Murine Leukemia Virus-infected Mouse Research Articles

Retroviral Infection and Commensal Bacteria Dependently Alter the Metabolomic Profile in a Sterile Organ.

Both viruses and bacteria produce "pathogen associated molecular patterns" that may affect microbial pathogenesis and anti-microbial responses. Additionally, bacteria produce metabolites, while viruses could change the metabolic profiles of the infected cells. Here, we used an unbiased metabolomics approach to profile metabolites in spleens and blood of murine leukemia virus-infected mice monocolonized with Lactobacillus murinus to show that viral infection significantly changes the metabolite profile of monocolonized mice. We hypothesize that these changes could contribute to viral pathogenesis or to the host response against the virus and thus open a new avenue for future investigations.

Autoantibodies Against the Exocrine Pancreas in Autoimmune Pancreatitis: Gene and Protein Expression Profiling and Immunoassays Identify Pancreatic Enzymes as a Major Target of the Inflammatory Process

Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. The objective was to identify novel markers of disease and to unravel the pathogenesis of AIP. To explore key targets of the inflammatory process, we analyzed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, western blot, and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus-infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared with those of 8 patients with non-AIP chronic pancreatitis. Expression profiling showed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry showed a near-loss of trypsin-positive acinar cells, which was also confirmed by western blotting. The serum of AIP patients contained high titers of autoantibodies against the trypsinogens PRSS1 and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals, we found similar protein patterns and a reduction in trypsinogen. These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.

Effects of LP-BM5 murine leukemia virus infection on errors and response time in a two-choice serial reaction time task in C57BL/6 mice

Human immunodeficiency virus type 1 (HIV-1) infection is often accompanied by cognitive, motor, and behavioral dysfunction. Cognitive function diminishes in indices of attention, psychomotor speed, and learning and memory. These are collectively termed acquired immunodeficiency syndrome dementia complex (ADC or neuroAIDS). Inoculation with the LP-BM5 murine leukemia virus (MuLV) causes profound immunosuppression (murine acquired immunodeficiency syndrome, or MAIDS) in C57BL/6 mice. Previous studies show that the LP-BM5 MuLV impairs learning and memory without gross motor impairment. Since learning and memory performance deficits can be related to attention deficits, we assessed the effect of LP-BM5 MuLV infection on sustained attention performance using a two-choice serial reaction time task. This task required the animals to detect a visual stimulus presented randomly on the right or the left unit and respond by a nose-poke in the illuminated hole within a 5 s period for water reward. The LP-BM5 MuLV infected group, like the control group, improved sustained attention performance until 7 weeks of virus infection in all measures including choice accuracy, response omission, and correct response time. However, during the late stage of infection, LP-BM5 MuLV infected mice showed selective sustained attention performance deficits. From 8 weeks after LP-BM5 MuLV infection, the virus infected mice started to lose their improved sustained attention performance in response omission and began to make correct responses more slowly than the control mice when the duration of stimulus light was 5 s. Moreover, at 13 and 14 weeks after LP-BM5 MuLV infection, the virus infected group made correct choices significantly less accurately than the control group when duration of stimulus light was shortest (1 s). These data show that LP-BM5 MuLV infection causes not only the previously reported learning and memory deficits but also produces sustained attention performance deficits in mice.

Immunomodulatory effect of gold sodium thiomalate on murine acquired immunodeficiency syndrome.

Induction of IL-2 production and increased expression of CD25 were observed in C57BL/10 mice after weekly treatment with gold sodium thiomalate (GST). LP-BM5 murine leukemia virus (MuLV) infected mice treated with GST survived longer, had less cervical lymph node swelling, lower spleen weight, and fewer abnormalities in the expression of the cell surface markers, CD4, CD8a and CD45R/B220 on spleen cells than those that were not treated with GST. Thus, GST treatment may be beneficial through a decrease in disease progression via IL-2 induction in MuLV infected mice. This may have application in human immunodeficiency virus-infected individuals.

LP-BM5 virus-infected mice produce activating autoantibodies to the AMPA receptor.

Autoantibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors may contribute to chronic hyperexcitability syndromes and neurodegeneration, but their origin is unclear. We examined LP-BM5 murine leukemia virus-infected mice, which manifest excitotoxic brain lesions and hypergammaglobulinemia, for the presence of AMPA-receptor Ab's. Endogenous IgG accumulated upon neurons in the neocortex and caudate/putamen of infected mice and interacted with native and recombinant AMPA-receptor subunits with the following relative abundance: GluR3 > or = GluR1 > GluR2 = GluR4, as determined by immunoprecipitation. In a radioligand assay, IgG preparations from infected mice specifically inhibited [(3)H]AMPA binding to receptors in brain homogenates, an activity that was lost after preadsorbing the IgG preparation to immobilized LP-BM5 virus. These IgGs also evoked currents when applied to hippocampal pyramidal neurons or to damaged cerebellar granule neurons. These currents could be blocked using any of several AMPA receptor antagonists. Thus, anti-AMPA-receptor Ab's can be produced as the result of a virus infection, in part through molecular mimicry. These Ab's may alter neuronal signaling and contribute to the neurodegeneration observed in these mice, actions that may be curtailed by the use of AMPA-receptor antagonists.

Phenotyping of Evi1, Evi11/Cb2, and Evi12 Transformed Leukemias Isolated from a Novel Panel of Cas-Br-M Murine Leukemia Virus-Infected Mice

Cas-Br-M murine leukemia virus (MuLV) is a slow-transforming retrovirus that potently induces leukemias in mice and therefore is well suited for retroviral insertional mutagenesis. We used Cas-Br-M MuLV in NIH/Swiss mice to establish a new panel of mainly myeloid leukemias. All tumors found in leukemic animals were classified by gross pathology, morphology, and immunophenotype, as well as the incidence of known common virus integration sites (VISs) in MuLV-induced myeloid malignancies (i.e., Evi1, Evi11/Cb2, Evi12, Fli1, and c-Myb). Interestingly, male mice were more susceptible than females to the induction of leukemia by Cas-Br-M MuLV. Seventy-four of the Cas-Br-M MuLV-inoculated mice developed a severe splenomegaly, sometimes in association with a thymoma. Although most of the immunophenotyped Cas-Br-M MuLV tumors were of myeloid origin (58%), numerous T-cell leukemias (21%) and mixed myeloid/T-cell leukemias (21%) were found. The myeloid leukemias and myeloid compartment of the mixed leukemias were further characterized by immunophenotyping with stem cell-, myeloid-, and erythroid-specific antibodies. The known Cas-Br-M MuLV common VISs (Evi1, Evi11/Cb2, and Evi12) were demonstrated in 19%, 12%, and 20% of the cases, respectively, whereas no Fli1 and c-Myb rearrangements were found. Integrations into Evi1 were restricted to myeloid leukemias, whereas those in Evi11/Cb2 and Evi12 were identified in myeloid as well as T-lymphoid leukemias. This panel of well characterized Cas-Br-M MuLV-induced hematopoietic tumors may be useful for the isolation and characterization of new proto-oncogenes involved in myeloid or T-cell leukemias.

Identification of directly infected cells in the bone marrow of neonatal moloney murine leukemia virus-infected mice by use of a moloney murine leukemia virus-based vector.

Early bone marrow infection of Moloney murine leukemia virus (M-MuLV)-infected mice was studied. Previous experiments indicated that early bone marrow infection is essential for the efficient development of T lymphoma. In order to identify the cellular pathway of infection in the bone marrow, infection of mice with a helper-free replication-defective M-MuLV-based retroviral vector was carried out. Such a vector will undergo only one round of infection, without spreading to other cells; thus, cells infected by the initially injected virus (directly infected cells) can be identified. For these experiments, the BAG vector that expresses bacterial beta-galactosidase was employed. Neonatal NIH/Swiss mice were inoculated intraperitoneally with ca. 10(6) infectious units of a BAG vector pseudotyped with M-MuLV proteins, and bone marrow cells were recovered 2 to 12 days postinfection. Single-cell suspensions were tested for infection by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) or by immunofluorescence with an anti-beta-galactosidase antibody. Two sizes of infected cells were evident: large multinucleated cells and small nondescript (presumptively hematopoietic) cells. Secondary stains for lineage-specific markers indicated that the large cells were osteoclasts. Some of the small cells expressed nonspecific esterase, which placed them in the myeloid lineage, but they lacked markers for hematopoietic progenitors (mac-1, gr-1, sca-1, and CD34). These results provide evidence for primary M-MuLV infection of osteoclasts or osteoclast progenitors in the bone marrow, and they suggest that known hematopoietic progenitors are not primary targets for infection. However, the subsequent spread of infection to hematopoietic progenitors was indicated, since bone marrow from mice infected in parallel with replication-competent wild-type M-MuLV showed detectable infection in small cells positive for mac-1 or CD34, as well as in osteoclasts.

