bluuebunny/crossref_metadata_2025 huggingface dataset:
Intercellular Transmission of Viral Populations with Vesicles
Authors: Altan-Bonnet Nihal , Chen Ying-Han
Published: October 2015

A common paradigm holds that during cell-to-cell transmission, viruses behave as lone soldiers. Recently, we discovered not only that enteroviruses are transmitted via vesicles as populations of viral particles but also that this type of transmission enhances their infection efficiency (Y. H. Chen et al., Cell
160:
619–630, 2015). This mechanism could be advantageous for the overall fitness of the viral population, promoting genetic interplay by enabling viral quasispecies to collectively infect a susceptible host cell. Here, we discuss these findings in the context of viral pathogenesis and also propose that this novel type of vesicular transmission is widespread among different virus families and includes populations of both viral particles and naked viral genomes.
Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle
Authors: Londrigan Sarah L. , Short Kirsty R. , Ma Joel , Gillespie Leah , Rockman Steven P. , Brooks Andrew G. , Reading Patrick C.
Published: October 2015

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle.

IMPORTANCE
Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages.
Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles
Authors: Wan Juan , Basu Kaustuv , Mui Jeannie , Vali Hojatollah , Zheng Huanquan , Laliberté Jean-François
Published: October 2015

Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K
2
plays an essential role in endomembrane alterations. Although 6K
2
-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K
2
tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

IMPORTANCE
Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.
Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells.
Authors: Lois R , Freeman L , Villeponteau B , Martinson H G
Published: October 2015

We report active, inappropriate transcription of the chicken beta A-globin gene in normal fibroblasts, cultured MSB cells, and brain. We were unable to detect ovalbumin gene transcription in these same tissues. Most of the globin gene transcripts were found to be truncated near the beginning of the gene, suggesting the existence of a premature termination process that is preferentially active under conditions of inappropriate transcription. The inappropriately transcribed beta A-globin gene chromatin remained DNase I resistant and highly methylated. Thus, the DNase I-sensitive conformation of erythrocyte beta A chromatin was correlated not with beta A transcription per se but with beta A expression. Although both transcribed and nontranscribed genes within the globin domain exhibited the same DNase I sensitivity in erythrocyte nuclei, a housekeeping gene active in erythrocytes exhibited a different level of DNase I sensitivity. However, this gene exhibited the same level of DNase I sensitivity in both erythrocytes and a cultured cell line. These observations are consistent with the proposal (G. Blobel, Proc. Natl. Acad. Sci. USA 82:8527-8529, 1985) that the DNase I sensitivity of a gene may reflect properties of chromatin related to cotranscriptional and posttranscriptional aspects of mRNA production rather than to transcription per se.
Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.
Authors: Kipling D , Kearsey S E
Published: October 2015

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.
Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex.
Authors: Brunt S A , Riehl R , Silver J C
Published: October 2015

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.
Cell type-specific protein-DNA interactions in the human zeta-globin upstream promoter region: displacement of Sp1 by the erythroid cell-specific factor NF-E1.
Authors: Yu C Y , Chen J , Lin L I , Tam M , Shen C K
Published: October 2015

The protein-DNA interactions of the upstream promoter region of the human embryonic zeta-globin gene in nuclear extracts of erythroid K562 cells and nonerythroid HeLa cells were analyzed by DNase I footprinting, gel mobility shift assay, methylation interference, and oligonucleotide competition experiments. There are mainly two clusters of nuclear factor-binding sites in the zeta promoter. The proximal cluster spans the DNA sequence from -110 to -60 and consists of binding sites for CP2, Sp1, and NF-E1. NF-E1 binding is K562 specific, whereas CP2 binding is common to both types of cells. Overlapping the NF-E1- and CP2-binding sites is a hidden Sp1-binding site or CAC box, as demonstrated by binding studies of affinity-purified Sp1. In the distal promoter region at -250 to -220, another NF-E1-binding site overlaps a CAC box or Sp1-binding site. Extract-mixing experiments demonstrated that the higher affinity of NF-E1 binding excluded the binding of Sp1 in the K562 extract. NF-E1 factors could also displace prebound Sp1 molecules. Between the two clusters of multiple-factor-binding sites are sequences recognized by other factors, including zeta-globin factors 1 and 2, that are present in both HeLa and K562 extracts. We discuss the cell type-specific, competitive binding of multiple nuclear factors in terms of functional implications in transcriptional regulation of the zeta-globin gene.
The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes.
Authors: Haynes S R , Raychaudhuri G , Beyer A L
Published: October 2015

