarithms of mass terms.
Constructing Chaotic Systems with Total Amplitude Control
Authors: Li Chunbiao , Sprott Julien Clinton , Yuan Zeshi , Li Hongtao
Published: September 2015

A general method is introduced for controlling the amplitude of the variables in chaotic systems by modifying the degree of one or more of the terms in the governing equations. The method is applied to the Sprott B system as an example to show its flexibility and generality. The method may introduce infinite lines of equilibrium points, which influence the dynamics in the neighborhood of the equilibria and reorganize the basins of attraction, altering the multistability. However, the isolated equilibrium points of the original system and their stability are retained with their basic properties. Electrical circuit implementation shows the convenience of amplitude control, and the resulting oscillations agree well with results from simulation.
Extension of Lorenz Unpredictability
Authors: Akhmet Marat , Fen Mehmet Onur
Published: September 2015

It is found that Lorenz systems can be unidirectionally coupled such that the chaos expands from the drive system. This is true if the response system is not chaotic, but admits a global attractor, an equilibrium or a cycle. The extension of sensitivity and period-doubling cascade are theoretically proved, and the appearance of cyclic chaos as well as intermittency in interconnected Lorenz systems are demonstrated. A possible connection of our results with the global weather unpredictability is provided.
On the Number of Limit Cycles for Discontinuous Generalized Liénard Polynomial Differential Systems
Authors: Jiang Fangfang , Shi Junping , Sun Jitao
Published: September 2015

In this paper, we investigate the number of limit cycles for a class of discontinuous planar differential systems with multiple sectors separated by many rays originating from the origin. In each sector, it is a smooth generalized Liénard polynomial differential system x′ = -y + g1(x) + f1(x)y and y′ = x + g2(x) + f2(x)y, where fi(x) and gi(x) for i = 1, 2 are polynomials of variable x with any given degree. By the averaging theory of first-order for discontinuous differential systems, we provide the criteria on the maximum number of medium amplitude limit cycles for the discontinuous generalized Liénard polynomial differential systems. The upper bound for the number of medium amplitude limit cycles can be attained by specific examples.