Showing papers by "David Baltimore published in 1989"

Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.

Abstract: The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.

N-terminal mutations activate the leukemogenic potential of the myristoylated form of c-abl.

Abstract: The two major forms of the c-abl gene differ from their activated counterpart, the v-abl oncogene of the Abelson murine leukemia virus by the replacement of their N-terminal sequences with viral gag sequences Overexpression of p150c-abl type IV in a retroviral vector similar to Abelson virus does not transform NIH 3T3 fibroblasts, even though it is expressed and myristoylated at levels comparable to pp160v-abl Members of a nested set of deletion mutations of the N-terminus of c-abl type IV in this expression system will activate abl to transform murine fibroblasts The smallest of these deletions, delta XB, efficiently transforms lymphoid cells in vitro and causes leukemia in vivo demonstrating that gag sequences are not necessary for abl-induced leukemogenesis The delta XB mutation defines an N-terminal regulatory domain, which shares a surprising homology with chicken oncogene v-crk and phospholipase C-II Although overexpression of the myristoylated form of c-abl does not transform cells, it nonetheless has a profound effect on cell growth

Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1

Abstract: Human immunodeficiency virus type 1 (HIV-1) contains an open reading frame called nef at the 3' end of its genome. The nef gene product has been reported to down-regulate viral growth by suppressing viral transcription through interaction with the long terminal repeat region. We have compared two isogenic HIV-1 (HIV-1-WI3) strains, one of which lacks nef expression, and found little difference between them in in vitro growth. We tested effects on viral entry, DNA synthesis, and RNA expression by measuring HIV-specific low molecular weight DNA and RNA after infection. The qualitative and quantitative aspects of DNA and RNA synthesis were comparable between the nef+ and nef- strains. The effects on viral growth were also examined by following changes in reverse transcriptase activity during the course of infection. The presence of the nef gene product failed to slow viral growth in several different cell types tested, including the human T-lymphocyte cell lines H9 and CEM-SS, human primary T cells enriched for CD4+ cells, and human monocytic cell lines U-937 and THP-1. On the contrary, the nef+ strain grew more efficiently in some cell types than the nef- strain. The same results were obtained with nef+ and nef- strains of a different virus, HIV-1-432, whose Nef had been reported to have a negative effect on viral growth. Our data suggest that the Nef protein does not act as a negative factor, at least in the experimental systems employed in our studies.

A novel, inducible and T cell-specific enhancer located at the 3' end of the T cell receptor alpha locus.

Abstract: A novel, powerful and T-cell-specific enhancer element has been identified at 3 kb 3' of the mouse T cell receptor (TCR) C alpha (constant) gene. A previous report of an intragenic enhancer could not be confirmed. The unique organization of the TCR alpha locus and transcriptional data of the alpha promoter suggest that this enhancer can act over 69 kb. This feature of the alpha enhancer may lead to deregulation of c-myc and other oncogenes translocated to this locus in many T cell leukemias, resulting in T cell neoplasia. The enhancer was localized to 230 bp of DNA and has been found to be inducible in an immature T lymphoid cell line. Deletions localizing critical regions and T-cell-specific binding proteins of this enhancer are described.

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

Abstract: 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.

Repression of the IgH enhancer in teratocarcinoma cells associated with a novel octamer factor.

Abstract: Embryonal carcinoma (EC) cell lines are models for early cells in mouse embryogenesis. A 300-base pair fragment of the heavy chain enhancer was inactive in F9 EC cells, unlike in other nonlymphoid cells where it has significant activity. Alterations of the octamer motif increased enhancer activity. Nuclear extracts from F9 cells contained an octamer binding protein (NF-A3) that was unique to EC cells; the amount of NF-A3 decreased upon differentiation. It is proposed that NF-A3 represses specific regulatory sequences that contain the octamer motif. Thus, the same DNA sequence mediates either negative or positive transcriptional effects, depending on the cell type.

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

Abstract: 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.

Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation

Abstract: To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

ACTIVITION OF NF-kB PRECURSOR

Abstract: Activation of an NF-kB precursor which includes NF-kB and an inhibitor and control of expression of the kappa light chain gene and of human immunodeficiency virus (HIV) DNA by preventing or inducing dissociation of the inhibitor from NF-kB.

Recombination activating gene (RAG-1)

Abstract: Recombination activating gene of mammalian origin (RAG-1), cDNA of RAG-1 of mammalian origin, mRNA expressed by RAG-1, the encoded recombinase and antibodies specific for the recombinase, as well as the use of the same for a diagnostic or therapeutic purpose.

Purified bovine NF-kappa B recognizes regulatory sequences in multiple genes expressed during activation of T- and B-lymphocytes.

Abstract: A crucial event in the differentiation of B-lymphocytes is the transcription of the immunoglobulin light-chain gene which leads to expression of immunoglobulin antigen receptor on the surface of the cell. In an apparently separately regulated arm of the immune response, antigenic stimulation causes proliferation of T-lymphocytes by transcriptional activation of the IL-2 gene and the IL-2 receptor gene. Previous studies have shown that a lymphoid-specific enhancer element plays an important role in achieving high-level transcription of the kappa light-chain gene [1-4]. Molecular genetic dissection of this enhancer has revealed that a DNA sequence which binds to a nuclear factor, NF-κB, is essential for its function [5-8]. NF-KB binding activity is constitutively present only in mature B-lymphocytes and exceptional T-lymphocyte lines [5, 13]. Its binding may be induced in cells early in the B-lymphoid lineage, T-lymphocytes, and in nonlymphoid cells by various treatments such as bacterial lipopolysaccharide, cycloheximide, lectins, and phorbol esters [9]. Previously we have shown that NF-κB binding is critical for the kappa enhancer activity that is constitutively present in mature B-lymphocytes and inducible by lipopolysaccharide or phorbol esters in pre-B cells [7].

The Recombination Activating Gene and Lymphoid Differentiation

Abstract: Lymphoid cell differentiation is like the differentiation of any other cell in the body: a series of events occur that turn an undifferentiated cell into one that carries out a range of specific functions. The most characteristic proteins of lymphoid cells are immunoglobulin (Ig) and the T cell receptor (TCR). These products are formed through a differention event apparently unique to the lymphoid cell series: DNA rearrangement.

ACTIVATION DU PRECURSEUR NF-kB

Abstract: On active un precurseur de NF-kB comprenant le NF-kB ainsi qu'un inhibiteur, et on regule l'expression du gene de la chaine lumineuse de kappa ainsi que l'ADN du virus d'immunodeficience humaine (VIH) en empechant ou en induisant la dissociation de l'inhibiteur du NF-kB.