Showing papers in "Journal of Cellular Physiology in 1967"

A cytological study of the capacity for differentiation of normal hemopoietic colony-forming cells.

Abstract: A two-stage procedure has been used to obtain hemopoietic spleen colonies derived from single precursor cells containing radiation-induced chromosomal markers. Of a total of 46 colonies examined, 17 were found to contain cells with abnormal karyotypes. In each of the 17 marked colonies, 90% or more of the dividing cells in the colony carried the same marker. Cell suspensions prepared from each of the individual colonies were tested for their content of dividing cells possessing recognizable differentiated functions. Metaphase cells with peroxidase-positive granules in their cytoplasm were considered to be members of the granulopoietic series, while metaphase cells which contained Fe55 were considered to be members of the erythropoietic series. Results were obtained for 12 of the marked colonies, and in nine of these, the percentage of metaphases lacking the marker was less than the percentage of metaphases which were scored as erythropoietic, and also was less than the percentage of metaphases scored as granulopoietic. This is the result which would be expected if the marker were present in both erythropoietic and granulopoietic cells. These results provide support for the view that colony forming hemopoietic stem cells are multipotent, and that differentiation along more than one pathway can occur during the formation of macroscopic splenic colonies.

Properties of mitotic cells prepared by mechanically shaking monolayer cultures of Chinese hamster cells.

Abstract: A technique has been developed for the selective detachment of mitotic cells from monolayer cultures of Chiness hamster cells with a simple reciprocating shaking machine. Cultures prepared by the shake treatment and placed in spinner flasks are routinely obtained, in which the mitotic fraction drops from 0.95–0.05 in 19 minutes. Some properties of mitotic cells prepared by this technique are described, along with a simple procedure for producing large quantities of mitotic cells. Cells chilled immediately after collection from a series of shake treatments complete mitosis synchronously upon subsequent resuspension in warm medium.

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Abstract: Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.

Erythrocyte membrane sulfhydryl groups and cation permeability

Abstract: Reaction of the slowly penetrating organic mercurial compound parachloromercuribenzene sulfonate (PCMBS) with intact erythrocytes has been characterized. Addition of concentrations of PCMBS which result in binding within the interior of the membrane of more than 1.9 × 10−18 moles/cell produces alterations in Na+ and K+ permeability, but does not affect choline permeability. However, the increased cation permeability is observed only after a lag period of over two hours. After ten hours, a spontaneous slow “recovery” to normal rates of K+ leakage occurs at 25°C but not at 2°C. Subsequent to the effects on cation balance, increasing degrees of hemolysis occur, interpreted as colloid osmotic lysis. The relationships between the binding of the agent and its effects are as follows: a small, rapid initial uptake does not affect cation permeability; the subsequent slower uptake is associated with increased leakage of K+ and Na+; and the recovery at 25°C is associated with desorption of about half of the PCMBS due to competition by soluble thiol substances released into the medium from the cells. Desorption and “recovery” can be mimicked at any time by addition of small amounts of protein in the medium. The half of the PCMBS that cannot be desorbed is assumed to be bound by the hemoglobin inside the cell. The sulfhydryl groups involved in control of cation permeability constitute only a fraction of the total within the membrane (4–18%). They are located within the interior of the membrane separated from the medium and from the interior of the cell by diffusion barriers to PCMBS.

Studies on carcinogenesis by avian sarcoma viruses. V. Requirement for new DNA synthesis and for cell division

Abstract: Partially synchronized secondary cultures of chicken embryo fibroblasts were exposed to Rous sarcoma virus (RSV) at various times in the cell cycle. The initiation of normal virus production could be inhibited by treatment with excess thymidine or with cytosine arabinoside without any effect on mitosis. This result is consistent with the hypothesis that the provirus of RSV is DNA, although the virion of RSV contains only RNA. It was, also, found that treatment which prevented or interferred with the first mitosis after exposure to virus prevented the initiation of normal virus production. Later mitoses did not appear to be necessary. A corollary of this finding is that virus production is synchronized by mitosis and that the length of the latent period depends upon when in the cell cycle a cell is exposed to virus.

The beginning of a genetic analysis of recombination proficiency.

