Decreased vascular relaxation in hypertension. Relaxation of spirally cut aortic strips was diminished in vessels from both spontaneously hypertensive rats and renal hypertensive rats. Aortic relaxation was decreased in response to the cyclic nucleotides and the beta adrenergic stimulant, isoproterenol, in both models, of hypertension. Defective aortic relaxation also occurred with two other vasodilators, nitroglycerin and adenosine. Further evidence for a reduced relaxant ability of blood vessels from hypertensive rats was obtained by measuring aortic relaxation after exposure and subsequent removal of vascular contractile agonists. The time for aortic preparations from spontaneously hypertensive rats to relax to base-line tension after maximum contraction with norepinephrine, serotonin and potassium chloride was significantly prolonged compared to recovery time for vessels from Kyoto Wistar normotensive rats. Treatment of the spontaneously hypertensive rat with reserpine, but not hydralazine, resulted in an improved ability of aortic preparations to relax. Based on these data, we propose that defects in vascular relaxation may contribute to hypertension and that some antihypertensive drugs may improve or facilitate vascular relaxation.


Analysis of pairs of individual Ia-E.P.S.P.S in single motoneurones. 1. Recordings of individual e.p.s.p.s evoked by the action of single medial gastrocnemius Ia fibres have been made from medial gastrocnemius motoneurones. In many motoneurones the action of two Ia fibres has been observed and the properties of the e.p.s.p.s compared. 2. For sixty-three pairs of averaged e.p.s.p.s, each from the same motoneurone, the ratio of half-widths was plotted against the ratio of rise times. These results were compared with theoretical values derived from the Rall compartmental model. It was found that variations in synaptic current time courses and differences in the termination of localized synaptic terminals were not sufficient to account for all the data. 3. Amplitude and rise time were inversely related but the correlation coefficient was very low. For pairs of e.p.s.p.s in the same motoneurone the e.p.s.p. with the fast rise time was larger than that with the slow rise time in forty-eight of sixty-three cases. 4. In a given motoneurone individual e.p.s.p.s evoked by the action of different Ia fibres did not vary greatly in amplitude. The ratio of peak amplitudes was less than 3 for 86% of the pairs of e.p.s.p.s examined, and the maximum was 4-8. 5. Amplitude histograms were constructed for individual e.p.s.p.s at thirty-three synapses. Twenty-two of them could be shown to satisfy the Poisson law. The others satisfied the binomial law or neither. 6. Within a given motoneurone the amplitude of an e.p.s.p. is closely related to the mean number of quanta released but not to the amplitude of the unit e.p.s.p. produced by the action of a single quantum of transmitter.


Cyclic AMP in sheep ovarian follicles: site of production and response to gonadotrophins. This study was undertaken (i) to establish a relationship between cyclic AMP (cAMP) production and the degree of LH and FSH stimulation; (ii) to determine the effects of various gonadotrophins on follicular formation of cAMP; and (iii) to identify the precise intrafollicular site of cAMP formation. The formation of cAMP increased rapidly in follicles exposed to LH. Maximum concentrations were reached after 90 min and were maintained for 180 min. Extracellular release of cAMP increased steadily throughout the 180-min experimental period. Tissue levels of cAMP increased proportionally and significantly when LH concentrations in the medium were increased from 0 to 200 mi.u. ml-1. Tissue levels of cAMP were significantly increased by HCG, prostaglandin E-2 and noradrenaline, but not by prolactin, prostaglandin F-2alpha, serotonin or melatonin. Cyclic AMP formation occurred predominantly in the thecal compartment; the membrana granulosa contributed less than 3% of the total amount of cAMP formed after gonadotrophic stimulation. A significant amount of cAMP from the thecal cells was released into the extracellular compartment and appeared to pass into the granulosa cells.


Effect of LH on the release of cyclic AMP by the rabbit ovary perfused in vivo and in vitro. After perfusion of 10 rabbit ovaries in vitro with a modified Krebs bicarbonate buffer containing dextran and glucose, the concentration of cAMP in the perfusion medium was significantly increased 2-5 min after stimulation with 10 mug LH/ml medium and was higher at 15 and 30 min. Intravenous injection of 100 mug LH/rabbit caused a significant increase of cAMP concentrations in the ovarian venous blood from 8 ovaries 10 min after the injection and the cAMP concentrations were higher after 15 and 30 min. The ovarian blood flow was not changed after the LH injection. It is concluded that perfusion techniques can be useful in analysis of the mechanisms and physiological significance of release of cAMP from the ovary after hormonal stimulation.


Natural estrogen and anti-thrombin III activity in postmenopausal women. Anti-thrombin III activity was measured in normal pre- and postmenopausal female controls: during late pregnancy, in young women taking oral contraceptives and in postmenopausal women treated with natural, conjugated estrogens. Both the pregnant patients and women on oral contraceptives had significantly lower anti-thrombin III levels when compared with the normal, untreated control group. Treatment with natural estrogens did not result in any alteration in anti-thrombin III activity. There was no significant difference in the anti-thrombin III values in the pre- and postmenopausal control subjects.


Adriamycin analogs. Periodate oxidation of adriamycin. Adriamycin is selectively cleaved in high yield at the C13-C14 bond by 1 equiv of sodium metaperiodate to yield carboxylic acid 3. The corresponding methyl ester 4 is obtained by Fischer esterification. Spectral studies indicate that 3 and 4 bind to calf thymus DNA in a manner similar to adriamycin and daunomycin but Tm measurements suggest that less stable complexes are formed. Ester 4 inhibits DNA and RNA synthesis in cultured L1210 cells at levels comparable to adriamycin but acid 3 is much less effective. Both new compounds are moderately effective as antitumor agents against P388 lymphocytic leukemia in the mouse.


Synthesis of some 1, 8- and 2, 8-disubstituted derivatives of adenosine cyclic 3', 5'-phosphate and their interaction with some enzymes of cAMP metabolism. 1, 8-Disubstituted derivatives of adenosine cyclic 3', 5'-phosphate (cAMP) were synthesized by N-oxidation or N-methylation of previously reported 8-substituted cAMP derivatives to yield 8-bromoadenosine cyclic 3', 5'-phosphate 1-oxide and 8-(benzylthio)-1-methyladenosine cyclic 3', 5'-phosphate. Substituents were introduced into the 8 position of 2-methyladenosine cyclic 3', 5'-phosphate and 2-butyladenosine cyclic 3', 5'-phosphate by bromination, followed by treatment with sodium benzylmercaptide, sodium p-chlorothiophenolate, or, in the former case, sodium azide. Each of the 1,8- and 2,8-disubstituted derivatives of cAMP was tested as activators of cAMP-dependent protein kinase and as substrates for the inhibitors of cyclic nucleotide phosphodiesterases. Depending on the substitutions, examples were found where the disubstituted derivatives were either more active, equally as active or less active than the monosubstituted parent compounds as protein kinase activators. For the compounds reported, 8-substitution completely or substantially eliminated the ability of 1- or 2-substituted derivatives of cAMP to serve as substrates for phosphodiesterase and diminished the ability of these latter derivatives to inhibit cAMP hydrolysis.


Genetic factors in amyloidosis.

Genetic factors in amyloidosis. In the absence of biochemical distinctions, the nosography of the inherited amyloidoses must at present depend largely upon clinical subdivisions. In the broad classification adopted here, the disorders have for convenience been grouped according to the anatomical system that is predominantly affected. It is evident that the amyloid syndromes display considerable heterogeneity. However, they overlap. Thus in the Iowa type classified with the hereditary amyloid neuropathies (van Allen et al, 1969; Gimeno et al, 1974), renal involvement was frequent and was the usual cause of death. In the English (Zalin et al, 1974) and Scandinavian (Andersson, 1970) families with neuropathy as the predominant feature, cardiac involvement was a common finding. In certain of the conditions discussed, such as medullary carcinoma of the thyroid and Down's syndrome, amyloid deposition is merely an incidental aspect of the disorder. In those conditions in which generalized or localized amyloid deposition occupies a more central position in the clinical syndrome, an autosomal dominant inheritance has been established or suggested in the majority. An autosomal recessive inheritance has so far only been recognized in familial Mediterranean fever. In the family with hereditary amyloid heart diseases reported by Fredricksen et al (1962), the disorder was confined to a single sibship, raising the possibility of recessive inheritance. This could also be true in sporadic examples of primary amyloidosis. The dominantly inherited amyloidoses comprise a number of geographically widely scattered families with clinical pictures that do not show consistent differences between some families. The families that do not show consistent differences are not necessarily harbouring nutations at the same locus, or the same mutation at any particular locus. However, many of these dominantly inherited clinical syndromes are sufficiently different from each other and the clinical manifestations of each sufficiently consistent to indicate that separate main genes are likely to be involved...


