Showing papers in "The EMBO Journal in 1990"
TL;DR: Using the hPR gene 5′‐flanking sequences as promoter region in chimeric genes, it is shown that a functional promoter directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15.
Abstract: The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5'-flanking region were cloned and sequenced. Comparison of the cDNA-deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5' region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5'-flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between -711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co-transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.
1,506 citations
TL;DR: Interestingly, NF‐IL6 was shown to bind to the regulatory regions for various acute‐phase protein genes and several other cytokine genes such as TNF, IL‐8 and G‐CSF, implying that NF‐ IL6 has a role in regulation not only for the IL‐6 gene but also for several other genes involved in acute‐ phase reaction, inflammation and hemopoiesis.
Abstract: NF-IL6 is a nuclear factor that specifically binds to an IL1-responsive element in the IL-6 gene. In this study the gene encoding NF-IL6 has been cloned by direct screening of a lambda gt11 library using NF-IL6 binding sequence as a ligand. The full-length cDNA encoded a 345 amino acid protein with a potential leucine zipper structure and revealed a high degree of homology to a liver-specific transcriptional factor, C/EBP, at the C-terminal portion. The bacterial fusion protein bound to the CCAAT homology as well as the viral enhancer core sequences as in the case of C/EBP. Recombinant NF-IL6 activated the human IL-6 promoter in a sequence-specific manner. Southern blot analysis demonstrated the high-degree conservation of the NF-IL6 gene through evolution and the existence of several other related genes sharing the DNA-binding domain. NF-IL6 mRNA was normally not expressed, but induced by the stimulation with either LPS, IL-1 or IL-6. Interestingly, NF-IL6 was shown to bind to the regulatory regions for various acute-phase protein genes and several other cytokine genes such as TNF, IL-8 and G-CSF, implying that NF-IL6 has a role in regulation not only for the IL-6 gene but also for several other genes involved in acute-phase reaction, inflammation and hemopoiesis.
1,453 citations
TL;DR: The sequence similarity of these RNA polymerases to RT suggests that these two enzymes evolved from a common ancestor, and thus RNA polymerase can be used as an outgroup to root the RT tree.
Abstract: To study the evolutionary relationship of reverse transcriptase (RT) containing genetic elements, a phylogenetic tree of 82 retroelements from animals, plants, protozoans and bacteria was constructed. The tree was based on seven amino acid domains totalling 178 residues identified in all RTs. We have also identified these seven domains in the RNA-directed RNA polymerases from various plus-strand RNA viruses. The sequence similarity of these RNA polymerases to RT suggests that these two enzymes evolved from a common ancestor, and thus RNA polymerase can be used as an outgroup to root the RT tree. A comparison of the genetic organization of the various RT containing elements and their position on the tree allows several inferences concerning the origin and evolution of these elements. The most probable ancestor of current retroelements was a retrotransposable element with both gag-like and pol-like genes. On one major branch of the tree, organelle and bacterial sequences (e.g. group II introns and bacterial msDNA) appear to have captured the RT sequences from retrotransposons which lack long terminal repeats (LTRs). On the other major branch, acquisition of LTRs gave rise to two distinct groups of LTR retrotransposons and three groups of viruses: retroviruses, hepadnaviruses and caulimoviruses.
1,302 citations
TL;DR: The physiological importance of these two proteins in cellular signal transduction is discussed, and partial purification of GSK‐3 activity from bovine brain results in the isolation of active alpha and beta proteins.
Abstract: Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.
1,293 citations
TL;DR: A mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in‐line attack of water as well as a mechanism for rate enhancement by GAP is proposed.
Abstract: The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.
1,042 citations
TL;DR: Data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures.
Abstract: An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron X-ray absorption measurements of crystals of the selenomethionyl proteins. Taken with reference to experience in the structural analysis of selenobiotinyl streptavidin by the method of multiwavelength anomalous diffraction (MAD), these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures.
