Purification and properties of poliovirus double-stranded ribonucleic acid.
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Adenine Nucleotides↗
Biomedical subjects
D Baltimore
Publications and source records attributed to D Baltimore.
An intermediate in the synthesis of poliovirus RNA.
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Centrifugation, Density Gradient↗
The effect of HeLa cell cytoplasm on the rate of sedimentation of RNA.
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Centrifugation, Density Gradient↗
Joining of V kappa to J kappa gene segments in a retroviral vector introduced into lymphoid cells.
V kappa to J kappa recombination occurred within a DNA construct introduced into cells in the form of a defective retrovirus. Analysis of one recombinant demonstrated that V kappa-J kappa joining can occur via inversion with a net loss of a few base pairs.
Animals↗
Double-stranded cleavage by cell extracts near recombinational signal sequences of immunoglobulin genes.
The genes encoding the variable regions of murine immunoglobulin light chains are present in the germ line in two separate segments, V and J. During B lymphocyte differentiation these segments are brought together to form a single unit (for review see ref. 1). Although much is known about the structures of V and J segments, both in germ-line configuration and after rearrangement, essentially nothing is known about the biochemical mechanism of V-J recombination. One possible step in proposed mechanisms of immunoglobulin gene rearrangement is endonucleolytic cleavage of the participating DNA segments before joining. In an attempt to detect such an activity, we have developed an assay for the detection of site-specific double- or single-strand endonucleolytic activity in crude soluble extracts. Using this assay we have detected an activity in extracts of nuclei from mouse B-lymphoid lines and from mouse L cells that is capable of introducing duplex breaks near the recombinational signal sequences of immunoglobulin JK segments. We report the activity here because of its intrinsic interest although we lack any direct evidence that it has a role in V-J recombination.
Abelson murine leukemia virus↗
Sexual preference of apparent gene conversion events in MHC genes of mice.
Polymorphisms exist at many genetic loci. At some loci, however, polymorphism is so high that tens and even hundreds of different alleles coexist in the population. Two such highly polymorphic systems are the immunoglobulin genes and the vertebrate major histocompatibility loci. The origin and maintenance of highly polymorphic loci remain open to debate but it seems likely that special mechanisms contribute to their variability and that their polymorphism serves important biological roles. The high degree of polymorphism at the H-2 class I major histocompatibility locus of the mouse has been documented by both tissue transplantation and serological methods. More recently, molecular cloning and DNA sequencing of some of the class I genes has shown that most of the sequence variability is concentrated in the first two domains and is often found in clustered regions within them. In addition, several groups have suggested that gene conversion events among the many class I genes may contribute to H-2 polymorphism; such events would have to occur during meiosis to produce heritable alterations. The strongest evidence for gene conversion comes from sequence analysis of mutant class I H-2 alleles where concerted changes at adjoining sites in DNA imply gene conversion by distant but closely related loci. We report here an analysis of these mutants indicating that the chromosomes containing loci that have experienced gene conversion originated from females. These data suggest a striking preference for mammalian meiotic gene conversion events during female rather than male gametogenesis.
Alleles↗
Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines.
The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.
Amino Acid Sequence↗
Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.
The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products (VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines that represent analogues of cells at early stages of B-lymphocyte differentiation. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.
Animals↗
Inversion for gene construction.
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Animals↗
A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.
Trans-acting factors that mediate B-cell specific transcription of immunoglobulin genes have been postulated based on an analysis of the expression of exogenously introduced immunoglobulin gene recombinants in lymphoid and non-lymphoid cells. Two B-cell-specific, cis-acting transcriptional regulatory elements have been identified. One element is located in the intron between the variable (V) and constant (C) regions of both heavy and kappa light-chain genes and acts as a transcriptional enhancer. The second element is found upstream of both heavy and kappa light-chain gene promoters. This element directs lymphoid-specific transcription even in the presence of viral enhancers. We have sought nuclear factors that might bind specifically to these two regulatory elements by application of a modified gel electrophoresis DNA binding assay. We report here the identification of a human B-cell nuclear factor (IgNF-A) that binds to DNA sequences in the upstream regions of both the mouse heavy and kappa light-chain gene promoters and also to the mouse heavy-chain gene enhancer. This sequence-specific binding is probably mediated by a highly conserved sequence motif, ATTTGCAT, present in all three transcriptional elements. Interestingly, a factor showing similar binding specificity to IgNF-A is also present in human HeLa cells.
