Showing papers by "David Baltimore published in 1987"

An inducible transcription factor activates expression of human immunodeficiency virus in T cells

Abstract: Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III and art genes. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-kappa B, with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-kappa B acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility

Abstract: Individual protein-binding sites within the mouse immunoglobulin heavy chain and kappa light chain gene enhancers were altered, making it possible to examine the functional role of the sites during transcription. The E motifs, which bind factors that are present in many if not all cells, mostly behave as transcriptional activating sites. The only known heavy chain enhancer site that binds a lymphocyte-specific factor, the "octamer" site, plays a critical role in transcription but only in a truncated form of the enhancer. In the full enhancer, no one site is crucial because of an apparent functional redundancy. The site in the kappa enhancer that binds a factor specific to mature B cells, kappa B, was crucial to the constitutive activity of the enhancer in B cells. This factor is also inducible in pre-B cells, and the site was necessary for inducibility of the kappa enhancer. Thus, the sites defined by protein binding are important for the functional activity of immunoglobulin enhancers, with the sites that bind proteins restricted in their cellular distribution playing the most important roles.

An octamer oligonucleotide upstream of a TATA motif is sufficient for lymphoid-specific promoter activity.

Abstract: The octamer sequence ATGCAAAT or its inverse complement ATTTGCAT is well-conserved in all immunoglobulin gene promoters and has been implicated in promoter function by deletion analysis. Although immunoglobulin promoters are tissue-specific, the octamer is also a functional element in non-tissue-specific upstream regions--like those controlling U1 and U2 small nuclear RNA and histone H2B genes--where it is associated with additional canonical elements. Specific interactions occur between the octamer motif and both lymphoid-specific and ubiquitous proteins. By using a synthetic octamer oligonucleotide inserted upstream of the beta-globin TATA box we show here that the octamer element by itself is sufficient for directing lymphocyte-specific RNA synthesis when within 70 base pairs of the start site of transcription. We also demonstrate that mutations in any position of the conserved motif interfere with this function.

Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart

Abstract: Neuronal cells express a pp60c-src variant that displays an altered electrophoretic mobility and a different V8 peptide pattern relative to pp60c-src expressed in tissues of non-neuronal origin. To determine whether the neuronal form of pp60c-src is encoded by a brain-specific messenger RNA, a mouse brain complementary DNA (cDNA) library was screened with a chicken c-src probe and a 3.8-kilobase c-src cDNA clone was isolated. This clone encodes a 60-kilodalton protein that differs from chicken or human pp60c-src primarily in having six extra amino acids (Arg-Lys-Val-Asp-Val-Arg) within the NH2-terminal 16 kilodaltons of the molecule. S1 nuclease protection analysis confirmed that brain c-src RNA contains an 18-nucleotide insertion at the position of the extra six amino acids. This insertion occurs at a position that corresponds to a splice junction in the chicken and human c-src genes. The isolated c-src cDNA clone encodes a protein that displays an identical V8 peptide pattern to that observed in pp60c-src isolated from tissues of neuronal origin.

Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemia virus enhancer.

Abstract: Binding sites for six distinct nuclear factors on the 75-base-pair repeat of the Moloney murine leukemia virus enhancer have been identified by an electrophoretic mobility shift assay combined with methylation interference. Three of these factors, found in WEHI 231 nuclear extracts, which we have named LVa, LVb, and LVc (for leukemia virus factors a, b, and c) have not been previously identified. Nuclear factors that bind to the conserved simian virus 40 corelike motif, the NF-1 motif, and the glucocorticoid response element were also detected. Testing of multiple cell lines showed that most factors appeared ubiquitous, except that the NF-1 binding factor was found neither in nuclear extracts from MEL cells nor in the embryonal carcinoma cell lines PCC4 and F9, and core-binding factor was relatively depleted from MEL and F9 nuclear extracts.

Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector.

Abstract: The role of terminal deoxynucleotidyl transferase (TdT) in the insertion of N regions into the junctional sites of immunoglobulin genes was investigated. Pre-B-cell lines capable of continuous rearrangement of immunoglobulin light-chain genes and differing only in the presence or apparent absence of TdT were derived by infecting cells with a TdT retroviral expression vector or a control vector. The cell lines were then superinfected with a retrovirus-based artificial immunoglobulin gene rearrangement substrate. The substrate was allowed to rearrange in the cell lines and the rearranged proviruses were rescued from the cell lines. Nucleotide sequence analysis of the V-J junctions of the proviral rearranged genes showed a fivefold greater frequency of N-region insertion in proviruses rescued from the TdT+ cell lines than in those rescued from the TdT- cell lines, so that at least 50% of the rearrangements that occurred in the presence of TdT had N regions. It is thus evident that TdT can stimulate N-region insertion, and the enzyme is presumably directly responsible for adding nucleotides at V-J and other immunoglobulin and T-cell receptor gene junctions.