1,042 citations
TL;DR: The distribution of the BDNF mRNA in the adult mouse brain is reported on, using quantitative Northern blot analysis and in situ hybridization, to suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.
Abstract: Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.
986 citations
TL;DR: It is proposed that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggested that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein.
Abstract: Point mutations in the p53 gene are the most frequently identified genetic change in human cancer. They convert murine p53 from a tumour suppressor gene into a dominant transforming oncogene able to immortalize primary cells and bring about full transformation in combination with an activated ras gene. In both the human and murine systems the mutations lie in regions of p53 conserved from man to Xenopus. We have developed a monoclonal antibody to p53 designated PAb240 which does not immunoprecipitate wild type p53. A series of different p53 mutants all react more strongly with PAb240 than with PAb246. The PAb240 reactive form of p53 cannot bind to SV40 large T antigen but does bind to HSP70. In contrast, the PAb246 form binds to T antigen but not to HSP70. PAb240 recognizes all forms of p53 when they are denatured. It reacts with all mammalian p53 and chicken p53 in immunoblots. We propose that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggest that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein. This conformational change abolishes T antigen binding and promotes self-oligomerization. These results are consistent with a dominant negative model where mutant p53 protein binds to and neutralizes the activity of p53 in the wild type conformation.
972 citations
TL;DR: In this paper, the retention motifs of transmembrane endoplasmic reticulum (ER) proteins were identified as a retrieval signal that brought proteins back from a sorting compartment adjacent to the ER.
Abstract: Several families of transmembrane endoplasmic reticulum (ER) proteins contain retention motifs in their cytoplasmically exposed tails. Mutational analyses demonstrated that two lysines positioned three and four or five residues from the C-terminus represent the retention motif. The introduction of a lysine preceding the lysine that occurs three residues from the terminus of Lyt2 renders this cell surface protein a resident of the ER. Likewise, the appropriate positioning of two lysine residues in a poly-serine sequence confines marker proteins to the ER. Arginines or histidines cannot replace lysines, suggesting that simple charge interactions are not sufficient to explain the retention. The identified consensus motif may serve as a retrieval signal that brings proteins back from a sorting compartment adjacent to the ER.
911 citations
TL;DR: The results suggest that the synthesis of these two neurotrophic factors in the brain is regulated by neuronal activity via non‐NMDA glutamate receptors.
Abstract: The mRNAs of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) exhibit a similar, though not identical, regional and cellular distribution in the rodent brain. In situ hybridization experiments have shown that BDNF, like NGF, is predominantly expressed by neurons. The neuronal localization of the mRNAs of these two neurotrophic molecules raised the question as to whether neuronal activity might be involved in the regulation of their synthesis. After we had demonstrated that depolarization with high potassium (50 mM) resulted in an increase in the levels of both BDNF and NGF mRNAs in cultures of hippocampal neurons, we investigated the effect of a large number of transmitter substances. Kainic acid, a glutamate receptor agonist, was by far the most effective in increasing BDNF and NGF mRNA levels in the neurons, but neither N-methyl-D-aspartic acid (NMDA) nor inhibitors of the NMDA glutamate receptors had any effect. However, the kainic acid mediated increase was blocked by antagonists of non-NMDA receptors. Kainic acid also elevated levels of BDNF and NGF mRNAs in rat hippocampus and cortex in vivo. These results suggest that the synthesis of these two neurotrophic factors in the brain is regulated by neuronal activity via non-NMDA glutamate receptors.
900 citations
TL;DR: The recombinant tau isoforms were biologically active at micromolar concentrations, as assessed by their ability to promote microtubule assembly and the rates of assembly were 2.5–3.0 times faster for isoforms containing four repeats when compared with three‐repeat containing isoforms, with no significant contribution by the amino‐terminal insertions.
