PubMed Health⌕ Search

Biomedical subjects

K P Pauls

Publications and source records attributed to K P Pauls.

At least 19 recordsLinked to original sources
PubMed2005

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples.

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.

DNA, Plant↗
PubMed2003

Inheritance of plant regeneration from maize (Zea mays L.) shoot meristem cultures derived from germinated seeds and the identification of associated RAPD and SSR markers.

The inheritance of shoot regeneration through shoot-tip meristem culture derived from maize seedling was evaluated, and the markers (RAPD and SSR) associated with this regeneration character were identified both in a group of North American maize inbreds and a crossing population. A discrete distribution of percent regeneration and no. of shoots per explant was observed in the inbred group and the F(2) population. The results suggested that this regenerable trait was controlled by several major genes. Five RAPD markers were identified to be relevant to percent regeneration in maize shoot-tip culture system. One RAPD marker and three SSR markers were associated with no. of shoot per explant and its relevant traits. Of them marker BC603-1600 explained 18% of the variation for no. of shoot per explant and 16% of the variation for callus size. The BC603-1600 was sequenced and assigned in linkage group 7 based on a NCBI blast search. The information provided here should benefit to determine the genetic mechanisms involved in the maize regeneration response related to shoot meristem culture pathway and benefit to select high regenerable germplasm by using marker assisted selection.

Analysis of Variance↗
PubMed2002

Optimizing and quantifying fusion of liposomes to mammalian sperm using resonance energy transfer and flow cytometric methods.

BACKGROUND: Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. METHODS: Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL-alpha-phosphatidylcholine dipalmitoyl (PC)/DL-alpha-phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35 degrees C +/- 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time-dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) +/- 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome-to-sperm ratios (n = 4) and with sperm from different donors (n = 3). RESULTS: RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 x 10(6) sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). CONCLUSION: Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm-to-liposome ratio, and individual animals.

Animals↗
PubMed2002

Identification of putative genes in bean (Phaseolus vulgaris) genomic (Bng) RFLP clones and their conversion to STSs.

A set of 79 previously mapped bean (Phaseolus vulgaris) genomic (Bng) clones were partially sequenced. BLAST database searches detected homologies between 59 of these clones and genes from a variety of plants, especially Arabidopsis thaliana. Some matches in the database to the Bng clones included a putative P-glycoprotein-ABC transporter from Arabidopsis, an early nodulin-binding protein (ENBPI) from Medicago truncatula, a lon-protease protein from spinach, a branched-chain amino-acid aminotransferase from Arabidopsis, and a vacuolar sorting receptor (BP-80) from Pisum sativum. Additional matches were found for genes involved in isoprenoid biosynthesis, sulfur metabolism, proline biosynthesis, and floral development. Sequence tagged site (STSs) were produced for 16 of the clones, 2 of which contain simple sequence repeats (SSRs). Polymorphisms were detected for six of the STSs.

Base Sequence↗
PubMed2001

Mapping genetic factors affecting the reaction to Xanthomonas axonopodis pv. phaseoli in Phaseolus vulgaris L. under field conditions.

The objectives of the present study were to evaluate the field effects of Xanthomonas axonopodis pv. phaseoli (Xap), which causes common bacterial blight (CBB) on common bean (Phaseolus vulgaris L.), and to identify genetic factors for resistance to CBB using a linkage map constructed with random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP) markers. One hundred and forty-two F2:4 lines, derived from a cross between 'OAC Seaforth' and 'OAC 95-4', and the parents were evaluated for their field reaction to CBB. In the inoculated plots, the reaction to CBB was negatively correlated with seed yield, days to maturity, plant height, hypocotyl diameter, pods per plant, and harvest index. A reduction in seed yield and its components was observed when disease-free and CBB-inoculated plots were compared. The broad-sense heritability estimate of the reaction to CBB was 0.74. The disease segregation ratio was not significantly different from the expected segregation ratio for a single locus in an F2 generation. The major gene for CBB resistance was localized on linkage group (LG) G5. A simple interval mapping procedure identified three genomic regions associated with the reaction to CBB. One quantitative trait loci (QTL), each on LG G2 (BNG71Dra1), G3 (BNG21EcoRV), and G5 (PHVPVPK-1) explained 36.3%, 10.2%, and 42.2% of the phenotypic variation for the reaction to CBB, respectively. Together, these loci explained 68.4% of the phenotypic variation. The relative positions of these QTL on the core common bean map and their comparison with the previous QTL for CBB resistance are discussed.

