bluuebunny/crossref_metadata_2025 huggingface dataset:
Fused protein domains inhibit DNA binding by LexA.
Authors: Golemis E A , Brent R
Published: October 2015

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.
3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei.
Authors: Terns M P , Lund E , Dahlberg J E
Published: October 2015

We have identified and characterized a U6 small nuclear (sn) ribonucleoprotein particle (RNP) present in the nuclei of Xenopus laevis oocytes. The structure of this U6 snRNP was investigated by native gel shift analysis and a combination of RNA-protein UV cross-linking, RNase T1 fingerprinting, and immunoprecipitation assays. These analyses demonstrate that certain forms of U6 snRNA associate with the 50-kDa nuclear antigen La both in vivo and in vitro. The La protein binds the stretch of uridylates at the 3' hydroxyl end of newly synthesized U6 snRNA. La does not bind to mature U6 snRNAs that have 2',3'-cyclic phosphate (greater than p) groups at their 3' ends (E. Lund and J. E. Dahlberg, Science 255:327-330, 1992) or to U6 snRNAs in anti-Sm-precipitable U4/U6 snRNPs. We propose that 3'-end modification, including posttranscriptional UMP addition, modulates the binding of La protein to U6 snRNA which, in turn, may affect the function of this RNA.
Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes.
Authors: Ucker D S , Obermiller P S , Eckhart W , Apgar J R , Berger N A , Meyers J
Published: October 2015

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.
Transcriptional repression by a novel member of the bZIP family of transcription factors.
Authors: Cowell I G , Skinner A , Hurst H C
Published: October 2015

We describe here a novel member of the bZIP family of DNA-binding proteins, designated E4BP4, that displays an unusual DNA-binding specificity which overlaps that of the activating transcription factor family of factors. When expressed in a transient transfection assay with a suitable reporter plasmid, E4BP4 strongly repressed transcription in a DNA-binding-site-dependent manner. Examination of a series of deletion mutants revealed that sequences responsible for the repressing potential of E4BP4 lie within the carboxyl-terminal region of the protein. No similarity was found between this region and the repressing domains of other known eukaryotic transcriptional repressors.
The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein.
Authors: Kumar S , Rabson A B , Gélinas C
Published: October 2015

The v- and c-Rel oncoproteins bind to oligonucleotides containing kappa B motifs, form heterodimers with other members of the Rel family, and modulate expression of genes linked to kappa B motifs. Here, we report that the RxxRxRxxC motif conserved in all Rel/kappa B family proteins is absolutely required for v-Rel protein-DNA contact and its resulting transforming activity. We also demonstrate that serine substitution of the cysteine residue conserved within this motif enables v-Rel to escape redox control, thereby promoting overall DNA binding. These mutant proteins retained the ability to competitively inhibit kappa B-mediated transcriptional activation of the human immunodeficiency virus long terminal repeat but failed to efficiently transform chicken lymphoid cells both in vitro and in vivo. Our data indicate that reduction of the conserved cysteine residue in the RxxRxRxxC motif may be required for optimal DNA-protein interactions. These results provide direct biochemical evidence that the DNA-binding activity of v-Rel is subject to redox control and that the conserved cysteine residue in the RxxRxRxxC motif is critical for this regulation. These studies suggest that the DNA-binding, transcriptional, and biological activities of Rel family proteins may also be subject to redox control in vivo.
Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity.
Authors: Annweiler A , Müller-Immerglück M , Wirth T
Published: October 2015

Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position.
Growth-regulated expression of rhoG, a new member of the ras homolog gene family.
Authors: Vincent S , Jeanteur P , Fort P
Published: October 2015

Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.
In vitro cell cycle arrest induced by using artificial DNA templates.
Authors: Kornbluth S , Smythe C , Newport J W
Published: October 2015

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.
Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins.
Authors: Aboussekhra A , Chanet R , Adjiri A , Fabre F
Published: October 2015

Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination. These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells. The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding. The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa. Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis. The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses.
An activation-dependent, T-lymphocyte-specific transcriptional activator in the mouse mammary tumor virus env gene.
Authors: Miller C L , Garner R , Paetkau V
Published: October 2015

Transcription of the complete mouse mammary tumor virus (MMTV) proviral genome in mouse cells is controlled by a strong promoter in its long terminal repeat. In the mouse T lymphoma EL4, there is a second, activation-dependent transcriptional initiation site within the envelope (env) gene, from which a short mRNA is generated, encoding the open reading frame of the long terminal repeat. We now report the isolation of a segment of the MMTV env gene (called META, for MMTV env transcriptional activator) which has the expected transcription-activating properties seen in EL4.E1 cells. Namely, it induces activation-dependent, T-lymphocyte-specific transcription of a chloramphenicol acetyltransferase reporter gene. It is active in mouse or human T-helper lymphocyte lines when they are stimulated to transcribe lymphokine genes but is inactive in unstimulated T-helper cells, fibroblasts, a cytotoxic T-lymphocyte line, and a mastocytoma cell line. Its activity is inhibited by cyclosporin A, a specific inhibitor of lymphokine transcription. Several forms of the META have been isolated from EL4.E1 cells, a mouse T-helper cell hybridoma, and from BALB/c spleen cells. Linked to the heterologous thymidine kinase promoter, a 400-bp portion of it is an inducible, orientation-independent, and cyclosporin A-sensitive transcriptional activator in T-helper cells.
Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.
Authors: Feldheim D , Rothblatt J , Schekman R
Published: October 2015

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.
Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.
Authors: Izquierdo M , Downward J , Graves J D , Cantrell D A
Published: October 2015

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.
Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins.
Authors: Veals S A , Schindler C , Leonard D , Fu X Y , Aebersold R , Darnell J E , Levy D E
Published: October 2015

Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex. We suggest that other IRF family members may participate in signaling pathways by interacting with as yet unidentified regulatory subunits analogous to ISGF3 alpha.
Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene.
Authors: von Lindern M , van Baal S , Wiegant J , Raap A , Hagemeijer A , Grosveld G
Published: October 2015

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Both transcripts contain the open reading frame for a putative SET protein with a predicted molecular mass of 32 kDa. The set-can fusion gene is transcribed into a 5-kb transcript that contains a single open reading frame predicting a 155-kDa chimeric SET-CAN protein. The SET sequence shows homology with the yeast nucleosome assembly protein NAP-I. The only common sequence motif of SET and DEK proteins is an acidic region. SET has a long acidic tail, of which a large part is present in the predicted SET-CAN fusion protein. The set gene is located on chromosome 9q34, centromeric of c-abl. Since a dek-can fusion gene is present in t(6;9) acute myeloid leukemia and a set-can fusion gene was found in a case of acute undifferentiated leukemia, we assume that can may function as an oncogene activated by fusion of its 3' part to dek, set, or perhaps other genes.
Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.
Authors: Widom R L , Rhee M , Karathanasis S K
Published: October 2015

The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXR alpha together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXR alpha together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAI gene responsiveness to RXR alpha and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXR alpha. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.
Transcriptional repression of the E2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1 gene.
Authors: Hamel P A , Gill R M , Phillips R A , Gallie B L
Published: October 2015

