The isolation and restriction mapping of a miniplasmid from the Actinomycete Nocardia corallina

Abstract A variety of plasmids has been identified as covalently closed circular and linear DNA in certain Actinomycetes, such as Streptomyces . This paper describes the first isolation and characterisation of a plasmid from the genus Nocardia . The plasmid pKU100 isolated from Nocardia corallina is a cccDNA molecule, 2.7 kb in length. This plasmid has been mapped with a wide variety of restriction enzymes and contains a number of unique restriction sites making it suitable for development as a cloning vector.     

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     

Proximal and distal transformation during intercalary regeneration in the planarianDugesia(S)mediterranea

Summary Studies on intercalary regeneration in several organisms have shown that a regenerate is formed when surfaces of different positional value along the proximo-distal axis are opposed. One of the main problems posed by this phenomenon is to know which piece contributes to the building of the regenerate. In the present work we have studied this problem in planarians using chimaeras made between pieces of different body levels, irradiated or not, of the sexual and asexual races ofDugesia(S)mediterranea that differ in a chromosomal marker.The results found show very clearly that intercalary regenerates in planarians are formed by cells coming from both pieces (stumps), and that irradiated pieces keep the positional values and interact with non-irradiated pieces to restore the missing parts. This means that distal and proximal transformation do actually occur at the same time during intercalary regeneration in planarians. The implications of these results as regards to the origin of cells in the regenerate and to present models of intercalary regeneration are discussed.     

The majority of independently transformed BHK cell clones share a single functional lesion which determines anchorage independence and influences tumorigenicity

Summary Expression of the anchorage-independent transformed phenotype in BHK 21/13 cells generally behaves as a recessive trait. When chemically induced and spontaneously arising transformants are fused to the nontransformed parent line, transformation is initially suppressed, reappearing after extended growth of the hybrids. In this paper, complementation for the expression of anchorage independence was sought among a large group of such transformants, all independently derived from BHK 21/13 cells. Tumorigenicity studies on selected hybrids and parental lines indicated that the in vitro trait of anchorage independence is an accurate indicator of in vivo neoplasia for these cells. Seventeen of the 18 clones tested did not complement one or more of three tester strains. This result indicates that anchorage independence arose in these clones as a result of lesions in the same genetic function and suggests that the final step in the progressive changes of carcinogenesis may frequently be restricted to lesions at a single locus. This investigation was supported by National Institutes of Health grant CA27306.     

Effects of incubation in an atmosphere of 20% CO2 in air on the syrian hamster embryo clonal transformation assay

Summary An atmosphere containing 10% CO₂ has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO₂ may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO₂ in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO₂, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO₂, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO₂ in air. The data also show that 20% CO₂ increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO₂ data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations of CO₂ concentrations. In some instances, the CO₂ levels indicated by incubator flow meters vary considerably from analytically determined CO₂ values. To prevent these CO₂ discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO₂ environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO₂ is a preferred environment for the conduct of this assay.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Carbohydrate-binding proteins of tumor lines with different growth properties

Summary A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weights of 80 kDa, 70 kDa, 22 kDa, 18 kDa and 16 kDa and of a fucose-binding protein at apparent molecular weight of 42 kDa. The -glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5–8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5–8#1 and 5–20#20, whereas the metastatic variant 5–20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.     

Regeneration in Hydrozoa: distal versus proximal transformation in Hydractinia

Summary Polyps of mature colonies of Hydractinia echinata obey the rule of distal transformation by regenerating heads but not stolons. However, this rule is not valid for young polyps as these regenerate stolons from proximal cut ends. Also, small cell aggregates and even small fragments excised from full-grown polyps are capable of stolon formation. Aggregates produced from dissociated cells undergo either distal or proximal transformation depending on their size, speed of head regeneration in the donor used for dissociation and the positional derivation of the cells. The latent capability of stolon formation is released under conditions that cause loss of morphogens and depletion of their sources. However, internal regulative processes can also lead to gradual proximal transformation: regenerating segments of polyps sometimes form heads at both ends and the distal pattern is duplicated. Subsequently, all sets of proximal structures, including stolons, are intercalated. In contrast to distal transformation, proximal transformation is a process the velocity of which declines with the age and size of the cell community.     

Dispersion and Distribution of Pratylenchus scribneri and Hoplolaimus galeatus in Soybean Fields

Examination of dispersional characteristics of Pratylenchus scribneri and Hoplolaimus galeatus indicated that there were patches within soybean fields in which both survival and reproduction wexe enhanced in spite of apparent homogeneity of soil type and topography. Treatment with carbofuran reduced the patchiness (or increased the dispersion) for H. galeatus while it had the opposite effect for P. scribneri. P. scribneri was less highly dispersed in conventional tillage plots than in the zero tillage plots. Populations from quadrats contained entirely within the patches could be described by the normal distribution (in the case of P. scribneri) or by the Poisson distribution (in the case of H. galeatus), while populations from quadrats contained entirely outside the patches could be described by the Poisson distribution for both nematodes. None of the distributions tested (Poisson, normal, negative binomial, Neyman''s) gave an adequate fit when populations from both inside and outside the patches were considered together. In all instances, log₁₀ and ln transformations reduced the goodness of fit of the data to all of the distributions tested. Even with logarithmic transformations, parametric statistics were not appropriate for analysis of data in most instances.