bluuebunny/crossref_metadata_2025 huggingface dataset:
An alternative helix in the 26S rRNA promotes excision and integration of the Tetrahymena intervening sequence.
Authors: Woodson S A , Emerick V L
Published: October 2015

A highly conserved ribosomal stem-loop immediately upstream of the Tetrahymena splice junction can inhibit both forward and reverse self-splicing by competing with base pairing between the 5' exon and the guide sequence of the intervening sequence. Formation of this unproductive hairpin is preferred in precursor RNAs with short exons and results in a lower rate of splicing. Inhibition of self-splicing is not observed in longer precursors, suggesting that additional interactions in the extended exons can influence the equilibrium between the productive and unproductive hairpins at the 5' splice site. An alternative pairing upstream of the 5' splice site has been identified and is proposed to stabilize the active conformer of the pre-rRNA. Nucleotide changes that alter the ability to form this additional helix were made, and the self-splicing rates were compared. Precursors in which the proposed stem is stabilized splice more rapidly than the wild type, whereas RNAs that contain a base mismatch splice more slowly. The ability of DNA oligomers to bind the RNA, as detected by RNase H digestion, correlates with the predicted secondary structure of the RNA. We also show that a 236-nucleotide RNA containing the natural splice junction is a substrate for intervening sequence integration. As in the forward reaction, reverse splicing is enhanced in ligated exon substrates in which the alternative rRNA pairing is more stable.
Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei.
Authors: Bringaud F , Baltz T
Published: October 2015

A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It is composed of two 80% homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose. These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2. In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucose-dependent expression of THT2.
Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.
Authors: Brabletz T , Pfeuffer I , Schorr E , Siebelt F , Wirth T , Serfling E
Published: October 2015

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.
In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.
Authors: Klucher K M , Sommer M , Kadonaga J T , Spector D H
Published: October 2015

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.
Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.
Authors: Vincent C K , Gualberto A , Patel C V , Walsh K
Published: October 2015

Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle.
Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes.
Authors: Spencer C A , Kilvert M A
Published: October 2015

Both transcription initiation and transcription elongation contribute to the regulation of steady-state c-myc RNA levels. We have used the Xenopus oocyte transcription assay to study premature transcription termination which occurs in the first exon and intron of the human c-myc gene. Previous studies showed that after injection into Xenopus oocytes transcription from the c-myc P1 promoter resulted in read-through transcripts whereas transcription from the stronger P2 promoter resulted in a combination of prematurely terminated and read-through transcripts. We now demonstrate that this promoter-specific processivity results from the overall amount of RNA polymerase II transcription occurring from either promoter. Parameters that reduce the amount of transcription from P1 or P2, such as decreased concentration of template injected or decreased incubation time, result in a reduction in the ratio of terminated to read-through c-myc transcripts. Conversely, when transcription levels are increased by higher concentrations of injected template, increased incubation time, or coinjection with competing template, the ratio of terminated to read-through transcripts increases. We hypothesize that an RNA polymerase II processivity function is depleted above a threshold level of transcription initiation, resulting in high levels of premature transcription termination. These findings account for the promoter-specific effects on transcription elongation previously seen in this assay system and suggest a mechanism whereby limiting transcription elongation factors may contribute to transcription regulation in other eukaryotic cells.
Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes.
Authors: Bonner W M , Mannironi C , Orr A , Pilch D R , Hatch C L
Published: October 2015

Histone H2A.X is a replication-independent histone H2A isoprotein species that is encoded by a transcript alternatively processed at the 3' end to yield two mRNAs: a 0.6-kb mRNA ending with the stem-loop structure characteristic of the mRNAs for replication-linked histone species, and a second, polyadenylated 1.6-kb mRNA ending about 1 kb further downstream (C. Mannironi, W. M. Bonner, and C. L. Hatch, Nucleic Acids Res. 17:9113-9126, 1989). Of the two, the 0.6-kb H2A.X stem-loop mRNA predominates in many cell lines, indicating that the presence of two types of mRNA may not completely account for the replication independence of H2A.X protein synthesis. The ambiguity is resolved by the finding that the level of the 0.6-kb H2A.X mRNA is only weakly downregulated during the inhibition of DNA replication and only weakly upregulated during the inhibition of protein synthesis, while the levels of other replication-linked mRNAs are strongly down- or upregulated under these two conditions. Analysis of the nuclear transcription rates of specific H2A genes showed that while the rates of transcription of replication-linked H2A genes decreased substantially during the inhibition of DNA synthesis and increased substantially during the inhibition of protein synthesis, the rate of H2A.X gene transcription decreased slightly under both conditions. These differences in transcriptional regulation between the H2A.X gene and other replication-linked histone genes are sufficient to account for the differences in regulation of their respective stem-loop mRNAs.
V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites.
Authors: Sheehan K M , Lieber M R
Published: October 2015

