Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization

We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.     

Global bifurcations of a periodically forced nonlinear oscillator

The effects of periodic pulsatile stimulation on a simple mathematical model of biological oscillations, called the radial isochron clock (RIC), are investigated as a function of stimulus frequency and amplitude. This system can be reduced to a two parameter, one-dimensional circle map. Numerical and topological methods are used to give a very detailed picture of the observed bifurcations over the complete range of parameters. The bifurcations are generic for a class of models which generalize the RIC.     

Epidermal cell migration on laminin-coated substrates

Summary Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for the migrating wound epithelium. Laminin, a protein that has been implicated as an epithelial-specific adhesin, was a moderately good migration substrate. Type-IV collagen, fibrinogen and fibronectin, however, were significantly better. Fetuin, myoglobin, and casein all proved to be very poor substrates, allowing practically no migration. The inability of fetuin, myoglobin, and casein to support migration is further evidence that the considerable migration that occurs on collagen (Donaldson et al. 1982) fibrinogen and fibronectin (Donaldson and Mahan 1983) and the moderate migration on laminin, is a relatively specific response to these proteins and is therefore of special significance. The fact that laminin is a poorer migration substrate than collagen, fibrinogen or fibronectin suggests that the absence of cell surface laminin that has been associated with epithelial movement in several studies (Stanley et al. 1981; Clark et al. 1982; Madri and Stenn 1982; Gulati et al. 1983) may promote motility by allowing epithelial cells to interact directly with other extracellular macromolecules.     

Association of gap junctions with endoplasmic reticulum in rat parotid glands

Summary Examination of the parotid gland of the rat has shown specific associations of cisterns of the endoplasmic reticulum with gap junctions. About 20% of the junctions are so intimately associated with cisterns of the endoplasmic reticulum that in freeze fractured material the cisternal membranes remain attached to the junctional membrane faces, obscuring most of the junctional array except for a thin ring of telltale particles. This association was seen only in the parotid gland of the rat, but not that of the other species examined.     

Autoradiographic localization of ornithine decarboxylase in mouse kidney by use of radiolabeled α-difluoromethylornithine

Summary Ornithine decarboxylase, a key enzyme in polyamine biosynthesis and cell growth, has been localized in mouse kidney by autoradiography after administration of radiolabeled -difluoromethylornithine. This drug is an enzyme-activated irreversible inhibitor of ornithine decarboxylase and forms a covalent bond with the enzyme. It was found that ornithine decarboxylase is present in all cell types studied but that the highest content occurs in the proximal convoluted tubules followed by the distal convoluted tubules and the collecting tubules. The majority of the enzyme is located in the cytoplasm but about 10–15% is present in the nuclei (often associated with nucleolus-like components) of the cells of the proximal and distal convoluted tubules. The labeled ornithine decarboxylase was lost rapidly from both nucleus and cytoplasm of all the cell types examined, and labeling by radioactive -difluoromethylornithine was greatly reduced if the mice were pretreated for 5 h with cycloheximide to block protein synthesis. These results indicate that ornithine decarboxylase turns over rapidly in all of the cells.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

The use of light green and organge II as quantitative protein stains,and their combination with the feulgen method for the simultaneous determination of protein and DNA

Summary The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO₂) and-Thionin(SO₂) method for the simultaneous determination of DNA and protein. — With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. — In addition, the Feulgen-Thionin(SO₂) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO₂) method. — When combining the Light Green staining with the Feulgen-Pararosanilin(SO₂) procedure and the Orange II staining with Feulgen-Thionin-(SO₂), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. — When the Feulgen-Pararosanilin(SO₂) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO₂) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.This work was supported by the Dutch Cancer Foundation Koningin Wilhelmina Fonds grant NUKC 1981-15     

Effects of aplysiatoxin and debromoaplysiatoxin on growth and differentiation of normal human bronchial epithelial cells

The effects of aplysiatoxin and debromoaplysiatoxin on the clonal growth rate, cross-linked envelope formation and plasminogen activator secretion of normal human bronchial epithelial cells were studied. Neither compound was mitogenic over a wide range of concentrations (10^–13 to 10^–7M). Both aplysiatoxin and debromoaplysiatoxin inhibited clonal growth rate with 50% inhibitory concentrations of 3 × 10^–11M and 10^–10M, respectively. Both compounds induced the formation of cross-linked envelopes and increased plasminogen activator secretion with equal potency. These data are similar to those previously obtained with 12-0-tetradecanoylphorbol-13-acetate and teleocidin B and suggest that aplysiatoxin and debromoaplysiatoxin induce terminal squamous differentiation in normal human bronchial epithelial cells.Abbreviations TPA 12-0-tetradecanoylphorbol-13-acetate - NHBE Normal Human bronchial epithelial - ID₅₀ 50% inhibitory concentration (dose) - PA Plasminogen activator - CLE Cross-linked envelope - LHC Laboratory of Human Carcinogenesis     

The natural heterogeneity of trypanosoma cruzi: Biological and medical implications

Trypanosoma cruzi is a heterogeneous group of parasites. The imposition of natural or artificial pressures can result in the selection of subsets of the population with concomitant changes in characteristics used to evaluate the group. In order to ascertain the extent of heterogeneity, stocks of single-cell clones were prepared from various sources. Selected cell biological, biochemical, immunochemical, parasitological, and histopathological parameters of these clones have been studied. A ten-fold difference in the rate of growth of the epimastigote stage of T cruzi clones has been observed. The extracellular growth rates of the clones correlate with the rate of growth of the obligate intracellular amastigote stage and consequently, the length of intracellular cycle of the parasite. A 40% difference in the amount of total DNA/parasite has been found between clones. Although the amount of DNA/kinetoplast and nucleus varies between clones, the major contribution to the differences in total DNA/parasite appears to be the nucleus. From 16 to 35 antigens have been demonstrated in the T cruzi clones assayed to date. Five to seven of these antigens are common to all of the stocks assayed. However, both isolate- and clone-specific antigens have also been demonstrated. The susceptibility of inbred strains of mice to T cruzi clones varies with the clone of the parasite. These data imply that the genetics of the parasite as well as the host modulate both the course and outcome of a T cruzi infection. The influence of monosaccharides on the receptor-mediated infection of vertebrate cells by trypomastigotes of T cruzi also varies between clones. The implications of these findings upon our concept and understanding of present and future problems in Chagas disease are discussed.