Abstract: Recombination-deficient mutants of Escherichia coli have been isolated as members of a class showing reduced ability to mother recombinants when crossed with a given Hfr strain. All those showing less than one-tenth the ability of their immediate ancestors in mothering merodiploids have been eliminated from consideration, as have those strains of reduced fertility with only one donor strain. The remainder are all more sensitive to ultraviolet light (UV) than were their immediate ancestors; differences in levels of sensitivity are evident. Mutants have been isolated from two distantly related ancestors, and the sets of mutants from each are divisible into three phenotypic groups. The strains in the first group are the least UV-sensitive (UVs), and lambda lysogens of these show normal spontaneous induction. The strains in the second and third groups are more UVs than those in Group 1, and lysogens of these show abnormally low spontaneous induction. Groups 2 and 3 are separable by their levels of fertility with the Hfr strain (KL16) found to transfer the rec+ allele of some of the strains early in conjugation. The strains more fertile with KL16 are in Group 2, and those less fertile are in Group 3. Two mutants (JC1553 and AB2463) of different parentage but both in phenotypic Group 2 have been analyzed by transduction; and a single mutation probably determines both their mutant UVs and Rec− phenotypes. When the mutants of different parentage are arranged together, a continuous ranking of fertilities is observed. Preliminary complementation tests show no complementation between those mutations carried by strains in Group 2 and Group 3 which have been tested. Complementation has been observed between those mutations carried by strains in Group 1 and those carried by strains in Groups 2 and 3.

Studies on carcinogenesis by avian sarcoma viruses. VI. Differential multiplication of uninfected and of converted cells in response to insulin

Abstract: Insulin can replace the factor(s) in calf serum whose amount is limiting for multiplication in cell culture of chicken embryo fibroblasts and of chicken embryo fibroblasts infected and converted by avian sarcoma virus. In serum-free, insulin-containing medium, converted cells multiply more than do uninfected cells. It appears, therefore, that the increased multiplication in cell culture of converted cells as compared with uninfected cells results from a decreased requirement by the converted cells for an insulin-like activity found in serum.

Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum

Abstract: An analysis has been made of cell colonies developing in agar cultures from mouse bone marrow cells following stimulation either by neonatal kidney cell feeder layers or AKR lymphoid leukemia serum. Colonies arose by cell proliferation and were mixtures of granulocytic and mononuclear cells. Colonies stimulated by kidney feeder layers reached a mean size of 2000 cells by day 10 of incubation and remained predominantly granulocytic in nature. When bovine serum was substituted for fetal calf serum, cell colonies grew to a smaller size and lost their granulocytic nature, finally becoming almost pure populations of mononuclear cells. Colonies stimulated by AKR leukemic serum reached a mean size of 350 cells by day 10 of incubation. Although these colonies initially were granulocytic in nature, they finally became almost pure populations of mononuclear cells. The colony mononuclear cells actively phagocytosed carbon, and contained metachromatic granules probably derived from ingestion of agar. The mononuclear cells in these colonies may not have been members of the original colony, but may have been incorporated in the colony as it expanded in size, subsequently proliferating in the favourable environment of the colony.

Correlation between cell enlargement and nucleic acid and protein content of HeLa cells in unbalanced growth produced by inhibitors of DNA synthesis.

Abstract: HeLa cells in monolayer cultures were treated with the following inhibitors of DNA synthesis: mitomycin C, nitrogen mustard, fluorodeoxyuridine, hydroxyurea, arabinofuranosylcytosine and high concentrations of thymidine. The concentration of each inhibitor used was, in most cases, just sufficient to arrest cell multiplication and all produced unbalanced growth in the sense that the synthesis of RNA and protein were only partially inhibited while DNA synthesis stopped. This resulted in approximately 100% increases in RNA and protein content per cell in 48 hours and, since cell volume also increased by 100% during this time, the concentration of RNA and protein per unit cell volume remained constant. It was concluded that cell protein content may be used as an accurate index of variation in cell size in HeLa cells treated with inhibitors of DNA synthesis.