V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.
Isolation and characterization of DNA sequences that are specifically bound by wild-type p53 protein.
Authors: Foord O , Navot N , Rotter V
Published: October 2015

Wild-type p53 was shown to function as a transcription factor. The N-terminal region of the protein contains the transcription activation domain, while the C terminus is responsible for DNA binding. Localization of the DNA-binding domain of the p53 protein to the highly conserved carboxy-terminal region suggests that the interaction of p53 with DNA is important for its function. We have developed a strategy for studying the DNA sequence specificity of p53-DNA binding that is based on random sequence selection. We report here on the isolation of murine genomic DNA clones that are specifically bound by the wild-type p53 protein but are not bound by mutant p53 protein forms. The isolated p53 target gene contains the unique DNA-binding sequence GACACTGGTCACACTTGGCTGCTTAGGAAT. This fragment exhibits promoter activity as measured by its capacity to activate transcription of the chloramphenicol acetyltransferase reporter gene. Our results suggest that p53 directly binds DNA and functions as a typical transcription factor.
Regulated splicing of the Drosophila sex-lethal male exon involves a blockage mechanism.
Authors: Horabin J I , Schedl P
Published: October 2015

In Drosophila melanogaster, sex determination in somatic cells is controlled by a cascade of genes whose expression is regulated by alternative splicing [B. S. Baker, Nature (London) 340:521-524, 1989; J. Hodgkin, Cell 56:905-906, 1989]. The master switch gene in this hierarchy is Sex-lethal. Sex-lethal is turned on only in females, and an autoregulatory feedback loop which controls alternative splicing maintains this state (L. R. Bell, J. I. Horabin, P. Schedl, and T. W. Cline, Cell 65:229-239, 1991; L. N. Keyes, T. W. Cline, and P. Schedl, Cell 68:933-943, 1992). Sex-lethal also promotes female differentiation by controlling the splicing of RNA from the next gene in the hierarchy, transformer. Sosnowski et al. (B. A. Sosnowski, J. M. Belote, and M. McKeown, Cell 58:449-459, 1989) have shown that the mechanism for generating female transformer transcripts is not through the activation of the alternative splice site but by the blockage of the default splice site. We have tested whether an activation or a blockage mechanism is involved in Sex-lethal autoregulation. The male exon of Sex-lethal with flanking splice sites was placed into the introns of heterologous genes. Our results support the blockage mechanism. The poly(U) run at the male exon 3' splice site is required for sex-specific splicing. However, unlike transformer, default splicing to the male exon is sensitive to the sequence context within which the exon resides. This and the observation that the splice signals at the exon are suboptimal are discussed with regard to alternate splicing.
p53-mediated cell death: relationship to cell cycle control.
Authors: Yonish-Rouach E , Grunwald D , Wilder S , Kimchi A , May E , Lawrence J J , May P , Oren M
Published: October 2015

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.
Identification of a pentanucleotide telomeric sequence, (TTAGG)n, in the silkworm Bombyx mori and in other insects.
Authors: Okazaki S , Tsuchida K , Maekawa H , Ishikawa H , Fujiwara H
Published: October 2015

A pentanucleotide repetitive sequence, (TTAGG)n, has been isolated from a silkworm genomic library, using cross-hybridization with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and Bal 31 exonuclease experiments revealed major clusters of (TTAGG)n at the telomeres of all Bombyx chromosomes. To determine the evolutionary origin of this sequence, two types of telomeric sequence, (TTAGG)5 and a hexanucleotide repetitive sequence, (TTAGGG)4, which is conserved mainly among vertebrate and several invertebrate telomeres so far examined, were hybridized to DNAs from a wide variety of eukaryotic species under highly stringent hybridization conditions. The (TTAGGG)5 oligonucleotide hybridized to genomic DNAs from vertebrates and several nonvertebrate species, as has been reported so far, but not to any DNAs from insects. On the other hand, the Bombyx type of telomere sequence, (TTAGG)n, hybridized to DNAs from 8 of 11 orders of insect species tested but not to vertebrate DNAs, suggesting that this TTAGG repetitive sequence is conserved widely among insects.
Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells.
Authors: Watanabe S , Mui A L , Muto A , Chen J X , Hayashida K , Yokota T , Miyajima A , Arai K
Published: October 2015

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.
Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin.
Authors: Johnson P , Chung S , Benchimol S
Published: October 2015

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.
Mitotic arrest caused by the amino terminus of Xenopus cyclin B2.
Authors: van der Velden H M , Lohka M J
Published: October 2015

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.
A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.
Authors: Houtteman S W , Elder R T
Published: October 2015

Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.
Phosphatidylcholine hydrolysis and c-myc expression are in collaborating mitogenic pathways activated by colony-stimulating factor 1.
Authors: Xu X X , Tessner T G , Rock C O , Jackowski S
Published: October 2015

Stimulation of diglyceride production via phospholipase C (PLC) hydrolysis of phosphatidylcholine was an early event in the mitogenic action of colony-stimulating factor 1 (CSF-1) in the murine macrophage cell line BAC1.2F5 and was followed by a second phase of diglyceride production that persisted throughout the G1 phase of the cell cycle. Addition of phosphatidylcholine-specific PLC (PC-PLC) from Bacillus cereus to the medium of quiescent cells raised the intracellular diglyceride concentration and stimulated [3H]thymidine incorporation, although PC-PLC did not support continuous proliferation. PC-PLC treatment did not induce tyrosine phosphorylation or turnover of the CSF-1 receptor. The major protein kinase C (PKC) isotype in BAC1.2F5 cells was PKC-delta. Diglyceride production from PC-PLC did not target PKC-delta, since unlike phorbol esters, PC-PLC treatment neither decreased the electrophoretic mobility of PKC-delta nor increased the amount of GTP bound to Ras, and PC-PLC was mitogenically active in BAC1.2F5 cells in which PKC-delta was downregulated by prolonged treatment with phorbol ester. PC-PLC mimicked CSF-1 action by elevating c-fos and junB mRNAs to 40% of the level induced by CSF-1; however, PC-PLC induced c-myc mRNA to only 5% of the level in CSF-1-stimulated cells. PC-PLC addition to CSF-1-dependent BAC1.2F5 clones that constitutively express c-myc increased [3H]thymidine incorporation to 86% of the level evoked by CSF-1 and supported slow growth in the absence of CSF-1. Therefore, PC-PLC is a component of a signal transduction pathway leading to transcription of c-fos and junB that collaborates with c-myc and is independent of PKC-delta and Ras activation.
Nonimmune thyroid destruction results from transgenic overexpression of an allogeneic major histocompatibility complex class I protein.
Authors: Frauman A G , Chu P , Harrison L C
Published: October 2015

The overexpression of major histocompatibility complex (MHC) class I molecules in endocrine epithelial cells is an early feature of autoimmune thyroid disease and insulin-dependent diabetes mellitus, which may reflect a cellular response, e.g., to viruses or toxins. Evidence from a transgenic model in pancreatic beta cells suggests that MHC class I overexpression could play an independent role in endocrine cell destruction. We demonstrate in this study that the transgenic overexpression of an allogeneic MHC class I protein (H-2Kb) linked to the rat thyroglobulin promoter, in H-2Kk mice homozygous for the transgene, leads to thyrocyte atrophy, hypothyroidism, growth retardation, and death. Thyrocyte atrophy occurred in the absence of lymphocytic infiltration. Tolerance to allogeneic class I was revealed by the reduced ability of primed lymphocytes from transgenic mice to lyse H-2Kb target cells in vitro. This nonimmune form of thyrocyte destruction and hypothyroidism recapitulates the beta-cell destruction and diabetes that results from transgenic overexpression of MHC class I molecules in pancreatic beta cells. Thus, we conclude that overexpression of MHC class I molecules may be a general mechanism that directly impairs endocrine epithelial cell viability.
Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif.
Authors: Dobrzanski P , Ryseck R P , Bravo R
Published: October 2015

