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The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with “tracer” hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK+, EGFP+ stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with 18F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([18F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK+ tumor stroma with an average of 0.36 ± 0.24% ID/g [18F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF+ and CD31+ endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK+, GFP+ endothelial cells had limited proliferative capacity and a short life span of

hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.

In this study, the effectiveness of a 188Re labeled sulfur colloid with two particle size ranges was used to evaluate the effectiveness of this agent on melanoma tumors in mice in terms of animal lifespan.Two separate group of animals were used for investigating biodistribution and survival time. A total of 188 B16F10-melanoma-bearing BDF(1) mice were injected intraperitoneally with 3.7 MBq (0.1mCi)/2mL of radiolabeled sulfur colloid ten days after intraperitoneal inoculation of 5x10(5) B16F10 melanoma cells/2ml. For group 1, 30 mice were sacrificed at 1, 4, 24, 48 and 72 hours for biodistribution studies. In group 2, 158 mice were divided into 9 groups (n=16 approximately 18/groups)each receiving respectively tumor alone, tumor with normal saline, cold colloid or hot colloid with 16, 23, 31, 46, 62, or 124 MBq activity. Each of these colloid groups was further divided into two groups, one receiving smaller particle sizes (3 microm:80.4 +/-7.2%, colloid 1) and the other receiving larger particle sizes (3 microm:12.3+/-1.0%, colloid 2). The animals were checked daily until death and their survival recorded.Colloid 2 showed higher accumulation in almost all tissues, the highest accumulation organ was tumor ( approximately 40%), then spleen ( approximately 20%), stomach ( approximately 15%), diaphragm ( approximately 3%), and liver ( approximately 2%). There was a significant increase in survival time with increasing amount of the larger-particle-size colloid. Administered levels of 16-31 MBq/mouse were most efficacious and with higher amounts the survival times decreased significantly below that of the controls. There was a significant difference in the dose-response curves for the two preparations. Protection factors (1/Relative-risk) of nearly 5 were achieved using the larger colloid size, and nearly 30 using the smaller colloid size. An amount of 16-31 MBq of the colloid 2 was the optimal activity in these studies. On the one hand, the survival data agreed well with the biodistribution data, where higher accumulation was found in tumor with colloid 2.Rhenium-188 offers on-site availability, medium half-life, higher beta-particle energy of 2.12 MeV for therapy and emission of 155keV gamma photon suitable for imaging. The present study demonstrated that 188Re-sulfur colloid is an effective agent in controlling tumor cells in the abdominal cavity in animals.

E6/E7 genes of human papilloma virus type 16 were used to immortalize a primary culture of marginal cells (MC) from gerbils. One of the cloned lines was selected which demonstrated preservation of the main characteristics of the MC, both morphologically and physiologically. Electron microscopic examination showed well-developed junctional complexes and apical microvilli which suggested its epithelial origin. Polymerase chain reaction (PCR) demonstrated the incorporation of E6/E7 genes with the genome. Reverse transcription PCR revealed the existence of mRNA of the IsK channel, a unique marker of MC among the inner ear cells, in this clone. Flow cytometric analysis of this cell line's DNA content was diploid. Numerous large domes formed after confluence of the cell monolayer. Electrophysiologic studies displayed evidence of apical K+ and Na+ channels which were blocked by Ba2+ (2 mM) and amiloride (10(-5) M), respectively. Existence of basolateral Na,K-ATPase and Na+/Cl-/K+ cotransporter was shown by blockage by ouabain (10(-3) M) and bumetanide (50 microM), individually. Injection of the cell line to nude mice failed to induce growth of tumors. This cell line was serum-, density- and anchorage-dependent when cultured in plastic dishes. In conclusion, this cell line shows characteristics of well-differentiated MC maintaining the major ionic transport processes, and provides us a good model to study the possible mechanisms and regulating factors of endolymph production.

