Aspartate and asparagine tRNA genes in wheat mitochondrial DNA: a cautionary note on the isolation of tRNA genes from plants.

We have identified genes encoding a "native" tRNA(Asp) (trnD-GTC) and a "chloroplast-like" tRNA(Asn) (trnN-GTT) on opposite strands and 633 bp apart within a sequenced 1640 bp RsaI restriction fragment of wheat mtDNA. The trnD gene has been found previously at a different location in wheat mtDNA (P.B.M. Joyce et al. (1988) Piant Mol. Biol. 11, 833-843); the duplicate copies of this gene are identical within the coding and immediate flanking regions (9 bp downstream and at least 68 bp upstream), after which obvious sequence similarity abruptly disappears. The trnN gene is identical to its homolog in maize ctDNA; continuation of sequence similarity beyond the coding region suggests that this gene originated as promiscuous ctDNA that is now part of the wheat mitochondrial genome. In the course of this work, we have encountered some unexpected similarities between tRNA gene regions from wheat mitochondria and other sources. Detailed analysis of these similarities leads us to suggest that trnN genes reportedly from petunia nuclear DNA (N. Bawnik et al. (1983) Nucleic Acids Res. 11, 1117-1122) and lupine mtDNA (B. Karpińska and H. Augustyniak (1988) Nucleic Acids Res. 16, 6239) are, in fact, from petunia mtDNA and lupine ctDNA, respectively, whereas a putative wheat nuclear tRNA(Ser) (trnS-TGA) gene (Z. Szwekowska-Kulińska et al. (1989) Gene 77, 163-167) is actually from wheat mtDNA. In these instances, it seems probable that the DNA samples used for cloning contained trace amounts of DNA from another sub-cellular compartment, leading to the inadvertent selection of spurious clones.     

Macromolecular adducts of ethylene oxide: a literature review and a time-course study on the formation of 7-(2-hydroxyethyl) guanine following exposures of rats by inhalation

The results of efforts to identify and quantify macromolecular adducts of ethylene oxide (ETO), to determine the source and significance of background levels of these adducts, and to generate molecular dosimetry data on these adducts are reviewed. A time-course study was conducted to investigate the formation and persistence of 7-(2-hydroxyethyl)guanine (7-HEG; Fig. 1) in various tissues of rats exposed to ETO by inhalation, providing information necessary for designing investigations on the molecular dosimetry of adducts of ETO. Male F344 rats were exposed 6 h/day for up to 4 weeks (5 days/wk) to 300 ppm ETO by inhalation. Another set of rats was exposed for 4 weeks to 300 ppm ETO, and then killed 1–10 days after cessation of exposures. DNA samples from control and treated rats were analyzed for 7-HEG using neutral thermal hydrolysis, HPLC separation, and fluorescence detection. The adduct was detectable in all tissues of treated rats following 1 day of ETO exposure and increased approximately linearly for 3–5 days before the rate of increase began to level off. Concentrations of 7-HEG were greatest in brain, but the extent of formation was similar in all tissues studied. The adduct disappeared slowly from DNA, with an apparent half-life approx. 7 days. The shape of the formation curve and the in vivo half-life indicate that 7-HEG will approach steady-state concentrations in rat DNA by 28 days of ETO exposure. The similarity in 7-HEG formation in target and nontarget tissues indicates that the tissue specificity for tumor induction is due to factors in addition to DNA-adduct formation.     

Elevated uridine nucleotide pools in fluorouracil/fluorouridine resistant mutants ofNocardia lactamdurans

Summary Selection of spontaneous mutants ofNocardia lactamdurans MA2908 for resistance to 5-fluorouracil results in the simultaneous development of resistance to 5-fluorouridine. The resulting mutants fall into four distinct classes based on the amount of uracil accumulating in fermentation broths. An additional characteristic of these mutants is a reduction in the ability to incorporate exogenous uracil into nucleic acids even though transport and conversion to the nucleotide level appears normal. Finally, production of efrotomycin is increased in these mutants in both chemically defined and complex fermentation media to levels equivalent to those of MA4820, the first productivity mutant isolated in a conventional strain improvement program. Resistance development and uracil excretion are adequately explained by an elevation of the intracellular uridine nucleotide pool, in particular UMP. The role of the uridine necleotides in the efrotomycin fermentation is unknown.     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

Gastric colonization withCandida albicans

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.     

Diol sulfonamides: A potent and novel class of inhibitors of human renin

The syntheses and pharmacological activity of a series of diol sulfonamides which function as inhibitors of human renin are described. The most potent compound in this series, compound 20 (SQ 33,800), is a subnanomolar inhibitor of human renin (IC₅₀ = 0.35 × 10⁻⁹ M).     

Climbing Darwin's ladder

The work of Bartel and Szostak, in which RNA molecules were selected to enhance the ability to catalyze a reaction similar to a step in protein-catalyzed RNA replication, is discussed. An important aspect of this experiment was the ability to reach a high level of functional organization in ten evolutionary steps. Further steps necessary to obtain an RNA enzyme with RNA replicase activity include performing the reaction with mononucleoside 5'-triphosphates, generalizing the reaction to include a variety of sequences without loss of template-dependent specificity, and overcoming template self-structure that could prevent some regions from being copied efficiently.     

Evolution in vitro: analysis of a lineage of ribozymes

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.