Control of the uptake of amino acids by serum chick embryo cells, untransformed or transformed rous sarcoma virus. Forty to fifty minutes after removal of serum, the net total uptake of amino acids in growing secondary cultures of normal or virus-transformed chick embryo cells, stopped or proceeded only at a highly reduced rate. In both normal and transformed cells, the initial (0-40 min) rate of the above uptake was the same in the absence of serum as in its presence. The initial rate of the total uptake of amino acids in growing transformed cells was about the same as in growing normal cells. Neither in the normal nor in the transformed cells was the rate of the total uptake of amino acids reduced by cell confluence alone. In highly dense, hyperconfluent cultures of normal cells in which cell growth was arrested, the rate of uptake in the absence or in the presence of serum was four- to fivefold lower than the rate obtained in growing normal cells under similar conditions; in the absence of serum, the net uptake stopped after 40 min in the hyperconfluent cultures as well. It appears that cells growing in tissue culture require a serum factor for maintenance of the required high rates of uptake of amino acids and that the inhibition of growth at high cell densities is a result of depletion of this factor from serum, or the inability of the cells in a dense culture to respond to the factor. A serum factor is apparently also required for maintenance of the reduced rates of uptake of amino acids observed in hyperconfluent cultures.


Study of enzymes regulating vasopressin-stimulated cyclic AMP metabolism in separated mitochondria-rich and granular epithelial cells of toad urinary bladder. The epithelial cells of the toad urinary bladder are morphologically heterogenous. In order to relate the effect of vasopressin on cyclic AMP metabolism to cell type, the epithelial cells were separated by the density gradient technique of Scott, Sapirstein and Yoder (Science 184:797, 1974). The separation was verified by electron-microscopy and by observing that the band of cells enriched in mitochondria-rich cells was enriched in carbonic anhydrase activity compared to the band of granular cells. A large portion of cells collected from the gradient was considered to be nonviable, precluding further study of their function as intact cells. Vasopressin-stimulated adenylate cyclase activity in homogenates of granular cells was simular to that in homogenates of mitochondria-rich cells. Cyclic nucleotide phosphodiesterase activity was also similar in the two types of cell. Thus, the enzymes known to be involved in cyclic AMP metabolism in response to vasopressin appear to be located in both major cell types.


Selective inhibition of urea transport by oxidizing agents. Evidence for a site of inhibition behond the generation of cyclic AMP. Urea and water transport across the toad bladder epithelial cell  appears to take place through independent vasopressin-stimulated pathways. Agents such as chromate, for example, when added to the luminal bathing medium, inhibit urea transport without inhibiting osmotic water flow, providing evidence for such independent pathways. In the present study, selective inhibition of urea transport is shown for permanganate and methylene blue, which like chromate, are oxidizing agents. Permanganate inhibits urea transport irreversibly, while methylene blue acts reversibly. Not all oxidizing agents are inhibitory; perchlorate, peroxide and ferricyanide have no effect on urea transport or water flow. Permanganate and chromate both act at a point beyond the generation of cyclic AMP, since they continue to inhibit urea transport in bladders treated with exogenous cyclic AMP, 8-bromoadenosine 3', 5'-cyclic monophosphate, and a combination of cyclic AMP and theophylline. These findings suggest that selective inhibition of urea transport can be brought about by oxidation of  one or more components in its transport pathway, and that, in the case of chromate and permanganate, these components may be in the luminal membrane itself.


Characterization of mouse mammary tumor viruses from primary tumor cell cultures.I. Immunologic and structural studies. Primary cell cultures of mammary tumors from Rill, GR, DD, BALB/cfC3H, and BALB/c mice were prepared by trypsin-EDTA dissociation of tumors. Cultures from these strains contained predominantly cells of epithelial morphology which formed three-dimensional domelike structures. Cultures from Rill, GR, DD, and BALB/cfC3H tumors produced extra-cellular type-B mouse mammary tumor virus(es) (MuMTV), either in the absence of detectable type-C virus or with less  than 1% contamination with type-C virus. This was determined by radioimmunoassays for MuMTV and murine leukemia virus (MuLV) antigens. Only BALB/c cultures produced MuMTV with as much as 3%  contaminating MuLV. High levels of MuMTV surface antigen were also found in soluble form in culture supernatants. Virus polypeptide analyses by electrophoresis on polyacrylamide gels showed that the Rill BALB/cfC3H, DD, and BALB/c viruses all contained polypeptides characteristic of MuMTV. Primary cultures of mammary tumor cells make available a source of purified MuMTV antigens, structural proteins, and nucleic acids for comparative studies of MuMTV from various mouse strains.


Characterization of mouse mammary tumor viruses from primary tumor cell cultures. II. Biochemical and biophysical studies. Primary mammary tumor cultures of RIII, GR, DD, BALB/c, and BALB/cfC3H mice were examined for mouse mammary tumor virus (MuMTV) production. Levels of production of 12-32 mug virus protein/day/75-cm2 culture flask could be maintained for 30-50 days with daily virus harvests. The viruses from tumor cell cultures of these mouse strains contained DNA polymerase with a strong preference for Mg++ over Mn++ as the divalent cation, a characteristic of DNA polymerase of MuMTV from mouse milk. These viruses from tumor cell cultures were excellent sources of MuMTV 3H-complementary DNA (complexed to 60-70S RNA) and radioactive 60-70S RNA, sufficiently free of contaminating murine leukemia virus nucleic acids, that can be used in molecular hybridization experiments. The effects of several culture parameters on MuMTV production were also studied.


A murine Wilms' tumor as a model for chemotherapy and radiotherapy. We studied the effect on tumor volumes and host survival of twenty-four chemotherapeutic and radiotherapeutic regimens in rats bearing the Furth/Columbia murine Wilms' tumor. After 5 years and some 100 transfers, this tumor retained its sensitivity to suitable combinations of actinomycin D, vincristine, and X-ray, regimens in current use in the treatment of human Wilms' tumor. Cytoxan alone was highly inhibitory; adriamycin was modestly inhibitory; and lethal doses of methotrexate followed by leucovorin "rescue" were slightly inhibitory to the growth of the rat tumor. No other chemotherapeutic agent tested was effective by our criteria. Cytoxan, with or without adriamycin, and X-irradiation, were the most useful combinations studied. The chemotherapeutic agents given 3 days before irradiation were significantly more carcinostatic than they were when given after radiotherapy.


Airborne transmission of polyoma virus.

Airborne transmission of polyoma virus. Polyoma virus (PV) infection was transmitted through the air of an animal laboratory. Mice free of detectable antibodies to PV were exposed for 1 month to the airborne environment of laboratories housing naturally infected mice. The seroconversion rate was 75% (24/32), as measured by hemagglutination inhibition. Control mice, housed in the sterile atmosphere of a mass air flow cabinet (MAFC) in the same laboratory, had a seroconversion rate of 15.8% (3/19). Airborne transmission occurred bia PV aerosois, generated by the handling of contaminated bedding, cages, and mice during weekly housekeeping. Length of exposure to PV aerosols correlated with seroconversion. One- and 3-hour exposures resulted in seroconversion rates of 40% (6/15) and 72% (23/32), respectively. Seroconversion rates of mice continuously housed in MAFC totaled 5% (2/40). Checkerboarding mice free of detectable antibodies with mice given 10(5) mean tissue culture infective doses of PV ip resulted in an airborne infection rate of 50% (15/30) in a conventionally ventilated room during a 12-week study. The airborne transmission rate was 10% (3/30) when experiments were performed in a mass air flow room with a vertical air velocity of 30 feet/minute was used. Antibodies to PV could not be detected in any of 138 human sera, including sera from 29 animal-care technicians who handled PV-infected mice and 15 personnel who had worked with the virus.


Growth in semisolid agar medium of human cord leukocytes freshly transformed by Epstein-Barr virus. The semisolid agar method for the selective growth of transformed cells was applied to investigations of Epstein-Barr virus (EBV) transformation. When 1 X 10(5) HUMAn umbilical-cord  blood leukocytes were seeded in agar medium immediately after EBV exposure, about 100 colonies developed in each dish. The occurrence of colonies correlated well with dilutions of EBV inoculum. These colonies were composed of lymphoblasts, were positive for EBV-determined nuclear antigen immunofluorescence, and consistently resulted in the establishment of cell lines. In cord leukocyte cultures exposed to EBV, cells capable of growing in semisolid agar medium appeared early and increased rapidly, though the total numbers of leukocytes and control cultures decreased during the first several days.


Stimulatory effect of 3', 5'-adenosine monophosphate on protein synthesis in heat-shocked murine leukemia lymphoblasts. The murine leukemia lymphoblast line L5178Y was tested for its early molecular response to the presence of dibutyryl cyclic AMP. Protein synthesis was reversibly inhibited by short durations of hyperthermia, and against this reduced background of protein synthetic activity, early leucine incorporation (30-120 min)  was markedly and consistently stimulated by the presence of the cyclic nucleotide. The effect became one of inhibition with extended times of incubation (longer than 120 min).