Chromosome Mapping↗
PubMed2000

Ty1-copia-like retrotransposons of tomato (Lycopersicon esculentum Mill.).

We have used a PCR and cloning strategy to identify Ty1-copia-like retrotransposons in tomato, Lycopersicon esculentum Mill. Using degenerate oligonucleotide primers corresponding to conserved domains of the Ty1-copia retrotransposon reverse transcriptase (RT), fragments of about 260 bp were obtained by PCR amplification. Sequences of 20 cloned amplification fragments showed similarity to retrotransposon sequences. The copy number for total tomato Ty1-copia-like RT population was estimated to be approximately 2500 and may account for about 1.5% of the tomato genome. Copy numbers for four of the individual RT clones ranged from 20 to 1400 copies. A comparison of the conceptual translations of the RT sequences identified four clusters as well as three sequences which were ungrouped. When compared to RT sequences reported from several other sources, the tomato RT population was found to be widely dispersed with the majority of the RT sequences from Lycopersicon species delineated by the four tomato cluster groups. The gag region of a tomato retrotransposon was cloned from PCRs with primers based on the Tnt1 retrotransposon of tobacco. The tomato clone (pTom1.1) had 81% sequence similarity to the Tntl gag region. Several pTom1.1 sequences are present in other solanaceous species as indicated by Southern hybridization.

Amino Acid Sequence↗
PubMed1997

Characterization of somatic embryogenesis-related cDNAs from alfalfa (Medicago sativa L.).

Messenger RNAs from cultures of embryogenic and non-embryogenic alfalfa (Medicago sativa L.) genotypes were used to differentially screen a cDNA library prepared from embryogenic cell masses of somatic embryo cultures to identify early-stage embryo transcripts. The three alfalfa somatic embryogenesis-specific transcripts cDNAs (ASET1, ASET2 and ASET3) identified by this screen were enriched in RNA samples from embryogenic tissues of the embryogenic genotype but were absent from petioles or mature embryos of an embryogenic genotype as well from tissue cultures of a nonembryogenic genotype. The ASET clones did not cross-hybridize and showed different patterns of expression in northerns of RNA from various fractions of alfalfa somatic embryo cultures. The ASET clones did not hybridize with the soybean embryogenesis-specific clone (Sbh1) which was shown to be expressed in embryogenic and non-embryogenic alfalfa tissue cultures. Sequencing showed ASET1 to be a partial transcript 595 nucleotides long. ASET2 was a complete transcript of 1193 nucleotides. From a comparison of the predicted open reading frame with the GenBank protein database it was concluded that ASET2 was a novel transcript. The protein predicted by the ASET2 sequence has several potential membrane-spanning domains and a potential phosphorylation site. In addition, the ASET2 cDNA had a long 5' region that contained two upstream reading frames (URFs) which could potentially code for 30 and 6 amino acid polypeptides.

Amino Acid Sequence↗
PubMed1995

Plant biotechnology for crop improvement.

The typical crop improvement cycle takes 10-15 years to complete and includes germplasm manipulations, genotype selection and stabilization, variety testing, variety increase, proprietary protection and crop production stages. Plant tissue culture and genetic engineering procedures that form the basis of plant biotechnology can contribute to most of these crop improvement stages. This review provides an overview of the opportunities presented by the integration of plant biotechnology into plant improvement efforts and raises some of the societal issues that need to be considered in their application.

Journal Article↗
PubMed1993

Identification of a RAPD marker associated with somatic embryogenesis in alfalfa.

The current study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to genes controlling somatic embryogenesis in alfalfa. Segregation analyses of the somatic embryogenesis trait and the RAPD markers in an F1 population of 83 plants, derived from a cross between embryogenic A70-34 and non-embryogenic Arrow36 alfalfa plants, identified a polymorphic band that is associated with somatic embryogenesis. Based on the assumptions that somatic embryogenesis in alfalfa is controlled by two dominant genes with complementary effects and that the genotypes of A70-34 and Arrow36 are AAaaBbbb and aaaabbbb, respectively, the segregation data for the marker and the somatic embryogenesis trait in the F1s indicate that the marker is linked to the A locus. The maximum recombination fraction estimated for the linkage between the marker and the gene is 36.3%.

Base Sequence↗
PubMed1992

Plant growth environment effects on rapeseed microspore development and culture : a flow cytometric study.

