Search PubMed⌕ Search

Biomedical subjects

E Rothenberg

Publications and source records attributed to E Rothenberg.

12 recordsLinked to original sources
PubMed1979

Synthesis and processing of molecules bearing thymus leukemia antigen.

Thymus-leukemia (TL) antigens are expressed in murine lymphocytes under strict developmental regulation. To elucidate the molecular basis of TL expression, we have identified the molecular species that react with TL antiserum. At least three species can be resolved by metabolic radiolabeling of thymocytes and ASL1 leukemia cells, lysis, immune precipitation, and sodium dodecyl sulfate-polyacrylamide. After a brief incubation with [35S]methionine, the only radioactive molecule recognized by TL antiserum is a homogeneous species with an apparent Mr of 45,000 daltons. This molecule, 45K TL, includes high-mannose-type carbohydrate attached to a 45,000 dalton glycosidase-resistant backbone. In this form, 45K, it is never exposed on the cell surface. If pulse-labeled cells are further incubated with nonradioactive methionine before lysis, however, radioactivity disappears from the 45K TL species and appears in the slower migrating species 46K and 48K TL. Thus, 46K and 48K appear to represent products generated from the 45K TL precursor by posttranslational modification. These TL forms are displayed on the cell surface; they lack high-mannose carbohydrate but evidently include acidic complex-type carbohydrate. Normal thymocytes from Qa:Tla-negative mice lack not only the surface forms of TL but also the intracellular 45K TL form. Peripheral lymphoid cells of Qa:Tla-positive mice synthesize none of these TL species. But the TL antiserum, which contains Qa antibody, recognizes a distinct gene product in spleen and thymus of Qa-Tla-positive mice. In its pulse-labeled form, this molecule, which may represent Qa-1, has an apparent Mr of 44,000 daltons, and consists of a glycosidase-resistant polypeptide core of only 35,000 daltons linked to more high mannose carbohydrate than 45K TL.

Animals↗
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↗
PubMed1977

In vitro synthesis of infectious DNA of murine leukaemia virus.

DNA synthesised in vitro by purified virions of murine leukaemia virus is infectious. Neither RNA nor protein is required for infectivity. Transfection with reverse trancriptase product shows a single-hit dose response and results in the production of complete, infectious virus.

Cell Line↗
PubMed1977

Increased length of DNA made by virions of murine leukemia virus at limiting magnesium ion concentration.

Conditions have been developed for reverse transcription by detergent-disrupted virions of Moloney murine leukemia virus which permit synthesis of molecules that appear to be complete transcripts of the 35S RNA subunits. At limiting Mg2+ concentration, DNA is synthesized in good yield, up to a maximum size of about 2.4 X 10(6) daltons. DNA larger than 2 X 10(6) daltons, taken from alkaline sucrose gradients, has no detectable self-complementarity and was protected from digestion by S1 nuclease to an extent of 90% by annealing to 70S RNA. All size classes of DNA made in these reactions are primed with RNA, because all are initiated with a pApdAjunction. To produce such long molecules, it is necessary to keep the concentration of Mg2+ in the reaction mixture below the total concentration of deoxyribonucleoside triphosphates. Under these conditions, degradation of the RNA template is minimized. The rate of DNA synthesis is also slowed by 30 to 50%, but products longer than 5,000 nucleotides, which are not found otherwise, are completed between 3 and 6h of reaction.

Cell-Free System↗
PubMed1975

Synthesis of long, representative DNA copies of the murine RNA tumor virus genome.

Virions of Moloney murine leukemia virus can synthesize two classes of DNA molecules complementary to their 70S RNA. One class consists of molecules about 200 nucleotides long, which are of limited sequence complexity; these molecules are formed preferentially if the dNTP concentration during the reaction is low. The second class consists of very heterogeneous DNA molecules with weight-average size of about 1,000 nucleotides containing at least 70% of the viral RNA sequences in approximately equal concentration. The longest of these molecules can be 5,000 nucleotides long. This second class of DNA is formed in large amounts only in reactions containing dNTP concentrations of 0.2 mM or higher. In such reactions after 24 h of incubation, at least 35% of the input RNA is represented in DNA copies. The ability to make long, representative DNA transcripts of tumor virus RNA provides a source of excellent probes for molecular hybridization.

Base Sequence↗