Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 181 records · Page 10Linked to original sources

PubMed1991

Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins.

The DNA binding activities of some basic region and putative helix-loop-helix (bHLH)-containing transcriptional factors can be inhibited by the Id protein. Because Id contains the HLH motif for dimerization but not the basic amino acid region for DNA binding, heterodimers of Id with bHLH transcriptional factors may not bind to DNA. We have isolated and characterized the gene and cDNA clones for a new Id protein, designated Id2. The Id2 protein contains a helix-loop-helix motif similar to that of the previously described Id protein (referred to here as Id1), but the two proteins are different elsewhere. Id1 and Id2 are encoded by two unlinked genes, as shown by chromosome mapping. The two Id proteins have similar inhibitory activities. They selectively bind to and inhibit the function of one set of bHLH proteins, typified by E2A.E47 and E2B.m3, but not that of the other set, including TFE3, USF, and AP4. The Id proteins also homodimerize poorly. Expression of both Id genes is down-regulated during differentiation in a variety of cell types.

Amino Acid Sequence↗

PubMed1991

Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants.

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants.

Animals↗

PubMed1991

B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits.

Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.

Animals↗

PubMed1991

Silencing of the expression of the immunoglobulin kappa gene in non-B cells.

Although the activating factor NF-kappa B can be present in the nucleus of many cell types, transcription and rearrangement of the immunoglobulin kappa chain gene is restricted to cells of the B lineage. Part of this specificity is determined by sequences within the major intron of the kappa gene that specifically silence gene expression in non-B cells (T cells and HeLa cells). These sequences are found in a 232-bp fragment located 5' of the NF-kappa B binding sequence of the enhancer. When this fragment is added back upstream of an active NF-kappa B site, it specifically decreases the expression of a linked gene by more than 10-fold in activated T cells but it has no effect on expression in B cells. The kappa silencer region acts in an orientation- and distance-independent manner and appears to be composed of multiple negative elements. The kappa silencer may act to restrict transcription and rearrangement of the C kappa locus to cells of the B lineage.

Animals↗

PubMed1990

A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA.

The existence of a computer-predicted cloverleaf structure for the first 100 nucleotides at the 5' end of poliovirus RNA was verified by site-directed mutagenesis and by chemical and RNAase probing. Mutations that modified the cloverleaf in the positive strand but not the negative strand were lethal to the virus. This RNA cloverleaf structure binds a cellular protein and the viral proteins 3Cpro and 3Dpol. Mutations in specific regions of the RNA cloverleaf prevented this binding. Mutations in either 3Cpro or the RNA that disrupted ribonucleoprotein complex formation inhibited virus growth and selectively affected positive strand RNA accumulation. Phenotypic reversion of these mutations restored the ability to form the complex. Thus, a cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production.

Base Composition↗

PubMed1990

Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal.

The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

Amino Acid Sequence↗

PubMed1990

Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression.

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1-long terminal repeat (HIV-1-LTR) CAT constructs transfected into monocyte/macrophage-like cell lines but not a T cell line. This effect appears to be mediated through the induction of nuclear factor kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrate that LPS induces a DNA binding activity indistinguishable from NF-kappa B in U937 and THP-1 cells. LPS is also shown to dramatically increase HIV-1 production from a chronically infected monocyte/macrophage-like cloned cell line, U1, which produces very low levels of HIV-1 at baseline. The stimulation of viral production from this cell line occurs only if these cells are treated with granulocyte/macrophage colony-stimulating factor (GM-CSF) before treatment with LPS. This stimulation of HIV-1 production is correlated with an increase in the level of HIV-1 RNA and and activation of NF-kappa B. LPS is not able to induce HIV-1 production in a cloned T cell line. The effect of LPS on HIV-1 replication occurs at picogram per milliliter concentrations and may be clinically significant in understanding the variability of the natural history of HIV-1 infection.

Base Sequence↗

PubMed1990

Cells nonproductively infected with HIV-1 exhibit an aberrant pattern of viral RNA expression: a molecular model for latency.

U1 and ACH-2 cells are subclones of HIV-1-infected monocyte/macrophage-like and T lymphocyte cell lines, respectively, which express the HIV-1 genome at very low levels. We have examined whether they might provide a model of HIV-1 latency. The patterns of HIV-1-specific RNA expressed in these cells consisted of singly and multiply spliced RNA species, with little or no full-length genomic RNA. Upon stimulation with agents that activate the HIV-1 long terminal repeat in these cells, a marked rise in the amount of small mRNAs, encoding the viral regulatory proteins, preceded the increase in the unspliced RNA. Thus, U1 and ACH-2 cells maintain HIV-1 in a state equivalent to the early phase of a lytic infection and, after stimulation, recapitulate the events of a single cycle infection of highly susceptible target cells.

Blotting, Northern↗

PubMed1990

RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination.

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3' untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction.

Amino Acid Sequence↗

PubMed1990

Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B.

Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.

Adenosine Triphosphate↗

PubMed1990

A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL.

It was previously shown that the chromosome 19 breakpoint of the t(1;19)(q23;p13.3) translocation, found in human pre-B cell acute lymphoblastic leukemias, is within the E2A transcription factor gene on chromosome 19. A cell line with this translocation contains two novel chimeric mRNAs, both with the same 5'E2A sequences but with different lengths of 3' sequence from a previously unrecognized gene dubbed prl, located on chromosome 1. The chimeric RNAs encode a protein that lacks 171 amino acids of E2A, including its DNA binding and dimerization motifs, but have instead a homeobox-related sequence from prl. Therefore, the production of a chimeric E2A-Prl protein may contribute to the acute lymphoblastic phenotype by directly altering the expression of genes normally responsive to the Prl homeoprotein.