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.
Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome.
Authors: Tazi J , Forne T , Jeanteur P , Cathala G , Brunel C
Published: October 2015

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.
T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.
Authors: Qian L , Theodor L , Carter M , Vu M N , Sasaki A W , Wilkinson M F
Published: October 2015

The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.
Expression of a gene duplication encoding conserved sperm tail proteins is translationally regulated in Drosophila melanogaster.
Authors: Schäfer M , Börsch D , Hülster A , Schäfer U
Published: October 2015

We have analyzed a locus of Drosophila melanogaster located at 98C on chromosome 3, which contains two tandemly arranged genes, named Mst98Ca and Mst98Cb. They are two additional members of the Mst(3)CGP gene family by three criteria. (i) Both genes are exclusively transcribed in the male germ line. (ii) Both transcripts encode a protein with a high proportion of the repetitive motif Cys-Gly-Pro. (iii) Their expression is translationally controlled; while transcripts can be detected in diploid stages of spermatogenesis, association with polysomes can be shown only in haploid stages of sperm development. The genes differ markedly from the other members of the gene family in structure; they do not contain introns, they are of much larger size, and they have the Cys-Gly-Pro motifs clustered at the carboxy-terminal end of the encoded proteins. An antibody generated against the Mst98Ca protein recognizes both Mst98C proteins in D. melanogaster. In a male-sterile mutation in which spermiogenesis is blocked before individualization of sperm, both of these proteins are no longer synthesized. This finding provides proof of late translation for the Mst98C proteins and thereby independent proof of translational control of expression. Northern (RNA) and Western immunoblot analyses indicate the presence of homologous gene families in many other Drosophila species. The Mst98C proteins share sequence homology with proteins of the outer dense fibers in mammalian spermatozoa and can be localized to the sperm tail by immunofluorescence with an anti-Mst98Ca antibody.
Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro.
Authors: Suen C S , Chin W W
Published: October 2015

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.
In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.
Authors: Cooper J A , Kashishian A
Published: October 2015

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential pp40I kappa B-beta inhibition of DNA binding by rel proteins.
Authors: Diehl J A , McKinsey T A , Hannink M
Published: October 2015

Regulation of gene expression by members of the NF-kappa B/rel transcription factor family is a central component of signal transduction pathways utilized by many cellular processes, including lymphocyte activation, embryonic development, and oncogenesis. The members of the NF-kappa B/rel transcription factor family are regulated by association with a family of inhibitor (I kappa B) proteins (I kappa B) proteins. To address the importance of the association between rel and I kappa B proteins for oncogenesis by rel proteins, we characterized rel-I kappa B interactions in chicken embryo fibroblasts (CEF) infected with retroviral vectors encoding the avian c-rel (p68c-rel), v-rel (p59v-rel), and I kappa B-beta (pp40I kappa B-beta) proteins. In these experiments, the p59v-rel:pp40I kappa B-beta ratio in coinfected CEF was nearly identical to the p59v-rel:pp40I kappa B-beta ratio in v-rel-transformed cells. The avian I kappa B-beta protein, pp40I kappa B-beta, was able to associate with both the nononcogenic p68c-rel and the oncogenic p59v-rel. Association of p68c-rel with pp40I kappa B-beta in coinfected CEF resulted in inhibition of the DNA-binding activity of p68c-rel. Anti-pp40I kappa B-beta serum was able to restore DNA binding to p68c-rel in the presence of high levels of pp40I kappa B-beta, indicating that pp40I kappa B-beta functions in a trans-acting manner to inhibit DNA binding by p68c-rel. In contrast, sequence-specific DNA binding by the oncogenic v-rel protein, p59v-rel, was not abolished by pp40I kappa B-beta in coinfected CEF. Anti-pp40I kappa B-beta serum did not immunoprecipitate the p59v-rel-DNA adduct or alter the electrophoretic mobility of the p59v-rel-DNA adduct, consistent with the idea that pp40I kappa B-beta and DNA are competitive inhibitors for the same or overlapping domains on rel proteins. Internal v-rel-derived sequences were identified that are responsible for loss of pp40I kappa B-beta-mediated inhibition of DNA binding by p59v-rel. Loss of pp40I kappa B-beta-mediated inhibition of DNA binding by recombinant v/c-rel proteins was not sufficient for oncogenic activation of c-rel. Instead, removal of C-terminal c-rel-derived sequences in addition to loss of pp40I kappa B-beta-mediated inhibition of DNA binding was required for oncogenic activation of c-rel. These results demonstrate the presence of an interaction between internal and C-terminal regions of the c-rel protein that is important for the ability of c-rel to regulate the proliferation of lymphoid cells.
cis regulation of the keratin 18 gene in transgenic mice.
Authors: Neznanov N S , Oshima R G
Published: October 2015

