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D Baltimore

Publications and source records attributed to D Baltimore.

At least 37 records · Page 2Linked to original sources
PubMed1979

Synthesis of a 600-nucleotide-long plus-strand DNA by virions of Moloney murine leukemia virus.

A discrete, 600-nucleotide-long plus-strand DNA has been identified among the products of reverse transcription by virions of Moloney murine leukemia virus. Its polarity was shown by hybridization to minus-strand DNA. It appears to be copied from the right end of minus-strand DNA because (i) its restriction endonuclease cleavage pattern corresponds to the redundant 600-base segment found at either end of the ultimate double-stranded reverse transcription products, (ii) its synthesis is actinomycin D sensitive, and (iii) its synthesis begins during the first hour of a reverse transcription reaction when only the right-hand end of minus-strand DNA is available as template. We therefore call this DNA plus-strong-stop DNA by analogy with the minus-strong-stop DNA copied from the left end of the viral RNA. Both strong-stop DNAs are made early during in vitro reactions and decline in concentration later, consistent with postulated roles as initiators of long minus- and plus-strand DNA. Unlike minus-strong-stop DNA, plus-strong-stop DNA remains as a double-stranded nucleic acid after its synthesis, as shown by S1 nuclease resistance. A primer to initiate plus-strong-stop DNA synthesis has not been identified; the product found thus far has no detectable RNA attached to it.

Chromosome Mapping↗
PubMed1979

Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide.

A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.

Centrifugation, Density Gradient↗
PubMed1979

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.

A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.

Animals↗
PubMed1979

Virus-like 30S RNA in mouse cells.

Uninfected JLS-V9 mouse cells are known to express high levels of viral sequences that hybridize to complementary DNA made by the BrdU-induced virus of JLS-V9 cells. The genome in the BrdU-induced virus has been found to consist mainly of an RNA species that migrates as 30S RNA material during electrophoresis through agarose gels. This virus-like 30S RNA, designated VL30 RNA, apparently represents a new class of endogenous defective retroviruses that are not generally evident because of their defectiveness and lack of biological function. Fingerprint analysis and hybridization studies show that VL30 RNA does not have homology with the standard nondefective murine leukemia viruses. Upon superinfection with a nondefective murine leukemia virus, or upon induction of endogenous virus with BrdU, VL30 RNA is rescued into virions by phenotypic mixing. When VL30 RNA is rescued by BrdU induction, the VL30 RNA is mainly organized as a 50S complex, but when VL30 is rescued by superinfection, VL30 is also found in 70S RNA. Rescued VL30 RNA sequences can be reverse transcribed by the virion-associated DNA polymerase in an endogenous reaction. Many mouse cells express the sequences, whereas heterologous cells such as rat or rabbit cells do not contain them. By using hybridization of a complementary DNA probe to cellular RNA immobilized on paper, no subgenomic RNA related to the VL30 RNA could be found in cells expressing the VL30 sequences. From 20 to 50 copies of these sequences were found to be contained in the mouse genome. VL30 RNA is probably present in most stocks of leukemia and sarcoma viruses made in mouse cells.

Animals↗
PubMed1979

Virus production by Abelson murine leukemia virus-transformed lymphoid cells.

Cell lines obtained by in vitro transformation of bone marrow with Abelson murine leukemia virus (A-MuLV) can be divided into three classes: producers, releasing reverse transcriptase-containing particles and infectious virus; nonproducers, releasing no viral particles; and defective producers, the most common phenotype, releasing particulate reverse transcriptase in the absence of infectious virus. When such cell lines were analyzed 1 to 2 weeks after their isolation, however, all produced infectious virus. Because these cell lines were carried in culture, many ceased to release infectious virus but produced defective virions. One defective producer, SWR4, has been extensively studied. The particles it produces have the same density as that of virions of Moloney murine leukemia virus (M-MuLV). The particles contain no 35 to 70S RNA, as determined by analysis of [3H]uridine-labeled particles, and exhibit no endogenous reverse transcriptase activity. Although the reverse transcriptase enzyme is of normal size, the major structural protein of the defective virions has a molecular weight of 28,000 (p28), in contrast to the p30 of M-MuLV, and no viral glycoprotein was evident. The defective particles do not appear to arise either from the helper virus or from Abelson virus. An alteration of the protein of the helper virus is an unlikely source of p28 because particles produced by lymphoid cells transformed with another strain of M-MuLV as helper (M-MuLV-TB) contained p28 with an unaltered cleavage pattern, although M-MuLV-TB p30 differs from M-MuLV p30. The A-MuLV genome lacks the capacity to code for the reverse transcriptase virions. Clones of fibroblasts infected with A-MuLV only occasionally produce defective particles. The defective particles therefore probably arose from an endogenous virus that is preferentially expressed in the class of lymphoid cells transformed by A-MuLV. This interpretation implies that the majority of A-MuLV-transformed lymphoid cells completely lose expression of the helper virus genome.