The Drosophila Hrb98DE locus encodes proteins that are highly homologous to the mammalian A1 protein, a major component of heterogeneous nuclear ribonucleoprotein (RNP) particles. The Hrb98DE locus is transcribed throughout development, with the highest transcript levels found in ovaries, early embryos, and pupae. Eight different transcripts are produced by the use of combinations of alternative promoters, exons, and splice acceptor sites; the various species are not all equally abundant. The 3'-most exon is unusual in that it is completely noncoding. These transcripts can potentially generate four protein isoforms that differ in their N-terminal 16 to 21 amino acids but are identical in the remainder of the protein, including the RNP consensus motif domain and the glycine-rich domain characteristic of the mammalian A1 protein. We suggest that these sequence differences could affect the affinities of the proteins for RNA or other protein components of heterogeneous nuclear RNP complexes, leading to differences in function.
The sum1-1 mutation affects silent mating-type gene transcription in Saccharomyces cerevisiae.
Authors: Livi G P , Hicks J B , Klar A J
Published: October 2015

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci. MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression. The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3). As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations. We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts. At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.
ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences.
Authors: Evans C F , Engelke D R , Thiele D J
Published: October 2015

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.
Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene.
Authors: Libri D , Goux-Pelletan M , Brody E , Fiszman M Y
Published: October 2015

The beta tropomyosin gene contains two internal exons which are spliced in a mutually exclusive manner. Exon 6B is specifically included in the mature transcripts expressed in skeletal muscle or cultured myotubes, while exon 6A is a myoblast- or smooth muscle-specific exon. The intron between them, which is never spliced in normal conditions, contains two characteristic features: first, the unusual location of the branch point at position -105 from the acceptor, and second, the presence of a very long pyrimidine stretch upstream of the skeletal muscle exon. In this study we designed a number of sequence modifications to investigate the role of these two elements and of a computer-predicted secondary structure in the mutually exclusive splicing of the two exons. We found that mutations in the skeletal exon as well as in the upstream intron could change in vivo the tissue-specific pattern as well as the mutually exclusive character of the two exons. Our results suggest that the unusual position of the branch point does not prevent the utilization of exon 6B in myoblasts and that the region around the acceptor site of exon 6B and the polypyrimidine tract have an important role in this control. Last, we discuss the possible implications of secondary structures.
Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae.
Authors: Kovari L , Sumrada R , Kovari I , Cooper T G
Published: October 2015

Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.
Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules.
Authors: Wallrath L L , Burnett J B , Friedman T B
Published: October 2015

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.
The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.
Authors: Witte M M , Dickson R C
Published: October 2015

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.
Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family.
Authors: Ahmad S , Ahuja R , Venner T J , Gupta R S
Published: October 2015

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.
Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA.
Authors: Cheng J , Fogel-Petrovic M , Maquat L E
Published: October 2015

The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations within the TPI gene that cause translation to terminate prematurely at or upstream of codon 189, within exon 6, result in a decreased level of TPI mRNA (I.O. Daar and L.E. Maquat, Mol. Cell. Biol. 8:802-813, 1988). For all mutations in this group, the decrease is to the same extent, i.e., to approximately 20% of the normal level. We show here that a second group of nonsense mutations that cause translation to terminate prematurely at or downstream of codon 208, in exon 6, did not affect TPI mRNA abundance. Deletion analysis demonstrated that the abundance of translationally active TPI mRNA is a function of both the distance and the polarity of the nonsense codon relative to the final intron in TPI pre-mRNA. Our results indicate that if translating ribosomes are unable to progress to at least a certain position within the penultimate exon relative to the final intron, then the level of the corresponding mRNA will be abnormally low. Studies inhibiting RNA synthesis with dactinomycin demonstrated that a block in translation does not affect the half-life of mature TPI mRNA. The simplest interpretation of our data is that the translation of TPI mRNA in the cytoplasm facilitates the splicing of TPI pre-mRNA or the transport of TPI mRNA across the nuclear envelope or both.
Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.
Authors: Szeberényi J , Cai H , Cooper G M
Published: October 2015