Synchronized mammalian cell cultures. I. Cell replication cycle and macromolecular synthesis following brief colcemid arrest of mitosis

Abstract: Chinese hamster fibroblasts in monolayer cultures were synchronized by accumulating mitotic cells in the presence of Colcemid, removing the mitotic cells with a brief trypsin treatment, and growing them in medium lacking Colcemid. Such cultures grew normally and exhibited no significant deviations from control cultures in their mitotic interval, generation time, DNA synthesis kinetics, or proliferative capacity. The macromolecular composition of 106 mitotic cells was chemically determined to be: DNA, 15 μg; RNA, 28 μg; and protein, 190 μg. In stock cultures, the corresponding values were about 60% to 70% of those for mitotic cells. The kinetics of DNA, RNA, and protein synthesis were measured throughout a 12-hour cell cycle by incorporation of tritiated precursors. DNA synthesis began two hours after, and continued until ten hours after Colcemid recovery, with 40 minute interruptions at five and eight hours. RNA synthesis commenced at one hour and continued linearly until the fifth hour, at which time the rate abruptly doubled. Protein synthesis began immediately after cell division (0.5 hour) and continued linearly until the sixth hour, at which time its rate also doubled. The simplest interpretation of the data suggests that most of the DNA involved in transcription was replicated in the first third of the DNA synthesis period. Thereafter, the rates of RNA and protein synthesis increased because of the doubling of the active template population in each cell.

K+-stimulated phosphatase of microsomes from gastric mucosa.

Abstract: The light microsomal fraction was isolated from homogenates of rabbit and bullfrog gastric mucosa. On examination with the electron microscope, the light microsomes appear as tubular membranous structures with morphology and dimensions similar to the elements of the smooth-surfaced endoplasmic reticulum seen in intact oxyntic cells. A K+-stimulated, Mg++-requiring p-nitrophenylphosphatase has been demonstrated in the gastric microsomes. Neither Na+ nor ouabain (10−6–10−3 M) altered the K+-stimulated phosphatase. SCN− was not very effective as an inhibitor of the gastric microsomal phosphatase, in contrast to the effect of this anion on the ATPase activity; however, the gastric phosphatase as well as the ATPase are destroyed by phospholipase C, thus showing the lipoprotein nature of these enzymes. Kinetics of the K+ activation of the microsomal phosphatase suggest that the K+-PNPP complex is the active substrate for the enzymic reaction. Rb+, NH4+ and Cs+ will substitute to some degree for K+ as an activator of the microsomal phosphatase. It is pointed out that K+ is an essential requirement for HCl secretion in intact gastric mucosa and the replacement of K+ with Rb+, Cs+ and NH4+ is discussed. The K+-stimulated phosphatase presented in this paper may play a role in the H+ secretion process.

Circular genetic maps

Abstract: The widespread occurrence of genetic circularity suggests a selective advantage to map circularity per se. Circularity permits gene clustering relations not possible in linear maps; that is, every gene in a circular map can have two nearest map neighbors, two next nearest, etc. It seems possible that map circularity is a consequence of the selective forces responsible for the clustering of genes of related function. Certain features of the pattern of crossing-over in various fungi suggest that circularity of linkage maps is there to be found. A concurrence of high frequencies of second-division segregation and negative chromatid interference across the centromere, both of which phenomena have been reported, is a feature of a simple hypothetical crossover pattern that generates circular linkage maps.

Enzyme activity during the growth and aging of human cells in vitro.

Abstract: Acid phosphatase, alkaline phosphatase, and lactic dehydrogenase activities have been compared in normal human diploid cell strains and in SV40-transformed heteroploid cell lines derived from them. A higher level of acid phosphatase activity was observed in diploid cultures derived from adult lung than in cultures derived from fetal lung of similar passage levels. The alkaline phosphatase activity of normal diploid fibroblasts was significantly higher than that of SV40-transformed cell lines derived from them. Generally, the lactic dehydrogenase activities of all these cell cultures were similar. Human diploid cells in culture “age,” in the sense that their ability to proliferate decreases with time during serial subcultivation. Evaluation of the activities of these three enzymes during the “aging” process showed that, although alkaline phosphatase and lactic dehydrogenase activities were similar in “young” and “senescent” cells, acid phosphatase showed a small but significant increase in the senescent cells.