Genetic analysis of mammary tumor induction and expression of mammary tumor virus antigen in hormone-treated ovariectomized GR mice. Early stages of mammary tumors (EMT) were induced with a combined treatment of progesterone (P) and estrone (E) in ovariectomized adult GRS/A (GR) mice, a strain of European origin and with a high incidence of mammary cancer. The mammary tumors were comparable to the pregnancy-dependent tumors of breeding females of this strain. The hormone treatment did not lead to EMT in a variety of other strains and only occasionally in the RIII an C3H strains. Treatment with P or E alone di not lead to EMT in GR mice, but treatment with the steroid compound 17 alpha-ethynyl-19-nortestosterone (ANT) did mimic the combined effe(t of P and E. Since EMT could be induced by ANT in all ovariectomized adult GR mice within 3 weeks, this tumor-induction method was suitable for analysis of the gene responsible for palpable, pregnancy-dependent mammary tumors of this strain. Another argument for the usefulness of this test for genetic analysis was the fact that, though mouse mammary tumor virus (MuMTV) of the GR strain was introduced into BALB/c and tmas mice by foster-nursing, ANT treatment did not lead to EMT. First and second backcross analyses showed that one gene was responsible for EMT induction. There was a strong (orrelation between the presence of EMT and MuMTV antigens in the mammary glands and milk of several first backcross populations between GR and other strains such as C57BL, BALB/c, DBAf, and C3Hf. This suggested that the expression of MuMTV antigens was also controlled by the EMT gene. Two types of resistance phenomena were observed. Neither type could prevent EMT after hormone treatment; however, they could delay EMT development. One resistance factor for EMT induction was noticeable and dominant in reciprocal hybrids of the GR and DBAf strains, whereas another resistance factor was detected in the backcross population only [i.e., in the C57BL X (C57BL X Gr) ba(kcross] and not in hybrids; therefore, this factor was recessive. Until now, linkage experiments with 18 markers to locate the gene for EMT induction in the map of the mouse were unsuccessful.


Amount of satellite DNA in four experimentally induced tumors of the central nervous system. Quantitative changes in a glioblastoma producing C-type particles. The quantitative preservation of satellite NA was studied in several central nervous system (CNS) neoplasms; four tumor lines deriveo from 3-methylcholanthrene implantation into the CNS of mice were compared with brain and tissue cultures of normal mouse cells by analytical centrifugation in cesium chlorie. Three tumors showed no detectable difference from normal cells; nuclear and whole cell preparations were comparable. Only a glioblastoma line proucing C-type particles (TC509) revealed a significant difference from normal cells and exhibited a decrease of approximately 20% in satellite DNA or 2% of the total DNA on repeated examination for 1 year. C-type RNA virus may be related to relative decreases in satellite DNA observed in TC509.


Development of a lymphocyte transformation microassay for murine leukemia virus. A lymphocyte transformation microassay (LTA) was developed from spleen harvests of 6- to 8-week-old BABL/cCr mice.  The optimal culture conditions for the microassay were established by measurement of lymphoblastogenesis in response to phytohemagglutin (PHA) and pokeweek mitogen. Immunization, as measured by the LTA, of adult BALB/cCr mice with formalin-inactivated, sucrose-banded, murine type-C viruses was achieved with a three-dose regimen of 200, 100, and 100 mug during 3 successive weeks (Freund's complete adjuvant was used with the first dose). The ip route of immunization induced the best responses in lymphocytes harvested 18 days after the last immunogen was given. The LTA was consistently reproducible, limited only by biological variability of the mouse and the standardization of the antigen preparation. In mice immunized with Rauscher murine leukemia virus (R-MuLV) or AKR MuLV vaccine, the LTA was specific for the C-type virus and could be used to distinguish viral subtypes, because R-MuLV elicited responses significantly different from a B-tropic BALB/c leukemia virus. This specificity was evident when the stimulating antigen was presented as UV-inactivated, sucrose-banded virus or as freeze-thaw extracts of cell infected with MuLV.


Biochemical studies on mitochondria isolated from Normal and Neoplastic Tissues of the Mouse Mammary Gland. Mitochondria isolated from spontaneous and transplanted mammary adenocarcinomas of two strains of mice were compared, by various  biochemical criteria, to mitochondria from mammary glands of midpregnant or hormonally stimulated, cancer-free mice. The specific activities of several mitochondrial enzymes including cytochrome oxidase, alpha-glycerophosphate oxidase, and succinate dehydrogenase were twofold to threefold lower, whereas the activity of monoamine oxidase was two fold higher in tumor mitochondria. Malate dehydrogenase, adenylate kinase, and NADH oxidase showed similar levels of activity in tumor and midpregnant mammary gland mitochondria. In addition, mitochondrial polypeptide composition was analyzed by electrophoresis on sodium dodecyl sulfate-urea polyacrylamide gels. Midpregnant mammary gland and mammary tumor mitochondria were similar in polypeptide composition; however, several differences were observed. A high-molecular-weight polypeptide, present in mid-pregnant mammary gland mitochondria was absent from tumor mitochondria. Also, tumor mitochondria contained an additional high-molecular-weight polypeptide not found in the midpregnant mammary gland. There were numerous differences in the relative proportions of many polypeptides common to both tumor and midpregnant mammary gland mitochondria.


Reexpression of the original tumor pattern by a human breast carcinoma cell line (MCF-7) in sponge culture. A stable cell line (MCF-7), derived from a pleural effusion of a patient with metastatic breast carcinoma, was maintained in these laboratories for more than 3 years in conventional monolayer culture. To further characterize the tumor origin of the MCF-7 line, cells were grown on collagen-coated cellulos sponges. On the three-dimensional sponge matrix, the cells formed clusters, ductlike structures, and lumina similar to the patterns observed in the antecedent primary tumor and in the pleural metastasis. The similarity between the original tumor and the cells grown in sponge suggested that the MCF-7 cells did in fact retain the potential to express the histologic patterns of tumor, even in the absence of stroma support. This study confirmed the utility of sponge culture for the investigation of the retention of tumor characteristics by cultured cells of neoplastic origin.


Specificity of human antibodies to intracytoplasmic type-A particles of the murine mammary tumor virus. Large-scale studies showed that antibodies previously detectable in women with proliferating mastopathy or breast cancer were directed to intracytoplasmic type-A particles (iAp) of mouse mammary tumor virus. Immunofluorescence revealed the human antibodies to be bound only by those tumors producing a certain amount of iAp clusters visible by light microscopy. The intensity of the reaction corresponded to the iAp content of every tumor tested as revealed by electron microscopy and rabbit antisera to iAp. The fluorescence patterns obtained with positive human sera were similar to those obtained with rabbit antisera specific for iAp and resembled the tissue distribution patterns of iAp inclusions stained by acid fuchsin. The reaction with human sera was entirely blocked by rabbit antisera to iAp and, less so, by rabbit or mouse antisera to B particles. The human antibody activity was exhaustively absorbed by purified iAp or purified and disrupted B particles, which indicated that the human antibodies were directed to antigenic components shared by iAp and B particles. Preliminary immunoperoxidase studies supported the assumption that the human antibodies were bound to the iAp membrane; technical details might have accounted for the finding that the human antibodies reacted with the iAp but not with B particles in situ.


Limitation of multinucleation by dibutyryl adenosine 3',5'-cyclic monophosphate in tumor cells treated with cytochalasin B. Hamster BHK21 tumor cells and human rhabdomyosarcoma (RD) cells responded to cytochalasin B (CB) by becoming highly multinucleated. Nearly 80% of the BHK cells contained four or more nuclei after 7 days of exposure to CB, and 35% of these had at least seven nuclei. Similar frequencies of multinucleation were obtained with RD cells. Administration of 10(-3) M theophylline and dibutyryl adenosine 3',5'-cyclic monophosphate (Bu2cAMP) at 5X10(-4) M or 10(-3) M to CB-treated RD or BHK cells greatly reduced the frequency of cells with five or more nuclei. The frequency of cells with seven or more nuclei was reduced to less than 5%. Along with this reduction in highly multinucleated cells was an increase in the incidence of binucleated cells. Bu2cAMP was toxic and caused many CB-treated BHK cells to detach from the culture surface, but not all cells were killed. Bu2cAMP had little toxic effect on CB-treated RD cells. These observations indicated that the inhibition of high degrees of multinucleation were not the result of nonspecific toxic effects of Bu2cAMP but that nuclear division was limited by it. The effect of Bu2cAMP on density-dependent inhibition of growth was also studied. Addition of only theophylline and Bu2cAMP to either BHK or RD cells resulted in growth to significantly lower saturation densities. The toxicity of Bu2cAMP on cells in crowded cultures apparently caused the limited propagation. Bu2cAMP resulted in significant cell killing or detachment but, once the lower saturation densities were reached, cell death was minimized. Thus Bu2cAMP did not restore contact inhibition per se. It was also found that untreated RD cells grew to lower concentration densities than expected from the microscopic inspection of cells in situ. Microscopic inspection revealed high concentration densities and numerous mitoses. This apparent contradiction was due to the ability of RD cells to fuse upon the attainment of confluence and to produce multinucleated cells without the aid of CB.