A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells.
Transformation of mouse BALB 3T3 cells by enterobacterial plasmid misrepair gene mucAB.
Authors: Tosu M , Tanooka H
Published: October 2015

The enterobacterial plasmid misrepair gene mucAB, ligated to the metal-inducible mammalian MT-1 promoter, was introduced into the genome of mouse BALB 3T3 cells. In the presence of zinc ions, MucA but not MucB protein was produced, and the whole-cell population of each mucAB+ clone started to show the transformation phenotype in a few days. Foci appeared in the transformed cell population after 4 weeks, and cells from the foci produced tumors in nude mice, indicating malignant transformation by the mucA product. Growth of mucAB+ cells was stimulated by zinc-induced expression of mucA. The transformation phenotype was reversed by removing zinc ions from the culture, indicating that the transformation was due not to MucA-mediated mutation in the mouse genome but to the direct transforming activity of MucA protein.
Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.
Authors: Wynford-Thomas D , Bond J A , Wyllie F S , Burns J S , Williams E D , Jones T , Sheer D , Lemoine N R
Published: October 2015

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.
Wild-type V(D)J recombination in scid pre-B cells.
Authors: Hendrickson E A , Schlissel M S , Weaver D T
Published: October 2015

Homozygous mutation at the scid locus in the mouse results in the aberrant rearrangement of immunoglobulin and T-cell receptor gene segments. We introduced a retroviral vector containing an inversional immunoglobulin rearrangement cassette into scid pre-B cells. Most rearrangements were accompanied by large deletions, consistent with previously characterized effects of the scid mutation. However, two cell clones were identified which contained perfect reciprocal fragments and wild-type coding joints, documenting, on a molecular level, the ability of scid pre-B cells to generate functional protein-coding domains. Subsequent rearrangement of the DGR cassette in one of these clones was accompanied by a deletion, suggesting that this cell clone had not reverted the scid mutation. Indeed, induced rearrangement of the endogenous kappa loci in these two cell clones resulted in a mixture of scid and wild-type V-J kappa joints, as assayed by a polymerase chain reaction and DNA sequencing. In addition, three immunoglobulin mu- scid pre-B cell lines showed both scid and wild-type V-J kappa joins. These experiments strongly suggest that the V(D)J recombinase activity in scid lymphoid cells is diminished but not absent, consistent with the known leakiness of the scid mutation.
The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain.
Authors: Bull P , Morley K L , Hoekstra M F , Hunter T , Verma I M
Published: October 2015

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.
Upstream activation and repression elements control transcription of the yeast COX5b gene.
Authors: Hodge M R , Singh K , Cumsky M G
Published: October 2015

The Saccharomyces cerevisiae COX5b gene is regulated at the level of transcription by both the carbon source and oxygen. To define the cis-acting elements that underlie this transcriptional control, deletion analysis of the upstream regulatory region of COX5b was performed. The results of the study suggest that at least four distinct regulatory sites are functional upstream of the COX5b transcriptional starts. One, which was precisely defined to a region of 20 base pairs, contains two TATA-like elements. Two upstream activating sequences (UAS15b and UAS2(5b)) and an upstream repression sequence (URS5b) were also found. Each of the latter three elements was able either to activate (UAS1(5b) and UAS2(5b)) or to repress URS5b) the transcription of a heterologous yeast gene. Further analysis revealed that UAS1(5b) is the site of carbon source control and may be composed of two distinct domains that act synergistically. URS5b mediates the aerobic repression of COX5b and contains two sequences that are highly conserved in other yeast genes negatively regulated by oxygen.
Cell type-specific mechanisms of regulating expression of the ornithine decarboxylase gene after growth stimulation.
Authors: Abrahamsen M S , Morris D R
Published: October 2015

Ornithine decarboxylase (ODC) mRNA is strongly induced by mitogenic activation of resting Swiss 3T3 fibroblasts and T lymphocytes. Nuclear run-on analysis revealed a low level of nascent transcripts in resting fibroblasts that was elevated upon activation. In contrast, there was a high level of transcription across the entire ODC gene in resting T cells, which remained unchanged upon activation. The stability of the mature ODC message was found to be unaffected by mitogenic stimulation. These results indicate that ODC mRNA levels are regulated transcriptionally in Swiss 3T3 cells and posttranscriptionally within the nucleus of T lymphocytes in response to mitogenic stimuli. In this unique situation, the mitogenic induction of a single gene, ODC, is regulated by two very distinct, cell-specific mechanisms.
A 22-amino-acid peptide restores DNA-binding activity to dimerization-defective mutants of the estrogen receptor.
Authors: Lees J A , Fawell S E , White R , Parker M G
Published: October 2015

