bluuebunny/crossref_metadata_2025 huggingface dataset:
Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.
Authors: Swanson M S , Dreyfuss G
Published: September 2015

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.
Cooperation between the polyomavirus middle-T-antigen gene and the human c-myc oncogene in a rat thyroid epithelial differentiated cell line: model of in vitro progression.
Authors: Berlingieri M T , Portella G , Grieco M , Santoro M , Fusco A
Published: September 2015

Two rat thyroid epithelial differentiated cell lines, PC Cl 3 and PC myc, were infected with the polyoma murine leukemia virus (PyMLV) carrying the Middle-T-antigen gene of polyomavirus. After infection, both cell lines acquired the typical markers of neoplastic transformation; however, the PC myc cells showed a greater malignant phenotype. Furthermore, the thyroid differentiated functions were completely suppressed in PC myc cells transformed by PyMLV, whereas they were, at least partially, retained in PC Cl 3 cells transformed by PyMLV, and in particular, thyroglobulin synthesis and secretion were not affected at all. Since no differences in the expression of the middle-T-antigen gene were observed in the two PyMLV-transformed cell lines, the different properties shown by these two infected cell lines must be ascribed to the expression of the c-myc oncogene.
Oxygen-dependent upstream activation sites of Saccharomyces cerevisiae cytochrome c genes are related forms of the same sequence.
Authors: Cerdan M E , Zitomer R S
Published: September 2015

In Saccharomyces cerevisiae, the two genes, CYC1 and CYC7, that encode the isoforms of cytochrome c are expressed at different levels. Oxygen regulation is mediated by the expression of the CYP1 gene, and the CYP1 protein interacts with both CYC1 upstream activation sequence 1 (UAS1) and CYC7 UASo. In this study, the homology between the CYP1-binding sites of both genes was investigated. The most noticeable difference between the CYC1 and CYC7 UASs is the presence of GC base pairs at the same positions in a repeated sequence in CYC7 compared with CG base pairs in CYC1. Directed mutagenesis changing these GC residues to CG residues in CYC7 led to CYC1-like expression of CYC7 both in a CYP1 wild-type strain and in a strain carrying the semidominant mutation CYP1-16 which reverses the oxygen-dependent expression of the two genes. Our results strongly support the hypothesis that the CYP1-binding sites in CYC1 and CYC7 are related forms of the same sequence and that the CYP1-16 protein has altered specificity for the variant forms of the consensus sequences in both genes.
Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells.
Authors: Masibay A S , Qasba P K , Sengupta D N , Damewood G P , Sreevalsan T
Published: September 2015

We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.
Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.
Authors: Akhurst R J , Flavin N B , Worden J
Published: September 2015

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.
Group II intron domain 5 facilitates a trans-splicing reaction.
Authors: Jarrell K A , Dietrich R C , Perlman P S
Published: September 2015

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.
Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation.
Authors: Lagasse E , Clerc R G
Published: September 2015

The cellular mechanisms involved in chronic inflammatory processes are poorly understood. This is especially true for the role of macrophages, which figure prominently in the inflammatory response. Two proteins, MRP8 and MRP14, which are expressed in infiltrate macrophages during inflammatory reactions but not in normal tissue macrophages, have been characterized. Here we report that MRP8 and MRP14 mRNAs are specifically expressed in human cells of myeloid origin and that their expression is regulated during monocyte-macrophage and granulocyte differentiation. To initiate the analysis of cis-acting elements governing the tissue-specific expression of the MRP genes, we cloned the human genes encoding MRP8 and MRP14. Both genes contain three exons, are single copy, and have a strikingly similar organization. They belong to a novel subfamily of highly homologous calcium-binding proteins which includes S100 alpha, S100 beta, intestinal calcium-binding protein, P11, and calcyclin (2A9). A transient expression assay was devised to investigate the tissue-specific regulatory elements responsible for MRP gene expression after differentiation in leukemia HL60 cells. The results of this investigation demonstrated that the cis-acting elements responsible for MRP expression are present on the cloned DNA fragment containing the MRP gene loci.
The S promoter of hepatitis B virus is regulated by positive and negative elements.
Authors: De-Medina T , Faktor O , Shaul Y
Published: September 2015