Abstract: We have expressed six previously cloned isoforms of human microtubule-associated tau protein in Escherichia coli and purified them to homogeneity in a biologically active form. They range from 352 to 441 amino acids in length and differ from each other by the presence of three or four tandem repeats in the carboxy-terminal half and by the presence or absence of 29 or 58 amino acid inserts in the amino-terminus. When mixed together they gave a set of six bands on SDS-PAGE gels with apparent molecular weights of 48-67 kd and with a characteristic pattern of spacings. Four of these bands aligned with the major tau bands found in adult human cerebral cortex following perchloric acid extraction and alkaline phosphatase treatment. They consisted of isoforms with three repeats and no insertions, four repeats and no amino-terminal insertions and three- and four-repeat containing isoforms with the 29 amino acid insertion. In fetal human brain extracts treated with alkaline phosphatase one of the two major tau bands aligned with the three-repeat containing isoform with no insertions, whereas the molecular nature of the second major tau band remains to be established. The recombinant tau isoforms were biologically active at micromolar concentrations, as assessed by their ability to promote microtubule assembly. The rates of assembly were 2.5-3.0 times faster for isoforms containing four repeats when compared with three-repeat containing isoforms, with no significant contribution by the amino-terminal insertions.
TL;DR: Results are compatible with a model whereby HIV‐1 can persist in a non‐productive extra‐chromosomal state in resting T cells until subsequent antigen‐induced or mitogen‐induced T cellactivation, virus integration and release, and agents that induce T cell activation may control the rate of HIV‐ 1 replication and spread during AIDS progression.
Abstract: During progression of the Acquired Immune Deficiency Syndrome (AIDS), the human immunodeficiency virus type 1 (HIV-1) is harbored in CD4+ T cells, which act as the primary reservoir for the virus. In vitro, HIV-1 requires activated T cells for a productive infection; however, in vivo, the number of circulating T cells in the activated state that are potential targets for HIV-1 infection is low. We have investigated the ability of HIV-1 to infect resting T cells, and the consequences of such an infection. T cell activation was not required for HIV-1 infection; however, viral DNA was unable to integrate in resting T cells and was maintained extrachromosomally. Subsequent T cell activation allowed integration of extrachromosomal forms and led to a productive viral life cycle. Extrachromosomal forms of viral DNA were found to persist for several weeks after infection of resting T cells and, following T cell activation, these forms maintained their ability to integrate and act as a template for infectious virus. Several lines of evidence, including temporal analysis of HIV-1 replication and analysis of an HIV-1 integrase deletion mutant, indicated that extra-chromosomal HIV-1 DNA genomes were transcriptionally active. These results are compatible with a model whereby HIV-1 can persist in a non-productive extra-chromosomal state in resting T cells until subsequent antigen-induced or mitogen-induced T cell activation, virus integration and release. Thus agents that induce T cell activation may control the rate of HIV-1 replication and spread during AIDS progression.
TL;DR: It is shown that IL‐1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum‐‐Golgi route, which may involve translocation of intracellular membranes and is increased by stress conditions.
Abstract: Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.
TL;DR: It is demonstrated that the agonistic effect of OHT on the whole hER is due to the cell‐type and promoter‐context dependent activity of TAF‐1, and it is confirmed that the wild type human oestrogen receptor has no ligand independent transcriptional activity.
Abstract: Various oestrogen responsive reporter genes and vectors expressing truncated or chimeric human oestrogen receptors (hER) containing either of the two independent hER transcriptional activation functions (TAF-1 and TAF-2) have been transfected into HeLa cells, chicken embryo fibroblast (CEF) or yeast cells to investigate the agonistic activity of the anti-oestrogen 4-hydroxytamoxifen (OHT). We demonstrate that the agonistic effect of OHT on the whole hER is due to the cell-type and promoter-context dependent activity of TAF-1. In similar experiments, we show that the anti-oestrogen, ICI 164,384, does not exhibit any oestrogenic activity and, therefore, acts always as a pure antagonist, even though it does not inhibit the activity of the isolated TAF-1. We also confirm that the wild type human oestrogen receptor has no ligand independent transcriptional activity. The implications of our results for the variable antagonist/agonist activity of anti-oestrogens in vivo are discussed.