Mechanism of cell killing by transfer of Herpes simplex virus type-1 thymidine kinase ( HSVtk ) and subsequent ganciclovir (GCV) treatment was examined in B16F10 murine melanoma model. While parental B16F10 melanoma cells were resistant to GCV at 100 μM or higher, HSVtk -transduced B16F10 melanoma cell clones became susceptible to GCV with IC 50 of 0.1 to 0.3 μM. By means of various parameters including characteristic morphological changes, in situ DNA end-labeling, DNA ladder pattern, flow cytometric detection of sub-G1 DNA content, and annexin V binding of inverted cell surface phosphatidylserine, apoptosis was shown to be associated with the cell killing of ganciclovir on HSVtk -transduced melanoma B16F10 cells. Kinetic analysis showed that the signs of apoptosis were observed not until 60 h of continued GCV treatment and preceded first by a rise in p53 protein level in 12 h and then by S-phase/G2-phase cell cycle arrest associated with corresponding increases in the level of cyclin B1 protein but no apparent change in protein level of Bax or Cdc2. These results suggest that apoptosis occurred as a result of ganciclovir-induced cell cycle arrests rather than direct chemical effect on HSVtk -transduced B16F10 melanoma cells.

Interleukin-3 (Il-3) is a glycoprotein produced by a CD4+CD8- subpopulation of T-lymphocytes. Il-3 has been associated with the proliferation of bone marrow stem cells and their differentiation to granulocytes, macrophages, basophil/mast cells, megakaryocytes, erythroid cells, and neutrophils. The pBOR-Il-3 transgenic mice were developed by pronuclear microinjection to study how chemical insults modulate transcription of the Il-3 gene driven by a long-terminal repeat (LTR) of an endogenous retrovirus and to determine the biological consequences of interleukin-3 expression. We injected 5-azacytidine, a demethylating agent, to increase the LTR-driven expression of Il-3. Upon 5-azacytidine treatment, both the pBOR-Il-3 and the FVB/N nontransgenic controls developed thymic lymphomas. The pBOR-Il-3 mice developed thymic lymphomas at a higher frequency than the FVB/N mice. The thymic lymphoma cells were of a T-cell origin, as determined by T-cell receptor gene rearrangement analysis, and, in most cases, were of monoclonal origin. According to flow cytometric analysis of CD3, CD4, and CD8 cell surface markers, the thymic lymphoma cells did not lose their ability to differentiate, but the differentiation process was aberrant. Flow cytometric analyses also revealed that in pBOR-Il-3 mice the thymic lymphomas are mostly of a CD8+CD4- origin, whereas in the FVB/N group, the predominant type of thymic lymphoma is of a CD4+CD8- origin.

As one of the most intensively studied probes for imaging of the cellular proliferation, [ 18 F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[ 123 I]Iodo arabinosyl uridine ([ 123 I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/μmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-( E )-iodovinyl arabinosyl uridine along with 2′-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [ 123 I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [ 18 F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [ 18 F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1- tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [ 18 F]FLT and HSV1-TK provides a synergistic imaging potency.

Cyclooxygenase-2 (COX-2) expression has been shown to correlate with the invasiveness of colon cancer cells. To further investigate this positive correlation and its possible therapeutic implications, a selective COX-2 inhibitor, etodolac, was tested on three variants of HT-29 colon cancer cell lines, HT-29/Inv1, HT-29/Inv2 and HT-29/Inv3, with graded increases of in vitro Matrigel invasive potential and COX-2 expression levels. HT-29 variants with higher invasive potential were found to be more sensitive to etodolac by in vitro growth inhibition assays, the estimated LD(50) being 0.5 mM for highly invasive HT-29/Inv2 and HT-29/Inv3 cells, 0.6 mM for slightly less invasive HT-29/Inv1, and 1.8 mM for the parental HT-29. Treatment of the highly invasive HT-29/Inv2 and Inv3 variants with as little as 0.1 mM etodolac in the growth medium produced signs of apoptosis, as detected by DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling) assay. In vivo experiments in SCID mice showed that etolodac inhibited the growth of subcutaneous tumors induced by HT-29/Inv3 cells significantly more than those by the parental HT-29 cells. These results suggest that COX-2 inhibitors have a potential role in prevention of tumor invasion in colon cancer patients.

Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and caspase-3, (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the FasL expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.

