Anion-exchange high-performance liquid chromatographic analysis of inositol phosphates

The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.     

Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene

We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed. dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.     

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.     

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.     

Analysis of antigens from membrane and soluble fractions of Entamoeba histolytica

Axenic cultures of Entamoeba histolytica strains HK-9, HM-1, and Rahman were fractionated to provide plasma membranes, internal, vesiculated membranes, and a soluble cytosol. Each particulate fraction was solubilized and all fractions were analyzed by techniques designed to demonstrate molecular complexity and serologic reactivity. The cytosol contained more antigenic moieties than either membrane fraction; however, the antigens associated with the membranes had very high reactivity and lower nonspecific activity than the cytosol.     

Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase.

Repeated passages of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1,000-fold. Analyses of viral protein synthesis by using [35S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase (EC 1.17.4.1) activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification which yielded greater than 90% of the induced ribonucleotide reductase activity in the fraction obtained by 35% saturation with ammonium sulfate resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [3H]thymidine into DNA earlier and at a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. In the absence of the drug, the attainment of a maximum viral DNA synthesis rate was accelerated after infection by drug-resistant isolates. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolates readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is a 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses.