bluuebunny/crossref_metadata_2025 huggingface dataset:
cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle.
Authors: Ebbole D J , Paluh J L , Plamann M , Sachs M S , Yanofsky C
Published: October 2015

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.
Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein.
Authors: Bouton A H , Kanner S B , Vines R R , Wang H C , Gibbs J B , Parsons J T
Published: October 2015

GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.
Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.
Authors: Stern D F , Zheng P , Beidler D R , Zerillo C
Published: October 2015

A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.
Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.
Authors: Bengal E , Flores O , Krauskopf A , Reinberg D , Aloni Y
Published: October 2015

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.
Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns.
Authors: Himmelspach M , Gattoni R , Gerst C , Chebli K , Stévenin J
Published: October 2015

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.
Multiple basal promoter elements determine the level of human c-fos transcription.
Authors: Runkel L , Shaw P E , Herrera R E , Hipskind R A , Nordheim A
Published: October 2015

Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.
Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.
Authors: Paciucci R , Pellicer A
Published: October 2015

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.
Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.
Authors: Young L S , Rivier D H , Sprague K U
Published: October 2015

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.
Relationship of avian retrovirus DNA synthesis to integration in vitro.
Authors: Lee Y M , Coffin J M
Published: October 2015

An in vitro integration system derived from avian leukosis virus-infected cells supports both intra- and intermolecular integration of the viral DNA. In the absence of polyethylene glycol, intramolecular integration of viral DNA molecules into themselves (autointegration) was preferred. In the presence of polyethylene glycol, integration into an exogenously supplied DNA target was greatly promoted. Analysis of integration intermediates revealed that the strand transfer mechanisms of both reactions were identical to those of retroviruses and some transposons: each 3' end of the donor molecule is joined to a 5' end of the cleaved target DNA. The immediate integration precursor appears to be linear viral DNA with the 3' ends shortened by 2 nucleotides. Finally, in the avian system, most cytoplasmic viral DNA appears to be incomplete and further DNA synthesis is required for integration in vitro.
Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein.
Authors: Araki S , Kaibuchi K , Sasaki T , Hata Y , Takai Y
Published: October 2015

smg p25A is a ras p21-like small GTP-binding protein which is implicated in the regulated secretory processes. We have recently found that bovine brain smg p25A is geranylgeranylated at its C-terminal region. In this study, we examined the function(s) of the C-terminal region of smg p25A. Limited proteolysis of bovine brain smg p25A with Achromobacter protease I produced an N-terminal fragment and a C-terminal tail. The Mrs of intact smg p25A, the N-terminal fragment, and the C-terminal tail were estimated to be about 24,000, 20,000, and less than 2,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal fragment contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities and showed these activities with kinetic properties similar to those of the intact protein but did not bind to plasma membranes or phosphatidylserine-linked Affigel under conditions in which the intact protein bound to them. The C-terminal tail neither contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities nor bound to plasma membranes or phosphatidylserine-linked Affigel. The GDP/GTP exchange protein specific for smg p25A, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of the intact smg p25A at a molar ratio of 1:1 and thereby inhibited its GDP/GTP exchange reaction but neither made a complex with the N-terminal fragment or the C-terminal tail nor affected the GDP/GTP exchange reaction of the N-terminal fragment. We expressed smg p25A in Escherichia coli and purified it to near homogeneity. This bacterial protein was not geranylgeranylated. Bacterial smg p25A did not bind to plasma membranes or phosphatidylserine-linked Affigel. smg p25A GDI neither made a complex with bacterial smg p25A nor affected its GDP/GTP exchange reaction. These results suggest that the N-terminal region of smg p25A has GDP/GTP-binding and GTPase activities but lacks the ability to interact with membranes and smg p25A GDI, that the C-terminal region of smg p25A plays important roles in its interaction with membranes and smg p25A GDI, and that some modifications of the C-terminal region, such as geranylgeranylation, which are absent in bacterial smg p25A, are important for these interactions.
A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization.
Authors: Kashles O , Yarden Y , Fischer R , Ullrich A , Schlessinger J
Published: October 2015