Antibody response to related leukosis viruses induced in chickens tolerant to an avian leukosis virus. White Leghorn chickens congenitally infected with a subgroup A avian leukosis virus ALV-F42 were challenged at various ages with the serologically related subgroup A avian leukosis viruses (ALV) RAV-1, RAV-3, and RAV-5 and their antibody responses and viremic status determined. Birds given injections of RAV-1 failed to produce neutralizing antibody to any of the pseudotype sarcoma viruses of the various ALV, whereas many birds challenged with RAV-3 or RAV-5 produced neutralizing antibody directed against the heterologous RAV-1 as well as the homologous viruses, but not against the tolerated virus ALV-F42. Virtually all birds remained viremic despite the presence of neutralizing antibody. The results suggested that a phenomenon akin to circumvention or abrogation of tolerance to ALV-F42 was effected, and that there are multiple determinants of the viral envelope antigens of ALV as measured by neutralization.


Growth suppression of subcutaneously inoculated hepatic tumors after injection of syngeneic liver cells into the subcutaneous tissue of rats. One week after the sc injection of minced tissues of the livers, kidneys, or spleens of W, Donryu, and MP rats into the left back of syngeneic rats, ascites-type tumor cells derived from rat hepatoma (AH 130) or Walker carcinosarcoma cells derived from rat breast tumor were inoculated sc into the right back. Ascites hepatoma cell growth was slowest in the group given the liver preparation, whereas no significant difference was noted in the growth of Walker carcinosarcoma in all groups. The results were similar in all strains.


Naturally occurring reticuloendotheliosis in turkeys: transmission. This report describes the natural occurrence and experimental transmission of reticuloendotheliosis in a turkey flock. In the naturally affected turkeys, lesions were present in the liver, spleen, heart, intestines, and peripheral nerves, and were composed mainly of lymphoreticular cells. The disease was experimentally reproduced in turkey poults with cellfree tissue extracts of infected birds. A type-C RNA virus similar to reticuloendotheliosis virus (strain T) was present in the tissues of turkey poults experimentally inoculated. This virus was unrelated to the type-C RNA viruses of the avian leukosis sarcoma complex.


Host-range properties of murine xenotropic and ecotropic type-C viruses. Host-range properties of xenotropic (x-tropic) and ecotropic mouse type-C viruses were determined. NB-tropic viruses replicated only in mouse and rat cells, whereas x-tropic viruses, which do not exogenously infect mouse cells, grew in cells from various other mammalian species. Great variability in susceptibilities to x-tropic viruses was demonstrated in cells from heterologous species and in cells from a single species (human). Although rat cells were susceptible to both types of viruses, hamster cells were uniformly resistant. In addition, the x-tropic viruses crossed class barriers to infect cells of avian species but not insect, fish, or reptile cells.


Prevention of tumor growth after intradermal injection of BCG extracts: a comparison of results in strain-2 guinea pigs from the National Institutes of Health and from the National Jewish Hospital and Research Center. Line 10, a transplantable hepatocellular carcinoma, was obtained originally from an NIH strain-2 male guinea pig fed diethylnitrosamine. The antitumor activity of BCG and BCG extracts was evaluated in strain-2 guinea pigs obtained both from NIH and the National Jewish Hospital and Research Center (NJH). Animals were immunized with these materials and then tested for their capacity to resist the growth of intradermally injected line-10 tumor cells. Tumor growth was not prevented in 18 NIH animals immunized with living BCG. No tumor growth occurred in 1 of 22 NIH animals immunized with a residue that remained after exhaustive methanol extraction of BCG and in 1 of 44 NIH guinea pigs immunized with BCG extracts. In contrast, tumor growth was prevented in 13 of 22 similarly immunized NJH guinea pigs.


IgM antibody to a tumor-associated antigen (AG-4) induced by herpes simplex virus type 2: its use in location of the antigen in infected cells. Rate-zonal centrifugation was used to separate 7S and 19S lg's from sera of patients with cervical cancer and of controls. The lg's were assayed for their ability to recognize AG-4, a tumor-associated antigen, and herpes simplex virus 2 (HSV-2) by various serologic assays including neutralization, immunofluorescence, and complement fixation. These studies indicated that antibody to AG-4 is a macroglobulin; antibody-fixing complement with HSV-2 is a 7S lg. The results of various serologic assays did not show absolute correlation; various tests preferentially identified some antigen-antibody reactions. We obtained evidence that AG-4 is the cytoplasm and is probably on the cell surface.


Antibody-dependent lymphocyte cytotoxicity against cells expressing Epstein-Barr virus antigens. Antibody-dependent lymphocyte cytotoxicity (ADLC) was demonstrated against Epstein-Barr virus (EBV)-infected RAJI cells with peripheral with peripheral blood lymphocytes from EBV-infected donors. No cytotoxic activity was detected against unifected RAJl cells. The results indicated that this antibody-mediated cytotoxic reaction was directed against the same EBV-induced membrane antigens (MA) previously defined by the membrane immunofluorescence (MF) assay. Antibody to EBV-associated early antigens did not participate in this in vitro reaction. Antibody titers to EBV-induced MA were significantly higher by the ADLC assay in comparison with the MF test. A preliminary study showed no relationship between high antibody titers and the presence of EBV-associated malignancies. The possible in vivo significance of this immune reaction was discussed.


Effect of partial hepatectomy on tumor incidence and metabolism of mice fed thioacetamide. Six-week-old male Swiss mice were given 0.03% thioacetamide (TAA) in the diet 24, 72, and 168 hours after partial hepatectomy. TAA-treated mice from all three groups were killed when they were 4, 9, and 13 months old. Intact and partially hepatectomized animals on normal diets served as controls. None of the controls evidenced neoplasms at any age. All three experimental groups developed liver tumors earlier than did intact mice treated with the TAA diet. Progressive metabolic studies on the livers or tumor tissues of treated mice showed that the levels of glucose-6-phosphatase, fructose-1,6-diphosphatase, and glycogen decreased significantly in the 4-month-old treated group when there was no significant alteration in liver histology. These parameters were lowest in the tumor tissues of treated mice.


Mixed-culture cytopathogenicity between KC and XC oncornavirus indicator lines and "virus-free" human choriocarcinoma cells. When rat (XC) cells transformed by Rous sarcoma virus and human (KC) cells were cocultivated with appropriate infected cells, cell fusion was extensive and rapid. Many human cell lines were screened, with negative results; however, several hormone-secreting human choriocarcinoma cells fused extensively with both KC and XC cells. No evidence of a virus involvement in this interaction was found by direct examination, transmission, or immunologic tests.


Vascular tumors induced by polyoma virus in pregnant rats. Female R rats mated with an R male and inoculated in utero with polyoma virus after "fetectomy" developed tumors. These tumors originated in the uterus and were of fetal origin (visceral yolk sac). Histologically, they were hemangiomas or hemangiosarcomas. The latter were transplantable and grew in tissue culture. Infectious polyoma virus could not be isolated from the tumor cells kept as transplantable lines or cultured in vitro. However, the tumor cells were positive for the polyoma-specific surface antigen, polyoma tumor-specific transplantation antigen(s), and polyoma nuclear T antigen.


Prostaglandin production in cultures of BALB/3T3 and SV3T3 mouse fibroblasts. The concentration of different prostaglandins (PG's) was determined in both cells and culture medium of growing BALB/3T3 and BALB/3T3 (3T3) cultures transformed by simian virus 40 (SV3T3). Most PG's were found in the culture medium rather than in the cells. Further, the larger PG measurements were PGE and a composite measurement of PGA and PGB. PGF was detected at lower levels. The sum of PGE and the composite measurement (PGA+PGB) was the best indication of PG production in culture. When 2-day medium collections from 3T3 and SV3T3 cells were measured by radioimmunoassay for PGs, higher concentrations of PG were detected in the media of SV3T3 cultures. This difference in PG production was not due merely to differences in cell density, since SV3T3 cells produced higher PG concentrations, even at equal cell densities. PG production for a 2-day interval was more a function of cell type than cell density.


Stimulation of growth of a transplantable hepatoma in rats by parenteral nutrition. Young BUF rats of similar ages were inoculated with the transplantable Morris hepatoma No. 7777. At 4 weeks after inoculation, 1 group was given total iv (parenteral) feeding at high caloric and nutritional levels for 2 weeks. Such total iv feeding (hyperalimentation) of rats stimulated a more rapid tumor growth in the host. In addition, the tumors from rats fed parenterally for 2 weeks had higher mitotic activity and larger areas of necrosis, which indicate that iv feeding caused the tumor to undergo faster cell turnover with greater cell production and cell loss. Analysis of organ weights showed that parenteral feeding caused atrophy of the intestines, whereas spleen weights of the hepatoma-bearing rats fed iv were greater than those of the orally fed hepatoma-bearing rats.


