bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms.
Authors: Downing J R , Roussel M F , Sherr C J
Published: October 2015

The turnover of the colony-stimulating factor 1 receptor (CSF-1R), the c-fms proto-oncogene product, is accelerated by ligand binding or by activators of protein kinase C (PKC), such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The mechanisms of ligand- and TPA-induced downmodulation were shown to differ by the following criteria. First, in cells in which PKC was downmodulated, CSF-1R reexpressed at the cell surface remained sensitive to ligand but was refractory to TPA-induced degradation. Second, a kinase-defective receptor containing a methionine-for-lysine substitution at amino acid 616 at its ATP-binding site failed to undergo ligand-induced downmodulation but remained responsive to TPA. Following CSF-1 stimulation, no intermediates of receptor degradation could be immunoprecipitated with polyvalent antisera to CSF-1R. In contrast, TPA induced specific proteolytic cleavage of the receptor near its transmembrane segment, resulting in the release of the extracellular ligand-binding domain from the cell and the generation of an intracellular fragment containing the kinase domain. Two-dimensional phosphopeptide mapping demonstrated no new sites of phosphorylation in response to TPA in either the residual intact receptor or the intracellular proteolytic fragment. Therefore, PKC appears not to trigger downmodulation by directly phosphorylating the receptor but, rather, activates a protease which recognizes CSF-1R as a substrate.
Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.
Authors: Wahl M I , Olashaw N E , Nishibe S , Rhee S G , Pledger W J , Carpenter G
Published: October 2015

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.
Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.
Authors: Oliphant A R , Brandl C J , Struhl K
Published: October 2015

We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases. The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4. The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding. Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C . G base pair. Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent. The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA.
Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the regulatory light chain.
Authors: Tafuri S R , Rushforth A M , Kuczmarski E R , Chisholm R L
Published: October 2015

Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.
A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.
Authors: Foiani M , Santocanale C , Plevani P , Lucchini G
Published: October 2015

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.
Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.
Authors: Radna R L , Caton Y , Jha K K , Kaplan P , Li G , Traganos F , Ozer H L
Published: October 2015

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.
Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis.
Authors: Cole G M , Schild D , Mortimer R K
Published: October 2015

The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/alpha cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of beta-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.
A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.
Authors: Staeheli P , Yu Y X , Grob R , Haller O
Published: October 2015

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.
Related proteins are phosphorylated at tyrosine in response to mitogenic stimuli and at meiosis.
Authors: Cooper J A
Published: October 2015

Forty-two-kilodalton proteins that contain phosphotyrosine in metaphase-arrested Xenopus laevis eggs are closely related to p42, a protein that is phosphorylated at tyrosine when somatic cells are exposed to mitogenic stimuli.
Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.
Authors: Huibregtse J M , Engelke D R
Published: October 2015

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.
Identification of multiple proteins that interact with functional regions of the human cardiac alpha-actin promoter.
Authors: Gustafson T A , Kedes L
Published: October 2015

5' Sequences of the human cardiac alpha-actin gene are involved in the tissue-specific and developmental regulation of the gene. Deletion analyses combined with transient expression experiments in muscle cells have demonstrated three primary regions of functional importance (A. Minty and L. Kedes, Mol. Cell. Biol. 6:2125-2136, 1986; T. Miwa and L. Kedes, Mol. Cell. Biol. 7:2803-2813, 1987), and we have previously demonstrated binding of a protein indistinguishable from serum response factor (SRF) to the most proximal region (T.A. Gustafson, T. Miwa, L.M. Boxer, and L. Kedes, Mol. Cell. Biol. 8:4110-4119, 1988). In this report, we examine protein interaction with the remainder of the promoter. Gel shift and footprinting assays revealed that at least seven distinct nuclear proteins interacted with known and putative regulatory regions of the promoter. The transcription factor Sp1 bound to eight sites, as demonstrated by footprinting assays and gel shift analysis with purified Sp1. Purified CCAAT box-binding transcription factor CTF/NF-I and Sp1 were shown to interact with the far-upstream regulatory element at -410, and footprint analysis showed extensive overlap of these two sites. Two unidentified proteins with similar but distinct footprints interacted with the second region of functional importance at -140, which contains the second CArG motif [CC(A + T rich)6GG], and these proteins were shown to be distinct from SRF. SRF was found to bind to the remaining three CArG boxes, two of which were closely interdigitated with Sp1 sites. In addition, CArG box 4 was found to interact with SRF and another distinct protein whose footprint was contained within the SRF-binding site. Sequences surrounding the TATA box were also shown to bind proteins. Sp1 was shown to bind to a site immediately downstream from the TATA box and to a site within the first exon. Thus, each of the three functional upstream regions, as defined by transfection assays, was shown to interact with five factors: Sp1 and CTF/NF-I at the upstream site, two unidentified proteins at the central site, and SRF at the most proximal site. These results suggest that expression of the cardiac actin gene in muscle cells is controlled by complex interactions among multiple upstream and intragenic elements.
Depletion of topoisomerase II in isolated nuclei during a glucose-regulated stress response.
Authors: Shen J W , Subjeck J R , Lock R B , Ross W E
Published: October 2015