RNA interference (RNAi) is a gene silencing process within living cells, which is controlled by the RNA-induced silencing complex with a sequence-specific manner. In flies and mice, the pseudogene transcripts can be processed into short interfering RNAs (siRNAs) that regulate protein-coding genes through the RNAi pathway. Following these findings, we construct an innovative and comprehensive database to elucidate siRNA-mediated mechanism in human transcribed pseudogenes (TPGs). To investigate TPG producing siRNAs that regulate protein-coding genes, we mapped the TPGs to small RNAs (sRNAs) that were supported by publicly deep sequencing data from various sRNA libraries and constructed the TPG-derived siRNA-target interactions. In addition, we also presented that TPGs can act as a target for miRNAs that actually regulate the parental gene. To enable the systematic compilation and updating of these results and additional information, we have developed a database, pseudoMap, capturing various types of information, including sequence data, TPG and cognate annotation, deep sequencing data, RNA-folding structure, gene expression profiles, miRNA annotation and target prediction. As our knowledge, pseudoMap is the first database to demonstrate two mechanisms of human TPGs: encoding siRNAs and decoying miRNAs that target the parental gene. pseudoMap is freely accessible at http://pseudomap.mbc.nctu.edu.tw/. Database URL: http://pseudomap.mbc.nctu.edu.tw/

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus—related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCI4 treatment, decreased protection was seen at potential regulatory cis-elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5′ of the consensus enhancer sequences) and the 3′ terminal portion of the polytropic class-specific enhancer-like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3–6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis-element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCI4-induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF-2, HNF-1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCI4 exposure are discussed.

Replication of Gross strain N-tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC4) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast-derived differentiated line (PCD1) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC4 and PCD1 cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (from III viral DNA) appeared at 4 hr, reached a maximal level at 8-9 hr, and declined rapidly thereafter, while two closed-circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8-24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD1 cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC4 cells. The Hirt supernatant DNAs prepared from PCD1 and PCC4 cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection, although one positive result with PCD1 cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentiated embryonal carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.

Fv-1 gene-specific resistance to ecotropic murine retroviruses can be transferred to permissive cells with RNA prepared from restrictive cells. The active RNA has a sedimentation coefficient of 20-22S at low ionic strength and multiple regions of activity (5-6S, 10-12S, 24-27S) at high ionic strength. The maximum level of resistance transfer cannot be enhanced with noninhibitory carrier RNA or facilitators of RNA uptake, and the maximum inhibition (40-60%) is independent of the MOI. The low efficiency of resistance transfer is, therefore, probably the result of a fraction of cells resistant to RNA uptake.

RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1n allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission.

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).

The decline in phytohemagglutinin (PHA) responsiveness of spleen cells from aged mice was studied in an effort to determine the nature of the depressed PHA reactivity. The reduced response of DNA synthesis (thymidine incorporation) to PHA stimulation could not be attributed to a decreased viability of spleen lymphocytes from old mice in in vitro culture conditions, to different PHA dose requirements, or to longer periods of culture time for old mouse spleen cells to reach maximal activity. Culture of cells for 24–48 h prior to addition of PHA gave patterns of thymidine incorporation similar to those of freshly prepared spleen cells. The elimination of red blood cells by a brief hypotonic shock significantly increased thymidine incorporation in spleen lymphocytes from both young and old mice.

We molecularly cloned and analyzed an N-tropic ecotropic retrovirus induced with iododeoxyuridine from RFM/Un mouse cell cultures. Based on the restriction map, the RFM/Un virus appears to be indistinguishable from other induced N-tropic retroviruses. A nucleotide sequence analysis of the long terminal repeat of an infectious clone revealed structural features characteristic of murine type C retrovirus long terminal repeats. The U3 region of the RFM/Un virus long terminal repeat, however, contained no short sequence duplication or insertion found in other murine leukemia virus isolates.

Formation of viral closed circular supercoiled DNA duplexes and production of progeny virus were both inhibited in cultured mouse cells treated with cycloheximide in the first 4 h of type C retrovirus infection. With different doses of cycloheximide to cause different degrees of inhibition, the number of viral supercoiled DNA duplexes detected in the cells at 11 h showed an apparent correlation with the amount of progeny virus produced in the 12- to 22-h period of infection. A slight accumulation of the full-genome linear duplex and an open circular duplex of viral DNA intermediate was observed in the cycloheximide-treated cells. Cycloheximide given to the cells during the time of conversion of viral DNA from linear to supercoiled duplex forms (6 to 11 h after virus inoculation) did not inhibit the conversion. These kinetic data suggest that a cycloheximide-sensitive metabolic process, probably early viral protein synthesis, is required for retrovirus replication and supercoiled viral DNA formation in the cell.