Animals↗
PubMed1979

Preparation of syngeneic tumor regressor serum reactive with the unique determinants of the Abelson murine leukemia virus-encoded P120 protein at the cell surface.

Antisera reactive with the Abelson murine leukemia virus (A-MuLV)-specified P120 (anti-AbT sera) were produced in C57L/J mice. Of many strains tested, only C57L/J reproducibly rejected syngenic A-MuLV-induced tumor cells; after multiple immunizations their sera would immunoprecipitate both P120 and Moloney-MuLV (M-MuLV) proteins. Using labeled A-MuLV-induced nonproducer cells, only P120 could be detected by anti-AbT sera, suggesting that it may be the only A-MuLV-specified protein. Reactivity of anti-AbT sera with P120 was not blocked by M-MuLV virion proteins, implying that the sera recognize a portion of P120 that is not homologous to any M-MuLV product. Anti-AbT sera stained the surface of live, A-MuLV-transformed nonproducer cells in a two-stage immunofluorescence assay, and such staining was not blocked by M-MuLV protein. Also, intact A-MuLV-transformed cells absorbed much of the reactivity of certain anti-AbT sera for P120. Thus a portion of P120 appears to be exposed on the surface of transformed cells. P120 lacks detectable carbohydrate, is not affected by endoglycosidase H, and cannot be labeled by lactoperoxidase-catalyzed iodination. Thus P120 is an unusual surface protein.

Abelson murine leukemia virus↗
PubMed1978

Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine.

Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labeled with 32P) in 0.5% sodium dodecyl sulfate or 6 M guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5' end of poliovirion RNA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5'-terminal uridine. After acid hydrolysis of [3H]tyrosine-labeled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labeled with either 32P or [3H] tyrosine yields tyrosine-phosphate. There appears to be only 1 tyrosine residue per VPg molecule.

Binding Sites↗
PubMed1978

Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5.

The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.

Animals↗
PubMed1978

Terminal transferase as a predictor of initial responsiveness to vincristine and prednisone in blastic chronic myelogenous leukemia: a co-operative study.

We undertook a prospective trial to evaluate terminal deoxynucleotidyl transferase activity as a predictor of responsiveness to vincristine and prednisone in 22 Philadelphia-chromosome-positive patients with blastic chronic myelogenous leukemia. Thirteen patients were transferase positive, and nine negative. None of the nine negative patients responded, whereas eight of the 13 positive (P = 0.004) responded with complete clearing of peripheral blood blast cells and a return of normal marrow cellularity with less than 5 per cent blast cells. Among transferase-positive patients under 50 years of age the response rate was 78 per cent. Blast-cell morphology (i.e., lymphoblastic versus myeloblastic) had no significant correlation with either responsiveness or terminal transferase activity. The results of this study suggest that responsiveness to vincristine and prednisone in blastic chronic myelogenous leukemia is confined to patients whose leukemic cells are transferase positive.

Adolescent↗
PubMed1978

The effect of helper virus on Abelson virus-induced transformation of lymphoid cells.

Abelson murine leukemia virus (A-MuLV)-transformed fibroblast nonproducer cells were used to prepare A-MuLV stocks containing a number of different helper viruses. The oncogenicity of the A-MuLV stocks was tested by animal inoculation and their ability to transform normal mouse bone marrow cells was measured in vitro. All of the A-MuLV stocks transformed fibroblast cells efficiently. However, only A-MuLV stocks prepared with helper viruses that are highly oncogenic were efficient in vivo and in vitro in hematopoietic cell transformation. In addition, inefficient helpers did not establish a stable infection in lymphoid nonproducer cells. Thus, helper virus has a more central role in lymphoid cell transformation than in fibroblast cell transformation.

Animals↗
PubMed1978

Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma.

Neoplastic cells from 253 patients with leukemia and 46 patients with malignant lymphoma were studied for the presence of terminal deoxynucleotidyl transferase (TdT) by biochemical and fluorescent antibody technics. TdT was detected in circulating blast cells from 73 of 77 patients with acute lymphoblastic leukemia, 24 of 72 patients with chronic myelogenous leukemia examined during the blastic phase of the disorder and in cell suspensions of lymph nodes from nine of nine patients with diffuse lymphoblastic lymphoma. Blast cells from six of 10 patients with acute undifferentiated leukemia were TdT positive, but the enzyme was found in only two of 55 patients with acute myeloblastic leukemia. TdT was not detected in other lymphocytic or granulocytic leukemias or in other types of malignant lymphomas. The fluorescent antibody assay for TdT permits rapid and specific identification of the enzyme in single cells. The TdT assay is clinically useful in confirming the diagnosis of acute lymphoblastic leukemia, evaluating patients with blastic chronic myelogenous leukemia, and distinguishing patients with lymphoblastic lymphoma, whose natural history includes rapid extranodal dissemination, from patients with other poorly differentiated malignant lymphomas.