Conditions, such as anoxia or glucose starvation, which induce the glucose-regulated set of stress proteins also lead to resistance to adriamycin (J. Shen, C. Hughes, C. Chao, J. Cai, C. Bartels, T. Gessner, and J. Subjeck, Proc. Natl. Acad. Sci. USA 84:3278-3282, 1987) and etoposide. We report here that chronic anoxia, glucose starvation, 2-deoxyglucose, the calcium ionophore A23187, glucosamine, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and tunicamycin (all specific inducers of the glucose regulated system) lead to a rapid and selective depletion of topoisomerase II from isolated nuclei of Chinese hamster ovary cells. This effect precedes a decline in tritiated thymidine incorporation and a redistribution of cells from S into G1/G0. The depletion of the enzyme is not accompanied by a decline in mRNA levels. We have also examined the mutant Chinese hamster K12 cell line which is temperature sensitive for expression of glucose-regulated proteins. When nuclei were isolated from K12 cells incubated at the nonpermissive temperature, a loss of topoisomerase II was again observed in congruence with the expression of stress proteins and cellular resistance to etoposide. These changes were not obtained in parental Wg1A cells incubated at the same temperature. These studies indicate that topoisomerase II is highly sensitive to glucose-regulated stresses and that its depletion from the nucleus, with the associated changes in cell cycle parameters, may represent general characteristics of the glucose-regulated state. Since anoxia and glucose starvation can occur during tumor development, this pathway for expression of drug resistance may have clinical ramifications.
Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.
Authors: Harrigan M T , Baughman G , Campbell N F , Bourgeois S
Published: October 2015

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.
The efficiency of 3'-end formation contributes to the relative levels of different histone mRNAs.
Authors: Liu T J , Levine B J , Skoultchi A I , Marzluff W F
Published: October 2015

Sequences at both the 5' and 3' ends of mouse histone genes contribute to the expression of individual genes. The 3' sequences required for high expression of the mouse H2a-614 gene are the same as the sequences required for 3'-end formation. When these sequences were substituted for the 3' end of the poorly expressed H2a-291 gene, expression of the H2a-291 gene was increased fivefold. A 65-nucleotide fragment containing the H2a-614 3' processing signal increased expression of the H2a-291 gene when it was placed in the proper orientation downstream of the H2a-291 3' end. The only mRNAs that accumulated from this gene ended at the H2a-291 3' end, which suggests that the transcript is sequentially processed. In an in vitro processing system, the different histone 3' ends showed different processing efficiencies, which correlated with their expression in cells. These results suggest that the efficiency of processing is important in determining the steady-state levels of individual mouse histone mRNAs.
Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.
Authors: Dhar V , Skoultchi A I , Schildkraut C L
Published: October 2015

To investigate whether a switch in the transcriptional activity of a gene is associated with a change in the timing of replication during the S phase, we examined the replication timing of the beta-globin genes in two different types of somatic cell hybrids. In mouse hepatoma (Hepa 1a) x mouse erythroleukemia (MEL) hybrid cells, the beta-globin gene from the MEL parent is transcriptionally inactivated and is later replicating than in the parental MEL cell line. In human fibroblast (GM3552) x MEL hybrid cells, the human beta-globin gene is transcriptionally activated, and all of the sequences within the human beta-globin domain (200 kilobases) we have examined appear to be earlier replicating than those in the parental fibroblast cell line. The chromatin configuration of the activated human beta-globin domain in the hybrids is relatively more sensitive to nucleases than that in the fibroblasts. Furthermore, major nuclease-hypersensitive sites that were absent in the chromatin flanking the distal 5' region of the human beta-globin gene cluster in the parental fibroblast cell line are present in the transcriptionally activated domain in the hybrid cell line. These results suggest that timing of replication of globin genes has been altered in these hybrid cells and thus is not fixed during the process of differentiation.
Control of beta-interferon expression in murine embryonal carcinoma F9 cells.
Authors: Francis M K , Lehman J M
Published: October 2015