Recent studies provide evidence that defective receptors can function as a dominant negative mutation suppressing the action of wild-type receptors. This causes various diminished responses in cell culture and developmental disorders in murine embryogenesis. Here, we describe a model system and a potential mechanism underlying the dominant suppressing response caused by defective epidermal growth factor (EGF) receptors. We used cultured 3T3 cells coexpressing human wild-type receptors and an inactive deletion mutant lacking most of the cytoplasmic domain. When expressed alone, EGF was able to stimulate the dimerization of either wild-type or mutant receptors in living cells as revealed by chemical covalent cross-linking experiments. In response to EGF, heterodimers and homodimers of wild-type and mutant receptors were observed in cells coexpressing both receptor species. However, only homodimers of wild-type EGF receptors underwent EGF-induced tyrosine autophosphorylation in living cells. These results indicate that the integrity of both receptor moieties within receptor dimers is essential for kinase activation and autophosphorylation. Moreover, the presence of mutant receptors in cells expressing wild-type receptors diminished the number of high-affinity binding sites for EGF, reduced the rate of receptor endocytosis and degradation, and diminished biological signalling via EGF receptors. We propose that heterodimerization with defective EGF receptors functions as a dominant negative mutation suppressing the activation and response of normal receptors by formation of unproductive heterodimers.
Interleukin-7 retroviruses transform pre-B cells by an autocrine mechanism not evident in Abelson murine.
Authors: Overell R W , Clark L , Lynch D , Jerzy R , Schmierer A , Weisser K E , Namen A E , Goodwin R G
Published: October 2015

In this study, we have constructed retroviral vectors expressing the interleukin-7 (IL-7) cDNA and have used infection with these retroviruses to express this cytokine endogenously in an IL-7-dependent pre-B-cell line. Infection with IL-7 retroviruses, but not with a control retrovirus, resulted in the conversion of the cells to IL-7 independence. The frequency at which this occurred, together with data on vector expression levels, indicated that secondary events were required for factor independence in this system. Southern analysis showed that the IL-7-dependent clones harbored unrearranged copies of the vector proviruses. The factor-independent cells produced variable quantities of IL-7 as measured by an IL-7-specific bioassay, and their proliferation could be substantially inhibited by a neutralizing antibody directed against IL-7, indicating that a classical autocrine-mechanism was responsible for their transformation. These IL-7-independent cells were tumorigenic, in contrast to the parental IL-7-dependent cells or those infected with a control vector. These results showed that IL-7 could participate in the malignant transformation of pre-B cells. However, neither of two Abelson murine leukemia virus (A-MuLV)-transformed pre-B-cell lines expressed detectable IL-7 mRNA, at a level of sensitivity corresponding to less than one molecule of mRNA per cell. Moreover, the proliferation of the A-MuLV transformants was unaffected by addition of the IL-7 antisera under conditions in which parallel experiments with IL-7 virus-infected cells resulted in greater than 70% growth inhibition. Thus, transformation of pre-B cells by A-MuLV was not associated with a demonstrable autocrine loop of IL-7 synthesis. These results show that IL-7 can participate in the malignant transformation of pre-B cells and suggest studies aimed at assessing the role of autocrine production of IL-7 in the generation of human leukemias and lymphomas.
The tumor suppressor p53 is bound to RNA by a stable covalent linkage.
Authors: Samad A , Carroll R B
Published: October 2015

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.
Isolation, characterization, and expression of the murine Wilms' tumor gene (WT1) during kidney development.
Authors: Buckler A J , Pelletier J , Haber D A , Glaser T , Housman D E
Published: October 2015

The human Wilms' tumor predisposition gene, WT1, is a Cys-His zinc finger polypeptide which appears to be a transcription factor controlling gene expression during embryonic kidney development. In order to analyze the role of the WT1 gene in nephroblast differentiation, we have isolated the murine homolog of human WT1. An extremely high level of amino acid sequence conservation (greater than 95%) extends throughout all regions of the predicted mouse and human WT1 polypeptides. Two alternative splices within the WT1 transcript have been conserved between mice and humans, suggesting that these have functional significance. Expression of the mouse WT1 mRNA in fetal kidney increases during late gestation, peaks just prior to or shortly after birth, and declines dramatically by 15 days postpartum. Developmental regulation of WT1 expression appears to be selective for the kidney. The restriction of WT1 expression to a limited number of tissues is in contrast to previously described tumor suppressor genes. In addition, the narrow window of time during which WT1 is expressed at high levels in the kidney is consistent with the origin of Wilms' tumor from primitive nephroblasts and the postulated role of this gene as a negative regulator of growth.
Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene.
Authors: Yu D H , Scorsone K , Hung M C
Published: October 2015

The adenovirus type 5 early region 1A (E1A) gene was introduced into neu-transformed B104-1-1 cells. Cells that expressed E1A possessed reduced transforming activity in vitro and reduced tumorigenicity in nude mice. These results demonstrate that the E1A gene products can act negatively to suppress the transformed phenotype in neu-transformed cells.
The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.
Authors: Varnum B C , Ma Q F , Chi T H , Fletcher B , Herschman H R
Published: October 2015

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.
Common factor 1 is a transcriptional activator which binds in the c-myc promoter, the skeletal alpha-actin promoter, and the immunoglobulin heavy-chain enhancer.
Authors: Riggs K J , Merrell K T , Wilson G , Calame K
Published: October 2015