The S promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of mRNA for surface antigen. Transient expression studies revealed that this promoter is highly active in the Alexander hepatoma cell line but not in SK-Hep1 and HeLa cells. We found that a distal element of the promoter (-103 to -48) confers this cell-type-specific behavior through a mechanism in which the promoter activity is repressed in HeLa and SK-Hep1 cells but increased in Alexander cells. By using an inhibitor of protein synthesis, we obtained evidence that a labile repressor(s) confers the negative effect in SK-Hep1 cells. We also found an enhancerlike activity associated with a small DNA segment of the S promoter (-27 to + 30). This proximal element was active in HeLa and SK-Hep1 cells only in the absence of the distal negative element. Finally, analysis of S promoter deletion mutants demonstrated that the -27 to -17 region of the S promoter is crucial for its activity.
Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.
Authors: Jahng K Y , Ferguson J , Reed S I
Published: September 2015

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.
In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences.
Authors: Gottlieb J , Muzyczka N
Published: September 2015

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.
Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.
Authors: Hegemann J H , Shero J H , Cottarel G , Philippsen P , Hieter P
Published: September 2015

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.
Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.
Authors: Noguchi T , Fukumoto H , Mishina Y , Obinata M
Published: September 2015

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.
Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.
Authors: Hahn M , Hayward W S
Published: September 2015

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.
Diversity among beta-tubulins: a carboxy-terminal domain of yeast beta-tubulin is not essential in vivo.
Authors: Katz W S , Solomon F
Published: September 2015

Sequences of genes for beta-tubulins from many different organisms demonstrate that they encode highly conserved proteins but that these proteins diverge considerably at their carboxyl termini. The patterns of interspecies conservation of this diversity suggest that it may have functional significance. We have taken advantage of the properties of Saccharomyces cerevisiae to test this hypothesis in vivo. The sole beta-tubulin gene of this species is one of the most divergent of all beta-tubulins and encodes 12 amino acids which extend past the end of most other beta-tubulin molecules. We have constructed strains in which the only beta-tubulin gene is an allele lacking these 12 codons. We show here that this carboxy-terminal extension is not essential. The absence of these 12 amino acids had no effect on a number of microtubule-dependent functions, such as mitotic and meiotic division and mating. It did confer dominant supersensitivity to a microtubule-depolymerizing drug.
Leader length and secondary structure modulate mRNA function under conditions of stress.
Authors: Kozak M
Published: September 2015

Simian virus 40-based plasmids that direct the synthesis of preproinsulin in cultured monkey cells were used to study the effects of mRNA structure on translational efficiency. Lengthening the leader sequence enhanced translation in this system. The enhancement was most obvious when an unstructured sequence (two, four, or eight copies of the oligonucleotide AGCTAAGTAAGTAAGTA) was inserted upstream from a region of deliberate secondary structure; the degree of enhancement was proportional to the number of copies of the inserted oligonucleotide. Lengthening the leader sequence on the 3' side of a stem-and-loop structure, in contrast, did not offset the potentially inhibitory effect of the hairpin structure. Both the facilitating effect of length and the inhibitory effect of secondary structure were demonstrated most easily under conditions of mRNA competition, which was brought about by an abrupt shift in the tonicity of the culture medium. These experiments suggest a simple structural basis for the long-recognized differential response of viral and cellular mRNAs to hypertonic stress. The fact that the translatability of structure-prone mRNAs varies with changes in the environment may also have general implications for gene expression in eucaryotic cells.
Cloning and characterization of a second member of the mouse mdr gene family.
Authors: Gros P , Raymond M , Bell J , Housman D
Published: September 2015

The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript. The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor. Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length. The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB. This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process. However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established. Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.
Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum.
Authors: Katz K S , Ratner D I
Published: September 2015

We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.
Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences.
Authors: Sheng M , Dougan S T , McFadden G , Greenberg M E
Published: September 2015

