Abstract. Myeloma cells derived from BALB/c and C3H mice show evidence of infection by a murine leukemia virus. The immunoglobulin-producing myelomas secrete an RNA-containing virus with a density of 1.20 to 1.22 gm/cm3. RNA with a sedimentation coefficient of 74 S in 0.1 M sodium chloride has been isolated from secreted virus particles and has a base composition similar to that found for other murine leukemia virus RNA. An intracellular virus particle has been partially purified and has a density of 1.29 to 1.32 gm/cm3. Both extracellular and intracellular virus particles contain the leukemia virus group-specific antigen.Ultrastructural characteristics of viruslike particles in the cytoplasm of mouse plasma cell tumors have been described.1-' Here we identify the extracellular and intracellular particles from cultures of myeloma cells secreting immunoglobulin, and from spontaneous variants of these cell lines which secrete only the heavy or light chains of immunoglobulins, as viruses related to the murine leukemia viruses.Materials and Methods. Cell lines: C1 cells were derived from a myeloma of a C3H mouse; the other tumor lines were derived from oil-induced myelomas of BALB/c mice (see Table 1). Cell lines were cloned in the spleen or in agar,4 and grown as suspensions in fortified Eagle's medium'0 supplemented with 10 to 20% horse serum. All experiments were performed on exponentially growing cultures with doubling times of 18 to 24 hr.Virus purification: (a) Extracellular virus: Cells and debris were removed from the medium by two centrifugation steps (150 g for 5 min and 12,000 g for 10 min). The medium was layered over 2 ml 15% potassium tartrate containing 0.1 M Tris-HCl buffer (pH 8.0) and centrifuged in a Spinco SW25.3 rotor at 25,000 rpm for 60 min. The pellet was resuspended in 0.1 M Tris-HCl buffer (pH 8.0) and layered over a 4.8-ml gradient of 15 to 60% potassium tartrate containing 0.1 1I1 Tris-HCI buffer (pH 8.0)," and centrifuged in a Spinco SW65 rotor at 35,000 rpm for 60 min.(b) Intracellular virus: Cells were washed twice in NET buffer (0.1 M NaCl, 0.05 M Tris-HCl (pH 7.4) 0.001 ill EDTA), collected by low speed centrifugation, resuspended in NET buffer (approx. 106 cells/ml), and stored at -15'C until used.After thawing, Nonidet P40 was added to cell suspensions to a final volume of 1%, the solution pipetted vigorously to ensure lysis of all cells, and then centrifuged at 12,000 g for 10 min. The supernatant was layered over 2 ml 15% sucrose containing NET and centrifuged for 60 min in a SW65 rotor at 30,000 rpm. The pellet was resuspended in 0.1 M Tris-HCl buffer (pH 8.0) and analyzed by tartrate density-gradient centrifugation.CsCl-equilibrium sedimentation: Virus solutions were layered over 5 ml previously formed CsCI gradients (1.08-1.43 gm/cm3) and centrifuged for 20 hr at 15'C in a Spinco SW65 rotor at 50,000 rpm.
High titered anticarbohydrate antibodies were used to identify cell surface carbohydrates during different stages in histogenesis of mouse cerebellum in a micro tissue-culture system which mimics selected features of in vivo cerebellum development. Blockage of fiber formation within the first few days in vitro and inhibition of cell migrations by carbohydrate-specific antibodies served as an assay system for possible contributions of surface carbohydrates to the behavior of developing cerebellar cells. Microbial strains were selected on the basis of carbohydrate structures of their cell wall antigens, and anticarbohydrate antibodies were raised against treated whole bacteria and yeast in rabbits. We found that antibodies to mannan were active at all stages of development tested (embryonic day 13, E13; the day of birth, PO; and postnatal day 7, P7). Antibodies to sialic acids prepared against strains B and C of Neisseria meningitidis distinguish different subterminal structures: anti-B reacted with E13 and PO cerebellar cells, and anti-C mostly with cells older than P7. Antifetuin antibody recognized E13 and PO but not P7 cell populations. Pneumococcus C strain R36A-specific antibodies were effective only after coating cells to C type carbohydrate before application of the antibody. The results demonstrate that antimicrobiol carbohydrate antibodies cross-react with mammalian cell surface carbohydrate structures and therefore can be used as a powerful tool in tissue culture to analyse those structures which might control cell behaviors pertinent to cerebellar development.
Monoclonal antibodies specific to HLA antigens and the fluorescence-activated cell sorter were used to analyze the changes in the density of human histocompatibility antigens HLA-AB and HLA-DR on the surface of synchronously growing WI-L2 cells (a human B cell line) progressing through the cell cycle. The WI-L2 cells were synchronized by densitydependent arrest in GI, and samples from Go, G1, late S and late G2 phases were used to determine the frequency distribution of cell volume, DNA content, and the relative amounts of cell surface HLA antigens; the observed density changes were calculated from these values. The HLA-AB density remained nearly constant throughout the cell cycle, whereas the HLA-DR density increased sharply at the G2-M stage. These results suggest a cell cycle-dependent differential control of the expression of these two sets of histocompatibility antigens on B cells.
The frequency distribution of DNA content of human sperm was measured in an automated flow microfluorometer. The flow method measures the DNA content by quantifying the amount of fluorescence emitted by the fluorescent Feulgen stained DNA of single sperm cells suspended in microdroplets. The variability in the mean value for the haploid amount of DNA in sperm from 15 randomly chosen donors was less than 1%. Statistical tests on the observed frequency distribution data indicated that each sperm population probably conisists of two homogenous components present in almost equal proportions but differing in mean DNA content. The difference in their modal values for DNA is within the range of known values of DNA difference between the two sex chromosomes.Sperm of donors segregating balanced translocations, when compared to the random samples as a class, showed greater variability in the mean DNA content.Sperm population in ejaculates may be regarded as a statistical sample of all meiotic products in the male (1, 2). With the availability of the rapid flow microfluorometer (3, 4) it may become possible to detect population heterogeneity by the direct examination of individual sperm cells as haploid segregants. The genetic information obtained through this procedure should be fruitful in interpreting certain cytogenetic observations associated with abnormal transmission ratios (5, 6). In addition one may now ask, what is the extent of variability in the segregation of DNA in meiosis compared to mitosis? Does somatic cell differentiation or replication intrinsically select for least variability in the DNA content (3), whereas meiosis (7) in contrast, purposefully provides mechanisms to achieve more variability in gametes?In this report we describe the population heterogeneity of human sperm in the haploid DNA content, and the extent to which the variability could be determined by the segregation of specific chromosomes with structural rearrangements.
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