Immune reactivity in the Moloney strain of murine sarcoma virus oncogenesis: requirement of thymus-derived lymphocytes for in vivo protection. To study the function of different lymphocyte populations in the Moloney strain of murine sarcoma virus (M-MuSV) tumorigenesis, we gave M-MuSV injections to CBA mice selectively deprived of thymus (T) lymphocytes by thymectomy, X-rradiation, and syngeneic bone marrow injection. Although no tumors appeared in the control group, 80% of the derived mice had tumors that grew progressively and ultimately killed them. In deprived mice, grafted with a syngeneic thymus (reconstituted mice) before or after an M-MuSV injection, tumors regressed or did not develop. Histologically, the lymph nodes and spleens of reconstituted mice, compared to those of deprived animals, showed repopulation of the thymus-dependent areas and prominent follicles in the cortex. Moreover, tumor tissue of reconstituted mice was extensively infiltrated by lymphocytes. To evaluate the number of lymphoid cells needed to prevent or regress M-MuSV tumors, we injected varying amounts of lymphoid cells into deprived mice. Even low lymphocyte numbers (10(6) cells) were sufficient to exert, in some cases, protection against M-MuSV tumorigenesis. This effect was not abolished by subsequent splenectomy or antilymphocyte serum treatment. Finally, deprived mice, given repeated injections of antiserum (hyperimmune) against M-MuSV, had tumors which appeared only after a prolonged latency. From these results, it is concluded that T-cell population integrity is important in affording total host protection against the M-MuSV tumors.


Detection of complement-dependent antibody to tumor cells in sera of strain-2 guinea pigs cured of their tumors by BCG Treatment. Sera of strain-2 guinea pigs (cured of line-10 tumor by BCG therapy) were tested for complement-dependent, cytotoxic antibody. About 30% of the sera tested contained significant cytotoxic acitivity with the addition of human, but not syngeneic, complement. Using papain pretreated line-10 cells, we detected antibody in about 50% of the sera with syngeneic sera as the source of complement. Antibody to line-10 was also demonstrated in selected sera by indirect fluorescence and the C1 fixation and transfer test.


Typical type-C virus in human leukemia.

Typical type-C virus in human leukemia. The virus HL-23, recently isolated from a patient with myelogenous leukemia, was compared by electron microscopy with virus-like particles observed in human tissues and with several mammalian type-C virus particles. It closely resembled the typical type-C virus particles of murine, feline, and nonhuman primate species, and differed from all particles previously reported in human tissues, including the atypical human placental particle.


Lack of infectivity of bovine leukemia (C-type) virus to rats. Sprague-Dawley rats were given ip injections of bovine culture and sheep cultures of bovine leukemia virus (BLV) and Gross passage-A leukemia virus [MuLV(G)]. Sera were tested to BLV antigens. BLV did not induce tumors in Sprague-Dawley rats, but the rats were susceptible to MuLV(G) at low doses.


Relationship of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate to growth of dimethylbenz(a)anthracene-induced mammary tumors in rats. Alteration of growth of dimethylbenz[a]anthracene-induced mammary tumors was caused by removal of estrogen (ovariectomy), or insulin (diabetes), or by inhibition of prolactin secretin (treatment with an ergoline derivative). The levels of cyclic AMP (cAMP) and cGMP were measured in carcinomas classified as growing, static, and regressing. The amount of cAMP, expressed as pmoles/mg tumor weight or pmoles/mg protein, was lowest in growing tumors, intermediate in static tumors, and highest in those regressing. No correlation was seen between tumor growth and cGMP levels. Cyclophosphamide-induced tumor stasis did not elevate cAMP levels. The data suggest a role of cAMP in arrest of hormone-induced tumor growth.


Malignant transformation of hamster brain cells in vitro by human papovavirus Bk. Human papovavirus BK caused the malignant transformation in vitro of brain cells prepared from newborn hamsters. The transformed cells produced BK virus T antigen and grew as tumors after sc inoculation ito hamsters that developed antibodies to BK virus T antigen. The histopathology of the tumors revealed an undifferentiated glioma.


Hepatic cytologic and neoplastic changes in mice given benzidine dihydrochloride. The administration of benzidine dihydrochloride in water to mice for up to 39 weeks resulted in hemosiderosis of the spleen, focal vacuolization of the transitional epithelium of the urinary bladder, and chronic inflammation and cytologic and neoplastic changes in the liver. Basophilic and acidophilic cytologic foci appeared in the liver as two distinct entities and both appeared preneoplastic. Benign and malignant liver tumors were also seen. Both types of cytologic changes and malignant liver tumors were more common in the female than male mouse.


Epstein-Barr virus in an American patient with Burkitt's lymphoma: detection of viral genome in tumor tissue and establishment of a tumor-derived cell line (NAB). Epstein-Barr virus (EBV) DNA (17.7 genome equivalents/cell) was found in tumor tissue from an American patient with Burkitt's lymphoma who had never traveled outside the United States. A lymphoid cell line (NAB) containing the EBV genome was established from tumor tissue from this patient; characteristics of this cell line were described. Previous Burkitt's tumors found in Americans and examined by molecular hybridization were negative for EBV DNA. Our results suggested that EBV is associated with at least some American Burkitt's tumors.


Lymphoproliferative diseases of fowl: chromosome breaks caused in lymphocytes by JM-V herpesvirus. Chromosome preparations were made of bone marrow cells and peripheral lymphocytes isolated from chicks that developed leukemia following infection with JM-V herpes-virus. Karyotypic analysis revealed a high frequency of chromosome breaks and aneuploidy, as well as some pulverization of chromosomes. The number of chromosome breaks began to increase at 2-3 days post infection, and by 5 days post infection it reached 12.7% of bone marrow cells and 17.2% of peripheral lymphocytes. Similarly, the number of aneuploid metaphase figures increased rapidly and reached 12% of bone marrow cells and 19% of peripheral lymphocytes at 5 days post infection. Some specificity was observed in the chromosomes that were affected.


Neoplastic transformation in tissues of rats exposed to monocrotaline or dehydroretronecine. Male Sprague-Dawley rats received sc injections biweekly of either the pyrrolizidine alkaloid monocrotaline or its metabolite dehydroretronecine for 1 year. The animals were then observed for an additional 12 months for the induction of neoplasms. Of 60 rats that received dehydroretronecine, 39 developed rhabdomyosarcomas at the injection site, and 5 of these neoplasms metastasized. In the 60 monocrotaline-treated rats, 31 widely dispersed tumors of various cell types were recorded. The reason suggested for the variation in tissue response was that the metabolite dehydroretronecine is a proximate carcinogen, whereas monocrotaline must first be metabolized before its carcinogenic potential is realized.


Effect of phosphonoacetate on Marek's disease virus replication. Phosphonoacetate (PA), but not any of its analogues tested, effectively inhibited avian herpesvirus replication and viral DNA synthesis in cell cultures. At 100 mug/ml culture medium, PA completely inhibited the replication of Marek's disease virus (MDV), herpesvirus of turkeys, and owl herpesvirus, but had no measurable effect on normal cell growth. PA also inhibited DNA polymerases induced by these avian viruses. Enzyme inhibition was 50% at a PA concentration of 0.2 mug/ml. At a concentration of 3-6 mug/ml, the compound also effected a 50% inhibition of alpha (maxi) enzyme of the host DNA polymerase. It had no effect on the host beta (mini) enzyme. When administered to chickens, PA did not inhibit the replication of MDV, nor did it prevent the development of lymphoma.


Regression of established tumors and induction of tumor immunity by intratumor chemotherapy. The inoculation of a mixture of drugs and guinea pig hepatoma cells (line-10) induced tumor-specific immunity in about 20% of guinea pigs. When guinea pigs with established intradermal tumors were given various drugs ip, no cures were observed; in contrast, multiple intralesional injections of actinomycin D, 1,3-bis(2-chlorethyl)-1-nitrosourea, adriamycin, mitomycin C, and melphalan were effective in curing animals of their intradermal tumors at a time when there were tumor cells in the draining lymph nodes; dimethyl-triazenoimidazole carboxamide, methotrexate, 5-fluorouracil, and 6-mercaptopurine were not effective. More than 80% of the cured animals were immune to rechallenge with 10(6) line-10 tumor cells.


Epithelial cell cultures from normal and cancerous human tissues. Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal.


Virus-like particles in geometric tubuloreticular structures. Tubuloreticular structures of the geometric type were observed in dog osteosarcoma cells (D17a) before and after cocultivation with human placenta cells and before and after passage of the cocultivated cells through NIH nude mice. After passage through NIH nude mice, the reestablished cultures regularly produced conventional murine type-C particles and displayed budding virus-like particles (VLP) within the tubuloreticular structures. VLP, tentatively considered an aberrant form of type-C particles, were presumably of murine origin since they were not observed in the osteosarcoma cells before passage through NIH nude mice.