Gliosis in the LP-BM5 murine leukemia virus-infected mouse: an animal model of retrovirus-induced dementia

Mice infected with the LP-BM5 murine leukemia virus (MuLV) mixture develop severe immunosuppression, neurotransmitter abnormalities and cognitive impairments in the absence of significant viral or macrophage invasion of the CNS. The time-course of the changes in glial activation have been characterized in an effort to understand the cellular basis of the neurobehavioral abnormalities observed in these mice. Glial activation was determined by measuring the relative changes in F4/80 protein and GFAP immunoreactivity using immunoblots. Augmented F4/80 expression preceded that of GFAP, with global elevations of 4–6-fold at 3 weeks, sustained for up to 12 weeks after inoculation. GFAP immunoreactivity increased 2-fold only in the cerebral cortex and striatum 5 weeks postinfection, declining to control levels by 12 weeks. Immunohistochemistry revealed significant increases in microglial size and staining intensity in the cortex, corpus callosum and striatum, with the development of a unique population of highly ramified, intensely stained microglia and microglial nodules in the corpus callosum and striatum. No evidence of ameboid microglia was found. Astrocyte size and degree of ramification was increased in the hippocampus, cortex, striatum and corpus callosum. Thus, microgliosis is an early event in LP-BM5 infection, preceding astrocytosis, neurotransmitter loss, and development of cognitive deficits. Activated microglia may secrete neurotoxins leading to the neurochemical alterations and cognitive deficits observed in these mice. Because gliosis and microglial nodule formation are hallmarks of HIV-1 encephalopathy, LP-BM5 MuLV-infected C57/B16 mice may afford insights into the mechanisms contributing to the early stages of this syndrome.

A multistep process of leukemogenesis in Moloney murine leukemia virus-infected mice that is modulated by retroviral pseudotyping and interference

Mixed retroviral infections frequently exhibit pseudotyping, in which the genome of one virus is packaged in a virion containing SU proteins encoded by another virus. Infection of mice by Moloney murine leukemia virus (M-MuLV), which induces lymphocytic leukemia, results in a mixed viral infection composed of the inoculated ecotropic M-MuLV and polytropic MuLVs generated by recombination of M-MuLV with endogenous retroviral sequences. In this report, we describe pseudotyping which occurred among the polytropic and ecotropic MuLVs in M-MuLV-infected mice. Infectious center assays of polytropic MuLVs released from splenocytes or thymocytes of infected mice revealed that polytropic MuLVs were extensively pseudotyped within ecotropic virions. Late in the preleukemic stage, a dramatic change in the extent of pseudotyping occurred in thymuses. Starting at about 5 weeks, there was an abrupt increase in the number of thymocytes that released nonpseudotyped polytropic viruses. A parallel increase in thymocytes that released ecotropic M-MuLV packaged within polytropic virions was also observed. Analyses of the clonality of preleukemic thymuses and thymomas suggested that the change in pseudotyping characteristics was not the result of the emergence of tumor cells. Examination of mice infected with M-MuLV, Friend erythroleukemia virus, and a Friend erythroleukemia virus-M-MuLV chimeric virus suggested that the appearance of polytropic virions late in the preleukemic stage correlated with the induction of lymphocytic leukemia. We discuss different ways in which pseudotypic mixing may facilitate leukemogenesis, including a model in which the kinetics of thymic infection, modulated by pseudotyping and viral interference, facilitates a stepwise mechanism of leukemogenesis.

72 Cation influence on survival and lymphoma in murine immuno-deficiency deficiency disease (MAIDS): Effects of lithium and magnesium [formula omitted]: VS Gallicchio, ML Cibull, NK Hughes, KF Tse, and H Gaines. Hematology/Oncology Division, Departments of Medicine, Clinical Sciences and Pathology, University of Kentucky Medical Center and Department of Veterans Affairs, Lexington, KY, USA

