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Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 199 records · Page 11Linked to original sources
PubMed1990

An intragenic revertant of a poliovirus 2C mutant has an uncoating defect.

A revertant was isolated from a temperature-sensitive poliovirus 2C mutant, 2C-31, which is defective in viral RNA synthesis. This revertant, called 2C-31R1, grew well at 39 degrees C and was not defective in RNA synthesis. However, in contrast to its parental mutant, 2C-31R1 was cold sensitive and could hardly grow at all at 32 degrees C. Analysis of a single-cycle growth revealed that 2C-31R1 was defective in virion uncoating at 32 degrees C, and a substantial amount (more than 30%) of input viruses could be recovered as infectious particles from an infected cell lysate up to 6 h postinfection. The uncoating defect and the inability to grow at cold temperatures could be overcome by a brief incubation at the permissive temperature (39 degrees C) before the infection was continued at 32 degrees C. cDNA cloning and mix-and-match recombination experiments indicated that the defect in uncoating was the result of two secondary point mutations, seven nucleotides apart, in the 2C-coding sequence downstream of the inserted linker which is the original mutation in the parental 2C-31 genome. Another revertant, 2C-31R3, isolated from the same 2C-31 stock, was not defective in uncoating and appeared to be a secondary revertant that contained an intragenic suppressor for the uncoating defect. The uncoating defect of 2C-31R1 could be complemented by type 2 poliovirus. These results suggested that protein 2C, in addition to its role in viral RNA synthesis, has a function in determining virion structure.

Base Sequence↗
PubMed1990

The promoter of the human interleukin-2 gene contains two octamer-binding sites and is partially activated by the expression of Oct-2.

The gene encoding interleukin-2 (IL-2) contains a sequence 52 to 326 nucleotides upstream of its transcriptional initiation site that promotes transcription in T cells that have been activated by costimulation with tetradecanoyl phorbol myristyl acetate (TPA) and phytohemagglutinin (PHA). We found that the ubiquitous transcription factor, Oct-1, bound to two previously identified motifs within the human IL-2 enhancer, centered at nucleotides -74 and -251. Each site in the IL-2 enhancer that bound Oct-1 in vitro was also required to achieve a maximal transcriptional response to TPA plus PHA in vivo. Point mutations within either the proximal or distal octamer sequences reduced the response of the enhancer to activation by 54 and 34%, respectively. Because the murine T-cell line EL4 constitutively expresses Oct-2 and requires only TPA to induce transcription of the IL-2 gene, the effect of Oct-2 expression on activation of the IL-2 promoter in Jurkat T cells was determined. Expression of Oct-2 potentiated transcription 13-fold in response to TPA plus PHA and permitted the enhancer to respond to the single stimulus of TPA. Therefore, both the signal requirements and the magnitude of the transcription response of the IL-2 promoter can be modulated by Oct-2.

Animals↗
PubMed1990

Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.

Amino Acid Sequence↗
PubMed1990

Involvement of a second lymphoid-specific enhancer element in the regulation of immunoglobulin heavy-chain gene expression.

To determine whether enhancer elements in addition to the highly conserved octamer (OCTA)-nucleotide motif are important for lymphoid-specific expression of the immunoglobulin heavy-chain (IgH) gene, we have investigated the effect of mutating the binding site for a putative additional lymphoid-specific transcription factor, designated NF-microB, in the murine IgH enhancer. We demonstrate that the NF-microB-binding site plays a critical role in the IgH enhancer, because mutation of the microB DNA motif decreased transcriptional activity of the IgH enhancer in cells of the B-cell lineage but not in nonlymphoid cells. This effect was comparable to or even stronger than the effect of a mutation in the OCTA site. Moreover, combined mutation of both microB and OCTA sites further reduced enhancer activity in lymphoid cells. Interestingly, alteration of either the microB or E3 site in a 70-base-pair fragment of the IgH enhancer that lacks the binding site for OCTA abolished enhancer activity in lymphoid cells completely. Nevertheless, a multimer of the microB motif alone showed no enhancer activity. DNase footprinting analysis corroborated the functional data showing that a lymphoid-specific protein binds to the microB DNA motif. Our results suggest that the microB element is a new crucial element important for lymphoid-specific expression of the IgH gene but that interaction with another enhancer element is essential for its activity.

