Showing papers by "David Baltimore published in 1982"
TL;DR: It is suggested that this added sequence is a product of the activity of terminal deoxynucleotidyltransferase at the D/JH (and probably the VH/D) joints and that it represents a new element of heavy chain gene structure, the N region.
Abstract: A chromosomal segment with a unique structure around the immunoglobulin heavy chain joining region (JH) has been molecularly cloned from an Abelson murine leukemia virus-transformed cell line. Attached to JH3 in the cloned DNA, in inverted sequence, is the DNA from JH1 to the JH2 recognition sequence. The inverted segment is attached at its other end to the 5' recognition sequence of a diversity segment (D). To form this structure, three joining events must have occurred on the same chromosome. One of these events could have been a normal D-JH joining but the others must have been irregular events including ones that result in inversions. One of the joining events left fused recognition elements from JH2 and a D whose sequence shows that, during joining, reciprocal joinings of the recognition elements must occur to fuse the heptameric elements back to back. Because joined D and JH undergo deletion of terminal coding sequence during recombination but the joined heptameric recognition sequences do not contain the deleted sequence, joining must be a nonreciprocal event. Also, extra nucleotides are inserted between D and JH as part of the joining process; it is suggested that this added sequence is a product of the activity of terminal deoxynucleotidyltransferase at the D/JH (and probably the VH/D) joints and that it represents a new element of heavy chain gene structure, the N region.
676 citations
TL;DR: The implications of multiple rearrangements on a single chromosome and of the maintenance of reciprocal fragments are considered in the context of a model that postulates that the V kappa and J kappa segments are not all organized in the DNA in the same transcriptional direction, leading to inversions rather than deleting during joining.
232 citations
TL;DR: Data support the concept that heavy-chain allelic exclusion, like that of light chains, is not mediated by control at the DNA or RNA levels but is probably a consequence of feedback control from cytoplasmic mu chains.
Abstract: Lymphoid cells transformed by Abelson murine leukemia virus (A-MuLV) contain three classes of RNA transcripts from immunoglobulin mu genes. P mu-mRNAs (productive) correspond to the normal 2.7-kilobase (kb) membrane (mu m) and 2.4-kb secreted (mu s) mu mRNA species both in size and coding capacity and occur at approximately equal abundance in most mu-positive (pre-B-like) A-MuLV transformants. A mu-mRNAs (aberrant) generally fall into one of two categories--aberrantly small 2.3-kb mu m and 2.0-kb mu s mRNAs which encode aberrantly small mu polypeptide chains, or normal-sized, V H-containing mu RNAs which do not encode immunologically identifiable mu polypeptide chains. In one case, the latter type of A mu-mRNA was demonstrated to result from an in-phase termination codon in the D segment of the mu mRNA. Also, most, if not all, A-MuLV transformants express members of a 3.0 to 1.9-kb set of C mu-containing, but V H-negative S mu-RNAs (for sterile), the expression of which may occur simultaneously with but independently of P mu-mRNAs or A mu-mRNAs. The S mu-RNA sequences do not encode immunologically identifiable mu chains and can be produced by cells with unrearranged heavy-chain alleles, such as T-lymphocytes, although the structure of the S mu-RNAs from T-lymphoid cells appears to be different from that of B-lymphoid cell S mu-RNAs. Certain A-MuLV transformants also express gamma-RNA sequences that are probably analogous to the three different forms of mu RNA. These data support the concept that heavy-chain allelic exclusion, like that of light chains, is not mediated by control at the DNA or RNA levels but is probably a consequence of feedback control from cytoplasmic mu chains.
182 citations
TL;DR: A cell line that switches from μ to γ2b synthesis during growth in culture uses the same VH region for both heavy chains but retains two copies of the Cμ gene, suggesting that the μ toγ2b class switch can occur, at least in part, by an RNA processing mechanism.
Abstract: A cell line that switches from mu to gamma 2b synthesis during growth in culture uses the same VH region for both heavy chains but retains two copies of the Cmu gene. This suggests that the mu to gamma 2b class switch can occur, at least in part, by an RNA processing mechanism. Regulatory variants of this cell line lose constitutive mu-chain synthesis but simultaneously acquire lipopolysaccharide (LPS)-inducible synthesis of that chain. This co-variation is allele-specific and is correlated to a large deletion of DNA in the JH--Cmu intron.
146 citations
TL;DR: A functionally rearranged murine kappa light chain immunoglobulin (Ig) gene is introduced into an Abelson murine leukemia virus-transformed lymphoid cell line and levels of kappa mRNA and polypeptide increased, showing regulated expression of the introduced kappa gene.
Abstract: We have introduced a functionally rearranged murine kappa light chain immunoglobulin (Ig) gene into an Abelson murine leukemia virus-transformed lymphoid cell line. Plasmid pSV2gpt-kappa 41, containing the kappa light chain gene from the myeloma MOPC41 and the selectable marker gene gpt, was introduced into 81A-2 cells by the calcium phosphate coprecipitation technique. Cells expressing the gpt gene were selected by growth in medium containing mycophenolic acid. One transfected cell line, kappa-2, was shown to make kappa mRNA and polypeptide chains and to assemble the kappa chain product with gamma 2b heavy chains to form an apparently complete IgG2b. When bacterial lipopolysaccharide was added to the growth medium, levels of kappa mRNA and polypeptide increased, showing regulated expression of the introduced kappa gene.