Humoral immune responses of cats to feline leukemia virus: comparison of responses to the major structural protein p30 and to a virus-specific cell membrane antigen (FOCMA). Radioimmunoprecipitation was used to test cat sera for ability to bind to the purified major internal protein p30 of feline leukemia viurs (FeLV), to the endogenous cat virus (RD-114), and to murine leukemia virus (MuLV). The data were compared with results of tests for antibody to the feline oncornavirus-associated cell membrane antigen FOCMA and for the presence of viremia. In contrast to the general lack of free antibody to FeLV p30 in a random sample of healthy cats, high levels of antibody to FeLV p30 and FOCMA were found in normal animals from high-leukemia-cluster households. Titers of greater than or equal to 200 for p30 and greater than or equal to 32 for FOCMA were found in nonviremic animals; a percentage of animals with high FOCMA titers and lower or no p30 binding activity were viremic. Animals with neoplasms were low or negative for FOCMA antibody and did not have high titers of free p30 antibody. The p30 binding activity could be divided into three main categories: high binding with FeLV p30 and much lower activity with RD-114 and MuLV p30's, as seen with hyperimmune sera; high binding with FeLV and RD-114 p30's and low activity with MuLV p30, possibly indicative of specific antibody to both of the aforementioned proteins; and low level binding to all three p30's.


Hamster ependymomas produced by intracerebral inoculation of a human papovavirus (MMV). Ependymomas were produced in 3 of 11 newborn hamsters inoculated intracerebrally with a human papovavirus (MMV) isolated from a malignant lymphoma of the brain of a child with Wiskott-Aldrich syndrome. One of the tumors was serially transplanted into weanling hamsters. Cells from the transplanted tumors and from cell cultures derived from these tumors contained an intranuclear "T" antigen that reacted with simian virus 40 T antibody.


Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA. Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues.


Endonucleases and simian virus 40 virions: origin of a virion-associated endonuclease. The origin and role of the endonuclease activity associated with purified virions of simian virus 40, previously described by this and other laboratories, have been further investigated. We found that the enzymatic activity from virions of temperature-sensitive (ts) mutants is not more heat labile than that from wild-type virions. This result was obtained for a variety of ts mutants, including three of the tsA class, and in experiments in which the enzyme was tested in both the presence and absence of viral particles. Comparison of the viron enzyme with endonucleases prepared from either serum or nuclei of uninfected cells reveals a similarity between the viron and serum enzymes based on chromatographic behavior and relative activity with different cations. Virus particles prepared free of this endonuclease were still infectious. We were unsuccessful in uncovering endonuclease in such preparations upon disruption. These data emphasize the necessity for caution in interpreting the role of particle-associated enzymes.


Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus. The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle.


DNA synthesis in polyoma virus infection. IV. Mechanism of formation of closed-circular viral DNA deficient in superhelical turns. A marked reduction in the rate of viral DNA synthesis is accompanied by an alteration to the superhelicity of progeny DNA in polyoma virus-infected cells in which protein synthesis has been inhibited by cycloheximide. Viral DNA molecules formed in the presence of cycloheximide consist predominantly of closed-circular monometric species (referred to as form Ic) characterized by a decreased superhelix density, corresponding to deltasigmao = 0.0195, as compared to form I DNA by propidium diiodide-cesium chloride isopycnic analysis. Form Ic is synthesized on pre-existing form I templates without the intervention of progeny form I as an intermediate. It is concluded that inhibition of protein synthesis results in the alteration of some process in the closure of daughter DNA that leads to a marked reduction of superhelical turns of progeny molecules. About two-thirds of form Ic molecules return to the form I conformation upon reversal of cycloheximide inhibition by a mechanism independent of DNA replication.


DNA synthesis in polyoma virus infection. V. Kinetic evidence for two requirements for protein synthesis during viral DNA replication. Protein synthesis in polyoma virus-infected cells was inhibited by 99% within 4 min after exposure to 10 mug of cycloheximide per ml. Subsequent to the block in protein synthesis, the rate of viral DNA synthesis declined via inhibition of the rate of initiation of new rounds of genome replication (Yu and Cheevers, 1976). This process was inhibited with complex kinetics: within 15 min after the addition of cycloheximide, the rate of formation of closed-circular viral DNA was reduced by about one-half. Thereafter, DNA synthesis in cycloheximide-treated cells declined more slowly, reaching a level of 10% of untreated cells only after approximately 2 h. Protein synthesis was also required for normal closure of progeny form I DNA: in the presence of cycloheximide, DNA synthesis was diverted from the production of form I to form Ic, a monomeric closed-circular DNA component deficient in superhelical turns (Yu and Cheevers, 1976). Form I is replaced by Ic with first-order exponential kinetics. It is concluded that at least two proteins are involved in the control of polyoma DNA replication. One is apparently a stoichiometric requirement involved in the initiation step of viral DNA synthesis, since this process cannot be maintained at a normal rate for more than a few minutes in the absence of protein synthesis. The second protein requirement, governing the closure of newly synthesized progeny DNA, is considered distinct from the "initiation" protein on the basis of the kinetic data.


Lack of infectivity of the endogenous avian leukosis virus-related genes in the DNA of uninfected chicken cells. The infectivity of the avian leukosis virus-related genes in the DNA of four genetically distinct types of chicken cells was determined. Infectious DNA of Rous-associated virus-O(RAV-O) was obtained from V- chicken cells which were experimentally infected with RAV-O and from V+tvbs chicken cells, which spontaneously produced RAV-O and were sensitive to exogenous RAV-O infection. However, infectious DNA of RAV-O was not obtained from uninfected V- chicken cells or from V+tvbr chicken cells, which spontaneously produced a low titer of RAV-O but were resistant to exogenous RAV-O infection. No detectable amplification of the RAV-O related DNA sequences in the V+tvbs cells was found by hybridization of RAV-O 125I-labeled RNA to the DNAs of V+tvbs and uninfected V- cells. These results indicate that the endogenous avian leukosis virus-related genes in uninfected V- and V+tvbr cells differ from the RAV-O proviruses in RAV-O-infected V- and V+tvbs cells. The lack of infectivity of the DNA of V+tvbr cells is consistent with the hypothesis that the endogenous RAV-O genome in V+tvbr cells is linked to a cis-acting control element, which results in its inefficient expression.


Effect of medium of lowered NaCl concentration on virus release and protein synthesis in cells infected with reticuloendotheliosis virus. When cultures producing reticuloendotheliosis virus were incubated for 24 h in medium of lowered NaCl concentration, virus production was inhibited. The extent of inhibition increased as the salt concentration of the medium was decreased. The inhibition was rapidly reversed by replacement of low-salt medium with normal medium. During the first hour after the inhibited cultures were returned to normal medium, virus was released at an accelerated rate, making the total amount of virus released by inhibited and control cultures the same. After 1 h in normal medium, the rate of virus production in the previously inhibited cultures was the same as in the control cultures. Incubation of infected cells in low-salt medium resulted in a 60% decrease in the overall rate of protein synthesis. Although returning the cells to normal medium rapidly reversed the inhibition of virus production, it did not rapidly increase the rate of protein synthesis. These results suggest that host cell-directed protein synthesis is preferentially inhibited by the low-ionic-strength medium, whereas that required for virus production continues.


Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells. Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.


Anatomy of herpes simplex virus DNA. V. Terminally repetitive sequences. Native DNA from four strains of herpes simplex virus 1 (HSV-1) circularized after digestion with the lambda exonuclease, indicating that the molecules were terminally repetitious. In two strains, the terminal repetition was evident in nearly 50% of the DNA molecules. Maximal circularization was observed when only 0.25 to 0.5% of the DNA was depolymerized by the exonuclease, suggesting that the minimal size of the terminally repetitious regions is in the range of 400 to 800 bases pairs. More extensive exonuclease treatment resulted in a reduction in the frequency of circularization. To determine whether the terminally repetitive regions themselves contained self-annealing sequences that were precluding circularization of more extensively digested DNA, the terminal fragments from HinIII restriction endonuclease digests were isolated, denatured, and tested for their ability to self-anneal. The results of hydroxyapatite column chromatography and electron microscope examination of the terminal regions are consistent with this hypothesis.


Persistent reovirus infection of CHO cells resulting in virus resistance. We obtained a persistently infected line of Chinese hamster ovary cells by selection for resistance to reovirus infection. The cells were persistently infected by a population of viruses that were (i) cytopathic for parental chinese hamster ovary cells and (ii) similar to wild-type reovirus in molecular characteristics. The growth rate, plating efficiency, and morphology of the cells were altered. A large majority of the cells in the population were infected. There was no detectable interferon present in the medium. The cells were relatively resistant to a wide range of viruses.