Amino Acid Sequence↗

PubMed1990

Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor.

Friend spleen focus-forming virus (SFFV) is a defective murine C-type retrovirus which causes a multi-stage erythroleukaemia in mice and erythroblastosis in bone marrow cultures. The SFFV env gene encodes a membrane glycoprotein, gp55, which is located on the cell surface and in the rough endoplasmic reticulum and is essential both for the induction of leukaemia in vivo and erythroblast proliferation in vitro. The mechanism by which gp55 causes increased erythroblastosis and ultimately leukaemia is unknown, but a reasonable suggestion is that gp55 can mimic the action of erythropoietin by binding to its receptor (Epo-R), thereby triggering prolonged proliferation of erythroid cells. To test this possibility, we have co-expressed gp55 and the murine Epo-R in a fibroblast cell line. We show here that in such cells, the SFFV glycoprotein binds directly to Epo-R. Furthermore, when an interleukin-3 (IL-3)-dependent lymphoid cell line was co-infected by SFFV and a virus that carries the Epo-R gene, it could grow without IL-3. We suggest that through direct binding to Epo-R, gp55 can stimulate the receptor and by-pass the normal requirement for Epo, causing prolonged proliferation of infected erythroid cells. This could be the first step of leukaemogenesis induced by Friend virus.

Animals↗

PubMed1990

Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.

In tumor cells from virtually all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the P210bcr/abl protein. The protein-tyrosine kinase activity and hybrid structure of P210bcr/abl are similar to the oncogene product of the Abelson murine leukemia virus, P160gag/v-abl, which induces acute lymphomas. To determine whether P210bcr/abl can induce chronic myelogenous leukemia, murine bone marrow was infected with a retrovirus encoding P210bcr/abl and transplanted into irradiated syngeneic recipients. Transplant recipients developed several hematologic malignancies; prominent among them was a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P210bcr/abl. These results demonstrate that P210bcr/abl expression can induce chronic myelogenous leukemia. Retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.

Animals↗

PubMed1990

A human cell factor is essential for HIV-1 Rev action.

To examine the restriction of HIV growth in murine cells, we infected NIH 3T3 cells with HIV pseudotyped by Moloney murine leukemia virus. The virus, which carried a dominant selectable marker under the control of the HIV LTR, gave large numbers of resistant clones, showing that murine cells are permissive for HIV uncoating, reverse transcription, nuclear transport and integration. However, we found that several murine cell lines, as well as CHO cells, could not support the function of rev, the viral regulatory gene which, in human cells, induces the cytoplasmic expression of the incompletely spliced class of HIV mRNAs that encode the viral structural proteins. Transfection of the HIV-infected murine cells with a HTLV-1 rex-expressing vector failed to rescue the rev- phenotype, indicating that the block extended to rex function. Most importantly, we could complement the rev defect by fusing the infected murine with uninfected human cells. We conclude that HIV tropism is partly a consequence of a trans-acting cellular factor critical for Rev function.

Animals↗

PubMed1990

Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID.

Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.

Adenovirus Early Proteins↗

PubMed1990

Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains.

A common DNA binding and dimerization domain containing an apparent "helix-loop-helix" (HLH) structure was recognized recently in a number of regulatory proteins, including the E47 and E12 proteins that bind to the kappa E2 motif in immunoglobulin kappa gene enhancer. The effect of site-directed mutagenesis on E47 protein multimerization and DNA binding was examined. Mutations in either putative helix domain disrupted protein dimerization and DNA binding. No DNA binding was observed when mutations were introduced in the basic region, but these mutants were able to dimerize. These basic region mutants were not able to bind to DNA as heterodimers with the wild-type E47 proteins, demonstrating that two functional basic regions are required for binding to DNA. Therefore the basic region mutants are "transdominant."

Amino Acid Sequence↗

PubMed1990

Factors affecting cellular tropism of human immunodeficiency virus.

To evaluate the basis of the slow growth of many human immunodeficiency virus strains in monocytes/macrophages, various stages of the virus life cycle have been studied for their possible contribution to viral tropism. Although we found that monocytic U937 cells had a higher percentage of CD4-positive cells than T-lymphoid H9 cells, the human immunodeficiency virus strain grew much less efficiently in the monocytic line. Viral tropism was primarily determined during the early stages of the virus cycle, that is, sometime between binding of the virus to the cell surface and reverse transcription of viral genomic RNA. Once the virus entered the host cell, reverse transcription, use of the long terminal repeat, RNA expression, and production of virus particles was about as efficient in monocytes as in T cells. Thus, during viral entry into the host cell cytoplasm there is a major limiting event that is particularly inefficient in U937 cells and possibly in all monocytes/macrophages.

CD4 Antigens↗

PubMed1990

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region.

The poliovirus mutant 5NC-11 has a 4-base insertion at position 70 within the 5' untranslated region and is deficient in RNA synthesis. Revertants from 5NC-11 were isolated, showing a partial recovery of wild-type levels of RNA synthesis. The 5' noncoding region of those revertants contained the mutation intact; mix-and-match experiments with the cDNA from these revertants revealed that a restricted region within the 3C gene was the site of the suppressing mutations in the revertants. The suppressors were point mutations, confirmed by introducing them into the 3C gene by site-directed mutagenesis. Although complementation studies indicated that the suppressors were cis active, we believe that protein changes rather than RNA sequence alterations are responsible for the suppression because RNA changes that did not alter protein sequence had no effect, whereas various protein alterations were suppressive. The results therefore imply that protein 3C interacts with the 5' end of the RNA and may play a role in RNA replication.

Amino Acid Sequence↗