Human immunodeficiency virus type 1 (HIV-1) infection is often accompanied by cognitive, motor, and behavioral dysfunction. Cognitive function diminishes in indices of attention, psychomotor speed, and learning and memory. These are collectively termed acquired immunodeficiency syndrome dementia complex (ADC or neuroAIDS). Inoculation with the LP-BM5 murine leukemia virus (MuLV) causes profound immunosuppression (murine acquired immunodeficiency syndrome, or MAIDS) in C57BL/6 mice. Previous studies show that the LP-BM5 MuLV impairs learning and memory without gross motor impairment. Since learning and memory performance deficits can be related to attention deficits, we assessed the effect of LP-BM5 MuLV infection on sustained attention performance using a two-choice serial reaction time task. This task required the animals to detect a visual stimulus presented randomly on the right or the left unit and respond by a nose-poke in the illuminated hole within a 5 s period for water reward. The LP-BM5 MuLV infected group, like the control group, improved sustained attention performance until 7 weeks of virus infection in all measures including choice accuracy, response omission, and correct response time. However, during the late stage of infection, LP-BM5 MuLV infected mice showed selective sustained attention performance deficits. From 8 weeks after LP-BM5 MuLV infection, the virus infected mice started to lose their improved sustained attention performance in response omission and began to make correct responses more slowly than the control mice when the duration of stimulus light was 5 s. Moreover, at 13 and 14 weeks after LP-BM5 MuLV infection, the virus infected group made correct choices significantly less accurately than the control group when duration of stimulus light was shortest (1 s). These data show that LP-BM5 MuLV infection causes not only the previously reported learning and memory deficits but also produces sustained attention performance deficits in mice.

Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.

Differentiation of Abelson murine leukemia virus-infected promonocytic leukemia cells.

Virus-producing, tumorigenic, promonocytic leukemia cell lines were derived from Abelson murine leukemia virus-infected mice. This study shows that, of these 25 cloned lines, 22 were capable of extensive differentiation. It also shows that granulocyte-macrophage colony-stimulating activity increased the proportion of differentiating cells in 17/22 lines. Cells from agar colonies with a diffuse colony morphology had an increased expression of mature macrophage phenotypic characteristics, and a reduced proliferative capacity in vitro, compared to cells from agar colonies with a compact colony morphology. Cells from diffuse colonies also produced less Abelson virus, and were less tumorigenic in vivo than cells from compact colonies. Together, these results suggest some Abelson virus-producing leukemic cells are not blocked in their capacity to differentiate and are capable of reversing the transformed phenotype.

Design of a retrovirus-derived vector for expression and transduction of exogenous genes in mammalian cells.

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5' and 3' LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt(+) colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5' LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3' LTR gave an 80% reduction in activity. Thus, both 5' and 3' LTR sequences are essential for optimal gpt expression, although the 5' LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt(+) phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5' and 3' LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt(+) colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5' LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3' LTR gave an 80% reduction in activity. Thus, both 5' and 3' LTR sequences are essential for optimal gpt expression, although the 5' LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt(+) phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

Retrovirus gene expression during the cell cycle. I. Virus production, synthesis, and expression of viral proteins in Rauscher murine leukemia virus-infected mouse cells.

Synchronized mouse cells (JLS-V9) chronically infected with Rauscher murine leukemia virus were used to study virus production, the synthesis of gag and env precursor proteins, and the expression of env protein on the cell surface during the cell cycle. The amount of virus released into the medium by synchronized cells during a 30-min interval was determined by using the XC plaque assay and by measuring reverse transcriptase activity. The results show that virus production occurs during mitosis. Labeling of the cell surface of synchronized cells with 125I or with fluorescein-conjugated antiserum shows that the amount of gp 70env on the cell surface parallels cellular growth. Therefore, the cell cycle-dependent release of virus is not accompanied by similar variations in the amount of viral envelope protein on the cell surface. Immunoprecipitation of cells labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was used to measure viral protein synthesis during the cell cycle. The rate of synthesis of gag precursor proteins show three maximums corresponding to the G1, middle S, and late S to G2 phases of the cell cycle. The rate of synthesis of env precursor proteins does not change, suggesting that in these cells the synthesis of these two gene products is controlled separately.

The quantitation of cell surface antigens by antibody-complement mediated cytotoxicity: application to murine leukemia virus-infected mouse cells

51Cr-antibody-complement mediated cytotoxicity (ACC) can be used to quantify nanogram amounts of cell surface antigens. When pure antigen is available for calibration, inhibition of ACC by known amounts of antigen and by whole cells yields an estimate of the number of antigenic equivalents per cell. ACC is more suitable for this purpose than radioimmunoprecipitation assay. By ACC the number of antigenic equivalents of the oncornaviral proteins gp70 and p30 has been determined on the surface of various cells infected with Rauscher and Gross murine leukemia viruses.