TL;DR: A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted ‘flower specific’ cDNA library by utilizing the somatic and germinal instability of defA‐1 mutants.
Abstract: Deficiens (defA+) is a homeotic gene involved in the genetic control of Antirrhinum majus flower development Mutation of this gene (defA-1) causes homeotic transformation of petals into sepals and of stamina into carpels in flowers displaying the 'globifera' phenotype, as shown by cross sections and scanning electronmicroscopy of developing flowers A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted 'flower specific' cDNA library The identity of this cDNA with the defA+ gene product has been confirmed by utilizing the somatic and germinal instability of defA-1 mutants According to Northern blot analyses the defA+ gene is expressed in flowers but not in leaves, and its expression is nearly constant during all stages of flower development The 11 kb long mRNA has a 681 bp long open reading frame that can code for a putative protein of 227 amino acids (mol wt 262 kd) At its N-terminus the DEF A protein reveals homology to a conserved domain of the regulatory proteins SRF (activating c-fos) in mammals and GRM/PRTF (regulating mating type) in yeast We discuss the structure and the possible function of the DEF A protein in the control of floral organogenesis
TL;DR: Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta supergene family, DPP of Drosophila and Vg-1 (vegetal protein) of Xenopus.
Abstract: Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.
TL;DR: It is shown that the ectopic expression of Oct‐4 in HeLa cells is sufficient for transcriptional activation from the octamer motif, indicating thatOct‐4 is a transcription factor that is specifically expressed in cells participating in the generation of the germline lineage.
Abstract: Oct-4 is a maternally expressed octamer-binding protein encoded by the murine Oct-4 gene. It is present in unfertilized oocytes, but also in the inner cell mass and in primordial germ cells. Here we show that the ectopic expression of Oct-4 in HeLa cells is sufficient for transcriptional activation from the octamer motif, indicating that Oct-4 is a transcription factor. Therefore, Oct-4 is the first transcription factor described that is specific for the early stages of mouse development. The spatial and temporal expression patterns were further determined using in situ hybridization. With this technique Oct-4 expression is detected in the oocyte, in the blastocyst and before gastrulation in the embryonic ectoderm. After day 8 Oct-4 expression decreases and is restricted to primordial germ cells from about day 8.5 onwards. Therefore Oct-4 is a transcription factor that is specifically expressed in cells participating in the generation of the germline lineage. Linkage analysis using B X D recombinant inbred mouse strains demonstrates that Oct-4 maps to chromosome 17 in or near the major histocompatibility complex. Several mouse mutants in the distal region of the mouse t-complex affecting blastocyst and embryonic ectoderm formation also map to this region.
TL;DR: Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross‐linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen.
Abstract: The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.
TL;DR: The complete cDNA cloning of two human genes previously designated flg and bek is reported, indicating that bek and flg bind either aFGF or bFGF with dissociation constants of (2‐15) x 10(‐11) M.
Abstract: The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin-like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2-15) x 10(-11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor-receptor interactions.
TL;DR: It is shown that purified recombinant c‐sis/PDGF can induce a DNA binding protein which binds to a conserved sequence upstream of the human c‐fos gene, which is termed SIF, for sis‐inducible factor.
