Search PubMed⌕ Search

Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 307 records · Page 17Linked to original sources
PubMed1983

Molecular basis of a mouse strain-specific anti-hapten response.

The response of C57BL/6 and BALB/c mice to immunization with proteins coupled to (4-hydroxy-3-nitrophenyl)acetyl (NP) is dominated by distinctly different sets of antibodies. The VH gene family previously shown to be involved in the C57BL/6 response has now been shown to have highly homologous counterparts in BALB/c but of five sequenced BALB/c VH regions, none appeared likely to be able to encode an NP-binding protein. The active VH region from a BALB/c hybridoma making a characteristic anti-NP antibody was recovered and sequenced and shown to be quite different from the VH gene family involved in the C57BL/6 response. Comparison of the variation of the closely related VH regions between the two mouse strains showed that there are separate types of evolutionary pressures on the framework and complementarity-determining regions. The molecular basis for strain-specific immune responses appears to be that the structural divergence of VH regions between mouse strains is great enough that different strains use different VH regions for making the predominant class of antibodies to a specific hapten.

Amino Acid Sequence↗
PubMed1983

Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.

A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.

Chromosome Deletion↗
PubMed1983

Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation.

The role of various segments (gag or v-abl) of the Abelson murine leukemia virus (A-MuLV) genome in both lymphoid cell and fibroblast transformation was examined by deletion of areas from cloned, plasmid DNA representations of the genome. The deleted plasmids were tested by transfection into fibroblasts and by infection of bone marrow cells using virus stocks derived from the fibroblast transfectants. Deletion of gag coding sequence from the A-MuLV protein did not affect fibroblast transforming activity but abolished lymphoid transforming activity. The gag- A-MuLV genomes were very unstable in transformed fibroblasts leading to large secondary deletions in v-abl sequences. The gag- A-MuLV proteins also had lower autophosphorylation than their gag+ counterparts although cells transformed by gag- virus had a normal elevation of protein-linked phosphotyrosine. Systematic deletion of v-abl sequences showed that only 45,000 to the 130,000 molecular weight of v-abl sequence in the A-MuLV protein is needed for fibroblast transformation and, at most, slightly more is needed for lymphoid cell transformation.

Abelson murine leukemia virus↗
PubMed1983

Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: facilitation by fluorescence-activated flow sorting of metaphase chromosomes.

Human neuroblastoma IMR-32 cells have large homogeneously staining regions (HSRs), primarily in the short arms of chromosome 1. We have constructed a recombinant phage library that is enriched for DNA present in the HSR of this chromosome by using fluorescence-activated flow sorting for initial chromosome purification. Eleven distinct cloned DNA segments were identified that showed significantly greater hybridization to IMR-32 genomic DNA, detected by Southern blotting, than to normal human genomic DNA. These sequences have also been localized to the HSR of chromosome 1 by in situ hybridization. Based on an approximate 50-fold sequence amplification for each cloned segment and a total HSR size of 150,000 kilobases, the amplified unit in the HSR is estimated to be 3,000 kilobases. Sequences homologous to all cloned HSR DNA segments were mapped to human chromosome 2 by using human-mouse hybrid cells. Further work using in situ hybridization demonstrated that cloned HSR segments were localized in the short arm of chromosome 2 in both normal and IMR-32 cells. Thus, the amplification of these sequences in IMR-32 cells may have involved transposition from chromosome 2 to chromosome I.

Base Composition↗
PubMed1983

Molecular cloning and characterization of hepatitis A virus cDNA.

Double-stranded cDNA was synthesized from hepatitis A virus (HAV) RNA and inserted into the Pst I site of pBR322. Restriction endonuclease digestion and cross-hybridization of fragments yielded a map of overlapping cloned cDNAs that included at least 99% of the viral genome. Molecular clones containing HAV cDNA were identified by hybridizing cloned cDNA to electrophoretically resolved RNA from uninfected and HAV-infected tissue culture cells. Cloned cDNA probes specifically hybridized to RNA from infected cells, and the predominant species identified had the characteristic genomic length of picornaviral RNA (approximately equal to 7,500 nucleotides). A partial sequence from the 3' end of the genome revealed 414 bases in an open reading frame followed by two closely spaced stop codons, a 60-base noncoding region, and a tract of poly(A).

Amino Acid Sequence↗
PubMed1983

Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells.

VPg is a virus-encoded protein covalently attached to the 5' end of poliovirus virion RNA. We have used antibody prepared against chemically synthesized VPg to detect two forms of VPg in infected cells. Both forms were specifically immunoprecipitated from lysates of infected cells labeled with [3H]-leucine. One appears to be unmodified VPg because it had the same electrophoretic mobility as synthetic VPg. The other had a larger apparent molecular weight than VPg and could be labeled in vivo with 32Pi. Its structure is VPg-pUpU, the UMP dinucleotide being attached to VPg via a phosphodiester bond to tyrosine, the third amino acid from the NH2 terminus of VPg. This structure is identical to that found at the 5' end of virion and minus-strand RNA.

Genes, Viral↗
PubMed1983

Cloning and analysis of reverse transcript P160 genomes of Abelson murine leukemia virus.

Circular duplex reverse transcripts of the genome of a strain of Abelson murine leukemia virus that encodes a 160,000-molecular-weight protein were isolated, cleaved with HindIII restriction endonuclease, and cloned into the unique HindIII site of lambda phage Charon 21A. Recombinant phage clones, some of which were infectious in transfection assays, were found to contain a 789-base-pair region specific for Abelson murine leukemia virus; this region is not found in other strains of this virus. The extra sequence was localized by restriction endonuclease and electron microscopic heteroduplex analysis. Sequence analysis showed no homology at the ends of the extra sequence, implying that it was deleted by an event that did not utilize sequence homology. The sequence of this unique region has an open reading frame through its entirety.

