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Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 235 records · Page 13Linked to original sources
PubMed1988

Nuclear factor NF-kappa B can interact functionally with its cognate binding site to provide lymphoid-specific promoter function.

Enhancers and promoters, cis-acting regulators of mammalian gene expression, are modular units containing multiple short binding sites for specific trans-acting transcription factors. To investigate if factors binding to enhancer sequences are functionally different from promoter-binding factors, we asked if a short DNA sequence element in the immunoglobulin kappa (kappa) light chain enhancer that binds to the nuclear factor NF-kappa B could also serve as a functional promoter element. A synthetic oligonucleotide containing this binding site was placed in either orientation upstream of the beta-globin TATA-element. In myeloma cells, the NF-kappa B binding site efficiently directed transcription. The promoter activity was directly correlated with the presence of the nuclear factor NF-kappa B: there was no transcription in fibroblasts or in unstimulated pre-B cells where the factor was absent. Transcription could be stimulated in pre-B cells by treatments known to activate NF-kappa B. Thus, the same nuclear factor can act as a positive activator of both enhancer and promoter function, suggesting that the two functions involve similar events in the transcription process.

Animals↗
PubMed1988

NF-kappa B protein purification from bovine spleen: nucleotide stimulation and binding site specificity.

The activity of the enhancer for the kappa immunoglobulin light chain gene critically depends on the presence in the nucleus of the NF-kappa B protein. We purified NF-kappa B over 50,000-fold and identified two protein species, 42 and 44 kDa, that could be eluted and renatured from a sodium dodecyl sulfate/polyacrylamide gel to give specific DNA-binding activity. Binding of the purified bovine NF-kappa B as well as that from human and murine B- or T-lymphoid cell extracts was dramatically stimulated by nucleoside triphosphates. This effect distinguished NF-kappa B from a related factor, H2-TF1. Purified NF-kappa B interacted efficiently with regulatory sequences that function during either B- or T-lymphocyte activation, including the human immunodeficiency virus enhancer and a NF-kappa B binding site we detected in the interleukin 2 enhancer.

Animals↗
PubMed1988

Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein.

The P210bcr/abl protein is associated with virtually every case of human chronic myelogenous leukemia. Unlike the related P160gag/v-abl oncogene product of Abelson murine leukemia virus, P210bcr/abl does not transform NIH 3T3 fibroblasts. To assess whether P210bcr/abl might transform hematopoietic cell types, retroviral constructs encoding P210bcr/abl were used to infect the bone marrow-derived interleukin 3-dependent Ba/F3 cell line. As for P160gag/v-abl, cell lines expressing P210bcr/abl were growth factor independent and tumorigenic in nude mice. No evidence for autocrine production of interleukin 3 by factor-independent cell lines was found. These experiments establish that P210bcr/abl can transform hematopoietic cell types to tumorigenicity.

Abelson murine leukemia virus↗
PubMed1988

Oligonucleotide that binds nuclear factor NF-kappa B acts as a lymphoid-specific and inducible enhancer element.

The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction.

B-Lymphocytes↗
PubMed1988

T-cell-specific expression of interleukin 2: evidence for a negative regulatory site.

To understand the basis for T-cell-specific induction of interleukin 2 (IL-2), we have analyzed nuclear factors from the Jurkat T-lymphoid leukemia cell, which can be induced to secrete IL-2. We have used an electrophoretic mobility shift assay to examine binding of proteins to the upstream regulatory region, before and after activation with mitogens. We find two types of binding sites. One resembles an inducible enhancer element, but the protein that recognizes it is found in non-T cells and is unlikely to determine T-cell-specific expression of IL-2. A second site negatively regulates expression in resting T cells. A complex that binds to a DNA fragment containing this site is modified only when IL-2 is expressed, and it lies near a specific inducible DNase hypersensitive region. We suggest that negative regulation at this site, mediated by its associated protein(s), may contribute to the cell-specific expression of IL-2.

Animals↗
PubMed1988

Octamer-binding proteins from B or HeLa cells stimulate transcription of the immunoglobulin heavy-chain promoter in vitro.

The B-cell-type specificity of the immunoglobulin (Ig) heavy-chain and light-chain promoters is mediated by an octanucleotide (OCTA) element, ATGCAAAT, that is also a functional component of other RNA polymerase II promoters, such as snRNA and histone H2B promoters. Two nuclear proteins that bind specifically and with high affinity to the OCTA element have been identified. NF-A1 is present in a variety of cell types, whereas the presence of NF-A2 is essentially confined to B cells, leading to the hypothesis that NF-A2 activates cell-type-specific transcription of the Ig promoter and NF-A1 mediates the other responses of the OCTA element. Extracts of the B-cell line, BJA-B, contain high levels of NF-A2 and specifically transcribe Ig promoters. In contrast, extracts from HeLa cells transcribed the Ig promoter poorly. Surprisingly, addition of either affinity-enriched NF-A2 or NF-A1 to either a HeLa extract or a partially purified reaction system specifically stimulates the Ig promoter. This suggests that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell-specific transcription of this promoter, at least in vitro, is partially due to a quantitative difference in the amount of OCTA-binding protein. Because NF-A1 can stimulate Ig transcription, the inability of this factor to activate in vivo the Ig promoter to the same degree as the snRNA promoters probably reflects a difference in the context of the OCTA element in these two types of promoters.

B-Lymphocytes↗
PubMed1988

The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains.