Resume Le RNA genomique 35S des retrovirus de type C est present au niveau des polysomes 4 heures apres infection dans des lignees cellulaires non differenciees et differenciees derivees du teratocarcinome de la souris. Une proteine specifique virale de 65.000 daltons est alors produite dans les cellules infectees. Ces resultats montrent que le genome viral fonctionne comme RNA messager pour la synthese du precurseur des proteines gag Pr65 a une etape precoce du cycle infectieux des retrovirus ecotropiques murins.

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.

Histidyl transfer ribonucleic acid (tRNA) from 17 day mouse placenta was fractionated into six components by reversed-phase plaskon chromatography. Codon specificity of three components (tRNA2His, tRNA4His and tRNA6His) was studied by binding to mouse liver ribosomes in the presence of poly(AC) and poly(ACU). All three tRNAs respond equally to each polymer indicating a dual in vitro recognition of CAC and CAU. At present the data do not support changes in coding as a rationale for the quantitative variation shown by placental His-tRNAs during pregnancy.

A recombinant DNA clone, named AL10, that contains murine leukemia virus (MuLV) related sequences was isolated from BALB/c mouse chromosomal DNA and examined in detail. Restriction endonuclease mapping revealed that the 10.5 kbp EcoRI insert consists of a 3.6 kbp left flanking cellular DNA region and a 6.9 kbp MuLV-related region that has a typical proviral LTR-gag-pol-env structure up to the EcoRI site in the env gene region. Comparison of the AL10 map with ecotropic and xenotropic virus isolates revealed many common restriction sites in the LTR and pol gene regions, but much fewer in the leader and gag regions. A stretch of 1,700 nucleotides containing the cellprovirus junctional region was sequenced and revealed transcriptional consensus signals and other structural features characteristic of MuLV LTRs, as well as two distinctive features: (a) a sequence of approximately 170 bp with direct and inverted terminal repeats not seen in infectious MuLV LTRs was identified in the U3 region between the "enhancer" region and the "CAT" box. This novel segment or its homologous sequences appear to be present in most of the endogenous MuLV-related LTRs and in other chromosomal locations of the mouse (b) The tRNA primer binding site is not complementary to proline tRNA, the primer for all known MuLVs, but is a 17/18 match with rat glutamine tRNA. The integration site of AL10 provirus was in a unique DNA region but contained an "Alu"-like short interdispersed repeat in the 5' adjacent cellular region. The AL10 proviral integration found in BALB/c was also apparent in RFM, AKR and SENCAR mouse cells but not in cells of NFS/N, C3H, HRS/J, SC-1, and a California Lake Casitas wild mouse.

The DNA polymerase activities in the spleens of young adult (3–8 months) and senescent (24–30 months) female BALB/c mice have been examined. Comparisons were made by direct assay of activated DNA-dependent and (rA) n · (dT) n - dependent activities in extracts from cytoplasmic and nuclear fractions and also by sucrose gradient sedimentation analysis of the two major forms of DNA polymerases in the subcellular fractions. The high molecular weight DNA polymerase activity was equivalent in the spleens of young and old BALB/c mice. The low molecular weight DNA polymerase activity was decreased in the spleens of old BALB/c mice. This decreased activity was reflected most markedly in the nuclear fraction of the spleens. Mixing experiments did not indicate the presence of inhibitors in the nuclear extract from the old mouse spleens. Stepwise extractions of isolated nuclei with increasing NaCl concentrations showed consistent results without revealing any difference in extractability of the low molecular weight DNA polymerase from the old mouse chromatin. Isolated low molecular weight DNA polymerase preparations from the young and old BALB/c mouse spleens are similar in chromatographic migration, sucrose gradient sedimentation, heat lability in vitro and K m of substrates. The low molecular weight DNA polymerase activity was not found to decline in livers and kidneys of 24 to 30-month-old BALB/c mice, nor in spleens of 30 to 34-month-old BC3F 1 mice.

Different mouse plasma cell tumors have been shown to produce different homogenous species of myeloma immunoglobulin in large amounts and are currently thought to consist of monoclonal cells ( Potter et al. , 1965 ; Lennox and Cohn, 1967 ). We have previously observed multiple isoaccepting tRNA's in a tumor of this type, MOPC 31C ( Yang and Novelli, 1968a ). In the present work, comparative studies of isoaccepting tRNA's by co-chromatography on a reversed-phase chromatographic column have been performed on cell lines of transplantable mouse plasma cell tumors that produce different kinds of myeloma immunoglobulin. Similar chromatographic patterns were observed for most of the aminoacyl-tRNA's examined in these tumors. A marked difference, however, was found between the seryl-tRNA's of an immunoglobulin-A producer (MPC 62) and those of immunoglobulin-G producers (MPC 47 and MOPC 31C).

