Sequence, Genomic Distribution and DNA Modification of a Mu1 Element from Non-Mutator Maize Stocks

The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F2 individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification.     

New and noteworthy plants from Great Barrier Reef sand cays,Australia

Observations are presented on new and critical plants from the northern sand cays on the Great Barrier Reef, Queensland, Australia, based mainly on recent collections made by David R. Stoddart and Ralf Buckley. New species and varieties are described:Lepturus stoddartii (Poaceae),Boerhavia fistulosa var.fistulosa and var.puberuliflora (Nyctaginaceae),Boerhavia albiflora var.heronensis, Spermacoce everistiana (Rubiaceae), andSpermacoce buckleyi; a new combination is made:Diospyros ferrea var.compacta (R. Br.) (Ebenaceae); and additional taxonomic notes are given onBoerhavia, Euphorbia (Euphorbiaceae), andAbutilon (Malvaceae).     

Characterization of kinetochores in multicentric chromosomes

Long-term cultures of certain rat and mouse cell lines carry several dicentric and some multicentric chromosomes. Using antikinetochore antibodies obtainable from serum of scleroderma (var. CREST) patients we studied the number of kinetochores formed along the length of these chromosomes. The rat cells displayed as many kinetochores as there were centromeres. However, mouse cells showed the synthesis of only one kinetochore in dicentric and multicentric chromosomes which had been in the culture for a period of 1 year or more. When translocations were induced by bleomycin in mouse L cells, the newly formed dicentric chromosomes showed the formation of two kinetochores. It is not known when the accessory centromeres lose their capacity to assemble kinetochore proteins. Possibly, in the rat the latent kinetochores lack a specific component which renders them ineffective for microtubule binding. The reason for the formation of only one kinetochore in mouse multicentric chromosomes is not clear. It may be due to the accumulation of mutations, modification of the kinetochore protein so that it lacks the antibody binding component, or a more effective regulatory gene than in the rat.     

Use of alternate substrates to probe the order of substrate addition to dopamine beta-hydroxylase

In order to determine the order of substrate binding to dopamine beta-hydroxylase during catalysis, the effect of alternate substrates upon kinetic parameters was examined. The V/K value for ascorbate was unchanged when tyramine, phenylpropylamine, p-Cl-phenethylamine, p-CH3O-phenethylamine, or phenethylamine was the hydroxylated substrate. The V/K values for tyramine and oxygen were similarly unchanged when ferrocyanide was used as the reductant in place of ascorbate. In order to use ferrocyanide as reductant it was necessary to include copper to alleviate the substrate inhibition seen with this substrate. The pattern of substrate inhibition observed with ferrocyanide was consistent with a small amount of free cyanide present in the ferrocyanide. With ferrocyanide as reductant and [2,2-2H2]tyramine as substrate, there was a measurable isotope effect on the V/K value for oxygen, but none on the values of Vmax or V/K for tyramine. These results are consistent with a ping-pong mechanism in which tyramine binds to the enzyme after the release of oxidized ascorbate. Subsequently, oxygen binds to form a ternary complex.     

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.     

Inhibition of cyclooxygenase activity and platelet aggregation by epoxyeicosatrienoic acids. Influence of stereochemistry

Certain epoxyeicosatrienoic acids (EETs) that were not cyclooxygenase substrates were effective cyclooxygenase inhibitors. Both (+/-)-14,15-cis-EET and (+/-)-8,9-cis-EET inhibited purified enzyme at concentrations from 1 to 50 microM; (+/-)-11,12-cis-EET was ineffective at concentrations below 100 microM. For the case of 14,15-cis-EET, only the (14R,15S)-stereoisomer was active. Other isomers including (14S,15R)-cis-EET, (14R,15R)-trans-EET, (14S,15S)-trans-EET, and the erythro and threo vicinal 14,15-diols were inactive. In addition to their effects on isolated enzyme preparations, cyclooxygenase activity in platelet suspensions, reflected by thromboxane B2 formation, was also inhibited by (14R,15S)-cis-EET and (+/-)-8,9-cis-EET but not by the other isomers. Thus potency and stereospecificity requirements were maintained for cyclooxygenase within intact platelets. Unlike the stereospecific inhibition of the cyclooxygenase enzyme, platelet aggregation induced by arachidonic acid was inhibited by all EET isomers at concentrations from 1 to 10 microM with no evident stereospecificity. Inhibition of aggregation was not uniformly associated with inhibition of thromboxane B2 formation; ordinarily, these two parameters correlate closely. This dissociation was not maintained for another biochemical process involved in platelet activation. For instance, there was a uniform correlation between inhibition of phosphorylation of a 40-kDa platelet protein and inhibition of aggregation. Our results suggest that effects of EET may originate from either stereospecific or nonspecific mechanisms. Definition of such mechanisms may be important to appreciate any physiological relevance of these substances.     