The protein product of the retinoblastoma susceptibility gene, p110RB1, is a nuclear phosphoprotein [W.H. Lee, J.Y. Shew, F.D. Hong, T.W. Sery, L.A. Donoso, L.J. Young, R. Bookstein, and E.Y. Lee, Nature (London) 329:642-645, 1987] with properties of a cell cycle regulator (K. Buchkovich, L.A. Duffy, and E. Harlow, Cell 58:1097-1105, 1989; P.L. Chen, P. Scully, J.Y. Shew, J.Y. Wang, and W.H. Lee, Cell 58:1193-1198, 1989; J.A. DeCaprio, J.W. Ludlow, D. Lynch, Y. Furukawa, J. Griffin, H. Piwnica-Worms, C.M. Huang, and D.M. Livingston, Cell 58:1085-1095, 1989; and K. Mihara, X.R. Cao, A. Yen, S. Chandler, B. Driscoll, A.L. Murphree, A. TAng, and Y.K. Fung, Science 246:1300-1303, 1989). Although the mechanism of action of p110RB1 remains unknown, several lines of evidence suggest that it plays a role in the regulation of transcription. We now show that overexpression of p110RB1 causes repression of the adenovirus early promoter EIIaE and the promoters of two cellular genes, c-myc and RB1, both of which contain E2F-binding motifs. Mutation of the E2 element in the c-myc promoter abolishes p110RB1 repression. We also demonstrate that a p110RB1 mutant, which is refractory to cell cycle phosphorylation but intact in E1a/large T antigen-binding properties, represses EIIaE with 50- to 80-fold greater efficiency than wild-type p110RB1. These data provide evidence that hypophosphorylated p110RB1 actively represses expression of genes with promoters containing the E2F-binding motif (E2 element).
Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.
Authors: Hosokawa N , Hirayoshi K , Kudo H , Takechi H , Aoike A , Kawai K , Nagata K
Published: October 2015

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.
The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site.
Authors: Grayhack E J
Published: October 2015

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.
Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events.
Authors: Dowd D R , Miesfeld R L
Published: October 2015

WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways.
Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.
Authors: Traber P G , Wu G D , Wang W
Published: October 2015

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.
Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.
Authors: Kim D G , Kang H M , Jang S K , Shin H S
Published: October 2015

Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.
Positive and negative elements regulate a melanocyte-specific promoter.
Authors: Lowings P , Yavuzer U , Goding C R
Published: October 2015

Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed.
Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.
Authors: Dominguez I , Diaz-Meco M T , Municio M M , Berra E , García de Herreros A , Cornet M E , Sanz L , Moscat J
Published: October 2015

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.
Characterization of the promoter region of the src family gene lyn and its trans activation by human T-cell leukemia virus type I-encoded p40tax.
Authors: Uchiumi F , Semba K , Yamanashi Y , Fujisawa J , Yoshida M , Inoue K , Toyoshima K , Yamamoto T
Published: October 2015

The src family gene lyn is expressed preferentially in B lymphocytes but very little in normal T lymphocytes. Transcription of the lyn gene in T lymphocytes was shown to be induced by the p40tax protein encoded by human T-cell lymphotropic virus type I. For determination of the mechanism of p40tax-mediated trans activation, the transcriptional promoter region of the lyn gene was characterized. By endonuclease S1 mapping, the transcriptional initiation sites were identified within the 770-bp EcoRI-SacI fragment of the 5'-terminal portion of the human lyn gene. This fragment showed promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into various cell lines. Nucleotide sequence analysis revealed that the lyn promoter region contained four GC box-like sequences but not a TATA or CCAAT box. In addition, it contained sequences characteristic of a cyclic AMP-responsive element, octamer-binding motif, PEA3-like motifs, and NF kappa B-binding motif-like sequence. Mutational analysis suggested that the octamer-binding motif sequence is of primary importance for the lyn promoter activity but that the other elements are not. Cotransfection of various chloramphenicol acetyltransferase constructs containing different length of the lyn promoter together with p40tax expression plasmids into Jurkat T cells showed that the sequence responsible for p40tax-induced transcription is present around the transcription initiation sites.
NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.
Authors: Lee W C , Zabetakis D , Mélèse T
Published: October 2015

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.
Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.
Authors: Pietrzkowski Z , Sell C , Lammers R , Ullrich A , Baserga R
Published: October 2015

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.
Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.
Authors: Jehn B , Costello E , Marti A , Keon N , Deane R , Li F , Friis R R , Burri P H , Martin F , Jaggi R
Published: October 2015

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.
Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.
Authors: Molloy C J , Fleming T P , Bottaro D P , Cuadrado A , Aaronson S A
Published: October 2015

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.
A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.
Authors: Osada S , Mizuno K , Saido T C , Suzuki K , Kuroki T , Ohno S
Published: October 2015

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.