Erythrocyte membrane sulfhydryl groups and the active transport of cations

Abstract: Parachloromercuribenzenesulfonate (PCMBS) has two opposing effects on the influx of Rb+ into red blood cells. The ouabain-insensitive component, indicative of passive permeability of the membrane to cations, is increased whereas the ouabain-sensitive or active transport component is inhibited. The increased passive permeability is paralleled by the action of PCMBS on net K+ efflux into a K+-free medium. Inhibibition of active transport of Rb+ is paralleled by the inhibitory action of PCMBS on net Na+ efilux from Na+-rich cells. Both effects are reversed rapidly by the penetrating sulfhydryl compound, cysteine, and considerably more slowly by the non-penetrating sulfhydryl compound, ovalbumin. Measurements of PCMBS binding and of elution in the presence of cysteine and albumin indicate that only a fraction of the total sulfhydryl groups of the membrane is involved in either the effect on permeability or transport, and that the affected groups are located within the interior of the membrane.

The cytogenetic effects of mycoplasma in human leukocyte cultures

Abstract: To evaluate the possible role of mycoplasma contamination of tissue cultures in virus induced chromosome breakage, human leukocyte cultures were inoculated with three mycoplasma strains; M salivarium, M hominis type 2 and M fermentans All three strains caused mitotic inhibition when an inoculum of approximately 106 CFU was used, the effect of M fermentans being less severe than the one produced by the two other strains Using a lower inoculum of 103 CFU an increase of chromosome breakage could be produced with M salivarium when the leukocytes were cultivated for five days No chromosome changes were seen with M hominis type 2 and M fermentans The mitotic inhibition and chromosome breaks induced by M salivarium were discovered to be related to an arginine deficiency of the culture medium produced by the mycoplasma This conclusion is dervied from the fact that arginine addition was able to inhibit the mitotic inhibition and the increase of chromosome breakage, and secondly, that similar changes could be produced in leukocytes grown in argining deficient medidum without mycoplasma The growth of M salivarium was inhibited in leukocyte cultures treated with kanamycin, but the mycoplasma induced mitotic inhibition was still present, indicating that replication of mycoplasma organisms was not required for the production of arginine deficiency

The cellular composition of hemopoietic spleen colonies

Abstract: The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83–98% of the dividing cells within 14-day spleen colonies were derived from single precursors.

Pairing at the chromosomal level.

Abstract: The discovery of the phenomenon of nonhomologous pairing in the female of Drosophila melanogaster provided a genetic tool for analyzing chromosome behavior, which has made it possible to establish a temporal order of meiotic events that includes two types of pairing, one preceding exchange (exchange pairing) and one following exchange (distributive pairing). Studies with free X-duplications support the assumption that the initial pairing for exchange involves parasynapsis rather than short, effectively paired regions. Recognition at exchange pairing is correlated with the extent of euchromatic homology in the duplication up to a physical length between 11 μ and 25 μ of the salivary gland chromosome, at which point full recognition (equivalent to the same length of homology carried on a normal X chromosome) is achieved. Recognition at distributive pairing is correlated with total size of the chromosome, is independent of homology, and is restricted to chromosomes which have not undergone exchange with an independent homologue. Temperature-treatment and labeling studies indicate that there is a rough coincidence between the temperature-sensitive period for recombination and that of DNA replication. Temperature-induced recombinants have been shown to be meiotic, not oogonial, in origin; and marked sensitivity to temperature likewise appears to reside in the early oocyte rather than in the oogonia. The temperature responses of interstitial and proximal chromosomal regions reveal a chronological and a quantitative difference, with the later and most pronounced response in the region spanning the centromere. If temperature acts directly, exchange pairing is initiated at the interphase stage; whereas the excellent correlation between segregation behavior and mitotic metaphase length of noncrossover chromosomes suggests that distributive pairing occurs between greatly condensed chromosomes, late in the cycle.