The influence of donor plant growth conditions on microspore embryogenesis in rapeseed (Brassica napus) was studied for plants grown at 23/18 degrees C (16/8 hours) under continuous light, 23/18 degrees C (16/8 hours) with a light/dark (16/8 hours) cycle, 15/12 degrees C (16/8 hours) under continuous light and 15/12 degrees C (16/8 hours) with a light/dark (16/8 hours) cycle. Significantly higher embryo yields were obtained from microspore cultures initiated from donor plants grown at 15/12 degrees C instead of 23/18 degrees C. Flow cytometric measurements of the microspores isolated from 2.5- to 5.0-millimeter buds showed that the microspores isolated from low-temperature-grown plants had significantly lower log 90-degree light scatter to forward angle light scatter and log 90-degree light scatter to time of flight ratios than those isolated from high-temperature-grown plants, suggesting that the former are more translucent than the latter. Thus, the effect of donor plant growth temperature on microspore embryogenesis may be mediated by a change in the physiology of the microspore cell, which results in the reduction of its cytoplasmic granularity and/or exine density.

Journal Article↗
PubMed1991

Flow cytometric characterization and sorting of cultured Brassica napus microspores.

A flow cytometric procedure was used to characterize and sort embryogenic Brassica napus microspore cultures. Embryogenic microspores continued to increase in size throughout the culture period and fluoresced when stained with fluoresecein diacetate (FDA). However, most of the cells in culture (greater than 95%) lost their viability over the culture period and the correlation between the percentage viable cells in a culture and the productivity of the culture was not significant until day 3. By sorting large fluorescent cells on day 1 and day 3 of the culture period populations of cells that were 7-18-fold more embryogenic than sorted mixed cells were obtained. The flow cytometric procedure would be useful for rapidly assessing the effects of a large variety of culture and media conditions on embryogenesis is rapeseed microspore cultures. Furthermore, the populations of potentially embryogenic microspores isolated by flow cytometry might be useful for studies of the early cellular and molecular changes that occur during androgenesis in rapeseed.

Brassica↗
PubMed1991

Effects of chilling on the biochemical and functional properties of thylakoid membranes.

The mechanism of chilling resistance was investigated in 4-week-old plants of the chilling-sensitive cultivated tomato, Lycopersicon esculentum Mill. cv H722, and rooted cuttings of its chilling-resistant wild relative, L. hirsutum Humb. and Bonpl., which were chilled for 3 days at 2 degrees C with a 14-hour photoperiod and light intensity of 250 micromoles per square meter per second. This chilling stress reduced the chlorophyll fluorescence ratio, stomatal conductance, and dry matter accumulation more in the sensitive L. esculentum than in the resistant L. hirsutum. Photosynthetic CO(2) uptake at the end of the chilling treatment was reduced more in the resistant L. hirsutum than in L. esculentum, but recovered at a faster rate when the plants were returned to 25 degrees C. The reduction of the spin trap, Tiron, by isolated thylakoids at 750 micromoles per square meter per second light intensity was taken as a relative indication of the tendency for the thylakoids to produce activated oxygen. Thylakoids isolated from the resistant L. hirsutum with or without chilling treatment were essentially similar, whereas those from chilled leaves of L. esculentum reduced more Tiron than the nonchilled controls. Whole chain photosynthetic electron transport was measured on thylakoids isolated from chilled and control leaves of the two species at a range of assay temperatures from 5 to 25 degrees C. In both species, electron transport of the thylakoids from chilled leaves was lower than the controls when measured at 25 degrees C, and electron transport declined as the assay temperature was reduced. However, the temperature sensitivity of thylakoids from chilled L. esculentum was altered such that at all temperatures below 20 degrees C, the rate of electron transport exceeded the control values. In contrast, the thylakoids from chilled L. hirsutum maintained their temperature sensitivity, and the electron transport rates were proportionately reduced at all temperatures. This sublethal chilling stress caused no significant changes in thylakoid galactolipid, phospholipid, or protein levels in either species. Nonchilled thylakoid membranes from L. hirsutum had fourfold higher levels of the fatty acid 16:1, than those from L. esculentum. Chilling caused retailoring of the acyl chains in L. hirsutum but not in L. esculentum. The chilling resistance of L. hirsutum may be related to an ability to reduce the potential for free radical production by close regulation of electron transport within the chloroplast.