Transcription of the c-fos proto-oncogene is rapidly induced in the rat pheochromocytoma PC12 cell line by a wide variety of stimuli, including polypeptide growth factors, phorbol esters, and calcium ion fluxes. We have mapped the upstream sequence requirements for this activation in PC12 cells by analysis of promoter deletion mutants in a transient expression assay. Two distinct pathways of c-fos induction are defined that differ in their requirement for cis-acting DNA sequences. Calcium activation of c-fos transcription is dependent on a DNA element located approximately 60 base pairs upstream of the transcription start site. This region is highly conserved between human, mouse, and chicken c-fos genes and contains a sequence that resembles the consensus for a cyclic AMP response element. The dyad symmetry element at position -300, which is necessary for serum responsiveness of c-fos, appears to be unimportant for calcium activation of the gene. The dyad symmetry element is, however, an essential cis-acting sequence for c-fos inducibility by nerve growth factor, epidermal growth factor, fibroblast growth factor, and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. Studies in vivo and in vitro with various mutants of the dyad symmetry element indicate that c-fos activation by polypeptide growth factors and 12-O-tetradecanoyl activation by polypeptide growth factors and 12-O-tetradecanoyl phorbol-13-acetate is mediated by a common transcription factor, and that this factor is identical to the previously described serum response factor. In vitro DNA-binding assays suggest that the quantity of serum response factor-binding activity remains unchanged during c-fos transcriptional activation.
A highly conserved mouse gene with a propensity to form pseudogenes in mammals.
Authors: Heller D L , Gianola K M , Leinwand L A
Published: September 2015

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.
Expression of the X-CGD gene during induced differentiation of myeloid leukemia cell line HL-60.
Authors: Barker K A , Orkin S H , Newburger P E
Published: September 2015

The expression of the X-CGD gene, which encodes the heavy-chain subunit of the phagocyte cytochrome b, was studied during induced myeloid differentiation of HL-60 cells. Incubation of the cells with a combined regimen of retinoic acid and dimethyl formamide resulted in granulocytic morphological differentiation and acquisition of nitroblue tetrazolium reduction, a measure of superoxide generation. During the 5-day course of induced differentiation, the levels of X-CGD mRNA transcripts rose 13-fold, with a 2-fold increase detectable within 3 h of exposure to retinoic acid. Relative transcription rates for the X-CGD gene, determined by nuclear runoff, increased two- to eightfold after 24 to 72 h of induced differentiation. However, the greater change in X-CGD mRNA levels than that in transcription rates implies the involvement of posttranscriptional regulation as well. Fractionation by centrifugal elutriation into phases of the cell cycle showed expression of X-CGD transcripts predominantly in G1 cells before induction and in all phases of the cell cycle 24 h after induction. Thus the rapid increase in X-CGD expression in induced cells reflects the acquisition of functional competence and not the concomitant cessation of proliferation or shift in cell cycle distribution.
Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.
Authors: Guan J L , Ruusala A , Cao H , Rose J K
Published: September 2015

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.
Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene.
Authors: Sternberg E A , Spizz G , Perry W M , Vizard D , Weil T , Olson E N
Published: September 2015

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.
A new RAS mutation that suppresses the CDC25 gene requirement for growth of Saccharomyces cerevisiae.
Authors: Camonis J H , Jacquet M
Published: September 2015

In the yeast Saccharomyces cerevisiae, the activation of adenylate cyclase requires the products of the RAS genes and of CDC25. We isolated several dominant extragenic suppressors of the yeast cdc25 mutation. They did not suppress a thermosensitive allele of the adenylate cyclase gene (CDC35). One of these suppressors was a mutated RAS2 gene in which the transition C/G----T/A at position 455 resulted in replacement of threonine 152 by isoleucine in the protein. The same mutation in a v-Ha-ras gene reduces the affinity of p21 for guanine nucleotides (L.A. Feig, B. Pan, T.M. Roberts, and G.M. Cooper, Proc. Natl. Acad. Sci. USA 83:4607-4611, 1986). These results support a model in which the CDC25 gene product is the GDP-GTP exchange factor regulating the activity of the RAS gene product.
Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.
Authors: Defeo-Jones D , Tai J Y , Wegrzyn R J , Vuocolo G A , Baker A E , Payne L S , Garsky V M , Oliff A , Riemen M W
Published: September 2015