Dynamic adhesion and separation of cells in vitro II. Interactions of cells with hydrophilic and hydrophobic surfaces

Abstract: Experiments made on the passage of cells through untreated and siliconized glass beads, and on the adhesion and spread of cultured cells on glass and Teflon surfaces show that, in the absence of serum and in its presence in low concentrations, cell adhesion and spread is sensitive to substratum wettability. On the other hand, in the presence of 100% serum, no differences in adhesive parameters are detectable. It is concluded that arguments correlating cell adhesion to surface wettability, and, by inference, surface free energy, are unsubstantiated in 100% serum, which may well approximate to the in vivo situation. The results also show no correlation between parameters of cell adhesion and cell separation, and thereby support the hypothesis that these are different processes.

Regeneration of sialic acid on the surface of Chinese hamster cells in culture. II. Incorporation of radioactivity from glucosamine-1-14C.

Abstract: Cultured Chinese hamster cells incorporated radioactivity from glucosamine-1-14C into surface sialic acid and into trypsin-removable material distinct from the surface sialoglycans. Cells prelabeled with glucosamine-1-14C and then transferred to medium containing unlabeled glucosamine progressively lost counts to the medium for many hours. Such chase experiments suggested a more rapid turnover of trypsinremovable material than of surface-bound sialic acid. Further studies of the regeneration of surface sialic acid showed that the actinomycin D-resistant portion of the process involved emergence of an intracellular precursor onto the cell surface. An earlier portion of the process was inhibited by actinomycin D, and at least three steps were inhibited by puromycin or cycloheximide.

Recombination in bacteriophage T4

Abstract: Several multifactor crosses have been performed in which all segregating genotypes were identified. The genetic map of T4 and mapping techniques are re-evaluated in light of the finding that double-mutant recombinants occur less frequently than those identified as wild-type. This difference is attributable to heterozygotes which exaggerate the latter class. Analysis of the crosses suggests that the mathematical mapping functions so far devised overcorrect for clustering of multiple crossovers, and in so doing predict more crossovers than actually occur. An 8-factor rII cross in which crossover estimates were based exclusively on double-mutant recombinants shows that the high negative interference (HNI) observed in mass lysates is not an artifact of the method used to enumerate recombinants. Experiments to investigate the cause of HNI include parallel 8-factor rII crosses between alternating point mutations and deletions. One cross was performed in the presence of inhibitory concentrations of fluorouracil deoxyriboside, the other in its absence. Based on double-mutant recombination frequencies, neither case showed a difference in the coefficient of coincidence for insertion of a point mutant between two deletions and for insertion of a deletion between two point mutants. Either insertion heteroduplexes are not involved in formation of double crossovers, or deletion heteroduplexes (not observed among progeny phages) are efficiently repaired. Analysis of single-burst progenies of cells infected with a partial phage genome and a helper phage shows a 6.8-fold increase in recombination frequency near the ends of the partial phage genome. It is suggested that the HNI observed in mass lysates is a population genetics artifact accounted for by the increased frequency of recombination near ends of DNA molecules.

Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells.

Abstract: The temperature sensitivity of the ATPase enzyme systems in a muscle microsomal preparation from the crayfish, Astacus pallipes, was studied Preincubation of the enzyme preparation in the range 33–36°C produced a marked inactivation of the ATPases; the Mg++-dependent ATPase was very much more sensitive to this treatment than the Na+-K+-Mg++-dependent ATPase Thus, the Arrhenius μ for the inactivation of the Mg++-dependent ATPase produced by eight minute preincubation is > 100 Kcals These results are compared with the changes that are observed during the heat death of the whole animal, where exposure to 35°C produces a dramatic change in Na+ permeability within five minutes Arrhenius μ for heat death is also > 100 Kcals and operates over the identical critical temperature range It is suggested that the Mg++-dependent ATPase controls passive permeability in these excitable cells and the results also confirm the view that Mg++ and Na+-K+-Mg++ ATPases are separate enzymes

Molecular mechanisms of genetic recombination in bacteriophage: Joint molecules and their conversion to recombinant molecules†‡