Ubiquitously expressed transcription factors play an integral role in establishing and regulating patterns of gene transcription. Common factor 1 (CF1) is a ubiquitously expressed DNA-binding protein previously identified in our laboratory. We show here that CF1 recognizes sites in several diverse transcription elements, and we demonstrate the ability of the c-myc CF1 site to activate transcription of a basal promoter in both B cells and fibroblasts.
Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger.
Authors: Halvorsen Y D , Nandabalan K , Dickson R C
Published: October 2015

The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: [Formula: see text]. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.
BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias.
Authors: Muller A J , Young J C , Pendergast A M , Pondel M , Landau N R , Littman D R , Witte O N
Published: October 2015

The c-abl proto-oncogene encodes a cytoplasmic tyrosine kinase which is homologous to the src gene product in its kinase domain and in the upstream kinase regulatory domains SH2 (src homology region 2) and SH3 (src homology region 3). The murine v-abl oncogene product has lost the SH3 domain as a consequence of N-terminal fusion of gag sequences. Deletion of the SH3 domain is sufficient to render the murine c-abl proto-oncogene product transforming when myristylated N-terminal membrane localization sequences are also present. In contrast, the human BCR/ABL oncogene of the Philadelphia chromosome translocation has an intact SH3 domain and its product is not myristylated at the N terminus. To analyze the contribution of BCR-encoded sequences to BCR/ABL-mediated transformation, the effects of a series of deletions and substitutions were assessed in fibroblast and hematopoietic-cell transformation assays. BCR first-exon sequences specifically potentiate transformation and tyrosine kinase activation when they are fused to the second exon of otherwise intact c-ABL. This suggests that BCR-encoded sequences specifically interfere with negative regulation of the ABL-encoded tyrosine kinase, which would represent a novel mechanism for the activation of nonreceptor tyrosine kinase-encoding proto-oncogenes.
The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation.
Authors: Naldini L , Vigna E , Ferracini R , Longati P , Gandino L , Prat M , Comoglio P M
Published: October 2015

Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.
Biosynthesis of human fibroblast growth factor-5.
Authors: Bates B , Hardin J , Zhan X , Drickamer K , Goldfarb M
Published: October 2015

We have analyzed the biosynthesis of human fibroblast growth factor-5 (FGF-5) at the translational and posttranslational levels. FGF-5 RNA synthesized in vitro can be translated in rabbit reticulocyte lysates to yield a 29,500-Da protein, which is consistent with the molecular weight predicted from the coding sequence. The efficiency of FGF-5 translation is dramatically enhanced if an upstream open reading frame (ORF-1) in the RNA is deleted or if both AUG codons in ORF-1 are destroyed by point mutations, while partial enhancement is achieved by individual mutation of either ORF-1 AUG codon. These data suggest that FGF-5 synthesis requires the scanning of ribosomes past the two ORF-1 AUG codons. The introduction of these ORF-1 mutations into a eukaryotic FGF-5 expression vector increases its capacity to transform mouse NIH 3T3 cells up to 50-fold upon transfection. FGF-5 is secreted from transfected 3T3 cells and from human tumor cells as glycoproteins containing heterogeneous amounts of sialic acid. Glycosidase treatments suggest that the growth factor bears both N-linked and O-linked sugars.
Ecdysterone regulatory elements function as both transcriptional activators and repressors.
Authors: Dobens L , Rudolph K , Berger E M
Published: October 2015

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.
Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines.
Authors: Shah N P , Witte O N , Denny C T
Published: October 2015

The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.
Synergistic activation of a human promoter in vivo by transcription factor Sp1.
Authors: Anderson G M , Freytag S O
Published: October 2015

Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.
The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains.
Authors: D'Andrea A D , Yoshimura A , Youssoufian H , Zon L I , Koo J W , Lodish H F
Published: October 2015

The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism.
Cross-resistance to UV radiation of a cisplatin-resistant human cell line: overexpression of cellular factors that recognize UV-modified DNA.
Authors: Chao C C , Huang S L , Huang H M , Lin-Chao S
Published: October 2015

A human cell line selected for cisplatin resistance (CPR) was irradiated with UV light and showed cross-resistance to UV light. Applying a modified chloramphenicol acetyltransferase assay, we observed that CPR cells acquired enhanced host cell reactivation of a transfected plasmid carrying UV damage. Gel mobility shift analysis indicated that two nuclear factors that recognize UV-modified DNA were overexpressed in CPR cells. In addition, factors that bind UV-modified DNA were independent from the factors that bind cisplatin-modified DNA. The significance of the identified binding factors, possibly DNA repair enzymes, is discussed.
Autonomous DNA replication in human cells is affected by the size and the source of the DNA.
Authors: Heinzel S S , Krysan P J , Tran C T , Calos M P
Published: October 2015