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.
Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.
Authors: Avemann K , Knippers R , Koller T , Sogo J M
Published: September 2015

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.
Organization of the murine Mx gene and characterization of its interferon- and virus-inducible promoter.
Authors: Hug H , Costas M , Staeheli P , Aebi M , Weissmann C
Published: September 2015

Specific resistance of Mx+ mice to influenza virus is due to the interferon (IFN)-induced protein Mx. The Mx gene consists of 14 exons that are spread over at least 55 kilobase pairs of DNA. Surprisingly, the Mx gene promoter is induced as efficiently by Newcastle disease virus as it is by IFN. The 5' boundary of the region required for maximal induction by both IFN and Newcastle disease virus is located about 140 base pairs upstream of the cap site. This region contains five elements of the type GAAANN, which occurs in all IFN- and virus-inducible promoters. The consensus sequence purine-GAAAN(N/-)GAAA(C/G)-pyrimidine is found in all IFN-inducible promoters.
Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant.
Authors: Parker R , Simmons T , Shuster E O , Siliciano P G , Guthrie C
Published: September 2015

Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature. None of these RNAs associates with the Sm antigen. To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs. Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site. By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable. We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles. A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10. This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs.
Growth factor induction by the adenovirus type 5 E1A 12S protein is required for immortalization of primary epithelial cells.
Authors: Quinlan M P , Whyte P , Grodzicker T
Published: September 2015

The 12S protein encoded by the adenovirus E1A region induces cellular DNA synthesis in and proliferation and immortalization of primary rat epithelial cells in the presence or absence of serum. It also induces the production of a growth factor(s) that stimulates epithelial cell proliferation. We have undertaken a mutational analysis of the 12S gene to determine the sequences required for these functions. We found that a region near the C-terminus of the 12S protein was required for growth factor induction. No activities have been defined previously for this region. Furthermore, we show that growth factor production was necessary for epithelial cells to survive past their normal life span in culture and to become immortalized. The ability to induce growth factor production required prior expression of E1A activities encoded by the N-terminus of the 12S protein, including activation of quiescent cells into the cell cycle, and an unknown activity that required expression of the first 13 amino acids of the gene. In addition, examination of the subcellular localization of mutant 12S polypeptides suggested new regions that affect the nuclear localization of E1A proteins.
TATA box-dependent protein-DNA interactions are detected on heat shock and histone gene promoters in nuclear extracts derived from Drosophila melanogaster embryos.
Authors: Gilmour D S , Dietz T J , Elgin S C
Published: September 2015

We monitored protein-DNA interactions that occur on the hsp26, hsp70, histone H3, and histone H4 promoters in nuclear extracts derived from frozen Drosophila melanogaster embryos. All four of these promoters were found to be transcribed in vitro at comparable levels by extracts from both heat-shocked and non-heat-shocked embryos. Factors were detected in both types of extracts that block exonuclease digestion from a downstream site at ca. +35 and -20 base pairs from the start of transcription of all four of these promoters. In addition, factors in extracts from heat-shocked embryos blocked exonuclease digestion at sites flanking the heat shock consensus sequences of hsp26 and hsp70. Competition experiments indicated that common factors cause the +35 and -20 barriers on all four promoters in both extracts. The formation of the barriers at +35 and -20 required a TATA box but did not appear to require specific sequences downstream of +7. We suggest that the factors responsible for the +35 and -20 barriers are components whose association with the promoter precedes transcriptional activation.
Expression of heat shock proteins by isolated mouse spermatogenic cells.
Authors: Allen R L , O'Brien D A , Jones C C , Rockett D L , Eddy E M
Published: September 2015

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.
Fidelity of DNA synthesis in a mammalian in vitro replication system.
Authors: Hauser J , Levine A S , Dixon K
Published: September 2015