Journal Article↗
PubMed1987

Effect of a freeze-thaw cycle on properties of microsomal membranes from wheat.

A freeze-thaw cycle to -12 degrees C induced several physical and compositional changes in the microsomal membranes isolated from crown tissue of winter wheat (Triticum aestivum L. cv Frederick). Exposing 7-day-old, nonacclimated seedlings to a single freeze-thaw cycle prevented regrowth of the crown and resulted in increased membrane semipermeability. The phospholipid and protein content of microsomal membranes isolated from the crowns decreased by 70 and 50%, respectively. Microsomal membranes isolated after the lethal freeze-thaw stress, and liposomes prepared from total membrane lipids, exhibited greater microviscosity, measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The number of free thiol groups per milligram membrane protein, measured using the specific fluorescent probe, N-dansylaziridine, decreased after freezing. In contrast, acclimated wheat seedlings which showed increased freezing tolerance, as indicated by survival and ion leakage, suffered almost no effects from the freeze thaw treatment as determined by measurements of membrane microviscosity, phospholipid content, protein content, or danzylaziridine fluorescence. An examination of membranes isolated from frozen tissue showed that most of the changes occurred during the freezing and not during the thawing phase.

Journal Article↗
PubMed1986

Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine.

Solid gramicidin A and S and their interaction with DPPC bilayers were examined by 2H NMR as well as 31P NMR and differential scanning calorimetry (DSC). The deuterium spectra arose from deuterons associated with the peptide through chemical exchange in 2H2O. The spectra from both peptides were characterized by a quadrupolar splitting parameter, omega Q/2 pi approximately 150 kHz, and an asymmetry parameter, eta approximately 0.17. An additional 33 kHz, eta = 0 component arising from deuterons on mobile ornithine side chains was present in gramicidin S. In the gel phase of dipalmitoylphosphatidylcholine liposomes the gramicidins gave spectra that had components identical with those obtained from the solids. In the liquid-crystalline phase gramicidin A containing samples gave multicomponent spectra with a maximum quadrupolar splitting value of 133 kHz, eta = 0. A minimum in the T2e was observed, coinciding with the onset of the broadened phase transition measured by DSC and 31P NMR, due to the onset of axial rotation of the peptide in the bilayer. The different powder patterns in the liquid-crystalline spectra from gramicidin A probably arise from different amide sites along the transmembrane channel. The broad component of the 2H NMR spectra from gramicidin S in liposome preparations was not affected by the lipid-phase transition. The T2e was also constant over this temperature range. The results are consistent with a location of gramicidin S at the membrane surface.

Calorimetry, Differential Scanning↗
PubMed1985

Dynamic properties of the backbone of an integral membrane polypeptide measured by 2H-NMR.

The 2H-NMR spectrum of the exchangeable hydrogens of the synthetic amphiphilic polypeptide, lys2-gly-leu24-lys2-ala-amide, was measured for the solid peptide at room temperature and, as a function of temperature, for the peptide incorporated into hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. This study is a prototype of a similar class of experiments which can be carried out on integral membrane proteins to characterize, quantitatively, the dynamic properties of integral membrane proteins. At temperatures below the DPPC gel-liquid crystalline phase transition, the 2H NMR spectrum was very similar to that of the solid peptide indicating that the peptide was immobilized in the lipid bilayer on the time scale (approximately equal to 10(-5) s) of the 2H-NMR measurements. The 2H-NMR spectrum above the phase transition corresponded to that expected from a peptide in the alpha-helical conformation reorienting rapidly about the symmetry axis of the alpha-helix. Measurements of the quadrupolar echo relaxation time, T2e, gave a quantitative measure of the correlation time, tau c, for this motion. The value of tau c decreased rapidly with increasing temperature as the fraction of DPPC molecules in the liquid crystalline phase increased, reaching a value of 2 X 10(-7) s above the phase transition. The observation of a characteristic minimum in T2e as the temperature was raised provided a definitive, quantitative interpretation of the T2e measurements. Using the known geometry of the peptide and the theory of uniaxial rotational diffusion, a value of eta = 1.1 poise was obtained for the effective viscosity of the membrane in close agreement with values obtained previously from transient linear dichroism measurements.

Calorimetry, Differential Scanning↗
PubMed1984

Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons.

Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus vulgaris. The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H(2)O(2) only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (O(2) (-), H(2)O(2), OH) are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo.

Journal Article↗