Genomic complexities of murine leukemia and sarcoma, reticuloendotheliosis, and visna viruses. The genetic complexities of several ribodeoxyviruses were measured by quantitative analysis of unique RNase T1-resistant oligonucleotides from 60-70S viral RNAs. Moloney murine leukemia virus was found to have an RNA complexity of 3.5 x 10(6) daltons, whereas Moloney murine sarcoma virus had a significantly smaller genome size of 2.3 x 10(6). Reticuleondotheliosis and visna virus RNAs had complexities of 3.9 x 10(6), respectively. Analysis of RNase A-resistant oligonucleotides of Rous sarcoma virus RNA gave a complexity of 3.6 x 10(6), similar to that previously obtained with RNase T1-resistant oligonucleotides. Since each of these viruses was found to have a unique sequence genomic complexity near the molecular weight of a single 30-40S viral RNA subunit, it was concluded that ribodeoxyvirus genomes are at least largely polyploid.


Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B. The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.


Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy. We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.


High-titer replication of nondefective Sendai virus in MDBK cells. Egg-grown Sendai virus was adapted to growth in a bovine kidney cell line (MDBK cells) by serial passage under defined conditions. The adapted virus contained only 50S RNA and was highly infectious for MDBK cells. Infection of these cells with a high multiplicity of adapted virus resulted in a yield of 10(8) MDBK-infectious units/ml by 18 h, accompanied by severe cytopathic changes in the host. Cell fusion did not occur. Examination of the proteins of the adapted virus revealed that despite the high infectivity of this virus for MDBK cells the virions contained considerable quantities of Fo, the precursor to the F glycoprotein that is responsible for cell fusion and high infectivity in other systems.


Kinetics of utilization of Sendai virus RNA and protein in the process of virion assembly. The synthesis of the 50S genomic RNA and strucural proteins of Sendai virus was examined with respect to their utilization in virus assembly. It was found that during a single cycle of infection, 50S RNA was synthesized before the structural proteins and that both RNA and protein were synthesized 2 to 4 h before their appearance in released virions. Pulse-chase labeling indicated that the NP and P proteins synthesized early and the M and F proteins synthesized late were preferentially incorporated into virus relative to the other viral proteins. The kinetics of incorporation of pulse-labeled NP protein suggested that it was withdrawn from a relatively large pool whereas the M protein appeared to be present in a relatively small pool in the cytoplasm. Further, it was possible to chase pulse-labeled M protein, but not NP protein, from the cell during an 8-h time period.


Simian virus 40 DNA replication: characterization of gaps in the termination region. A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I. A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4). These pDNA II molecules have been isolated and further characterized. They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase. After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-[32P]dATP is incorporated per mol of DNA II. Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region. alpha-[32P]dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules. This indicates the presence of gaps in both the newly synthesized plus the minus strands. Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J. pDNA II molecules have the following properties. There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule. Nicked pDNA II molecules cannot be detected. The two molecules that arise by segregation contain gaps in both of the complementary strands. Based on the amount of alpha-[32P]dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides. Models for termination of DNA synthesis are proposed based on these findings.


Immunocytochemical localization of simian virus 40 T antigen with peroxidase-labeled antibody fragments. T antigen was localized in simian virus 40 lytically infected and transformed cells by Fab' antibody conjugates at the ultrastructural level. This virus-specific protein appeared in the cytoplasm of permissive cells as early as 3 h after infection. At later time intervals, the T antigen was localized in both the cytoplasm and nucleus and finally (24 h) in the nucleus. These results suggest a synthesis of T antigen on cytoplasmic ribosomes, with subsequent transfer to the nucleus.


Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen. The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.


Proposed structure of two defective viral DNA oligomers produced in 3T3 cells transformed by the ts-a mutant of polyoma virus. The various oligomeric viral DNA species produced at 32 C by two related syblines of ts-a-transformed mouse 3T3 cells were characterized. Results from the analysis of the cleavage products observed after digestion with restriction endonucleases from Haemophilus parainfluenzae, Escherichia coli RI, and Haemophilus suis are consistent with the assumption that in both sublines, the major oligomeric component is a dimer from which a segment of different length is deleted. The major oligomeric (27S) component in subline 1 was estimated to be 1.77 times the size of the viral monomer, and the major (25.5S) component in subline 15 was estimated to be 1.54 times the size of the viral monomer. These size estimates were confirmed by electron micrograph measurements. The larger oligomers produced by both sublines were found to be multiples of the major oligomeric component of each subline.


Nuclear membrane changes in herpes simplex virus-infected BHK-21 cells as seen by freeze-fracture. The freeze-fracture technique, which produced high-resolution replicas of large internal faces of membranes, was used for an ultrastructural study of the nuclei of herpes simplex virus-infected BHK-21 cells and mock-infected controls. Crystalline arrays of viral nucleocapsids were found in the nucleoplasm of infected cells, and numerous nuclear membrane "blebs" and protrusions were observed. The numerous areas of membrane distortions were not found to contain nuclear pores. In addition, specific areas of normal protein intramembranous particles are deleted from certain areas of the nuclear membrane as a result of herpes simplex virus, type 2, infection.


DNA-negative temperature-sensitive mutants of herpes simplex virus type 1: patterns of viral DNA synthesis after temperature shift-up. Temperature-sensitive mutants of herpes simplex virus type 1 belonging to four DNA- complementation groups exhibited two distinct patterns of viral DNA synthesis after shift-up to the nonpermissive temperature. In cultures infected with mutants belonging to complementation groups A, C, and D, little or no viral DNA was synthesized after shift-up. In cultures infected with a mutant in complementation group B, nearly normal amounts of viral DNA were synthesized after shift-up.


Herpesvirus proteins: induction of nucleoside phosphotransferase activity after herpes simplex virus infection. After herpes simplex virus infection of hamster kidney cells there is an induction of nucleoside phosphotransferase activity which can utilize AMP as phosphate donor. The activity is immunologically specific for the infected cell and is induced concomitantly with the virus-coded pyrimidine deoxynucleoside kinase activity. Phosphotransferase activity is not induced in cells lacking both thymidine and deoxycytidine kinase activity.


Complementation between BK human papovavirus and a simian virus 40 tsA mutant. Complementation tests between BK human papovavirus and SV40 temperature-sensitive mutants tsA58 and tsB11 were performed. Under the reported experimental conditions, BKV complemented the "early" mutant tsA58 but failed to complement the "late" mutant tsB11.


Polyoma and cell DNA synthesis in mouse L cells temperature sensitive for the replication of cell DNA. Polyoma (Py) virus multiplies, at 34 and 38.5 C, in wild-type (WT-4) and in ts A1S9 mouse L cells, which are temperature sensitive for growth and for DNA replication (R. Sheinin, 1976; L. H. Thompson et al., 1970). De novo synthesis of double-stranded, fully covalently closed Py DNA has been shown to proceed by semiconservative replication in WT-4 and ts A1S9 cells at the permissive and nonpermissive temperatures. Cell DNA is made late during infection, by both cell types and at both temperatures. Semiconservative replication of cell DNA proceeds in Py-infected WT-4 cells incubated at 34 or at 38.5 C and in Py-infected ts A1S9 cells incubated at 34 C. In virus-infected ts A1S9 cells incubated at 38.5 C, cell DNA synthesis appears to proceed almost entirely by a process analogous to repair replication. The inability of ts A1S9 cells to produce large-molecular-weight chromosomal DNA strands, at 38.5 C, by the normal mechanism is not overcome by Py infection.


Synthesis of complementary RNA containing polyadenylic acid by Sendai virions in vitro. Sendai virus synthesized, in vitro, [32P]AMP- and [3H]AMP-labeled RNA that ranged in size from 3 to 25S with major peaks at 7S and 13S. Both labeled products were predominantly single-stranded RNA and were complementary in base sequence to 50S virion RNA. Passage of the 3 to 25S in vitro RNA transcripts through a polyuridylic acid-cellulose column revealed that only the larger (predominantly 18S) RNA transcripts contained polyadenylic acid[poly(A)] segments capable of binding to the column. After treatment with a combination of RNase A and T1, the majority of the in vitro poly(A) sedimented at 6S although the product ranged in size from 3 to 9S. Proof that the RNase-resistant material was indeed poly(A) was obtained by nearest-neighbor analysis when 95% of the radioactivity was recovered in AMP.


Quantitation of the viral DNA present in cells transformed by UV-irradiated herpes simplex virus. Two cell lines biochemically transformed by UV-irradiated herpes simplex virus (HSV) each contain virus DNA. A comparison of the kinetics of reassociation of 3H-labeled HSV DNA in the presence and absence of either clone 139 (HSV-1 transformed) or clone 207 (HSV-2 transformed) DNA showed that the presence of transformed cell DNA increased the rate of reassociation of approximately 10% of the viral genome while having no effect on the remaining 90%. The Cot1/2 of this reaction was approximately 1,000 in each cell type, as compared to approximately 3,000 for the cellular unique sequences. These results suggest the presence of four to six copies of a 10% fragment of the virus DNA per cell. The DNA from a hamster fibroblast cell line morphologically transformed by UV-irradiated HSV-2 (333-8-9) did not affect the rate of reassociation of HSV-2 DNA, indicating that these cells had less than 3% of a viral genome present.