B-Lymphocytes↗
Distinct factors bind to apparently homologous sequences in the immunoglobulin heavy-chain enhancer.
The intron separating the variable- and constant-region exons of the rearranged immunoglobulin heavy-chain locus contains a lymphocyte-specific transcriptional enhancer. The enhancer is a member of a class of cis-acting, tissue-specific, transcriptional control elements which are characterized by orientation-independent and relatively position-independent function. In vivo analysis of the position of DNA-binding factors, by assessing the availability of specific bases to chemical modification, has identified four sequence clusters within the heavy-chain enhancer, denoted E1 to E4 (refs 3, 4). These sites are protected (that is, occupied) only in B lymphocytes. A consensus sequence relationship (consensus CAGGTGGC) between these four sites was suggested where three of the sites conformed to the consensus in seven of eight positions while the other was homologus in six of eight positions. We proposed that a single trans-acting factor might recognize all four sites. Using an assay involving gel electrophoresis of DNA-protein complexes to detect sequence-specific DNA binding factors that recognize these related motifs, we have now identified a mouse B-cell nuclear factor (NF-muE1) which binds specifically to one such motif within the mouse heavy-chain gene enhancer. This factor binds poorly, if at all, to the other related motifs, and other factors have been identified which interact preferentially with some of these latter motifs. Dimethyl sulphate interference experiments suggest that the NF-muE1 factor is in contact with at least the guanine residues in the sequence GATGGCCGATC. This factor seems to be present in both lymphoid and non-lymphoid cell lines.
Animals↗
A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes.
Immunoglobulin gene promoters are active only in lymphoid cells and this tissue-specific activity requires an octamer sequence, ATTTGCAT. Paradoxically, this same octamer motif seems to be a transcriptional control element in promoters which are active in all tissues. Using an electrophoretic mobility shift assay to identify DNA binding proteins, we have now detected two species of nuclear proteins which bind specifically to this octamer. One previously characterized form (NF-A1) was found in all cell lines tested while the other form (NF-A2) was restricted to lymphoid cell lines. NF-A2 was found in cell lines representing all stages of B-cell differentiation and in half of the T-lymphoma cell lines tested. The identification of a lymphoid-specific octamer binding protein may account for the lymphoid-specific activity of immunoglobulin promoters.
Animals↗
A family of unusually spliced biologically active transcripts encoded by a Drosophila clock gene.
Complementary DNA cloning of the transcripts of the Drosophila clock gene period reveals three distinct transcripts. These result from unusual splicing pathways, one involving a CG 3' splice site and one resulting in the use of two different reading frames in one exon, and they predict three separate proteins. Two of the cloned cDNAs can restore clock function to mutant arrhythmic flies.
Animals↗
An inducible transcription factor activates expression of human immunodeficiency virus in T cells.
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).
Base Sequence↗
Depletion of the predominant B-cell population in immunoglobulin mu heavy-chain transgenic mice.
The transgenic mouse line M54 was generated by introducing a functionally-rearranged immunoglobulin mu heavy-chain gene into the germ line of a C57B1/6 inbred mouse. Previous examination of the antibodies produced by B-cell hybridomas derived from transgenic M54 mice showed that the presence of the mu transgene grossly altered the immunoglobulin repertoire of unimmunized animals, suggesting that these mice suffer from a serious immunoregulatory perturbation. 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 antigens. 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, CD5 (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 peritoneum, 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.
Animals↗
Formation of disulphide-linked mu 2 omega 2 tetramers in pre-B cells by the 18K omega-immunoglobulin light chain.
Pre-B cells are precursors of B lymphocytes that contain intracellular heavy-chain protein (mu) and are either yet to rearrange their light-chain genes or are in the process of doing so. These cells have traditionally been considered to contain intracellular mu-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 omega, which forms disulphide-linked mu 2 omega 2 tetramers. This protein could be immunoprecipitated with mu-chain from pre-B lines, but not from T-cell and fibroblast lines that express transfected mu-genes, nor from a pre-B line that synthesizes a D mu-protein (which lacks a V domain). We view the omega-chain as being a pre-B specific surrogate light chain that may be essential for the important regulatory function that the mu-protein is believed to have at this stage of differentiation.
Animals↗
An octamer oligonucleotide upstream of a TATA motif is sufficient for lymphoid-specific promoter activity.
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.
Animals↗