Murine embryonal carcinoma F9 cells, a tissue culture model for early embryonic development, do not produce interferon (IFN) in response to poly(I-C), as determined by an antiviral assay. RNase protection analyses were used to examine total RNA extracted from the cells for the presence of beta-IFN RNA. Whereas F9 cells differentiated in vitro with retinoic acid produced a biologically active protein as well as beta-IFN RNA in response to poly(I-C), undifferentiated F9 cells produced no detectable beta-IFN RNA even in the presence of cycloheximide, an IFN-superinducing agent. These results show that undifferentiated embryonal carcinoma cells do not accumulate beta-IFN RNA in response to an IFN-inducing agent, suggesting a transcriptional regulatory mechanism. However, this control mechanism is altered upon differentiation, since the gene can be transcriptionally activated in retinoic acid-differentiated cells.
PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.
Authors: Banroques J , Abelson J N
Published: October 2015

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.
Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae.
Authors: Blinder D , Jenness D D
Published: October 2015

alpha-Factor pheromone inhibits division of yeast a cells. After prolonged exposure to alpha-factor, the cells adapt to the stimulus and resume cell division. The sst2 mutation is known to inhibit adaptation. This report examines adaptation in scg1 (also designated gpa1) and STE4Hpl (Hpl indicates haploid lethal) mutants that exhibit constitutive activation of the pheromone response pathway. Recovery of the STE4Hpl mutant was blocked by the sst2-1 mutation, whereas recovery of the scg1-7 mutant was not completely blocked by sst2-1. These results indicate that both SST2-dependent and -independent mechanisms regulate postreceptor events in the pheromone response pathway. Down regulation of receptors in response to alpha-factor was independent of the signal that was generated in the scg1 mutant.
Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.
Authors: Emanuel J R , Graw S , Housman D , Levenson R
Published: October 2015

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.
c-Ha-ras gene bidirectional promoter expressed in vitro: location and regulation.
Authors: Lowndes N F , Paul J , Wu J , Allan M
Published: October 2015

Increased transcriptional activity of the c-Ha-ras gene product is correlated with induction of several important human tumor types. For this reason, we have investigated the nature of the c-Ha-ras promoter and the factors that regulate its expression. Using S1 and primer extension analysis of c-Ha-ras RNA from EJ cells, we have identified 18 initiation sites within an upstream exon (exon -1) whose 3' end (the donor splice site [D]) is located 1,105 base pairs (bp) upstream of the ATG codon. The furthest-upstream initiation site is located -191 bp relative to D, and the furthest downstream is located -16 bp relative to D. Transient expression assays, in which a series of mutants spanning this region were ligated to a promoterless chloramphenicol acetyltransferase vector, functionally confirmed the position and extent of this promoter. Mutational analysis further located a 47-bp element located between -243 and -196 relative to D that up-regulated transcriptional activity of the promoter region by 20- to 40-fold. This region contained both a GC box known to bind SP1 and a CCAAT box. Insertion of a simian virus 40 enhancer 5' to the promoter up-regulated transcription from each initiation site by approximately 10- to 20-fold. We have also localized, both by chloramphenicol acetyltransferase assay and by S1 analysis, a strong promoter operating in the direction opposite that of the gene and originating immediately 5' to the 47-bp regulatory region. The reverse promoter was found to have nine initiation sites between -248 and -278 relative to D.
Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation.
Authors: Tsurimoto T , Fairman M P , Stillman B
Published: October 2015

A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.
Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator.
Authors: Becker D M , Hollenberg S M , Ricciardi R P
Published: October 2015

The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions.
Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins.
Authors: Reynolds A B , Kanner S B , Wang H C , Parsons J T
Published: October 2015