117 citations
TL;DR: The nucleotide sequence of two λ 1 gene from hybridomas and a λ 2 gene from a myeloma are determined to demonstrate that somatic mutation in λ genes can occur in both the framework and CDR residues.
Abstract: Studies of the murine λ1 light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 λ1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions1. Subsequently, one of these presumed somatic variants of the single λ1 V gene was characterized by DNA sequence analysis of the rearranged functional gene2. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of λ chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related κ chain variable region amino acid sequences, and more recently Vκ and VH genes8–12, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutation in λ and κ genes, we have determined the nucleotide sequence of two λ 1 gene from hybridomas and a λ 2 gene from a myeloma. These sequences demonstrate that somatic mutation in λ genes can occur in both the framework and CDR residues.
116 citations
TL;DR: The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits and an antibody response was elicited not only by the full-length synthetic VPg peptide, but also by a synthetic 14-amino acid carboxy-terminal peptide.
100 citations
TL;DR: The A-MuLV protein appears to be a tyrosine-specific protein kinase which is active in E. coli.
86 citations
TL;DR: Although synthesis of full length products occurs much more slowly than in the infected cell, this soluble system appears to mimic quite faithfully the initial steps of poliovirus replication.
64 citations
TL;DR: It is proposed that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci, and it is shown that A- MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.
Abstract: A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 104 transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3′ end of the genome was not present. The transforming gene was thus localized to the 5′ portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3′ ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.
63 citations
TL;DR: VP1 appears to be a neutralizing antigen for poliovirus and in its denatured form presents to the immune system its neutralizing determinants.
Abstract: Antibodies were raised in rats against the poliovirus type 1 capsid proteins, VP1, VP2, and VP3. Antibodies directed against VP1 from type 1 poliovirus (Mahoney) neutralized type 1 but not type 2 poliovirus. Antibodies raised against VP2 and VP3 failed to neutralize type 1 virus. Thus, VP1 appears to be a neutralizing antigen for poliovirus and in its denatured form presents to the immune system its neutralizing determinants.
TL;DR: A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant.
TL;DR: By analysis of a series of mouse x Chinese hamster hybrid celllines containing various mouse chromosomes, the C-abl gene is mapped to mouse chromosome 2.
Abstract: Abelson murine leukemia virus (A-MuLV) is a replication-defective retrovirus that transforms lymphocytes of the B-cell lineage. This virus is a recombinant between the parental Moloney murine leukemia virus and a cellular gene termed C-abl. By analysis of a series of mouse x Chinese hamster hybrid celllines containing various mouse chromosomes, we have mapped the C-abl gene to mouse chromosome 2.
TL;DR: Data indicates that the initiation of plus strong stop DNA synthesis by the virion RNA-dependent DNA polymerase is a precise event which occurs at a unique site on the retrovirus genome.
TL;DR: Anti-VPg antibodies inhibited host-factor-dependent RNA synthesis by the poliovirus replicase but not oligo(U)-primed synthesis, implicating VPg in the de novo initiation of replic enzyme products.
TL;DR: Antibodies raised against the p63 carboxy-terminal peptide inhibited poliovirus replicase and polyuridylic acid polymerase activities in vitro, providing strong support for earlier suggestions that these activities are a property of a single virus-specific polypeptide.
Abstract: A carboxy-terminal peptide of the poliovirus replicase protein (p63) was chemically synthesized, coupled to bovine serum albumin carrier, and injected into rabbits. The resulting antisera reacted with six virus-specific proteins from HeLa cells infected with poliovirus: NCVP 0b, NCVP 1b, NCVP 2, a protein of about 60,000 daltons, p63, and NCVP 6b. The identity of the 60,000-dalton protein is not known, but the other results were consistent with previous experimental approaches which demonstrated that p63 and the other four polypeptides have common coding sequences. An amino-terminal peptide of p63 failed to elicit an immune response in rabbits. Antibodies raised against the p63 carboxy-terminal peptide inhibited poliovirus replicase and polyuridylic acid polymerase activities in vitro, providing strong support for earlier suggestions that these activities are a property of a single virus-specific polypeptide.
TL;DR: External protease treatment apparently bypassed the lesion in these viral mutants, suggesting that their defect may involve a virus-specific protease.
Abstract: Cells infected with certain temperature-sensitive mutants of Moloney murine leukemia virus synthesize the virion precursor proteins but neither bud virions nor cleave precursor proteins to their mature form. Addition of proteases to cells infected with these mutants caused cleavage of the precursor proteins Pr65gag and Pr180gag-pol to their mature forms at the nonpermissive temperature. Concomitantly there was release from the cells of morphologically normal virions. The enzymatically inactive Pr180gag-pol was cleaved to active reverse transcriptase (p85), which was found in the released particles. External protease treatment apparently bypassed the lesion in these viral mutants, suggesting that their defect may involve a virus-specific protease.