Hematopoietic Stem Cells In Friend Murine Leukemia Virus-Infected Mice Undergoing Chemotherapy: Remission and Relapse of Erythropoietin-Independent Erythropoiesis Induced by Hydroxyurea2

Hydroxyurea (HU), given ip four times, each time at 500 mg/kg in 6-hour intervals, was used to treat DBA/2 mice with Friend murine leukemia virus-induced polycythemia (F-MuLV-P). In these mice a new cell type, found after virus infection, gave rise to erythropoietic colonies in vitro without addition of erythropoietin (Ep) and completely replaced Ep-dependent normal erythropoietic colonies in vitro. The colony-forming units in the spleen (CFUs), the colony-forming units in culture (CFUc), and the erythropoietic colony-forming units (CFUE) were studied. Two days after treatment, CFUs were reduced to about 20% in controls and F-MuLV-P-infected animals, and CFUc were reduced to 6-11% in controls and F-MuLV-P-infected animals. CFUE were not detectable. At day 4 after the first HU dose, when CFUs has regenerated to about normal levels, a sharp rise in Ep-dependent CFUE was seen in the marrow; this rise was not present before HU treatment. The subsequent fall at day 7 coincided with a regeneration of CFUE in the spleen, but in the spleen these CFUE were all Ep-independent. Possibly, the normal Ep-dependent CFUE during regeneration in the marrow might have derived from previously resting CFUs that were not killed by HU. The subsequent conversion to Ep independency could have been due to reinfection by F-MuLV-P persisting in the animal.

Electron microscopic evidence for splicing of Moloney murine leukemia virus RNAs.

Poly (A) containing RNA extracted from Moloney murine leukemia virus infected mouse cells was hybridized with long single-stranded complementary DNA, prepared in detergent disrupted virions. Visualization of the hybrids in the electron microscope revealed among the structures, circles and circles with tails. Measurements performed on the circular molecules revealed two major species with circumferences corresponding to 3 and 8.2 kilobases. The latter structures had identical size to circles obtained after annealing of cDNA with the viral genome, 35S RNA. Circularization of a small viral RNA (3 kb) from infected cells in the RNA-cDNA hybrids is a direct evidence that like the 35S RNA it shares similar nucleotide sequences at both the 5' and 3' ends. The presence of 5' end sequences common to the two RNA species indicates the existence of a spliced viral RNA. Furthermore, based on the circularization of viral RNA in the hybrids, we suggest a new way to quantitate and determine the lengths of spliced RNA in retrovirus infected cells.

Methylation pattern of genomic RNA from Moloney murine leukemia virus.

32P- and methyl-3H-labeled 70S Moloney murine leukemia virus RNA was purified from virions produced in Moloney murine leukemia virus-infected mouse embryo cells. Primer-free RNA subunits obtained by heat treatment and zonal centrifugation were digested with RNase T2, and methylated oligonucleotides were chromatographed on DEAE-Sephadex in 7 M urea. Approximately one molecule of RNase T2-stable oligonucleotide (-5 charge) was isolated per subunit. Structural analysis indicated that the sequence of the oligonucleotide is m7GpppGmpCp. Analysis of the mononucleotide fraction isolated by DEAE-Sephadex chromatography of the RNase T2 digest identified 15 to 23 internal N6-methyladenylic acid molecules per subunit.

In vitro uptake of tritium from uridine-5-H3 into RNA (and DNA) in normal and murine leukemia virus infected mouse embryo tissue.

The multiplication and release into the medium of a murine leukemic virus (GC) occurs in mouse embryo tissue cultures. Autoradiographic analysis of such infected and uninfected cultures after 24-h labeling periods with uridine-5-H3 demonstrated extensive nuclear and cytoplasmic activity. Such activity was always more extensive in infected cultures, indicating that they were more metabolically active than uninfected cultures. The cytoplasmic activity was apparently in single-stranded RNA, since it was completely removed with pancreatic RNase. A portion of the nuclear label which was RNase-resistant was in DNA, since RNase followed by DNase, or the reverse, removed all label.Autoradiographic analysis of cultures given pulse labels of uridine-5-H3 from 5 to 60 min revealed only nuclear label, always more extensive in infected cultures. This indicates that the synthesis of "rapidly labeled" RNA is enhanced by viral infection; this occurs even within 2 h after infection. Nuclear activity observed after 5- and 10-min pulses was removed by buffers containing RNase or DNase. Possibly this label was in diffusible low molecular weight species of RNA. After 30-min pulses, activity was exclusively associated with pancreatic RNase-sensitive single-stranded RNA. After 60-min labeling, tritium from uridine-5-H3 was taken up into nuclear DNA in addition to RNA.