Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

The development and ultrastructure of the testa and tracheid bar inErythrina lysistemon Hutch. (Leguminosae: Papilionoideae)

Summary The development of the testa was studied inErythrina lysistemon using both light and electron microscopy. Cells of the outer epidermis of the outer integument divide anticlinally and undergo radial elongation to form a palisade layer. The outer tangential walls are thickened at an early stage, and deposition of fluted thickenings on the radial walls occurs at maturity. Palisade cells in the hilar region differentiate from sub-funicular tissue, and at maturity the outer ends of the cells undergo extensive deposition of secondary walls and associated lignification. The light line occurs at the junction between the outer, thickened portions of the cells and the inner, less thickened portions. An electron-translucent (suberised) cap develops in the outer tangential walls of the palisade cells at a late stage. Microtubules and dictyosomes are closely associated with the developing thickenings in palisade and tracheid bar, and the microtubules run parallel to the wall microfibrils. Differentiation of the tracheid bar coincides with final secondary wall deposition and lignification in the hilar palisade. The cells of the tracheid bar are dead at maturity, but are surrounded by sheaths of elongate parenchyma.     

Characterization of a growth-inhibiting protein present in rat serum that exerts a differential effect onin vitro growth of nonmalignant rat liver cells when compared with Rous sarcoma virus-transformed rat liver cells

Summary We have previously reported the transformation by Rous sarcoma virus of a cloned epithelial cell line (BRL) established from Buffalo rat liver by H. Coon. The nontransformed (BRL) and transformed (RSV-BRL) cells grew at comparable rates in culture, whereas only the transformed cells were tumorigenic in vivo. We report here on the existence in rat and mouse sera of a growth inhibitor for the nontransformed BRL cells. The transformed BRL cells (RSV-BRL) were insensitive to this inhibitor. The inhibitory activity was not prominent in sera from other species of animals tested except for rabbit; this serum inhibited the growth of RSV-BRL cells more strongly than that of BRL cells. The growth inhibitor was partially purified from rat serum. It is a protein free of lipid and has a molecular weight of about 220 000. The inhibitor could be separated into three components of pI 4.6, 5.2 (major) and 5.6 by isoelectric electrophoresis. EDITOR'S STATEMENT Although compelling theoretical arguments sometimes can be made for the likely existence of growth-inhibitory substances of physical relevance in the control of cell proliferation, experiments aimed at identifying and studying such factors often are difficult to design and interpret, and little strong data exists to suggest that growth-inhibitory substances are important regulatorsin vivo. The information presented in this paper represents a start toward developing a useful system for studying growth-inhibitory factor. David W. Barnes     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

水稻二九南1号雄性不育系及相应保持系一些生化性状的比较

为研究高等植物雄性不育特性的遗传本质,我们以具有野败型不育细胞质、细胞核相同的水稻二九南一号(Oryza sativasubsp.Indica)雄性不育系、保持系为材料,分析了不同生长发育阶段正、负极向过氧化物酶、β-淀粉酶、酯酶同工酶和可溶性叶蛋白,结果初报如下。一、正、负极向过氧化物酶同工酶所分析的7个组织(根、茎、叶、芽鞘、胚轴、花药、胚乳)中不育系与保持     

Cell lines from imaginal discs ofDrosophila melanogaster

Summary New cell lines, designated as ML-DmDl≈10, were established from dissociated imaginal discs ofDrosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang’s M3(BF) medium, supplemented with 10% heat inactivated fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary culture supernatan, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums, imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to 10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae, they differentiated at high frequency into adult cuticular strutures in the early phase of the primary culture. This differentiation of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in vitro.     

大鼠非致瘤四倍体细胞系的建立及细胞遗传学和酶学特征

从大鼠自然诱发的肉瘤细胞中,我们建立了一个四倍体细胞系(4n=84),命名为RC(ratcell)。它具有典型的成纤维细胞外形,能在玻璃表面贴壁生长,但不能生长在琼脂半固体培养基中。该细胞在含15％小牛血清的RPMI 1640培养基中生长良好,至今已连续繁殖112世代,细胞群体倍增时间约为15小时。染色体G-带分析表明,RC为整四倍体细胞,它的1条X染色体在第32至34区为均染区。RC细胞核仁组织者(NORs)活性显然比大鼠二倍体细胞NORs活性的加倍还高(P<0.001)。这个具有非常高NORs活性的RC细胞系对于研究细胞18S+28S rRNA基因转录活性的调控、基因表达与基因剂量关系有一定的意义。RC细胞还有异常高的磷酸酯酶活性,而且它的同工酶谱也与大鼠肌肉细胞明显不同。体内接种实验和扫描电镜的观察表明,RC是非致瘤细胞。RC细胞各号染色体的C-带图样与大鼠二倍体细胞无明显的差异。     

Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.