Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

DNA markers tightly linked to a gall midge resistance gene (Gm2) are potentially useful for marker-aided selection in rice breeding

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select for Gall Midgeresistant rice lines under field conditions. The primers for the assay were designed on the basis of sequence information of two phenotype specific random amplified polymorphic DNA fragments which were found to be tightly linked to Gall Midge biotype-1 resistance gene (Gm2). The two RAPD fragments, F8₁₇₀₀ in the susceptible parent ARC6650 and F10₆₀₀ in the resistant parent Phalguna, were identified after screening 5450 loci using 520 random primers on genomic DNAs of ARC6650 and Phalguna. These primers, when used in a multiplexed PCR, amplified specifically a 1.7-kb and 0.6-kb fragment in the susceptible and resistant parents, respectively. When this assay was performed on genomic DNAs of 44 recombinant inbred lines derived from ARC6650 x Phalguna and 5 lines derived from other crosses where one of the parents was Phalguna, ARC6650 or their derivatives, the primers amplified a 1.7-kb fragment in all of the susceptible lines or a 0.6-kb fragment in all of the resistant ones. These markers can be of potential use in the marker-aided selection of Gall Midge biotype-1 resistant phenotypes. As screening for resistance can now be conducted independent of the availability of insects, the breeding of resistant varieties can be hastened.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

The chromosomal locations and linkage relationships of the structural genes for the prolamin storage proteins (secalins) of rye

Summary Rye secalins are a polymorphic mixture of polypeptides which are classified into four major groups. Previous studies have shown that the structural genes for two of the groups (the -secalins and 40K -secalins) are located on the short arm of chromosome 1R and those for a third group (the high molecular weight secalins) on the long arm of the same chromosome. Analysis of F₂ grain from crosses between inbred lines of S. cereale shows that the structural genes for the -secalins (designated Sec 1) and the high molecular weight secalins (designated Sec 3) are loosely linked (40.8 ±3.76% recombination, 57.4 ± 11.30 cM). Analysis of wheat rye addition lines shows that the structural genes for the 75K -secalins are present on chromosome 2R. This locus is provisionally designated Sec 2. These genes are probably derived from those for the 40K -secalins by duplication, divergence and translocation. Analysis of secalin fractions from wild species of rye shows that all contain 75K -secalins, indicating that the duplication and divergence, if not the translocation, occurred before speciation of the genus.     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

The response of a cytokinin resistant mutant is highly specific and permits a new cytokinin bioassay

We have previously isolated six independent cytokinin-resistant mutants of Nicotiana plumbaginifolia which define three complementation groups, zeal, zea2 and zea3. We report here the characterization of the phenotypic response to cytokinin treatment of the mutant 1–64, belonging to the zeal group, and the result of the study of the specificity of this response. The phenotype of this mutant grown in the presence of cytokinin concentrations higher than 0.1 M is characterized by a hypertrophy of the cotyledons and hypocotyl which results in an increase of plantlet fresh weight. This hypertrophy is correlated to cytokinin concentration in a range between 0.01 to 10 M. The specificity of this response has been verified by using adenine and urea type cytokinins, as well as enantiomers of methylzeatin and methylbenzyladenine which differ widely in their cytokinin activities. We show that the high specificity of the hypertrophic response to cytokinins can be used as a convenient bioassay to screen the cytokinin activity of adenine or urea type molecules.Abbreviations zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine] - iP isopentenyladenine [6-(3-methylbut-2-enylamino)purine] - BA benzyladenine [6-(benzylamino)purine] - (R)-(+)-MeZea [(R)--methylzeatin] - (S)-(–)-MeZea [(S)--methylzeatin] - (R)-(+)-MeBA [(R)--methylbenzyladenine] - (S)-(–)-MeBA [(S)--methylbenzyladenine] - CPPU N-(2-chloro-4-pyridyl)-N-phenylurea - thidiazuron N-(1,2,3-thiadiazol-5-pyridyl)-N-phenylurea The authors dedicate this paper to the memory of Jean-Pierre Bourgin, Director of the Laboratoire de Biologie Cellulaire, who died suddenly on October 29, 1994.     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Identification of a 4A/7R and a 7B/4R wheat-rye chromosome translocation

Summary By producing chromosome substitutions with Imperial rye chromosomes 4R (C) and 7R (D) in the wheat cultivar Chinese Spring two spontaneous translocation lines were obtained. One involves segments of wheat chromosome 4A and rye chromosome 7R, the other involves portions of wheat chromosome 7B and rye chromosome 4R     

Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers

Summary The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var Vogelsanger Gold and the spring var Alf. A map of chromosome 2 spanning 119cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley.     

