Search PubMed⌕ Search

Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 325 records · Page 18Linked to original sources

Continuing kappa-gene rearrangement in a cell line transformed by Abelson murine leukemia virus.

A cell line transformed by Abelson murine leukemia virus, called PD, is capable of carrying out kappa-gene rearrangement while growing in culture. Subclones of PD have diverse kappa-gene structures, and some derivatives show evidence of continued joining activity after as many as three subclonings. Analysis of PD sublineages has shown that a rearranged chromosome can undergo secondary kappa-gene rearrangements, producing either a new rearrangement or a deletion of C kappa. Although the PD line actively rearranges its kappa genes, its rearranged heavy-chain genes show little variation, and there is no rearrangement of lambda genes. In PD subclones, DNA fragments representing the reciprocal product of kappa-gene rearrangement are often evident, and they may undergo either further rearrangement or deletion. 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 deletions during joining.

Abelson murine leukemia virus↗

Antibodies against the chemically synthesized genome-linked protein of poliovirus react with native virus-specific proteins.

The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits. 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. All antisera reacted with virus-specific proteins from HeLa cells infected with poliovirus. Three of these proteins have previously been implicated by others as precursors of VPg. No free cytoplasmic VPg could be detected, and the antibodies did not react with radiolabeled proteins from uninfected cells.

Amino Acid Sequence↗

Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions.

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.

Animals↗

Isolated poliovirus capsid protein VP1 induces a neutralizing response in rats.

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.

Animals↗

Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line.

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.

Animals↗

Transfection of fibroblasts by cloned Abelson murine leukemia virus DNA and recovery of transmissible virus by recombination with helper virus.

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 10(4) 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.

Abelson murine leukemia virus↗

Antibody to a host protein prevents initiation by the poliovirus replicase.

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.

Antibodies↗

Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus-encoded proteins and inhibition of virus-specific polymerase activities in vitro.

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.

Animals↗

Protease bypass of temperature-sensitive murine leukemia virus maturation mutants.

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.

Animals↗

Multiple immunoglobulin heavy-chain gene transcripts in Abelson murine leukemia virus-transformed lymphoid cell lines.

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.

Abelson murine leukemia virus↗

Cloned poliovirus complementary DNA is infectious in mammalian cells.

A complete, cloned complementary DNA copy of the RNA genome of poliovirus was constructed in the Pst I site of the bacterial plasmid pBR322. Cultured mammalian cells transfected with this hybrid plasmid produced infectious poliovirus. Cells transfected with a plasmid which lacked the first 115 bases of the poliovirus genome did not produce virus.

Animals↗

Chromosomal location of structural genes encoding murine immunoglobulin lambda light chains. Genetics of murine lambda light chains.

To determine the chromosomal localization of murine lambda light (L) chain structural genes, DNA from a panel of 11 mouse x hamster somatic cell hybrids was scored for the presence of sequences homologous to cloned lambda DNA probe molecules. Six of the hybrids had detectable lambda I and lambda II gene sequences. In all six, the full complement of murine sequences was present, and in its germline configuration. The remaining hybrids lacked any detectable murine lambda L chain gene sequences. The only mouse chromosome present in all of the positive hybrids and absent from the negative ones was number 16, allowing the assignment of lambda L chain structural genes to this chromosome. Together with the previous assignments of the kappa L chain genes to chromosome 6 and heavy chain genes to chromosome 12, this finding completes the mapping of Ig structural genes in the mouse at the chromosomal level.

Animals↗

Dual expression of lambda genes in the MOPC-315 plasmacytoma.

The expression of two kappa light chain immunoglobulins in the MPC-11 mouse myeloma is well established, the two protein products being apparently from RNA transcripts derived from separate, rearranged kappa alleles in the MPC-11 genome. Recently, the characterization of kappa-related RNAs and protein products in several lambda-producing myelomas has indicated that multiple expression of light chain RNAs is a common event in myelomas and other cells of the B-lymphocyte lineage. These studies suggest that, although many light chain alleles may function to make RNA and protein in a given B-lymphocytic cell, only one complete, functional light chain is generally translated from the RNAs present in a single cell. The myeloma, MOPC-315, synthesizes and secretes an antibody which has an alpha heavy chain and a lambda II light chain. The DNA of MOPC-315 either has no kappa genes or has only a fragment of one, but it certainly has no kappa genes in the embryonic configuration. Rearrangement of its lambda genes has been observed but the exact nature of the rearrangement is not known. Because initial observations suggested that an immunoglobulin-related protein other than alpha and lambda II was present in MOPC-315 cells, we undertook to derive molecular cDNA clones from the MRNA in MOPC-315 tumour cells. Analysis of the clones has now identified two lambda chain mRNA species: a normal lambda II chain mRNA and another which directs the synthesis of a deleted form of a lambda I protein. The nucleotide sequence of the deleted lambda I mRNA shows that it resulted from a joining of the sequence encoding amino acid 31 of the variable region directly to the constant region coding sequence.

Amino Acid Sequence↗

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region.

To examine germ line genes of the heavy chain variable region (VH) that might contribute to formation of antibodies of the NPb family, we have derived cDNA clones from two hybridomas making NPb antibodies. One, B1-8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the VH region, had very different D segments and had identical J segments (J2). A set of closely related germ line VH genes was then cloned from a partial Eco RI library of C57Bl/6 DNA. By comparing the germ line VH regions to the cDNA VH regions, we identified seven potential candidates for encoding the VH regions of NPb antibodies. The seven VH regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1-8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1-8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other VH genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their VH regions from the same germ line gene. Certain of the sequenced VH genes contain crippling mutations; the repertoire of germ line VH genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hydridization.

Animals↗

Expression of J chain RNA in cell lines representing different stages of B lymphocyte differentiation.

During B cell differentiation to pentamer IgM secretion, synthesis of the pentamer joining component, the J chain, is initiated. We investigated the mechanism for initiating J chain synthesis by analyzing murine cell lines representing different stages in B cell differentiation. The expression of functional J chain mRNA was evaluated by cell-free translation and specific immunoprecipitation of a J chain product. The expression of precursor mRNA was examined by hybridization with a J chain probe obtained by molecular cloning of cDNA. No J chain-specific RNA could be demonstrated in a lymphoma line representative of an undifferentiated B lymphocyte, but three species of J chain RNA were identified in hybrid cell lines representative of IgM-secreting plasma cells: a mature message of approximately 1.5 kb and two minor components of 2.5 and 0.92 kb. The encounter of a B cell with antigen or mitogen must therefore trigger events that effect either transcription of J chain sequences or their intranuclear stabilization.

Animals↗