We have identified residues within the estrogen receptor that are required for dimerization and high-affinity DNA binding. A 22-amino-acid peptide encompassing these residues was sufficient to restore DNA-binding activity to a mutant receptor lacking most of the hormone-binding domain. Point mutagenesis of the fusion protein confirmed that this sequence continued to mediate dimerization in a manner similar to that within the native receptor, although its position relative to the DNA-binding domain was appreciably altered.
Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells.
Authors: Borellini F , Aquino A , Josephs S F , Glazer R I
Published: October 2015

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro. These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner.
Inhibition of c-fos transcription and phosphorylation of the serum response factor by an inhibitor of phospholipase C-type reactions.
Authors: Schalasta G , Doppler C
Published: October 2015

Phospholipase C activity is necessary for transcriptional c-fos activation by providing diacylglycerol as an activator of protein kinase C. We found that transcriptional activation of c-fos and the phosphorylation of its major transcription factor were inhibited by tricyclodecan-9-yl xanthogenate, which blocks phospholipase C-type reactions. Transcription of the c-ras and beta-actin genes in the same cells remained unaffected.
Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.
Authors: Wagner R W , Yoo C , Wrabetz L , Kamholz J , Buchhalter J , Hassan N F , Khalili K , Kim S U , Perussia B , McMorris F A
Published: October 2015

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.
Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.
Authors: Hanson R D , Ley T J
Published: October 2015

The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined.
Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.
Authors: Wojcik B E , Dermody J J , Ozer H L , Mun B , Mathews C K
Published: October 2015

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.
Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter.
Authors: Evans J L , Moore T L , Kuehl W M , Bender T , Ting J P
Published: October 2015

The function of c-Myb protein was revealed by transfecting an expression vector containing the entire c-Myb protein-coding sequence into the murine CTLL-2 T-cell line. Expressions of high levels of c-Myb protein did not alter the expression of several T-cell markers, c-fos mRNA expression, responses to interleukin-2, and growth characteristics of these cells. Interestingly, expression of the c-myc gene was drastically increased in this clone. Further, the c-myb expression plasmid, but not a frameshift mutant of c-myb, enhanced the expression of a hybrid construct of c-myc promoter linked to a reporter gene by 8- to 14-fold. These results demonstrate a role of c-Myb protein in c-myc gene expression.
In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins.
Authors: Nye S H , Scarpulla R C
Published: October 2015

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.
RNA processing is a limiting step for murine tumor necrosis factor beta expression in response to interleukin-2.
Authors: Weil D , Brosset S , Dautry F
Published: October 2015

We have previously reported that tumor necrosis factor beta (TNF beta) expression is induced by interleukin-2 (IL-2) in the murine lymphocytic T-cell line CTLL-2. In this study, we have characterized the nuclear and cytoplasmic TNF beta transcript and assessed their role in TNF beta gene expression. A unique feature of TNF beta expression was the accumulation of nuclear precursors, which reflected a slow nuclear RNA processing. As a consequence, there was a delay in the appearance of cytoplasmic messengers after the transcriptional induction of TNF beta by IL-2. We also found that two messengers, the fully spliced messenger and an intron 3-retaining messenger, were exported to the cytoplasm and actively translated. The same pattern of expression was observed in concanavalin A-stimulated splenocytes, although the level of expression was much lower than in CTLL-2 cells. The simple genetic structure and the high level of accumulation of nuclear precursors make TNF beta a particularly attractive model system to use for studies of RNA processing and cytoplasmic transport of partially spliced messengers.
Structure and evolution of the U2 small nuclear RNA multigene family in primates: gene amplification under natural selection?
Authors: Matera A G , Weiner A M , Schmid C W
Published: October 2015

The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.
Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation.
Authors: Darrow A L , Rickles R J , Pecorino L T , Strickland S
Published: October 2015

The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.