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.
Transcriptional suppression of cellular gene expression by c-Myc.
Authors: Yang B S , Geddes T J , Pogulis R J , de Crombrugghe B , Freytag S O
Published: October 2015

High levels of c-Myc in mouse 3T3-L1 cells specifically suppress the expression of three collagen genes. This effect is exerted through collagen promoter sequences and requires the leucine zipper motif of c-Myc. Our data suggest that an important aspect of c-Myc transforming activity is the ability to suppress specific cellular gene transcription.
Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells.
Authors: Defeo-Jones D , McAvoy E M , Jones R E , Vuocolo G A , Haskell K M , Wegrzyn R J , Oliff A
Published: October 2015

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.
The developmentally regulated shift from membrane to secreted mu mRNA production is accompanied by an increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency.
Authors: Peterson M L , Gimmi E R , Perry R P
Published: October 2015

To determine whether there are any developmental changes in the efficiencies of cleavage-polyadenylation or splicing reactions that could affect the usage of weak (suboptimal) processing signals and thus provide a basis for the regulated production of mu m versus mu s mRNA during B-lymphocyte maturation, we studied the expression of transfected mu genes in which the natural competition between cleavage-polyadenylation and splicing was replaced by alternative usage of tandem weak and strong poly(A) sites or by competition between suboptimal and optimal 5' splice junctions. Our results indicate that there is a 50 to 100% increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency as maturation proceeds from the B-cell to plasma cell stage.
Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.
Authors: Brown L E , Sprecher S L , Keller L R
Published: October 2015

The fate of exogenous DNA introduced into Chlamydomonas reinhardtii by electroporation was analyzed. With single and double electrical pulses, plasmids as large as 14 kb were introduced into cells with and without intact cell walls. Within hours after introduction, exogenous plasmid DNA was associated with nuclei isolated from cells; several weeks after introduction, exogenous DNA was stably integrated into the Chlamydomonas genome. These studies establish electroporation as a method for introducing DNA, and potentially other molecules, into C. reinhardtii.
Expression of different Jun and Fos proteins during the G0-to-G1 transition in mouse fibroblasts: in vitro and in vivo associations.
Authors: Kovary K , Bravo R
Published: October 2015

We have characterized the expression of c-Jun, JunB, JunD, c-Fos, and FosB proteins following serum stimulation of quiescent Swiss 3T3 cells by immunoprecipitation analyses. The synthesis of the three Jun proteins rapidly increases following stimulation, remaining at a significant level for at least 8 h. JunB protein presents the highest expression of all. FosB, like c-Fos, is transiently induced. Pulse-chase experiments show that all of the proteins except JunD are short-lived. We have shown that c-Fos and FosB form complexes in vivo with the different Jun proteins and that JunB complexes are predominant. In vitro association and competition experiments show that the affinities between the different Fos and Jun proteins are similar. This finding, together with the in vivo observations described above, suggests that the proportion of the different Jun/Fos heterodimers is governed by the concentration of the different components. The Fos and Jun proteins are phosphoproteins, and some remain relatively highly phosphorylated in their heterodimeric form.
Analysis of constitutive and noninducible mutations of the PUT3 transcriptional activator.
Authors: Marczak J E , Brandriss M C
Published: October 2015

The Saccharomyces cerevisiae PUT3 gene encodes a transcriptional activator that binds to DNA sequences in the promoters of the proline utilization genes and is required for the basal and induced expression of the enzymes of this pathway. The sequence of the wild-type PUT3 gene revealed the presence of one large open reading frame capable of encoding a 979-amino-acid protein. The protein contains amino-terminal basic and cysteine-rich domains homologous to the DNA-binding motifs of other yeast transcriptional activators. Adjacent to these domains is an acidic domain with a net charge of -17. A second acidic domain with a net charge of -29 is located at the carboxy terminus. The midsection of the PUT3 protein has homology to other activators including GAL4, LAC9, PPR1, and PDR1. Mutations in PUT3 causing aberrant (either constitutive or noninducible) expression of target genes in this system have been analyzed. One activator-defective and seven activator-constitutive PUT3 alleles have been retrieved from the genome and sequenced to determine the nucleotide changes responsible for the altered function of the protein. The activator-defective mutation is a single nucleotide change within codon 409, replacing glycine with aspartic acid. One activator-constitutive mutation is a nucleotide change at codon 683, substituting phenylalanine for serine. The remaining constitutive mutations resulted in amino acid substitutions or truncations of the protein within the carboxy-terminal 76 codons. Mechanisms for regulating the activation function of the PUT3 protein are discussed.