We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (pp130) and 110,000 (pp110) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp60c-src (pp60(527)F). Most if not all of the tyrosine-phosphorylated forms of pp130 and pp110 could be immunoprecipitated from lysates with any of several src protein-specific monoclonal antibodies directed against at least three spatially distinct epitopes. Consequently, of the more than 15 prominent phosphoproteins detected on immunoblots with phosphotyrosine-specific antibodies, pp130 and pp110 were selectively removed by src protein-specific immunoprecipitation, and their presence in the immunoprecipitates appears to have been due to a direct interaction with activated src proteins. src protein variants that induce different morphological phenotypes were altered in their ability to form detergent-stable complexes with pp130 and pp110 or with pp110 alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with pp110. Expression of src variants with mutations in the A box (pp60dl92/527F) or B box (pp60dl155/527F) of the src homology region induced differences in phosphorylation of pp130 and pp110, as well as changes in their association with variant src proteins. Sequences within the B-box region appeared to be necessary for stable complex formation with pp130 and pp110 and may be involved in the interaction of activated src proteins with cellular substrates.
Transcriptional mapping of the amplified region encoding the dihydrofolate reductase-thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs.
Authors: Kapler G M , Beverley S M
Published: October 2015

We have examined the transcriptional organization of the R region of the protozoan parasite Leishmania major. This region encodes the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and is frequently amplified as a 30-kilobase (kb) extrachromosomal circular DNA in methotrexate-resistant lines. Northern (RNA) blot analysis shows that the R region encodes at least 10 stable cytoplasmic polysomal poly(A)+ RNAs, ranging in size from 1.7 to 13 kb and including the 3.2-kb DHFR-TS mRNA. Transcriptional mapping reveals that these RNAs are closely spaced and collectively cover more than 95% of the 30-kb amplified R region. The organization is complex, including several overlapping RNAs 3' of DHFR-TS and two examples of antisense RNAs 5' of DHFR-TS. The R region RNAs can be grouped into two empirical domains, with eight contiguous RNAs transcribed in the same direction as that of DHFR-TS and two contiguous RNAs transcribed in the orientation opposite to that of DHFR-TS. The two 5'-most RNAs of the DHFR-TS-containing domain overlap the RNAs transcribed from the opposite strand. These data are relevant to models of transcription, including recent studies suggesting polycistronic transcription in trypanosomatids. The abundance of R region RNAs increases uniformly 10- to 15-fold in the amplified R1000-3 line relative to the wild type, and no new RNAs were observed. This suggests that all elements required in cis for DHFR-TS expression are contained within the 30-kb circular DNA. Quantitative analysis reveals that the steady-state DHFR-TS mRNA and protein levels are not growth phase regulated, unlike the monofunctional mouse DHFR. DHFR-TS is developmentally regulated, however, declining about fivefold in lesion amastigotes relative to promastigotes.
Homologous plasmid recombination is elevated in immortally transformed cells.
Authors: Finn G K , Kurz B W , Cheng R Z , Shmookler Reis R J
Published: October 2015

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.
Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site.
Authors: Pays E , Coquelet H , Pays A , Tebabi P , Steinert M
Published: October 2015

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.
The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc.
Authors: Stavnezer E , Brodeur D , Brennan L A
Published: October 2015

The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.
Isolation and characterization of three distinct cDNAs for the chicken c-ski gene.
Authors: Sutrave P , Hughes S H
Published: October 2015

Three types of c-ski cDNAs have been isolated from two different chicken cDNA libraries. Sequence comparisons suggest that the cDNAs derive from alternatively spliced mRNAs. A short stretch of sequence homology that exists between c-ski and avian leukosis virus may have played a role in viral transduction.
Cloning and disruption of Ustilago maydis genes.
Authors: Fotheringham S , Holloman W K
Published: October 2015

We have demonstrated that genes from Ustilago maydis can be cloned by direct complementation of mutants through the use of genomic libraries made in a high-frequency transformation vector. We isolated a gene involved in amino acid biosynthesis as an illustrative example and showed that integrative and one-step disruption methods can be used to create null mutations in the chromosomal copy of the gene by homologous recombination. The results of this investigation make it clear that one-step gene disruption will be of general utility in investigations of U. maydis, since simple, precise replacement of the sequence under study was readily achieved.
A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity.
Authors: Rozen F , Pelletier J , Trachsel H , Sonenberg N
Published: October 2015

Eucaryotic initiation factor 4A (eIF-4A) is a member of a family of proteins believed to be involved in the ATP-dependent melting of RNA secondary structure. These proteins contain a derivative of the consensus ATP-binding site AXXGXGKT. To assess the importance of the consensus amino acid sequence in eIF-4A for ATP binding, we mutated the consensus amino-proximal glycine and lysine to isoleucine and asparagine, respectively. The effect of the mutations was examined by UV-induced cross-linking of [alpha-32P]dATP to eIF-4A. Mutation of the lysine residue (but not of the glycine residue) resulted in the loss of [alpha-32P]dATP cross-linking to eIF-4A, suggesting that the lysine is an important determinant in ATP binding to eIF-4A.
Isolated aortitis: a rare cause of febrile illness
Authors: Fior Alberto , Barreto Paulo
Published: October 2015