Transcription of promoters of immunoglobulin genes is controlled by an octanucleotide sequence element. The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined. This protein specifically recognizes the octanucleotide element and is part of the previously identified NF-A2 family of proteins. The DNA-binding domain of Oct-2 is structurally related to the homeo box consensus and thus contains a potential helix-turn-helix sequence. Oct-2 also possesses a potential 'leucine zipper' domain, where four leucines are each separated by exactly seven residues. Comparisons of Oct-2 with protein Oct-1, which also recognizes the octanucleotide element but is constitutively expressed in all cell types, show high sequence conservation through the 60-residue DNA-binding domain, as well as an adjacent tract of 75 residues. The latter conserved region is also found in regulatory genes expressed in pituitary cells and nematodes and has been termed a POU box. Because two different cDNAs were isolated, it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns. Most interestingly, the oct-2 cDNA contains a second overlapping open reading frame, 278 residues in length, which might also specify a protein important for B-cell development.

Amino Acid Sequence↗
PubMed1988

Isolation of poliovirus 2C mutants defective in viral RNA synthesis.

Two poliovirus mutants were isolated that contain an oligonucleotide linker insertion in the 2C-coding region of the viral genome. One, 2C-31, has a strongly temperature-sensitive phenotype and the other, 2C-32, forms small plaques on HeLa cell monolayers at all temperatures. Both mutants have a severe temperature-sensitive defect in viral RNA synthesis but little effect on the types of viral protein that are made. Temperature shift experiments showed that the 2C function is continuously required for viral RNA synthesis to proceed. The 2C mutants could be complemented in trans by mutants with mutations in other viral proteins. Protein 2C is also the locus of the guanidine resistance and dependence mutants, a drug whose action also affects viral RNA synthesis. Thus, protein 2C is one that is needed continually for viral RNA synthesis and, at least with these temperature-sensitive alleles, can be provided in trans.

Genetic Complementation Test↗
PubMed1988

An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis.

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.

Animals↗
PubMed1988

Poliovirus mutant that contains a cold-sensitive defect in viral RNA synthesis.

By manipulating an infectious cDNA clone of poliovirus, we have introduced a single-codon insertion into the 3A region of the viral genome which has been proposed to encode a functional precursor of the virion-linked protein VPg. The resulting mutant was cold sensitive in monkey kidney cells. Viral RNA synthesis was poor at 32.5 degrees C, although no other function of the virus was obviously affected. The synthesis of both positive and negative strands was severely depressed. Temperature shift experiments suggest that a normal level of production of the affected function was required only during the early (exponential) phase of RNA synthesis. Analysis of viral polyprotein processing at the nonpermissive temperature revealed that some of the normal cleavages were not made, most likely as a consequence of the defect in RNA synthesis or as a result of the concomitant reduction in the level of virally encoded proteases.

Cold Temperature↗
PubMed1988

Recombinants within the tyrosine kinase region of v-abl and v-src identify a v-abl segment that confers lymphoid specificity.

The v-abl and v-src oncogenes encode protein-tyrosine kinases that possess different biological properties in spite of their high degree of amino acid conservation. To correlate functional differences with structural domains of the two oncogenes, we recombined v-abl and v-src just downstream of the lysines in their ATP-binding sites, within the kinase domain. The biological activity of the chimeric genes was studied and compared with that of v-src and v-abl. The v-src/v-abl recombinant shared with v-src and v-abl the ability to transform fibroblasts. In addition, like v-abl, it transformed lymphoid cells and relieved a hematopoietic cell line of its interleukin 3 requirement. In contrast, the reciprocal construct, v-abl/v-src, was transformation defective. Lack of biological activity correlated with formation of a stable complex between the chimeric protein and two cellular proteins and with low kinase activity. We conclude that the specificity within the kinase domain determines the particular biological behavior of protein-tyrosine kinase oncogenes.

Cell Transformation, Neoplastic↗
PubMed1988

Four murine c-abl mRNAs arise by usage of two transcriptional promoters and alternative splicing.

The c-abl gene in mice is transcribed into two major and at least two minor mRNAs which have different 5'-ends, but are otherwise colinear. Here we show that the two major mRNAs are initiated by separate promoters and that the minor transcripts arise by alternative splicing. Like in the human gene, one of the alternative murine 5' c-abl exons lies far upstream of the remaining exons. The major mRNA that begins with this exon has about 1275 nucleotides upstream of the abl coding region. Interestingly, this unusually long upstream mRNA segment contains multiple short open reading frames both in mouse and man and is highly conserved in sequence between these species. The 5'-most c-abl promoter contains several sequence motifs that are highly conserved between mouse and man. The downstream promoter is much less conserved.

Amino Acid Sequence↗
PubMed1987

The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1.

The recently described human immunodeficiency virus type 2 (HIV2) is significantly divergent in sequence from the more frequently isolated human immunodeficiency virus type 1 (HIV1). Both HIV1 and HIV2 encode a transactivator that is capable of strongly stimulating expression directed by the viral long terminal repeat (LTR). Here, we define the region of the HIV2 genome encoding the transactivator and show that the specificity of the transactivator differs from that of HIV1. By deletion analysis of the HIV2-LTR, we show that both HIV1 and HIV2 transactivators require sequences within 35 to 53 bp downstream of the start of transcription. However, in order to stimulate expression at full efficiency, the HIV2 transactivator further requires sequences unique to the HIV2-LTR between nucleotides +53 and +99. Hence, HIV2 poorly transactivates the LTR of HIV1, while two divergent isolates of HIV1 will efficiently transactivate the LTR of either HIV1 or HIV2. Nonetheless, in vivo competition between the transactivators of HIV1 and HIV2 suggests that they use a common mechanism.

Base Sequence↗
PubMed1987

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

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.

Abelson murine leukemia virus↗