Abstract: The c-fos proto-oncogene is rapidly and transiently induced by a variety of extracellular stimuli. We have previously shown that conditioned media from v-sis transformed NRK cells rapidly induces a DNA binding protein which binds to a conserved sequence upstream of the human c-fos gene. We now show that purified recombinant c-sis/PDGF can induce this binding activity which we have termed SIF, for sis-inducible factor. Oligonucleotides which bind to the SIF protein will confer sis/PDGF inducibility onto a truncated, unresponsive c-fos promoter. However, sequences lying between -100 and -57 of the c-fos gene are required for this induction. The sis-responsive element functions independently of a region of dyad symmetry previously identified as the serum responsive element (SRE). The time course of c-fos expression driven by the sis-responsive element is similar to that mediated by the SRE. Unlike the SRE, which can respond to signals generated by sis/PDGF, serum or phorbol esters, the SIF binding element mediates c-fos induction only in response to sis/PDGF. The SRE and SIF elements function in an additive manner to stimulate the transcription of the c-fos gene in response to sis/PDGF.
TL;DR: It is shown that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c‐kit coding sequence, which affect the c-kit associated tyrosine kinase to varying degrees.
Abstract: The proto-oncogene c-kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white-spotting locus (W) Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development The original W mutation and W37 are severe lethal mutations when homozygous In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics Wv and W41 are weak W mutations with dominant characteristics We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co-culture assay We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c-kit coding sequence (W37 E----K at position 582; Wv T----M position 660 and W41 V----M position 831), which affect the c-kit associated tyrosine kinase to varying degrees The c-kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c-kitW37 protein displays accelerated turnover characteristics The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c-kit protein A 125 kd c-kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The cDNA for TTF‐1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned and the protein encoded by the cDNA shows binding properties indistinguishable from those of TTF•1 present in nuclear extracts of differentiated rat thyroid cells.
Abstract: The cDNA for TTF-1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF-1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF-1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK-2 homeodomain. TTF-1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF-1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF-1 gene is not located within previously described clusters of homeobox-containing genes.
TL;DR: Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression, which suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.
Abstract: We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one 'pseudo-operator' O2 or O3 decreases repression by wild-type tetrameric Lac repressor approximately 2- to 3-fold; absence of both 'pseudo-operators' decreases repression greater than 50-fold. O1 alone represses under these conditions only approximately 20-fold. Dimeric active Lac repressor (iadi) represses the wild-type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.
TL;DR: This work cloned the genes for two novel ubiquitin‐conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae, and found that these enzymes mediate selective degradation of short‐lived and abnormal proteins.
Abstract: Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.
TL;DR: A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form and inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serineproteinase inhibitors.
Abstract: A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta-sheet wrapped around a five turn alpha-helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal 'trunk' (occupying the 'unprimed' subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.
TL;DR: The Myf‐6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix‐loop‐helix domain, and the different DNA binding properties in vitro might suggest that transactivation of gene expression by My f‐6 involves distinct binding sites and/or additional protein factors.
Abstract: The Myf-6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix-loop-helix domain. This sequence motif is a common feature of all Myf factors and other regulatory proteins. The new Myf gene is located on human chromosome 12, approximately 6.5 Kb upstream of the Myf-5 locus in a closely linked cluster of myogenic determination genes. Myf-6 cDNAs were isolated from human and mouse skeletal muscle, the only tissue in which expression of the corresponding mRNA was observed. In contrast to human primary muscle cell cultures which express moderate levels of Myf-6 mRNA, most established rodent muscle cell lines completely lack this mRNA. Myogenic 10T1/2 cells, however, induced by the expression of either pEMSV-Myf-4 or pEMSV-Myf-5 activate their endogenous mouse Myf-6 gene. Constitutive expression of Myf-6 cDNA in C3H 10T1/2 fibroblasts establishes the muscle phenotype at a similar frequency to the previously characterized myogenic factors. Moreover, muscle-specific CAT reporter constructs containing either the human myosin light chain (MLC) enhancer or the promoter of the embryonic myosin light chain gene are activated in NIH 3T3 fibroblasts or in CV1 kidney cells by cotransfection of Myf-6 expression vehicles. This transcriptional activation occurs in the absence of any apparent conversion of the cellular phenotype of the recipient cells. Glutathione-S-transferase fusion proteins with Myf-3, Myf-4 or Myf-5 specifically bind to a MEF-like consensus sequence present in the human MLC enhancer and the MLC1 emb promoter. In contrast, the Myf-6 hybrid protein interacts weakly with the same sequences showing lower affinity and reduced specificity. Since co-expressed pEMSV-Myf-6, nevertheless, is able to activate transcription of the MLC-CAT reporter constructs in non-muscle tissue culture cells, the different DNA binding properties in vitro might suggest that transactivation of gene expression by Myf-6 involves distinct binding sites and/or additional protein factors.