The gene coding for human keratin 18 (K18), a type I intermediate filament protein found in a variety of simple epithelia, is regulated correctly in transgenic mice but is promiscuously expressed after direct transfection into cell culture lines. We have begun an investigation of the mechanisms responsible for the correct regulation of K18 with a comparison of the chromatin state of K18 in permissive and nonpermissive transgenic mouse tissues to identify seven expression-specific, DNase-hypersensitive sites that correlate with known or potential regulatory regions of the gene. Four of these sites are associated with the proximal promoter region and the first intron that has been implicated previously in the transcriptional control of K18. Two hypersensitive sites are associated with a conserved Alu repetitive sequence located immediately upstream of the proximal promoter elements. Transcription of this Alu element in a direction opposite that of K18 was correlated with K18 expression in transgenic tissues. The final hypersensitive site was mapped to exon 6. The potential importance of this region for the expression of K18 was supported by the results of transient expression of the gene and various deleted constructions. In addition, exon 6 and the intron 1 regulatory region were distinguished from the remainder of K18 by differential DNA methylation in expressing and nonexpressing tissues. The CpG-rich proximal promoter and first exon regions remain unmethylated in both permissive and nonpermissive tissues. These results suggest that DNA methylation is not the primary mechanism of control of the gene. An Alu RNA polymerase III transcription unit and exon 6 are implicated in regulation of K18.
Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.
Authors: Bushman J L , Asuru A I , Matts R L , Hinnebusch A G
Published: October 2015

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.
A human mitochondrial transcriptional activator can functionally replace a yeast mitochondrial HMG-box protein both in vivo and in vitro.
Authors: Parisi M A , Xu B , Clayton D A
Published: October 2015

Human mitochondrial transcription factor A is a 25-kDa protein that binds immediately upstream of the two major mitochondrial promoters, thereby leading to correct and efficient initiation of transcription. Although the nature of yeast mitochondrial promoters is significantly different from that of human promoters, a potential functional homolog of the human transcriptional activator protein has been previously identified in yeast mitochondria. The importance of the yeast protein in yeast mitochondrial DNA function has been shown by inactivation of its nuclear gene (ABF2) in Saccharomyces cerevisiae cells resulting in loss of mitochondrial DNA. We report here that the nuclear gene for human mitochondrial transcription factor A can be stably expressed in yeast cells devoid of the yeast homolog protein. The human protein is imported efficiently into yeast mitochondria, is processed correctly, and rescues the loss-of-mitochondrial DNA phenotype in a yeast abf2 strain, thus functionally substituting for the yeast protein. Both human and yeast proteins affect yeast mitochondrial transcription initiation in vitro, suggesting that the two proteins may have a common role in this fundamental process.
The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae.
Authors: Conklin D S , Kung C , Culbertson M R
Published: October 2015

Eleven cobalt-tolerant mutants were found to belong to a single complementation group, cot2. In addition to cobalt, the cot2 mutants were found to tolerate increased levels of the divalent cations Zn2+, Mn2+, and Ni2+ as well. All of the cot2 mutants exhibited a wiener-shaped cellular morphology that was exacerbated by the carbon and nitrogen source but was unaffected by metals. The rate of glucose-dependent transport of cobalt into cells was reduced in strains that carry mutations in the COT2 gene. COT2 is not essential for growth. Strains that carry a COT2 allele conferring complete loss of function are viable and exhibit phenotypes similar to those of spontaneous cot2 mutations. The sequence of the COT2 gene shows that it is identical to GRR1, which encodes a protein required for glucose repression. The glucose dependence of the transport defect implies that cot2 mutations affect the link between glucose metabolism and divalent cation active transport.
Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae.
Authors: Türkel S , Farabaugh P J
Published: October 2015

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.