Modification of the properties of a translocation in the German cockroach by selection

Summary A stock of Blattella germanica bearing the interchange T(3; 12)/3;12 was subjected to close inbreeding with selection for random disjunction at metaphase I. After 3–4 generations of selection, interchange quadrivalent chiasma frequency decreased, variability in free bivalent chiasma frequency increased sharply, and individuals with either random or directed disjunction were present in the stock. Random disjunction was modified from a ratio of 2112 (adj.-1; alt.-1; adj.-2; alt.-2) to a ratio of 1111. After 7–8 generations of selection, chiasma frequency appeared to stabilize at lower than normal levels and variability decreased for both quadrivalents and free bivalents. Directed disjunction was modified from a ratio of 2114 to 1112, and no individuals with the original high level of directed disjunction were detected. Chains-of-four tended to orient randomly, especially in individuals where the ring quadrivalents showed directed disjunction. Relaxation of inbreeding, but not selection, produced an increase in chiasma frequency and variability in both free bivalents and quadrivalents, but the modified ratios for both random and directed disjunction were retained. These results are discussed with respect to inbreeding effects and genetic control of chiasma frequency and metaphase I disjunction in interchange quadrivalents.     

Use of recombinant substitution lines in the construction of RFLP-based genetic maps of chromosomes 6A and 6B of tetraploid wheat (Triticum turgidum L.)

RFLP-based genetic maps of chromosomes 6A and 6B of Triticum turgidum have been constructed using data obtained by the study of Triticum turgidum var durum cv Langdon-T. t. var dicoccoides recombinant substitution lines (RSLs) supplemented with data obtained from F₃ families derived from Langdon dicoccoides 6A and 6B disomic substitution lines. The average RFLP frequencies detected for the two chromosomes in a test of 45 DNA clones with six restriction enzymes were 56% and 53%, respectively, and a subset of 32 clones gave frequencies of 75% and 72%, respectively. Seventeen loci were mapped in 6A and 18 in 6B. With the possible exception of 5 loci in the centromeric region of 6A, all of the mapped 6A and 6B loci are located in the same arm as are homologous loci in hexaploid wheat, and the linear order of the loci is the same in the two chromosomes, except possibly close to the centromere. Major differences in genetic distances exist between homologous loci located in the proximal regions of the 6AL and 6BL linkage groups, however, the distances being much larger in the former than in the latter. The 6B maps that were constructed using data from both the RSL and the F₂ populations and using data from the RSL population alone closely resemble one another, indicating that the 6B RSL population, composed of 85 lines, can be reliably used for genetic mapping. Additional studies must be conducted before the utility of the 6A RSL population, composed of 66 lines, can be adequately assessed.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

Selective feeding by larval dytiscids (Coleoptera:Dytiscidae) and effects of fish predation on upper littoral zone macroinvertebrate communities of arctic lakes

Carbon and nitrogen stable isotopic data from the primary producers in mangrove ecosystems are needed to investigate trophic links and biogeochemical cycling. Compared with other mangrove species (e.g. Rhizophora mangle) very few measurements have been conducted on the white mangrove, Laguncularia racemosa. The carbon and nitrogen stable isotopic and elemental compositions of L. racemosa were analyzed and compared from Florida and Belize. ¹³C values of L. racemosa from Florida (mean = –26.4) were slightly higher than those from Twin Cays, Belize (mean = -27.4), which may be due to higher salinity in some parts of the Florida site. There was no difference between the ¹⁵N values from L. racemosa from these two sites (Florida mean = 0.6; Belize mean = 0.3), which are indicative of nitrogen derived from nitrogen fixation in a planktonic marine system. However, higher ¹⁵N values from L. racemosa at Man of War Cay in Belize (11.4 and 12.3), which is fertilized by roosting marine birds (14.0), illustrate that L. racemosa can sensitively reflect alternative nitrogen sources. Although the isotopic data could not distinguish between Avicennia germinans, R. mangle and L. racemosa in Belize the L. racemosa had considerably higher C/N ratios (46.5 – 116.1) compared with the Florida samples (42.2 – 76.0) or the other mangrove species. Unlike some previous findings from R. mangle, substrate characteristics (e.g. salinity, NH₄ ⁺, and H₂S) were not related to the isotopic or elemental composition of L. racemosa. ¹³C, ¹⁵N and C/N were analyzed for ecosystem components from L. racemosa habitats at Twin Cays, including other plants (e.g. R. mangle, A. germinans and seagrass), detritus, microbial mats and sediments. Results from mass-balance calculations show that mangrove detritus composes very little of the sediment, which is principally composed of microbial biomass (80 – 90%). Detritus at some sites is also influenced by sources other than that from L. racemosa, including seagrass leaves.     

Identification of the chromosomes of the rye translocation tester set

Summary Intercrossing the Wageningen translocation tester set of rye and the series of Imperial rye additions to Chinese Spring wheat of Sears yielded 29 chromosome disomic translocation hybrids. Observation of trivalents led to the identification of the chromosomes of the tester set in terms of the terminology system used in the Triticinae. The analysis was complicated by very low chiasma frequencies in some short chromosome segments in the hybrids. Nevertheless, it could be safely concluded that 1R=VII; 2R=III; 3R=II; 4R=IV; 5R=VI; 6R=V; 7R=I, which deviates slightly from previous classifications based on other methods.