Abstract: Density-gradient centrifugation analysis of the DNA extracted from Escherichia coli cells infected with 32P- and bromouracil-labeled phage and incubated with KCN showed the formation of linear molecules composed of two components de rived from the parental DNA molecules joined end-to-end by hydrogen bonds (joint molecules) However, when the infected cells were incubated under conditions which permitted limited synthesis of phage DNA, the components which had been derived from the parental DNA were joined by covalent bonds to form recombinant molecules It was also found that the structure of the majority of the chromosomes of mature phage T4, including heterozygous ones, could be represented by a duplex DNA molecule without an interruption in the strands Studies on the genetic control of recombination of phage T4 showed that gene 32 has to be expressed to form joint molecules, and that some other genes essential for DNA synthesis are involved in the transformation of joint molecules to recombinant molecules These results indicate that the process of genetic recombination is composed of several events: (a) formation of joint molecules, (b) their conversion to recombinant molecules, and (c) determination of chromosomal size followed by maturation Joint molecules isolated from the cells can be converted to recombinant molecules by an extract prepared from infected or uninfected cells In order for the reaction to proceed, deoxyribonucleotide triphosphates are essential

Measurement of the electrical potential difference across the membrane of the ehrlich mouse ascites tumor cell

Abstract: The electrical potential difference (PD) across the membrane of the Ehrlich mouse ascites tumor cell has been measured with intracellular microelectrodes. The mean for 111 cells in control Ringer solution was − 11.2 mV ± 0.29 (SE), interior negative. When sulfate replaced chloride in the external medium the PD fell to − 2.8 mV if measured as soon as possible after mixing the cells with a sulfate medium, but when nitrate replaced chloride the PD fell only to − 8.5 mV. Cells equilibrated in nitrate had the same PD as those in control Ringer. These results indicate that the PD is sensitive to changes in the external chloride concentration and that nitrate can substitute for chloride electrically. However, since the PD for chloride, based on the Nernst equation and calculated on the basis of 70% exchangeability of cell chloride, is three times greater than the measured PD, it is hypothesized that sodium contributes significantly to the membrane potential in addition to chloride. On the other hand, potassium does not influence the PD to any great extent.

Some physicochemical factors relevant to cellular interactions.

Abstract: Colloidal stability theory is discussed to accomodate the conditions imposed by biological systems. It is shown that to obtain potential curves with secondary minima, Hamaker's constant must be in the range of 1–5 × 10−14 ergs. The effect of increasing the dielectric constant is shown in theory to lower the surface potential and electrophoretic mobility but to increase the total energy of interaction. Calculations made from the theory predict the forces between model cells to be ca. 4.0 × 10−7 dynes. By cone-plate shearing of cell aggregates, the most successful of several techniques tried and discussed, at shear rates approaching 1 × 10−4 second−1 (1.5 × 10−4 dynes) semi-complete disaggregation was achieved although cell disruption was apparent; analysis of blood viscosity data indicates 5–10 × 10−7 dynes are required to separate red cells suspended in plasma. Colloidal stability theory, while not applicable to cell systems associated by special areas of attachment, seems to describe the physicochemical interaction of freely moving or reversibly adherent cells.

The rule of the ring.

Abstract: Different species of viruses contain linear or circular DNA molecules. The circular molecules are either single-chained or circular duplexes. The linear molecules from various species are always duplex. However, they may be either unique or circularly permuted collections of sequences. All species of linear duplexes that can be successfully tested can be shown to be terminally repetitious. The two temperate phages that have been studied (λ and P22) are unique and permuted collections, respectively. Shortly after infection both of these molecules form closed helical rings (superhelices). Certain virulent phages show no evidence of superhelix formation. How unique and permuted collections are produced at maturation is a puzzle. In this respect, it is of interest that P22 is a generalized transducing phage, whereas λ is a specialized transducing one.

The incorporation of microinjected 14C-amino acids into TCA insoluble fractions of the giant axon of the squid

Abstract: Giant squid axons were microinjected with serine, valine and leucine-C14 under controlled electrophysiological conditions. These amino acids are incorporated into TCA insoluble fraction in the isolated axon. This incorporation is higher in the stimulated axons as compared to non-stimulated ones. By processing separately the axoplasm and axon sheath, it was found that the last one is responsible almost entirely for the observed incorporation. Through differential centrifugation of homogenates of microinjected axons was shown that the highest incorporation occurred in the 1500 × g sediment, which probably corresponds to membranes. The incorporation of amino acids in stimulated axons, is strongly inhibited by chloramphenicol and actinomycin D.

