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Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 271 records · Page 15Linked to original sources
PubMed1986

Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction.

Poliovirus RNA polymerase requires a host factor to initiate RNA synthesis in vitro. The host factor was previously purified to near homogeneity from HeLa cells but was not assigned an enzymatic activity. This report describes the purification of a terminal uridylyltransferase that can act as host factor. By all criteria examined it is identical to the factor purified previously. It has the same molecular weight (68,000), chromatographic properties, and cellular localization. We present evidence that terminal uridylyltransferase can add uridine residues to the 3' poly(A) end of virion RNA and that these anneal back to the poly(A) and form a hairpin primer for polymerase.

HeLa Cells↗
PubMed1986

A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome.

We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, representing a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature.

Base Sequence↗
PubMed1986

B-Ly1 cells: immortal Ly-1+ B lymphocyte cell lines spontaneously arising in murine splenic cultures.

We have surveyed the molecular and functional properties of B-Ly1 cells, spontaneously occurring Ly-1+ cell tissue cultures lines established from murine spleen. Several features are surprising when compared to the conventional understanding of B cell physiology: In contrast to the major B cell subpopulation, these cells establish stable in vitro lines in the absence of nominal growth factors. This outgrowth is consistently accompanied by c-myc amplification and deregulation, and resistance to the effects of an autoregulatory IgM species which normally curtails the growth of B cells. These properties may be relevant to the disproportionate occurrence of Ly-1+ B cell malignancies in vivo. B-Ly1 cell lines consistently delete immunoglobulin constant region genes, and uniformly express lambda light chains, a rare murine isotype. These features may be causally related, and may reflect a novel recombinational activity (see this volume). Immunoglobulin expression can be modulated by conventional stimuli. However, the response is transient, and includes production of mature heavy chain isotypes ("class switching") without apparent switch deletion. Moreover, unstimulated B-Ly1 cells show transcriptional activity throughout the heavy chain locus, and a novel hypermutation activity affecting the immunoglobulin variable region. The mechanisms underlying this surprising pattern of immunoglobulin expression are unknown. However, one wonders whether this expression pattern, if common to Ly-1+ B cell in vivo, might provide modes to escape idiotypic or isotypic immunoregulation. If so, this may be relevant to the prevalence of autoantibody production by this subpopulation. Thus, we are hopeful that some of these unique properties, if confirmed in the Ly-1+ B cells in vivo, will provide more definitive markers for this subpopulation, and disclose mechanisms accounting for their distinctive physiology and pathophysiology.

Animals↗
PubMed1986

Genetic complementation among poliovirus mutants derived from an infectious cDNA clone.

We constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone. The derived viral strains carrying the mutations exhibited a variety of distinct plaque phenotypes. Thus, we were able to examine genetic complementation between different pairs of mutants by comparing the yields of progeny virus in mixed and single infections. Two mutants bearing lesions in the 2A and 3A regions of the genome, which are defective in the inhibition of host cell translation and the synthesis of viral RNA, respectively, could be rescued efficiently by genetic complementation; three replication-deficient mutants containing insertions in the 2B, 3D (replicase), and 3'-untranslated regions could not. Both the 2A and 3A mutants could be rescued by each other and by all of the other mutants tested. Because yield enhancement was apparent well before the completion of a single infectious cycle, it is likely that complementation of both mutants involved early diffusion of functional products. These data provide the first unambiguous evidence that the nonstructural portion of the poliovirus genome contains multiple complementation groups. The data also suggest that certain nonstructural functions act only in cis.

Cloning, Molecular↗
PubMed1986

Abelson virus abrogation of interleukin-3 dependence in a lymphoid cell line.

Among several tyrosine-protein kinases, only v-abl could abrogate interleukin 3 dependence of a lymphoblastoid cell line; v-src and v-fps proteins gave partial or no interleukin 3 independence, respectively. Lymphokine independence was achieved via a nonautocrine mechanism. Direct involvement of c-myc in this process was not evident.

Abelson murine leukemia virus↗
PubMed1986

The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19.

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase expressed in immature lymphocytes of the thymus and bone marrow, as well as certain leukemic cells. Chromosomal assignment of the gene coding for human TdT was accomplished by in situ hybridization of a 3H-labeled cDNA probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs. The human TdT gene was mapped to the region q23----q24 of chromosome 10. Breaks at this site have been reported in different translocations in human leukemias. The mouse TdT gene was assigned to chromosome 19 by Southern blot analysis of mouse X Chinese hamster somatic cell hybrids. This result adds a fourth locus to the conserved syntenic group on mouse chromosome 19 and human chromosome 10.

Animals↗
PubMed1985

Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus.

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.

Abelson murine leukemia virus↗
PubMed1985

Poliovirus replicase stimulation by terminal uridylyl transferase.