TL;DR: These results strongly favored the previous notion that the host factor was needed for the initiation step of viral complementary RNA synthesis.
Abstract: In vitro transcription of poliovirus RNA was catalyzed by the combination of a virus-coded polymerase and a host cell protein (host factor). Antibody to host factor inhibited template-dependent synthesis of complementary RNA where presumably RNA chain initiation occurred. On the contrary, elongation of already initiated RNA chains catalyzed by the replicase-template complex was not inhibited by anti-host factor antibody. These results strongly favored our previous notion that the host factor was needed for the initiation step of viral complementary RNA synthesis.
Patent•
14 Apr 1982
TL;DR: In this paper, a double-stranded virus of poliomyelitis is used to produce chimerical plasmids containing virus cDNA polio, which can be used for detecting the presence of the virus and for the production of antibodies against the virus.
Abstract: Procedes de production de ADNc representant des sequences virales de ARN, tel que ADNc a double fibre du virus de la poliomyelite, ses produits, et ses utilisations. CDNA production processes representative of viral sequences RNA as cDNA double-stranded virus of poliomyelitis, its products, and its uses. ADNc du virus de la poliomyelite est produit par exemple par transcription inverse de ARN du virus de la poliomyelite et ensuite par insertion de ADN complementaire (ADNc) du virus de la poliomyelite dans des plasmides bacteriens par des techniques de manipulation genetique. CDNA polio virus is produced for example by reverse transcription RNA virus of poliomyelitis and then by adding complementary DNA (cDNA) of the virus of poliomyelitis in bacterial plasmids by genetic manipulation techniques. Les bacteries transformees sont ensuite clonees et cultivees pour produire des plasmides chimeriques repliques contenant le virus de la poliomyelite de ADNc. The transformed bacteria are then cloned and cultivated to produce replicas chimerical plasmids containing virus cDNA polio. Un tel ADNc du virus de la poliomyelite est utile pour la detection en procedant a des essais de la presence du virus de la poliomyelite et pour la production d'anticorps contre le virus de la poliomyelite. Such cDNA polio virus is useful for detection by conducting tests of the presence of the virus of poliomyelitis and for the production of antibodies against the virus of poliomyelitis. Il s'est revele que ADNc du virus de la poliomyelite de longueur totale est infectieux, ce qui veut dire qu'il peut etre utilise pour produire des particules de virus alterees pour les vaccins. He revealed that cDNA of the virus of poliomyelitis in total length is infectious, which means it can be used to produce altered virus particles for vaccines.
01 Jan 1982
TL;DR: The availability of an infectious cDNA copy of poliovirus RNA should permit new genetic manipulations with this virus, and more reproducible numbers of infectious foci are obtained.
Abstract: Publisher Summary Poliovirus is the best-characterized member of the Picornavirus group, which includes positive-strand, non-segmented RNA animal viruses. It has been shown that a cloned, full-length copy of the poliovirus genome is infectious in mammalian cells. Using a procedure for transfection in suspension, more reproducible numbers of infectious foci are obtained. This chapter describes an experiment and its result to examine the RNAs and proteins of viruses that emerge from cells transfected with viral cDNA and presents their comparison to those of wild type poliovirus. Oligonucleotide fingerprinting was used to compare the RNA of transfection-derived virus with that of wild type virus. Large RNAse T1-resistant oligonucleotides were mapped on the viral RNA. These are scattered randomly across the genome. Therefore, the RNAse T1 oligonucleotide fingerprints can be used to compare a random sampling of the structure of the viral RNAs. The availability of an infectious cDNA copy of poliovirus RNA should permit new genetic manipulations with this virus.
Patent•
14 Apr 1982
TL;DR: Il s'est revele que ADNc du virus de the poliomyelite de longueur totale est infectieux, ce qui veut dire qu'il peut etre utilise pour produire des particules de virus alterees pour les vaccins.
Abstract: Procedes de production de ADNc representant des sequences virales de ARN, tel que ADNc a double fibre du virus de la poliomyelite, ses produits, et ses utilisations. ADNc du virus de la poliomyelite est produit par exemple par transcription inverse de ARN du virus de la poliomyelite et ensuite par insertion de ADN complementaire (ADNc) du virus de la poliomyelite dans des plasmides bacteriens par des techniques de manipulation genetique. Les bacteries transformees sont ensuite clonees et cultivees pour produire des plasmides chimeriques repliques contenant le virus de la poliomyelite de ADNc. Un tel ADNc du virus de la poliomyelite est utile pour la detection en procedant a des essais de la presence du virus de la poliomyelite et pour la production d'anticorps contre le virus de la poliomyelite. Il s'est revele que ADNc du virus de la poliomyelite de longueur totale est infectieux, ce qui veut dire qu'il peut etre utilise pour produire des particules de virus alterees pour les vaccins.