Animals↗
PubMed1989

The V(D)J recombination activating gene, RAG-1.

The RAG-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when introduced into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a RAG-1 genomic probe that detects a single 6.6-7.0 kb mRNA species in transfectants and pre-B and pre-T cells. RAG-1 genomic and cDNA clones were biologically active when introduced into NIH 3T3 cells. Nucleotide sequencing of human and mouse RAG-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence identity. RAG-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. RAG-1 may activate V(D)J recombination indirectly, or it may encode the V(D)J recombinase itself.

Amino Acid Sequence↗
PubMed1989

Alpha beta lineage-specific expression of the alpha T cell receptor gene by nearby silencers.

T cells expressing either the alpha beta or gamma delta antigen receptor (TCR) are distinct cell lineages. The single locus encoding the TCR alpha and delta genes requires special regulation to avoid alpha gene expression in gamma delta T cells. We show here that the minimal alpha enhancer is active in the gamma delta T cell lineage but gains alpha beta lineage specificity through negative cis-acting elements 3' of the C alpha gene that silence the enhancer in gamma delta T cells. The negative elements at the C alpha locus consist of several silencers that work in an orientation- and distance-independent fashion. These silencers also act on a retroviral enhancer that is normally ubiquitously expressed, restricting its activity to alpha beta cells. The alpha silencers are active in non-T cell lines, suggesting that the decision of a cell to differentiate into the alpha beta T cell lineage may involve specific relief from these silencers. Silencers are likely to be as important as enhancers in establishing lineage-specific gene expression in many systems.

Animals↗
PubMed1989

The gene for enhancer binding proteins E12/E47 lies at the t(1;19) breakpoint in acute leukemias.

The gene (E2A) that codes for proteins with the properties of immunoglobulin enhancer binding factors E12/E47 was mapped to chromosome region 19p13.2-p13.3, a site associated with nonrandom translocations in acute lymphoblastic leukemias. The majority of t(1;19)(q23;p13)-carrying leukemias and cell lines studied contained rearrangements of E2A as determined by DNA blot analyses. The rearrangements altered the E2A transcriptional unit, resulting in the synthesis of a transcript larger than the normal-sized E2A mRNAs in one of the cell lines with this translocation. These observations indicate that the gene for a transcription factor is located at the breakpoint of a consistently recurring chromosomal translocation in many acute leukemias and suggest a direct role for alteration of such factors in the pathogenesis of some malignancies.

Child↗
PubMed1989

HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus.

The products of the human immunodeficiency virus (HIV) gag gene exist in a highly multimerized state in the mature virion. For that reason, they may represent a particularly suitable target for the generation of dominant negative mutants. A number of HIV site-directed Gag mutants did show interference with the production of infectious viral particles from cells in which they were cotransfected with a wild-type proviral DNA. Furthermore, cells constitutively expressing such HIV Gag mutants had an impaired ability to support HIV replication when infected with wild-type virus. The block was localized to the late stages of the virus life cycle. Such Gag variants could constitute prototypes for the development of anti-HIV intracellular immunization.

Animals↗
PubMed1989

Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription.

We have developed a sensitive polymerase chain reaction assay for measuring the fraction of rearranged immunoglobulin kappa genes in a cell population. Using this assay with Abelson virus-transformed murine pre-B cells, we have found that bacterial lipopolysaccharide treatment, which activates transcription of the unrearranged kappa constant region gene, also activates kappa gene rearrangement. In addition, we have been able to detect kappa gene rearrangement in cell lines that do not produce a functional heavy chain gene product (mu protein). These results implicate transcription or transcription factor binding as a regulator of immunoglobulin gene rearrangement.

Animals↗
PubMed1989

The mouse type IV c-abl gene product is a nuclear protein, and activation of transforming ability is associated with cytoplasmic localization.

