Characterization of a frequent polymorphism in the coding sequence of the Tp53 gene in colonic cancer patients and a control population

Summary We describe a simple method for characterizing a frequent polymorphism (that subsitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population. We could find no evidence that this polymorphism is associated with a marked predisposition to colorectal cancer.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.     

Transposon Mutagenesis and Excision of R′ Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10⁻⁷ to 10⁻⁹. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg^r transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with ³²P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg^r both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg^r transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R′ plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Anaerobic C1 Metabolism of the O-Methyl-14C-Labeled Substituent of Vanillate

The O-methyl substituents of aromatic compounds constitute a C₁ growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C₁ substrate was determined by ¹⁴C radiotracer techniques. O-[methyl-¹⁴C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C₁ substrate. The data showed that for every O-methyl carbon converted to [¹⁴C]acetate, two were oxidized to ¹⁴CO₂. Quantitation of the carbon recovered in the two products, acetate and CO₂, indicated that acetate was formed in part by the fixation of unlabeled CO₂. The specific activity of ¹⁴C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO₂ and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-¹⁴C]vanillate by strain TH-001 can be described as follows: 3¹⁴CH₃OC₇H₅O₃ + CO₂ + 4H₂O → ¹⁴CH₃COOH + 2¹⁴CO₂ + 10H⁺ + 10e^- + 3HOC₇H₅O₃.     

Observation of manganese(II)-ligand superhyperfine couplings in complexes with proteins by electron spin-echo spectroscopy

Pulsed electron paramagnetic resonance spectroscopy has been used to detect Mn(II)-ligand superhyperfine couplings in complexes with creatine kinase and in the Mn(II) metalloprotein concanavalin A. Electron spin-echo envelopes from Mn(II), bound in these complexes, are modulated by superhyperfine interactions between Mn(II) and nearby, weakly coupled nuclear spins. The characteristic frequencies of the modulations were obtained by Fourier transformation of the three-pulse, spin-echo envelopes. In transition-state analogue complexes of creatine kinase (enzyme-MnIIADP-anion-creatine), superhyperfine interactions from the directly coordinated nitrogen of the thiocyanate ligand give envelope modulations. The source of the modulations was confirmed by measurements with the 14N and 15N forms of thiocyanate. On the other hand, the nitrogen of coordinated nitrate, which is two bonds removed from the paramagnetic center, does not produce detectable modulations. In spectra for Mn(II) concanavalin A, envelope modulations are detected due to the nitrogen of the coordinated histidine residue. Complexes prepared in 2H2O give strong signals due to weakly coupled 2H. For Mn(II)-doped single crystals of sodium pyrophosphate, signals are observed in the frequency domain spectra that are due to coupling from 31P. Phosphorus signals from the ADP ligand in complexes with creatine kinase show approximately the same coupling constant but have a much broader line width.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Phorbol 12,13-Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat

Phorbol 12,13-dibutyrate (PDBu) increased the production of 3,4-dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half-maximal at a concentration of approximately 0.1 microM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+-free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8-Br-cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 microM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion.     

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.