The first intron in the human c-abl gene is at least 200 kilobases long and is a target for translocations in chronic myelogenous leukemia.

Abstract: The c-abl protooncogene is unusual in two respects; it has multiple, widely space N-terminal coding exons transcribed by different promoters, and it is the target of the translocations that form the Philadelphia chromosome found in cells of chronic myelogenous leukemia patients. To understand the organization of the gene in normal and chronic myelogenous leukemia patient DNA we have mapped c-abl by pulsed field gradient gel electrophoresis. We find that one of the alternative 5' exons of the gene lies at least 200 kilobases upstream of the remaining c-abl exons, posing formidable transcription and splicing problems. The 5'-most c-abl exon includes an unusually long 1,276-base-pair segment that contains 15 ATG codons and multiple short open reading frames, upstream of the abl initiator codon. Its peculiar structure suggests that c-abl may be decapitated in most chronic myelogenous leukemia patients, and we demonstrate that this is the case in the chronic myelogenous leukemia cell line K562.

Formation of disulphide-linked mu 2 omega 2 tetramers in pre-B cells by the 18K omega-immunoglobulin light chain.

Abstract: Pre-B cells are precursors of B lymphocytes that contain intracel-lular heavy-chain protein (µ) and are either yet to rearrange their light-chain genes or are in the process of doing so1–4. These cells have traditionally been considered to contain intracellular µ-chain with no associated light chain. We demonstrate here that pre-B lymphoid lines synthesize a protein of relative molecular mass (Mr) 18,000 (18K), which we term ω, which forms disulphide-linked µ2ω2 tetramers. This protein could be immunoprecipitated with µ-chain from pre-B lines, but not from T-cell and fibroblast lines that express transfected µ-genes, nor from a pre-B line that synthesizes a Dµ-protein (which lacks a V domain). We view the ω-chain as being a pre-B specific surrogate light chain that may be essential for the important regulatory function that the µ-protein is believed to have at this stage of differentiation.

The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 fibroblasts.

Abstract: The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) is known to efficiently transform NIH/3T3 fibroblasts in vitro and to cause an acute lymphosarcoma in susceptible murine hosts. The role of its relative, the bcr/abl gene product, in the etiology of human chronic myelogenous leukemia (CML) remains speculative. To assess the transforming properties of the bcr/abl gene product, complementary DNA clones encoding the CML-specific P210 bcr/abl protein were expressed in NIH/3T3 fibroblasts. In contrast to the v-abl oncogene product P160, the P210 bcr/abl gene product did not transform NIH/3T3 cells. Cell lines were isolated that expressed high levels of the P210 bcr/abl protein but were morphologically normal. During the course of these experiments, a transforming recombinant of bcr/abl was isolated which fuses gag determinants derived from helper virus to the NH2-terminus of the bcr/abl protein. This suggests that a property of viral gag sequences, probably myristylation-dependent membrane localization, must be provided to bcr/abl for it to transform fibroblasts.

Depletion of the predominant B-cell population in immunoglobulin µ heavy-chain transgenic mice

Abstract: The transgenic mouse line M54 was generated by introducing a functionally-rearranged immunoglobulin µ heavy-chain gene into the germ line of a C57B1/6 inbred mouse1. Previous examination of the antibodies produced by B-cell hybridomas derived from transgenic M54 mice showed that the presence of the μ transgene grossly altered the immunoglobulin repertoire of unimmunized animals, suggesting that these mice suffer from a serious immunoregulatory perturbation2. Studies presented here introduce a new perspective on this functional defect. We show that the lymphoid tissues from these transgenic mice lack virtually all conventional bone-marrow-derived B cells, which constitute the predominant B-cell population in normal mice and which typically produce primary and secondary antibody responses to T-cell-dependent antigens3–6. Moreover, the bone marrow from transgenic M54 mice is depleted of pre-B lymphocytes, indicating a serious defect in early B-cell lymphopoiesis. In contrast, CDS (Ly-1) B cells, a second B-cell population displaying a characteristic set of cell surface markers which are derived from distinct precursors in the peritoneum3–7, are represented at normal frequencies in these transgenic mice. Thus, the presence of the rearranged immunoglobulin heavy-chain transgene in M54 mice results in an unexpected selective developmental defect that impairs the development of bone-marrow-derived pre-B and B cells without affecting Ly-1 B cells.

Myristoylation and the post-translational acquisition of hydrophobicity by the membrane immunoglobulin heavy-chain polypeptide in B lymphocytes.