The subcellular localization of the mouse type IV c-abl protein was determined by indirect immunofluorescence of nontransformed NIH 3T3 fibroblasts that overexpress the protein. Unlike the viral transforming protein p160gag/v-abl, which has cytoplasmic and plasma membrane localization, a large fraction of the c-abl (IV) protein is nuclear, with the remainder in the cytoplasm and plasma membrane. Deletion of a small N-terminal regulatory region of the c-abl (IV) protein, sufficient to activate its transforming potential fully, changes the distribution of the protein from the nucleus to the cytoplasm. Mapping of an amino acid sequence responsible for the nuclear localization of the c-abl (IV) protein reveals a nuclear localization signal similar to that of SV40 large T antigen.

Amino Acid Sequence↗
PubMed1989

The involvement of NF-kappa B in beta-interferon gene regulation reveals its role as widely inducible mediator of signal transduction.

The DNA binding protein NF-kappa B has been implicated in gene regulation in B and T lymphocytes. We have found that NF-kappa B also has a central role in virus induction of human beta-interferon (beta-IFN) gene expression. A critical virus-inducible element of this gene, PRDII, behaves interchangeably with the NF-kappa B binding site from the Ig kappa enhancer in both B lymphocytes and virus-infected fibroblasts. Single base substitutions that impair inducibility of the beta-IFN gene in vivo also reduce the binding of NF-kappa B to PRDII in vitro. Virus infection potently activates the binding and nuclear localization of NF-kappa B and, in pre-B lymphocytes, results in the expression of both the beta-IFN gene and the Ig kappa gene. The wide variety of cell types in which beta-interferon can be induced and the divergent set of gene induction processes involving NF-kappa B suggest that this transcription factor plays a broad role in gene regulation as a mediator of inducible signal transduction.

Binding Sites↗
PubMed1989

The "initiator" as a transcription control element.

Transcription of the lymphocyte-specific terminal deoxynucleotidyltransferase gene begins at a single nucleotide, but no TATA box is present. We have identified a 17 bp element that is sufficient for accurate basal transcription of this gene both in vitro and in vivo. This motif, the initiator (Inr), contains within itself the transcription start site. Homology to the Inr is found in many TATA-containing genes, and specific mutagenesis influences both the efficiency and accuracy of initiation. Moreover, in the presence of either a TATA box or the SV40 21 bp repeats, a greatly increased level of transcription initiates specifically at the Inr. Thus, the Inr constitutes the simplest functional promoter that has been identified and provides one explanation for how promoters that lack TATA elements direct transcription initiation.

Animals↗
PubMed1989

Separate lineages of T cells expressing the alpha beta and gamma delta receptors.

The T-cell antigen receptor is a heterodimer molecule composed of either alpha beta or gamma delta chains. The alpha beta receptor molecules are expressed mainly in CD4+ CD8- and CD4- CD8+ T cells (helper and killer T cells respectively), whereas the gamma delta receptor molecules are expressed mainly in CD4- CD8- T cells. CD4+CD8- and CD4-CD8+ T cells arise from a class of CD4-CD8- T cells during thymus development, raising the question of whether cells rearranging their gamma delta receptors later give rise to alpha beta T cells by further rearrangements of their receptor genes, or whether rearrangements and expression of the receptor genes occur in separate lineages. The delta-chain gene is located between the V alpha (variable) and J alpha (joining) gene segments, and when the rearrangements allowing alpha- and beta-receptors occur, the DNA between these segments is deleted as small circles which can be isolated from developing thymocytes. The rearrangement status of the delta-chain gene in the alpha-circles can therefore be investigated to see whether alpha-chain and delta-chain expression occur in parallel lineages or sequentially within a lineage. We find that the delta-chain gene in the T-cell receptor alpha-circles has a germline configuration, indicating that alpha beta and gamma delta T cells are distinct lineages.

Animals↗
PubMed1989

A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins.

Two cDNAs were isolated whose dimerized products bind specifically to a DNA sequence, kappa E2, located in the immunoglobulin kappa chain enhancer. Both cDNAs share a region of extensive identity to the Drosophila daughterless gene and obvious similarity to a segment in three myc proteins, MyoD, and members of the Drosophila achaete-scute and twist gene family. The homologous regions have the potential to form two amphipathic helices separated by an intervening loop. Remarkable is the stringent conservation of hydrophobic residues present in both helices. We demonstrate that this new motif plays a crucial role in both dimerization and DNA binding.

Amino Acid Sequence↗