Transfer RNAs from liver and brain of young and old BC3F1 mice were compared in regard to extent of aminoacylation and cochromatographic profiles of isoaccepting tRNA species on reversed-phase columns. Homologous synthetase preparations and optimal aminoacylation conditions were employed, having been determined for each amino acid and found to be the same for those from old and young mice. Small differences were found between tRNAs from young and old mice in the extent of acceptance for arginine and tyrosine in the liver and for aspartic acid in the brain. There were no differences observed between preparations from young and old mice in any of the cochromatographic profiles for the amino acids examined in this study, which included arginyl-, aspartyl-, glutamyl-, histidyl-, leucyl-, lysyl-, phenylalanyl-, seryl-, and tyrosyl-tRNAs from liver and arginyl-, aspartyl-, histidyl-, leucyl-, lysyl-, and seryl-tRNAs from brain. Comparisons of tRNA preparations from fetal and neonatal liver with those from adult liver did reveal both qualitative and quantitative differences. These results suggest that the postulated accumulation of errors as a result of age-related alterations in the translational mechanism does not occur in tRNA or aminoacyl-tRNA synthetases of these two tissues.

A high level of RNA-directed DNA polymerase activity is found in spleens of Balb c mice infected with Rauscher murine leukemia virus. The enzyme is associated with sedimentable subcellular fractions, can be separated from other cellular DNA polymerases, and is present in useful quantity for purification purposes. Complete solubilization of the enzyme, achieved and maintained by using a combination of detergent and high salt solution, is required for a satisfactory purification both in the initial extraction procedure and in subsequent column chromatography.

The tRNA synthesized by Escherichia coli during chloramphenicol treatment has altered chromatographic properties, as analyzed by reversed-phase chromatography. The data suggest that most of the tRNAs synthesized in the presence of chloramphenicol are immature, chromatographically distinct forms, capable of being converted to mature forms upon removal of chloramphenicol. Methylation of tRNA during chloramphenicol treatment appears to be quantitatively and qualitatively normal. The tRNA synthesized during chloramphenicol treatment differs from normal tRNA mainly in that it contains 60–70% less 4-thiouridine and dihydrouridine. Preliminary experiments indicate that these two minor bases are not required for normal aminoacylation. Phenylalanyl-, valyl-, isoleucyl- and lysyl-tRNAs from untreated and chloramphenicol-treated cells are equally functional in an in vitro hemoglobin-synthesizing system.

Characteristic long terminal repeats (LTR) of approximately 700 and 750 by were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp- gag-pol sequence with ACTGCCC repeat, a 113-bp mid- pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse spacies showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus , all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.

Using 125I-labeled red kidney bean phytohemagglutinin (125I-PHA), we have found that spleen cells from old BC3F1 mice bind this plant mitogen equally well, if not better, than spleen cells from young BC3F1 mice, although PHA-induced blastogenesis of spleen cells from old mice is sharply reduced. Analyses demonstrated that there is neither significant alteration of binding affinity nor decreased total number of membrane receptor sites for PHA in senescing mouse spleen cells. The amount of PHA which was initially bound to spleen cells in serum-free medium appeared to be insufficient for a subsequent full stimulation of blastogenesis ([3H]thymidine incorporation) in either young or old mouse spleen cells; when washed free of unbound extracellular PHA and upon cultivation in serum-containing culture medium, spleen cells rapidly released more than 90% of the bound PHA. Also, temperatures which change cell membrane morphology played a significant role in the binding and retention of PHA. However, no difference was observed between young and old mouse spleen cells in all these phenomena of PHA-cell membrane interaction.