Febrile illness often presents a challenge for the clinician. The main causes of febrile illness are infections, solid or haematological malignancies and connective tissue disorders, including vasculitis. A 49-year-old woman sought medical attention because of intermittent fever that lasted 2 weeks. She presented no further symptoms or physical signs to suggest the aetiology. The epidemiological context was irrelevant. Analyses revealed anaemia of chronic disease and significant elevations of inflammatory parameters. A comprehensive study was performed, which revealed presence of an aortitis. Investigation of infectious and immunological causes was negative. We arrived at the definitive diagnosis of isolated aortitis. She was treated with corticosteroid and methotrexate, with resolution of symptoms and clinical abnormalities.
Hyperreactio luteinalis encountered during caesarean delivery of an uncomplicated spontaneous singleton pregnancy
Authors: Sarmento Gonçalves Inês , Malafaia Sofia , Belchior Helena , Tiago-Silva Pedro
Published: October 2015

We report a rare case of hyperreactio luteinalis presenting as an incidental finding during caesarean section in an uncomplicated spontaneous singleton pregnancy. Caesarean section was performed due to failure to progress in labour, and delivered a healthy male neonate. The mother's analytical tests revealed a hyperandrogenic state without any other relevant alterations. Ovarian volume and testosterone serum concentration normalised in 2 months. The expectant management of this case permitted conservation of both ovaries in a young woman without adding morbidity.
A case of metastatic adenocarcinoma on a background of penoscrotal extramammary Paget's disease
Authors: Christodoulidou Michelle , Khetrapal Pramit , Mitra Anita , Muneer Asif
Published: September 2015

A 77-year-old man presented with a chronic lesion located in the left penoscrotal area. Apart from pruritus, bleeding and an occasional discharge from this area, he also reported reduced appetite and weight loss. Examination revealed an ulcerated skin lesion attached to a firm subcutaneous mass. Wide local excision of the lesion revealed invasive adenocarcinoma on a background of extramammary Paget's disease. Staging studies showed disseminated metastatic disease within the lymph nodes, and liver and bone metastases. He was treated with carboplatin and paclitaxel chemotherapy initially, but then continued only on Carboplatin chemotherapy due to side effects from Paclitaxel. Eleven weeks after the start of his chemotherapy, his restaging imaging showed reduced lymphadenopathy, unchanged liver metastasis and sclerosis of bone metastasis. With completion of chemotherapy, repeat imaging showed stable disease. The patient is currently on follow-up.
Perceptions of data processes in mobile-based versus paper-based health information systems for maternal, newborn and child health: a qualitative study in Andhra Pradesh, India
Authors: Nguyen Lillian H , LeFevre Amnesty E , Jennings Larissa , Agarwal Smisha , Mehl Garrett , Labrique Alain B , Chava Lakshmi Durga
Published: October 2015

Background

Many maternal, newborn and child health (MNCH) programmes have paired community health workers with mobile technologies to strengthen the ability of health information systems (HIS) to track women and children across time and beyond the clinical setting. However, little is known regarding the comparative effectiveness of using mobile technologies to enhance HIS data in resource-poor settings.

Methods

Focus group discussions were conducted with community health workers called Health Activists (HAs; n=30), Community Organisation Leaders (n=28), HA Trainers (n=21), district and tribal area officials (n=3) and State Officials (n=4). We analysed user perceptions along seven key HIS processes: data collection, transmission, processing, analysis, display, quality checking and feedback.

Results

The mobile-based health information system (mHIS) was found to be supportive of the MNCH continuum of care by improving the regularity and timeliness of access to robust data. Respondents noted that data errors were reduced in real time through automated error checking and data processing, which also reduced users’ workloads. The mHIS additionally enabled users to analyse both individual and aggregate data, allowing them to identify specific individuals in need of services or training as well as to identify general trends in service delivery. The system's data display and feedback mechanisms were viewed as improving data use for decision-making. The remaining challenges of the mHIS versus the paper-based HIS included resource, infrastructural and technological barriers that hindered efficient use over time.

Conclusions

As compared to paper-based HIS systems, mobile technologies can improve health information processes in resource-poor settings. More efforts are needed to ensure sufficient financial investment, training and use of mHIS data at all levels of the HIS.