In an in vitro poliovirus replication system, purified viral polymerase, plus sense virion RNA, and a host factor have been previously shown to be necessary for the transcription of minus strands. We have found that a partially purified eukaryotic initiation factor-2 (eIF-2) fraction from rabbit reticulocytes can replace HeLa host factor in the replicase reaction. This enzyme preparation contains eIF-2 and two other major proteins. In addition to eIF-2 activity, which does not appear to play a role in the replicase reaction, we find that the fraction contains terminal uridylyl transferase activity. The enzyme adds UMP moieties to the 3' end of primer RNA molecules. The number of UMP residues added depends on the primer. Although long tails of heterogeneous lengths (50 to 100 nucleotides) can be polymerized on the 3' end of oligo(U), a poly(A) primer accepts only four U's. The terminal uridylyl transferase activity requires only UTP, Mg2+, a sulfhydryl reagent, and an RNA primer for activity. It is partially associated with ribosomes. We provide preliminary evidence that it may be responsible for host factor-like activity. We present a model for minus strand synthesis by poliovirus replicase, based on the hypothesis that a terminal uridylyl transferase can participate in initiation.

Base Sequence↗
PubMed1985

DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes.

Immunoglobulin K genes are constructed during lymphocyte differentiation by the joining of two DNA elements, VK and JK, to form both a VKJK coding unit and a reciprocal recombination product. The two products formed in single VK-to-JK joining events can be directly isolated through the use of a retrovirally introduced recombination substrate. The structural analysis of a number of recombinants and the derivation of secondary recombination products define some of the basic features of the mechanism of immunoglobulin gene assembly.

Animals↗
PubMed1985

Localization of tyrosine kinase-coding region in v-abl oncogene by the expression of v-abl-encoded proteins in bacteria.

A series of plasmids containing different segments of the v-abl oncogene have been constructed to express different portions of the v-abl protein in bacteria. The tyrosine kinase activity of these proteins was determined by an in vitro assay employing histones or angiotensin II as substrates for the v-abl-encoded tyrosine kinase. These experiments show that the 5'-1.2 kilobases of v-abl is necessary and sufficient to produce an active tyrosine kinase which is functional as a monomeric soluble protein. The kinase-coding region corresponds to the minimal region of v-abl required for the transformation of fibroblasts. The kinase-coding region also coincides with the conserved protein sequences which are found in other tyrosine kinases. A compact domain of the v-abl protein including this kinase-coding region can accumulate to high levels in bacteria. The C-terminal region of the v-abl protein is not needed for the kinase activity and is rapidly degraded in bacteria.

Abelson murine leukemia virus↗
PubMed1985

Specific transforming potential of oncogenes encoding protein-tyrosine kinases.

Several chimeric murine retroviruses were constructed to test whether the gag sequence of Abelson murine leukemia virus (A-MuLV) could influence the in vitro specificity of two sarcoma-inducing oncogenes: src of Rous sarcoma virus and fps of Fujinami sarcoma virus. Although the src- or fps- containing chimerae could transform fibroblasts, they were unable to mimic the action of A-MuLV in causing lymphoid transformation in vitro. A-MuLV-derived gag sequences could, however, functionally replace the 5' end of src and restore the transformation potential of a 5'-truncated src gene. To investigate this functional similarity, we replaced the gag sequence of an A-MuLV virus with the 5' end of src. This recombinant virus behaved like the A-MuLV virus from which it was derived: it transformed both fibroblasts and lymphoid cells in vitro. Taken together, these results suggest that lymphoid transformation in vitro is a specific property of abl and not of src or fps. Furthermore, it shows that a functional homology exists between the gag sequence of A-MuLV and the 5' end of src.

Abelson murine leukemia virus↗
PubMed1985

Cell-type specificity of immunoglobulin gene expression is regulated by at least three DNA sequence elements.

The regulation of cell-type specificity of immunoglobulin (lg) mu heavy chain (H) gene expression was examined by introducing various hybrid genes containing lg gene sequences combined with portions of a tissue-nonspecific transcription unit into lymphoid and nonlymphoid cells. Replacing the lymphocyte-specific IgH enhancer with a viral enhancer did not affect tissue specificity of mu Ig gene expression. We identified two new regulatory regions that provide transcriptional tissue specificity. First, the V H promoter region between position -154 and +57 was shown to direct lymphocyte-specific transcription of a bacterial gpt gene, even in the presence of a viral enhancer. Second, mu intragenic sequences, lacking the IgH enhancer, were found to regulate the level of accumulated Ig transcripts in a tissue-specific fashion. These results demonstrate that tissue specificity of Ig gene expression is not solely regulated by the enhancer but that the promoter, and as yet undefined intragenic sequences, contain lymphoid-specific regulatory information.

Animals↗
PubMed1985

A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes.

Transgenic mice containing a microinjected rearranged immunoglobulin (Ig) mu heavy chain gene were examined for the effects on DNA rearrangement of the endogenous Ig genes. Abelson murine leukemia virus (A-MuLV) cell lines were isolated from pre-B cells of transgenic mice and of normal littermates. Microinjected mu gene RNA and a mu heavy chain protein were synthesized in every transgenic A-MuLV cell line. Only 10% of normal mouse A-MuLV transformants synthesized mu protein. A germ-line JH allele was observed in 40% of the transgenic lines, demonstrating that the block to endogenous Ig DNA rearrangement occurred at the first step of heavy chain DNA joining. All alleles were rearranged in normal mouse A-MuLV lines. Germline JH alleles were also detected in 10% of the transgenic hybridomas derived from proliferating B cells. Our results support a model of active prevention of rearrangement by the product of successfully rearranged mu genes.

Abelson murine leukemia virus↗