Soluble extracts of mouse cells with Fv-1 n or Fv-1 b gene alleles specifically and reciprocally inhibit infection of B- or N-tropic mouse leukemia viruses in permissive cell cultures. NB-tropic virus infection was not inhibited by either cell extract. Extracts from Fv-1 - cells did not inhibit infection by the three virus host-range types, but N- or B-tropic virus infection of Fv-1 - cells was inhibited by extracts of the nonpermissive cells, and Fv-1 nb cell extracts inhibited both viruses. The maximum degree of inhibition was 50-80% as determined by immunofluorescent or plaque assays, with extracts containing up to 500 μg/ml of nonpermissive cell protein. The inhibitor(s) is relatively unstable since activity is lost after 2 hr at 37° or 30 min at 56°. The inhibitor(s) was most effective if added 2 hr before or within 2 hr after infection, did not react with the virus directly, inhibit virus attachment, or inhibit the normal cell functions tested. These results indicate that nonpermissive mouse cells contain a product, possibly determined by the Fv-1 gene, which inhibits some early postpenetration event(s) in leukemia virus infection.

Unique 4S RNA species from AKR mouse embryo cells hybridize with AKR murine leukemia virus and avian myeloblastosis virus 35S RNAs in vitro . Analyses by reversed-phase column chromatography indicate that the major 4S species that hybridize with the two viral RNAs are probably the same. A 4S RNA species with similar chromatographic properties is a major component of the AKR viral 4S RNA which associates with the viral 70S RNA in vivo .

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.

Previously, in vitro recombinant DNA studies demonstrated that genetic determinants of N-tropism and B-tropism, or Fv-1-related host range properties of murine leukemia viruses, were located in a BamHI-HindIII DNA segment derived from the 5' portion of the cloned viral genome. We sequenced this segment and its immediate 5' region from cloned DNA of two BALB/c mouse C-type viruses (WN1802N and WN1802B) and found base differences at 12 positions out of the otherwise identical 1,390-base-pair sequences. Analysis of the most likely reading frame showed that 6 of the 12 base differences would result in four encoded amino acid changes, three of which occur at positions 109 (glutamine in WN1802N versus threonine in WN1802B), 110 (arginine in WN1802N versus glutamic acid in WN1802B), and 159 (glutamic acid in WN1802N versus glycine in WN1802B) of the p30 protein. The remaining one is located at position 36 (threonine in WN1802N versus isoleucine in WN1802B) of the viral polymerase protein. Significant conformational alteration of the p30 protein could be predicted from these amino acid changes.

We analyzed 15 recombinant DNA clones of the unintegrated closed circular DNA intermediate of the BALB/c endogenous ecotropic murine leukemia virus WN1802N. Thirteen of these clones had an insert which corresponded to the complete murine leukemia virus genome. Of these, six contained a single long terminal repeat (LTR) and seven contained two LTRs. The viral genomes in nine clones had an LTR of 520 base pairs (bp), one had an LTR of 570 bp, three had an LTR of 600 bp, and one had an LTR of 670 bp. Restriction endonuclease analysis demonstrated that the size variability resides in the U3 region. Seven of eight clones which yielded infectious virus by DNA transfection had the 520-bp LTR, and the other had a 600-bp LTR. More detailed examination of plasmid subclones of three isolates with different-sized LTRs revealed that the approximate position which varies in the U3 region corresponds to the 72-bp repeat region of Moloney sarcoma virus. Possible consequences of these variations are discussed.

Retrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.

Twenty molecular clones containing sequences homologous to the long terminal repeats (LTRs) of the endogenous ecotropic murine leukemia virus (MuLV) of the RFM/Un mouse were isolated from a library of RFM/Un mouse spleen DNA in phage λ Three of these LTRs were not associated with any viral structural genes. Nucleotide sequence analysis demonstrated that they were solitary LTRs which were flanked by 4-bp directly repeated cellular sequences and which lacked primer binding sites. Two of the three subclones were found to be identical except for their orientations in the vector pBR322. Unique-sequence regions on either side of the two nonidentical elements were used to characterize their integration sites in genomic DNA. The solitary LTRs and their flanking regions were found to be conserved in a number of inbred mouse strains, including three strains known not to harbor endogenous ecotropic MuLV-type proviruses. Comparison of cleavage by the methylation-sensitive restriction enzyme Smal and methylation-insensitive Kpn l at the characteristic LTR Sma l /Kpn l site suggested that at least one of these solitary LTRs is methylated to a lesser extent than are most endogenous proviral LTRs. These particular solitary LTRs, like endogenous proviral sequences, appear to be stably transmitted genetic elements.