We have used the simian virus 40 (SV40)-based shuttle vector pZ189 in a forward-mutation assay to determine the fidelity of DNA replication in the in vitro DNA replication system developed by J.J. Li and T.J. Kelly (Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984). We find that very few base substitution errors (approximately 1/180,000 bases incorporated) are made during in vitro replication of the pZ189 vector in a system derived from CV-1 monkey cells. This replication is completely dependent on added SV40 T antigen and presumably reflects synthesis that is initiated at the SV40 replication origin. The observed level of fidelity is far greater than that reported for in vitro replication of DNA by conventionally purified eucaryotic DNA polymerases alpha and beta. Thus, there must be additional cellular factors in the crude in vitro system that serve to enhance the fidelity of DNA replication.
Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized.
Authors: Brady H A , Wold W S
Published: September 2015

Complex transcription units encode multiple mRNAs which arise by alternative processing of a common pre-mRNA precursor. It is not known how the pre-mRNA processing pathways are determined or controlled. We are investigating this problem by using the E3 complex transcription unit of adenovirus as a model. Our approach is to construct virus mutants with lesions in E3 and then determine how the mutation affects the accumulation of E3 mRNAs in vivo. We report results which indicate that competition between splicing reactions and polyadenylation reactions occurs in vivo and that this plays an important role in alternative pre-mRNA processing.
tRNA(Tyr) genes of Drosophila melanogaster: expression of single-copy genes studied by S1 mapping.
Authors: Suter B , Kubli E
Published: September 2015

Six Drosophila melanogaster tRNA(Tyr) genes have been isolated and sequenced. They contained introns of different sequences and two size classes: 20 or 21 base pairs (bp) (five genes) and 113 bp (one gene). However, the sequences coding for the mature tRNA(Tyr) were identical in all six genes. The 113-bp intron-containing gene was a single-copy gene. Hence, its primary transcript could be traced by S1 mapping. The gene was turned on during embryogenesis and continually expressed to various degrees during the following developmental stages. Thus, S1 mapping is a feasible method to follow the transcriptional activity of individual genes with identical mature products, provided that their primary transcripts are unique. The six genes were organized in two clusters of three and two genes, respectively (each containing a 20- or a 21-bp intron; cytological localization, 85A), and a single-copy gene (113-bp intron; cytological localization, 28C). We show that four of the six tRNA(Tyr) genes characterized were localized in putative 5' control regions of developmentally controlled genes transcribed by polymerase II.
Entry of simian virus 40 is restricted to apical surfaces of polarized epithelial cells.
Authors: Clayson E T , Compans R W
Published: September 2015

The uptake of simian virus 40 (SV40) by polarized epithelial cells was investigated by growth of cells on permeable supports and inoculation on either the apical or the basolateral surface. Binding of radiolabeled SV40 occurred on the apical but not the basolateral surfaces of permissive polarized Vero C1008 cells and nonpermissive polarized MDCK cells. When similar experiments were performed on nonpolarized Vero or CV-1 cells, virus binding occurred regardless of the direction of virus input. Electron micrographs of Vero C1008 cells infected at high multiplicities revealed virions lining the surfaces of apically infected cells, while the surfaces of basolaterally infected cells were devoid of virus particles. Analysis of the binding data revealed a single class of virus receptors (9 x 10(4) per cell) with a high affinity for SV40 (Kd = 3.76 pM) on the apical surfaces of Vero C 1008 cells. Indirect immunofluorescence studies revealed that synthesis of viral capsid proteins in Vero C1008 cells occurred only when input virions had access to the apical surface. Virus yields from apically infected Vero C1008 cells were 10(5) PFU per cell, while yields obtained from basolaterally infected cells were less than one PFU per cell. These results indicate that a specific receptor for SV40 is expressed exclusively on the apical surfaces of polarized Vero C1008 cells.
trans activation of type 1 interferon promoters by simian virus 40 T antigen.
Authors: Hiscott J , Wong A , Alper D , Xanthoudakis S
Published: September 2015

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.