The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene.

The complete 648 amino acid sequence of the human raf oncogene was deduced from the 2977 nucleotide sequence of a fetal liver cDNA. The cDNA has been used to obtain clones which extend the human c-raf-1 locus by an additional 18.9 kb at the 5' end and contain all the remaining coding exons.     

Kinetics of expression of infectious ecotropic, xenotropic, and mink cell focus-forming murine leukemia virus after 5-iododeoxyuridine induction of cells from high- and low-leukemia mouse strains

Endogenous murine leukemia virus (MuLV) was induced with 5-iododeoxyuridine (IdUrd) from the high-leukemia mouse strain AKR and from two low-leukemia strains, C3H/He and BALB/c. A virus-free cell line from strain AKR readily gave rise to infectious, XC-positive MuLV upon treatment with IdUrd, whereas cells from strains C3H/He and BALB/c produced replication-deficient, XC-negative MuLV. IdUrd-induced cells also produced xenotropic and mink cell focus-forming MuLV. Xenotropic virus emerged at a higher titer from both AKR and BALB/c cells during two discrete time intervals, first at day 3 after induction and a second time during spread of the induced ecotropic MuLV. Xenotropic and mink cell focus-forming MuLVs were also produced by IdUrd-induced C3H/He cells but required another round of infection in Sc-1 cells for detection. The in vitro infectivity of endogenous ecotropic MuLV isolated by IdUrd induction from C3H/He cells correlated with pathogenicity in the Fv-1-compatible, leukemia-negative mouse strain NFS/N. Thus, the virulence of endogenous ecotropic MuLV may be an important factor in determining the leukemia incidence in these inbred strains of mice.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Properties of Hamster Embryo Fibroblasts Transformed In Vitro After Exposure to Ultraviolet-Irradiated Herpes Simplex Virus Type 2

An in vitro method which led to the transformation of hamster embryo fibroblasts after exposure to herpes simplex virus type 2 (HSV-2) inactivated with ultraviolet irradiation is described. The transformed cells (333-8-9) produced tumors when inoculated into newborn Syrian hamsters but not when injected into weanling Syrian hamsters of the same LSH inbred strain. However, after one in vivo passage, the 333-8-9 cells became highly oncogenic in weanling hamsters. No infectious virus was recovered from these cells. Herpes simplex virus antigens were detected in the transformed cells by the indirect immunofluorescence technique. Sera from tumor-bearing hamsters contained antibody with highly specific neutralizing activity against HSV-2. These studies indicate the continued involvement of the HSV-2 genome in an oncogenic cell line.     

1,3-Beta-glucan synthase in cell-free extracts from mycelium and protoplasts of Sclerotium glucanicum

Conditions for obtaining stable protoplasts from Sclerotium glucanicum and their reversion to hyphal growth were determined. 1,3-beta-Glucan synthase activity was detected in particulate enzyme fractions from mycelium and protoplasts of Scl. glucanicum. UDP-[U-14C]glucose was linearly incorporated into a beta-glucan for about 20 min at 25 degrees C. Optimum pH and temperature values, as well as thermal stabilities of the 1,3-beta-glucan synthase activity, were determined. High concentrations of EDTA were inhibitory. Enzyme activity was stimulated by ATP and GTP. The apparent Km value for UDP-glucose was 0.54 mM. The reaction product was characterized as 1,3-beta-glucan by 13C NMR spectroscopy and hydrolysis products of an exo-1,3-beta-glucanase.     

Susceptibility to raf and raf/myc retroviruses is governed by different genetic loci.

The susceptibility to tumors induced by raf and raf/myc retroviruses was investigated in BALB/c, C57BL/6, (BALB/c x C57BL/6)F1 and (BALB/c x C57BL/6) backcross mice. Newborn mice were susceptible to neoplasms generated by both viruses, but resistance to raf-induced leukemia developed rapidly in all mice as they matured. Older C57BL/6 mice were also resistant to raf/myc lymphomas, whereas BALB/c mice remained susceptible to the virus at all ages, indicating that different genes control susceptibility to raf and raf/myc tumors. From these data and the susceptibility of C x B recombinant inbred strains, it appears that very few genes (perhaps even a single gene) may govern susceptibility to raf/myc lymphomas and that resistance is the dominant trait.     

raf/myc-infected erythroid cells are restricted in their ability to terminally differentiate.

A comparison was made of the in vitro erythroid colony-forming abilities of v-raf-, v-myc-, and v-raf/v-myc-containing retroviruses. In methylcellulose, v-raf efficiently produced colonies of well-differentiated hemoglobin-synthesizing erythroid cells, whereas v-raf/v-myc-infected erythroid cells were inhibited from terminally differentiating but retained the ability to replicate extensively. In contrast, v-myc was unable to stimulate the formation of erythroid colonies.