Abstract: Membrane immunoglobulin heavy chain in pre-B and in B cells is initially synthesized as a relatively hydrophilic protein that is nonetheless stably anchored in the endoplasmic reticulum membrane. In B cells, but not in pre-B cells, the membrane immunoglobulin heavy chain is post-translationally converted to a relatively hydrophobic form that partitions into the oil phase when solubilized with the phase-separating detergent Triton X-114. Covalent myristoylation of the membrane and secretory forms of immunoglobulin heavy chains as well as of light chains was observed in B cells. Myristoylation of the membrane immunoglobulin heavy chain correlates with its transport to the cell surface and its post-translational conversion to a relatively hydrophobic form. This post-translational modification is hydroxylamine resistant and may be responsible for the assembly and transport of membrane immunoglobulin to the cell surface in B cells.

B lymphocyte-specific protein binding near an immunoglobulin kappa-chain gene J segment

Abstract: Nuclear extracts from pre-B and B cell lines contain a nuclear DNA binding protein (kappa locus protein, KLP) that specifically recognizes a DNA sequence in the immunoglobulin kappa light chain joining (J) segment gene region. KLP is not observed in mature B cells, T cells, or nonlymphoid cell types. Two tandem binding sites for KLP designated KI and KII have been identified by methylation interference analysis to be immediately proximal to the J kappa 1 nonamer-heptamer recognition sequences and separated by 38 base pairs from each other. Fragments of DNA containing KI and KII sites compete for binding to KLP, and both protein-DNA complexes have the same electrophoretic mobility. Other flanking sequences of immunoglobulin gene fragments do not bind to KLP. The position of KLP-DNA binding and its tissue-specific expression suggest that it may be involved in the regulation of lymphoid gene DNA rearrangements by targeting recombinase to the kappa-chain gene region.

Elevated myc expression and c-myc amplification in spontaneously occurring B lymphoid cell lines.

Abstract: Recently, a minor subpopulation of murine B lymphocytes, Ly-1+ B cells, has been distinguished by its unique ontogeny, tissue distribution, and prominence in certain autoimmune and neoplastic B cell diseases. We have previously described a simple murine spleen culture system that results in the spontaneous and exclusive outgrowth of long-term Ly-1+ B cell lines (B Ly-1 cells). Here, we report that the immortal growth property of B Ly-1 cells correlates with a 10-45-fold elevation of steady-state myc RNA and 2-10-fold amplification of the c-myc locus. While c-myc amplification has been observed in malignant cell lines derived from several tissues of origin, its occurrence in lymphoid cells has not been previously reported. The consistent c-myc amplification in B Ly-1 cells may reflect a unique state of this locus in the Ly-1+ B lymphocyte lineage, and contribute to the spontaneous immortalization of this B cell population in vitro, and its apparent predilection for malignant transformation in vivo.

Continuing rearrangement of immunoglobulin and T-cell receptor genes in a Ha-ras-transformed lymphoid progenitor cell line.

Abstract: The arrangement of immunoglobulin genes has been examined in a series of lymphoid cell lines transformed with the Harvey murine sarcoma virus. One cell line, HAFTL-1, expresses antigenic markers characteristic of B-lymphoid cells and undergoes frequent rearrangement at the JH locus (where J = joining and H = heavy chain) during propagation in culture. By molecular cloning and nucleotide sequence determination, these rearrangements were found to represent the earliest postulated step in heavy chain gene assembly: the joining of a diversity (D) segment to a JH segment. The HAFTL-1 cell line also undergoes infrequent D beta-to-J beta joining at the T-cell receptor beta locus in culture. The observations presented here suggest that the HAFTL-1 cell line represents the early stage of B-cell differentiation at which immunoglobulin gene rearrangement is initiated.

In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.

Abstract: Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

Characterization of endonuclease activities in Moloney murine leukemia virus and its replication-defective mutants.

Abstract: To study Moloney murine leukemia virus (M-MulV) proteins associated with the integration of proviral DNA into the host chromosome, we isolated endonuclease activities from purified virion preparations of the wild type and two of its replication mutants. A major endonuclease activity was identified in virions of M-MuLV; the enzyme catalyzed nicks in double-stranded DNA in the presence of either Mn2+ or Mg2+ and was stimulated by ATP. The endonuclease nicked DNA adjacent to all four nucleotides with some preference for G and C. The same enzyme, and in comparable amounts, was isolated from two virus replication mutants: dl2905, deficient in the processing of Pr65gag and Pr200gag-pol, and dl50401, deficient for the virus integration function. In the process of these experiments, the residual reverse transcriptase in mutant dl2905 was shown to be the mature size, implying that the uncleaved precursor lacks enzymatic activity. It appears that the major endonuclease activity found in virions of M-MuLV is not encoded by either the gag or pol genes.

Method of inducible gene expression

Abstract: Inducible gene expression can be obtained by placing a gene of interest under regulation of an enhancer sequence. Expression of the gene can be induced by stimulating production of enhancer binding factor.

Methode d'expression de gene inductible

Abstract: Une expression de gene inductible peut etre obtenue en placant un gene d'interet sous la regulation d'une sequence augmentatrice. L'expression du gene peut etre induite en stimulant la production d'un facteur de liaison augmentateur.