Adolescent↗
PubMed1978

An enzymatic activity in uninfected cells that cleaves the linkage between poliovirion RNA and the 5' terminal protein.

The 5' terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of heLa cells and is activated by Mg++. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5' terminal protein. The role of this enzyme in the uninfected cell is not known.

Base Sequence↗
PubMed1978

High frequency of aberrant expression of Moloney murine leukemia virus in clonal infections.

Clones of cells were isolated from single virus-single cell infections of NIH/3T3 cells with Moloney murine leukemia virus. Approximately one third of such clones aberrantly expressed viral gene functions. One clone produced virus with altered plaque morphology, while others failed to produce particles able to make plaques on XC cells. In addition, clones that made particles lacking reverse transcriptase were found, and these did not synthesize the reverse transcriptase precursor Pr180 gag-pol. One clone (M23) lacked any detectable glycoprotein or reverse transcriptase. Despite these defects, each clone released particles of type C morphology, suggesting that gag gene function alone may be sufficient for particle production. All the particles contained viral RNA of 60-70S that was composed of the normal 35S size subunits except for M23, which had a deletion in the viral genome of approximately 1000-1500 nucleotides. A variety of defective clones were also isolated following infection of rat cells with Moloney virus. It is apparent that the murine leukemia virus genome is ofter mutated by spontaneous processes generating a wide range of phenotypes.

Cell Line↗
PubMed1978

Analysis of a 5' leader sequence on murine leukemia virus 21S RNA: heteroduplex mapping with long reverse transcriptase products.

The majority of the mRNA that specifies retrovirus glycoproteins is known to be derived from the 3' half of the genome. To examine whether the glycoprotein mRNA of murine leukemia viruses (MuLVs) might consist of portions derived from both the 5' and 3' ends of the viral genome, we performed hybridization with a 5'-specific probe and heteroduplex analysis with long reverse transcribed DNA. A 5' probe was made by purifying a discrete 50 nucleotide-long reverse transcript attached to its tRNA primer. This probe was found to hybridize to RNA of the size of glycoprotein mRNA--21S, poly(A)-containing RNA--indicating that the mRNA could have a 5' leader sequence. The 5'-specific sequences were studied by electron microscopic examination of hybrids between 21S RNA and the two longest discrete cDNA species synthesized in the endogenous reverse transcriptase reaction. One of these species, 8.8 kb long, is only made in the absence of actinomycin D, but it does not contain any self-complementary sequences, and therefore appears to be a complete transcript of the viral genome. The shorter of the two species, 8.2 kb long, is synthesized whether or not actinomycin D is present; it must terminate 500--600 nucleotides internal to the 5' end of the template RNA. The structures observed in heteroduplexes of 21S RNA and these DNAs indicated the presence of a leader sequence approximately 500 nucleotides long at the 5' end of the 21S RNA. Sequences comprising this leader segment in the 21S RNA mapped at the 5' end of the genome RNA; the rest of the 21S RNA consisted of sequences from the 3' portion of the genome. Analysis of heteroduplexes with 8.2 kb DNA suggested that actinomycin D could block the reverse transcription of most of the sequence in the genome RNA that appears as a leader in the 21S RNA.

Base Sequence↗
PubMed1978

Heteroduplex analysis of the nonhomology region between Moloney MuLV and the dual host range derivative HIX virus.

The dual host range virus HIX has been previously characterized as an envelope gene recombinant between Moloney murine leukemia virus (Mo-MuLV) and an unidentified xenotropic murine leukemia virus. Using long reverse transcripts of Mo-MuLV, a region of nonhomology has been mapped by electron microscopic analysis of heteroduplexes formed with HIX 35S virion RNA. In this nonhomology region, the Mo-MuLV cDNA strand measured approximately 900 nucleotides, mapping between 1.6 and 2.5 kilobases from the 3' end. In a previous study, hybridization of Mo-MuLV 21S RNA with Mo-MuLV cDNA resulted in the formation of different heteroduplex structures diagnostic of a noncontiguously coded leader sequence at the 5' end of the 21S RNA. Following hybridization of poly(A)+ HIX 21S RAN with 8.2 kb Mo-MuLV cDNA, analogous heteroduplex structures were observed exhibiting the Mo-MuLV:HIX substitution loop in the DNA:RNA segment of the molecules. This analysis permitted more precise mapping of the nonhomology region with respect to the splice point in the 21S presumptive glycoprotein mRNA. The mapping of this nonhomology region in HIX virus provides an internal visual marker for the 3' end of the genome which may prove useful in future analyses of other deletion or substitution derivatives of Mo-MuLV.

Base Sequence↗