Mathematical modelling of primary production in Green Bay (Lake Michigan,USA), a phosphorus-and light-limited system

Application of mathematical models in the design and evaluation of lake restoration programmes must include due consideration of three basic concepts of model development; 1) that the model framework is appropriately matched to the intended management use, 2) that selection of the proper degree of model complexity is fundamental to the achievement of model credibility and 3) that field and laboratory studies must be designed and interpreted with the aid of the model to insure development of a comprehensive, integrated tool.These concepts are demonstrated for the case of lake restoration efforts in Green Bay (Lake Michigan, USA). Striking gradients in water quality (transparency, algal standing crop, hypolimnetic oxygen depletion) and trophic state occur along the major axis of the bay in response to phosphorus loaded from the Fox River. A simple model for gross primary production is developed to permit calculation of the relative importance of internal carbon production to the total organic carbon budget of the bay. Primary production varies from high rates over a limited photic depth in the turbid, phosphorus-rich waters of the eutrophic portions of the bay to low rates over an extensive photic depth in the transparent, phosphoruspoor reaches of the oligotrophic regions. Internal production accounts for approximately 90% of the total organic carbon loaded to the system over the summer growing season. Water quality management strategies must address the stimulation of primary production by phosphorus loaded from the Fox River in any attempt to lower the standing crop of nuisance algae, improve water clarity, and reduce rates of hypolimnetic oxygen depletion in Green Bay.     

Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

3-Phenylpropenes as mechanism-based inhibitors of dopamine beta-hydroxylase: evidence for a radical mechanism

A series of ring-substituted 3-phenylpropenes has been examined as mechanism-based inhibitors for the copper protein dopamine beta-hydroxylase. p-HO-, p-CH3O-, m-HO-, m-CH3O-, p-Br-, and p-CN-substituted phenylpropenes all inactivate the enzyme under turnover conditions, requiring ascorbate and oxygen. Replacement of the benzylic hydrogens in 3-(p-hydroxyphenyl)propene with deuterium results in a kinetic isotope effect of 2.0 on kinact/KO2 but in no effect on the partition ratio, Vmax/kinact, consistent with a stepwise mechanism for hydrogen abstraction and oxygen insertion. The partition ratio is unchanged in the pH range from 4.5 to 7.1. Determination of the kinetics of inactivation and the partition ratios for each of these ring-substituted phenylpropenes has allowed determination of the respective V/KO2 values. A linear free energy plot of these values as a function of sigma+ gives a rho value of -1.2, while the partition ratios show only a slight decrease upon going electron-withdrawing groups. The results are consistent with a mechanism for dopamine beta-hydroxylase in which a hydrogen atom is abstracted to form a benzylic radical, which then partitions between hydroxylation and enzyme inactivation.     

Leukotriene A4-hydrolase activity in guinea pig and human liver

Guinea pig and human liver homogenates transformed leukotriene A4 into leukotriene B4. In both species, the enzymatic activity was recovered in the 105000 X g supernatant, and it was found to be susceptible to heat treatment (56 degrees C, 1 h). Digestion with a proteolytic enzyme also resulted in loss of enzymatic activity. The formation of leukotriene B4 was pH-dependent, with an optimum between pH 7 and pH 8.5. In addition, two other organs from the guinea-pig, lungs and kidneys, contained leukotriene A4-hydrolase activity. The identity of leukotriene B4 was ascertained by high-performance liquid chromatography, ultraviolet spectrometry, gas chromatography-mass spectrometry and bioassay. We have recently demonstrated the presence of leukotriene A4-hydrolase activity in mammalian plasma (Fitzpatrick et al. (1983) Proc. Natl. Acad. Sci. USA 80, 5425-5429). The results of the present study suggest several possible origins of this plasma leukotriene A4 hydrolase.