TL;DR: Cloned a yeast gene is cloned, MIG1, which encodes a C2H2 zinc finger protein involved in glucose repression, which closely resemble the sequence recognized by the Egr proteins.
Abstract: We have cloned a yeast gene, MIG1, which encodes a C2H2 zinc finger protein involved in glucose repression. The fingers of MIG1 are very similar to those present in the mammalian Egr finger proteins, which are induced during the early growth response, and also to the finger protein encoded by a human gene that is deleted in Wilms' tumour cells. MIG1 protein binds to two sites in the upstream region of SUC2, a yeast gene that is repressed by glucose. The MIG1 sites closely resemble the sequence recognized by the Egr proteins. Thus, finger proteins that are similar in both amino acid sequence and DNA specificity are involved in the response of yeast to glucose, and in the mammalian early growth response.
TL;DR: The structural diversity of receptor‐like PTPases was examined by isolated human cDNA clones that cross‐hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non‐stringent hybridization conditions and found partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two P TPase‐like domains.
Abstract: Protein tyrosine phosphatases (PTPases), together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation. Previously, it has been recognized that both cytosolic PTPases and membrane associated, receptor-like PTPases exist. In order to examine the structural diversity of receptor-like PTPases, we isolated human cDNA clones that cross-hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non-stringent hybridization conditions. The cDNA clones thus isolated included LCA and six other novel receptor-like PTPases, named HPTP alpha, beta, gamma, delta, epsilon, and zeta. The cytoplasmic regions of HPTP alpha and epsilon are highly homologous, and are composed of two tandemly duplicated PTPase-like domains. The extracellular regions of HPTP alpha and epsilon are, respectively, 123 amino acids and 27 amino acids, and do not have obvious similarity to any known protein. The cytoplasmic region of HPTP beta contains only one PTPase domain. The extracellular region of HPTP beta, which is 1599 amino acids, is composed of 16 fibronectin type-III repeats. HPTP delta is very similar to leukocyte common antigen related molecule (LAR), in both the extracellular and cytoplasmic regions. Partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two PTPase-like domains. The PTPase-like domains of HPTP alpha, beta and delta expressed in Escherichia coli had tyrosine phosphatase activities.
TL;DR: The soluble forms of the TNF‐Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.
Abstract: Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross-react immunologically with two species of cell surface TNF receptors (TNF-R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine-rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF-R closely resemble those of TBPI. The COOH-terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF-R. CHO cells transfected with type I TNF-R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF-Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.
TL;DR: From a comparative study of the collagenase TORU and the analogous polyoma virus TORU, it is concluded that both the binding affinity of the PEA3 motif and the spacing between P EA3 and AP‐1 modulate transcription activation induced by oncogene expression.
Abstract: PEA3 is a transcription factor which binds to the polyoma virus enhancer and whose activity is regulated by the expression of a number of oncogenes. We show here that PEA3 also binds specifically to the collagenase and fos cellular promoters. On the collagenase promoter, PEA3 acts synergistically with AP-1 to achieve maximum levels of transcription activation by 12-O-tetradecanoylphorbol-13-acetate (TPA), and non-nuclear oncoproteins, thereby defining a TPA- and oncogene-responsive unit (TORU). From a comparative study of the collagenase TORU and the analogous polyoma virus TORU, we conclude that both the binding affinity of the PEA3 motif and the spacing between PEA3 and AP-1 modulate transcription activation induced by oncogene expression.