Abelson murine leukemia virus↗
PubMed1983

Cellular RNA homologous to the Abelson murine leukemia virus transforming gene: expression and relationship to the viral sequence.

To examine the expression of the cellular homolog of the Abelson murine leukemia virus transforming gene (the v-abl sequence), a DNA probe representing the v-abl sequence was prepared. The probe detected two cytoplasmic polyadenylic acid-containing c-abl RNAs of about 6.5 and 5.5 kilobases in a variety of rodent cells, and slightly larger RNAs were detected in human cells. These two RNA species were found in all normal tissues or cell lines examined, but at differing concentrations: liver cells had the least, fibroblastic cell lines had the most. By using a probe able to detect the cellular but not the viral gene, the two RNAs were shown to be present in Abelson murine leukemia virus-transformed cells at levels found either in their untransformed counterparts or in similar cell types transformed by other means. The target cells of the virus have a somewhat elevated level of the two RNAs although expression of the c-abl gene is not restricted to these cells. The v-abl sequence lacks 0.35 and 0.85 kilobases of the c-abl RNA on the 5' and 3' ends, respectively. Thus, the Abelson murine leukemia virus transforming gene is an internal fragment of the transcript of a normal cellular gene.

Abelson murine leukemia virus↗
PubMed1983

Structure and function of the Abelson murine leukemia virus transforming gene.

The oncogene (v-abl) of A-MuLV is a fragment of a normal mouse gene. Only 1.2 kb on the 5'-end of the v-abl is sufficient to encode a tyrosine-specific protein kinase whose activity is important in transformation. This A-MuLV protein kinase can be active in E. coli and it phosphorylates bacterial proteins. The lack of discrimination in its selection for substrates is puzzling. Because of the demonstration of such promiscuity, the identification of physiologically-relevant phosphorylation events must rely on evidence of functional modification rather than physical modification of proteins.

Abelson murine leukemia virus↗
PubMed1982

Chromosomal assignment of the endogenous proto-oncogene C-abl.

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.

Abelson murine leukemia virus↗
PubMed1982

Expression of an Abelson murine leukemia virus-encoded protein in Escherichia coli causes extensive phosphorylation of tyrosine residues.

A segment of the Abelson murine leukemia virus (A-MuLV) genome was inserted into an Escherichia coli plasmid designed to allow the expression of the protein encoded by the viral gene. Bacteria expressing the A-MuLV-encoded protein were isolated; they had new phosphorylated proteins in which the phosphate was linked to tyrosine residues. These proteins included many that must be E. coli protein. One phosphotyrosine-containing protein of 62,000 molecular weight had reactivity with antiserum specific for authentic A-MuLV protein. The A-MuLV protein thus appears to be a tyrosine-specific protein kinase which is active in E. coli.

Abelson murine leukemia virus↗
PubMed1982

Purification and properties of a host cell protein required for poliovirus replication in vitro.

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. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.

HeLa Cells↗
PubMed1982

In vitro copying of viral positive strand RNA by poliovirus replicase. Characterization of the reaction and its products.

Poliovirus replicase can be isolated in a form which depends on either oligo(U) or on a host cell protein for the initiation of copying of poliovirion (plus strand) RNA. The product of replicase reactions--initiated either with host factor or with oligo(U)--includes full length (35 S) RNA molecules, largely in double-stranded form, which contain the ribonuclease T1-resistant oligonucleotides of the poliovirus minus strand. For the oligo(U)-stimulated reaction, it is shown that the oligo(U) primer is covalently associated with full length product at its 5'-end. For either the host factor- or oligo(U)-dependent reactions, full length molecules appear only after 15 min of synthesis. The fraction of 35 S product is increased by raising the concentration of the limiting nucleoside triphosphate. The reaction is inhibited by as little as 100 mM salt, although it is stimulated by low (20 mM) salt concentrations. Zinc stimulates overall synthesis, but not the rate of appearance of full length molecules; the reaction is inhibited by agents which chelate zinc. 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.

Kinetics↗
PubMed1982

Somatic variants of murine immunoglobulin lambda light chains.

Studies of the murine lambda 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 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda 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 kappa chain variable region amino acid sequences, and more recently VK and VH genes, 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 mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.

Amino Acid Sequence↗
PubMed1982

Synthesis of murine leukemia virus plus strong stop DNA initiates at a unique site.

The 5'-terminal segment of plus strong stop DNA has been isolated from an endogenous virion reverse transcription reaction and characterized. The plus strong stop DNA is shown to initiate with the sequence 5'AATGAAAGA.... The 5'-terminal nucleotide is a deoxyribonucleotide. No residual protein or RNA moiety was found attached to the plus strong stop DNA. Thus, the mechanism which primes plus strand synthesis is still unclear. No evidence for multiple initiation sites or heterogeneous starts is observed. This 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.

Base Sequence↗
PubMed1982

Immunoglobulin heavy-chain expression and class switching in a murine leukaemia cell line.

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.

Animals↗
PubMed1982

Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA.

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 replicase products. Complexes of VPg-related polypeptide and newly made RNA could be immunoprecipitated by anti-VPg antibody from the host-factor-stimulated products of the replicase reaction. The complexes appeared to be covalently linked and involved 50 to 150 nucleotide chains of RNA that were RNAase-T1-resistant and could be largely poly(U).

Poliovirus↗