Factors involved in the generation and replication of rhabdovirus defective T particles. Previous indications that cloned B virions might be genetically predisposed to generate a particular defective T particle are shown to be inaccurate. T particle generation was found to be a much more random process than was previously believed. We show that the previously observed generation of particular sizes of T particles by B virion pools is due to the random generation of T particles during preparation of first-passage pools of cloned B virions, and these breed true during the additional passages needed to produce visible quantities of T particles. It is also shown that different host cell lines selectively amplify different T particles, suggesting a strong role of host cell factors in T particle replication. Surprisingly, our line of HeLa cells did not generate or replicate detectable T particles of vesicular stomatitis virus (VSV) Indiana after either serial undiluted passage or direct addition of T particles, even though the added T particles strongly interfered with B virion replication. In contrast to VSV, rabies virus generates large amounts of T particles during the first passage of cloned B virions, and every rabies-infected baby hamster kidney-21 cell culture evolves into a persistent carrier state. We find that T particle RNA is biologically inactive although T particle nucleocapsid ribonucleoprotein replicates and interferes in cells coinfected with B virions. Efforts to study the mechanism of T particle generation by in vitro attempts to generate T particles or modify their size (using sheared ribonucleoprotein or chemical or UV mutagenesis) were unsuccessful. The kinetics of UV and nitrous acid inactivation of T particles indicate a smaller target size relative to B virions, even after correcting for lengths of RNA molecules. The intercalating dye proflavine does not photosensitize VSV B virions or T particles when present during replication, indicating that there is little or no RNA base pairing in the helical nucleocapsids of either.


Further characterization of intracytoplasmic A particle-associated DNA. A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.


Quantitation of bovine papilloma viral DNA in viral-induced tumors. Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.


Genome localization of simian virus 40 RNA species. The topographical locations on the simian virus 40 (SV40) genome of the templates for virus-specific RNA species present late in the lytic infection were determined by RNA-DNA hybridization experiments with the Hind restriction enzyme fragments. Two classes of late virus-specific cytoplasmic mRNA's can be separated on the basis of either sedimentation properties in neutral sucrose or electrophoretic mobility in polyacrylamide gels. In the 16S class, two species of RNA were identified by hybridization experiments. One of these species was complementary to sequences of the early template on the minus (E) strand (0.175 to 0.655 map units), and the other more abundant species was complementary to sequences present in the late template on the plus (L) strand (0.655 to 0.175 map units). In addition two species were detected in the 16S class of late cytoplasmic virus-specific mRNA. One of these species was the major late RNA detected and consisted of a polyadenylated transcript complementary to the plus (L) DNA strands of Hind fragments K, F, J, and G (0.945 to 0.175 map units). This species appears to specify the major capsid protein (VP1). A less abundant nonpolyadenylated 16S RNA species complementary to the plus (L) strands of Hind fragments C, D, and E (0.655 to 0.945 map units) may result from post-transcriptional processing or nonspecific degradation of the 19S viral RNA complementary to the plus (L) strand.


Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells. Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis.


Analysis of structural proteins of purified murine cytomegalovirus. Murine cytomegalovirus propagated in mouse embryo fibroblasts was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 x g for 90 min. (ii) The concentrated virus was passed through a Bio-Rad Bio-Gel A-15m column to eliminate contaminating materials smaller than 15 x 10(6) daltons. Most of the virus was recovered in the void volume of the column. (iii) Two consecutive centrifugations through 20 to 50% potassium tartrate gradients were performed. After the second tartrate gradient centrifugation, symmetrical, coinciding peaks of plaque titer, protein, and radioactivity were found at a density between 1.20 g/cm3 and 1.21 g/cm3. To establish purification criteria, virus was purified from two different mixtures: [35S]methionine-labeled extracellular virus, mixed with an equal volume of unlabeled normal culture fluid, and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. At the end of the procedure, the extent of purification, as judged by the ratio of cellular to viral radioactivity was at least 70-fold. Virus proteins were analyzed by electrophoresis on a 5 to 20% gradient polyacrylamide gel slab. After gel electrophoresis,, Coomassie brilliant blue staining profiles and autoradiograms of the purified virus preparations were compared. At least 33 virus structural protein bands were present. The molecular weights of these proteins ranged from 11,500 to 255,000. The sum of the molecular weights of the virus structural proteins was 2,462,000. Autoradiograms obtained from electrophoresis of purified [14C]glucosamine-labeled virus showed that at lease 6 of the 33 viral structural proteins were glycoproteins.


Analysis of simian virus 40 wild-type and mutant virions by agarose gel electrophoresis. Intact wild-type simian virus 40 particles can be separated and resolved from a temperature-sensitive mutant and from a number of other viruses by agarose gel electrophoresis. The relative mobilities of the viruses appear to be a function of both virion size and surface composition. The virions of a temperature-sensitive strain of simian virus 40, tsB204, have significantly greater mobility than those of wild-type simian virus 40, when electrophoresis was conducted toward the cathode at pH 5.0. When electrophoresis was performed toward the anode at pH 7.0, TSB204 viruses have a slightly slower mobility as compared with that of the wild type. The data indicated that the virions of tsB204 have a greater positive charge at their surface than those of wild-type particles. No differences were detected in the finger print patterns of the tryptic peptides of VP1 and VP3 of these two virus strains. Although it was not possible to identify the structural polypeptide directly affected by the tsB204 mutation, we have shown that this mutation affects charge distribution on the surface of the virion.


Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus. Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.


Membrane proteins specified by herpes simplex viruses. I. Identification of four glycoprotein precursors and their products in type 1-infected cells. Polypeptide precursors to the major glycoproteins specified by herpes simplex virus type 1 were identified in immunoprecipitation experiments using antisera that reacted specifically with the viral glycoproteins and their precursors. The results demonstrate that the major glycosylated proteins detected in infected cells are derived from four antigenically distinct polypeptides. Three of these polypeptides become glycosylated in two discrete stages, yielding partially glycosylated intermediates and fully glycosylated products. The final products are the predominant species detected in cytoplasmic virions and in plasma membranes. The fourth polypeptide precursor appears to acquire very little carbohydrate and differs in several respects from the other three precursors.


DNA polymerase activities induced by polyoma virus infection of 3T3 mouse fibroblasts. Resting mouse 3T3 fibroblasts were stimulated to synthesize DNA either by infection with polyoma virus or by injection of fresh serum. Changes in the levels of DNA polymerase (alpha-, beta-, and gamma-enzymes) were measured in the cytoplasm and the cell nucleus. Both types of stimulation gave very similar increases for all three enzyme activities. In the cell nucleus, both alpha- and gamma-polymerases increased almost tenfold, whereas the beta-enzyme only was stimulated twofold. In the cytoplasm alpha- and gamma-polymerases increased two- to four-fold. Only insignificant amounts of the beta-enzyme were found in the cytoplasm.


Protein synthesis in bacteriophage ghost-infected cells. Escherichia coli B infected with T4 phage ghosts at 10 mM Mg2+ regains its protein synthesizing activity upon addition of ATP, GTP, and their generator to approximately 2% of the intact exponentially growing cells. In contrast to amino acid incorporation by intact cells, this system is sensitive to EDTA or low Mg2+. On the other hand, this system, differing from the regular cell-free system, does not respond to addition of soluble protein and ribonuclease. The ghost-infected cells were able to synthesize beta-galactosidase upon addition of the inducer isopropyl thiogalactoside. The initial rate of the induction was 2.6% of intact cells. For this induction, the addition of cyclic AMP, amino acids, ATP, GTP, UTP, CTP, and their generator was necessary. The induction of beta-galactosidase in these ghost-infected cells was very sensitive to the addition of EDTA, CaCl2, sulfhydryl blocking reagent, rifampin and chloramphenicol but insensitive to DNA synthesis inhibitors such as nalidixic acid and DNase.


Characterization of the type and group specificities of the immune response in mice to murine leukemia viruses. Normal sera from a variety of strains of inbred mice have precipitating antibodies to murine type C viruses that are detected by radioimmune precipitation assays. The results demonostrate that this humoral immune response is primarily directed against the AKR strain of murine leukemia virus (MuLV) proteins gp71, gp43, and p15(E). These sera also react with Friend- or Rauscher-MuLV in radioimmune precipitation assays. This reaction is not due to a separate immune response, but rather is primarily a consequence of the cross-reactivity of antibodies to the AKR strain of MuLV p15(E) with the p15(E) of these viruses. These data, using autogenous immune sera, emphasize the serological differences of the virion glycoproteins and the serological similarity of the p15(E) virion component of the viruses. Furthermore, based on the serological reactivities to the glycoproteins, the results suggest that the AKR strain of MuLV is endogenous to and expressed in mice, but that the Friend-Moloney-Rauscher virus group is not.