Ribonucleic acid within the cellular peripheral zone and the binding of calcium to ionogenic sites

Abstract: Ribonuclease was shown to reduce the electrophoretic mobility of a line of cultured mammalian cells (RPMI no. 41), and Ehrlich ascites tumour cells. No reduction was detected in the case of human, mouse or embryonic chick erythrocytes. These data, taken with the various controls, support the hypothesis that RNA is a structural component of the peripheries of two types of cells, but not of erythrocytes from three species. Calcium-binding was studied in RPMI no. 41 cells, Ehrlich ascites tumour cells, and human and mouse eryhrocytes, by measurement of reduction in cellular electrophoretic mobility in suspending solutions containing various concentrations of calcium chloride. The effect of treating cells with neuraminidase and/or ribonuclease on calcium-binding was also studied. The results suggest that less calcium binds to the carboxyl groups of peripheral sialic acids than to the phosphates of peripheral, structural RNA. However, calcium apparently binds most avidly to as yet unidentified anionic sites.

Replication of chromosomal and cytoplasmic DNA during mitosis and meiosis in the eucaryote Chlamydomonas reinhardi

Abstract: Both meiotic and mitotic replication of chromosomal and cytoplasmic DNAs in a unicellular green alga, Chlamydomonas reinhardi, have been studied by isotopic transfer experiments coupled with density-gradient centrifugation analysis. It has been shown that during the vegetative cycle (1) the replication mode of chloroplast DNA, as well as γ DNA, is semiconservative; (2) in synchronized culture these two cytoplasmic DNA satellites replicate coordinately with a high degree of synchrony; (3) the replication of chromosomal DNA is independent of and separable from that of the two cytoplasmic DNAs. It has been shown that during the sexual cycle (1) the chloroplast DNA replicates semiconservatively during zygote maturation, at which time there is neither chromosomal DNA replication nor nuclear division; (2) another DNA component (M-band DNA) replicates extensively and appears in large quantity; (3) meiosis occurs during zygote germination and is accompanied by one round of semiconservative chromosomal DNA replication; (4) the M-band DNA disappears in the early germination period, and the degraded substance is not incorporated into the newly replicated chromosomal DNA. A possible origin of the M-band DNA and a few properties of the cytoplasmic DNA satellites have been elucidated. Genetic recombination and regulation of incoordinate replication in an eucaryotic system have been discussed in terms of the data obtained.

Observations and measurements of sea urchin eggs with a centrifuge microscope.

Abstract: Eggs of the sea urchins, Arbacia punctulata and Lytechinus variegatus were observed with a centrifuge microscope. The protoplasmic viscosity calculated from the displacement velocity of the nucleus in a centrifugal field is about 30 poises. The surface forces of the unfertilized egg, which were determined from the relationship between the deformations of the egg in centrifugal fields and the magnitudes of the centrifugal forces, are 0.046 dyne/cm in Arbacia and 0.087 dyne/cm in Lytechinus (mean values). Both of these values increase as the deformation of the egg increases. The cleavage plane of the first cleavage of the egg in the centrifugal field (35–140 × g) is parallel to the direction of the centrifugal force. The flattening of the fertilized egg in a constant centrifugal field changes during development, probably owing to the change in the surface force. The flattening attains a minimum shortly before the onset of cleavage and another minimum during the cleavage, corresponding to a peak of the surface before the cleavage and another peak during the cleavage.

Colony stimulating activity of serum from germfree normal and leukemic mice

Abstract: Serum from germfree Swiss/HaM mice exhibited a reduced capacity to stimulate granulocytic and mononuclear cell colony formation by DBA/1 bone marrow cells in vitro when compared with serum from conventional Swiss/HaM mice. Sera from germfree preleukemic and leukemic AKR mice exhibited strong colony stimulating activity, indicating that the increased colony stimulating activity previously observed in the serum of conventional leukemic mice is not the consequence of bacterial or fungal infections supervening in leukemic animals with deficient immune responses.