Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

A comparative analysis of the development and quality of nursery plants derived from somatic embryogenesis and from seedlings for large-scale propagation of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Plants of Coffea arabica L. derived via somatic embryogenesis, namely, somaclones, were evaluated with C. arabica seedlings grown in the nursery. At the time of their transfer to the nursery, somaclones of C. arabica cvs. Caturra and Costa Rica 95 (Catimor) were smaller and less vigorous than seedlings of the same cultivars. Following an initial slow growth for a period of 10 weeks, somaclones began to grow faster than seedlings until both groups of plants were equal in size at 21 weeks (entire duration of growth in the nursery). Comparisons of aerial and root systems of 30-cm long somaclones and seedlings of two cultivars revealed that plants of somaclones were more vigorous than seedlings, based on the higher number of leaves (13–16 vs. 9), larger leaf area (1060–1280 vs. 730–890 cm²), and greater dry weight of aerial organs (8.5–12 vs. 7.0–7.5 g). For cv. Caturra, the root dry weight of somaclones was significantly greater than that of seedlings (2.7 vs. 1.9 g) and was attributable to the large diameter roots (>0.5 mm). Analysis of 176,000 F₁ hybrid somaclones revealed that these exhibited more heterogeneous growth than did the seedlings derived from zygotic embryos; moreover, there was a genotype effect. Almost 9–20% of somaclones required an additional 3–4 months of growth in the nursery, and 8–12% were culled for other undesirable horticultural attributes. Only 0.10–0.23% of somaclones displayed variant phenotypes. The observed somaclone vigor in the nursery was carried over to field performance as these plants were more precocious than seedlings and yielded coffee beans 1 year earlier than seedlings.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

Genetic integrity of somaclonal variants in tea (Camellia sinensis (L.) O Kuntze) as revealed by inter simple sequence repeats

Adoption of inter simple sequence repeats (ISSR) technique to analyze the genetic variability of somatic embryo derived tea plants was evaluated. Morphological characterisation of the field grown plants revealed no identical character aligning with the parent, UPASI-10. Out of 40 primers, 15 exhibited concurrent polymorphism were selected for the study. Genetic variability of somaclones derived from single line cotyledonary culture ranged from 33.0 to 55.0%. A unique fragment of 1.2Kb was visible in majority of the accessions whereas the fragments below the length of 0.6Kb were noticed only in 50% of the variants. Out of 120 interactions attempted using Pearson's coefficient correlation, only 9.2% of somaclones exhibited significant similarity at genetic level. Dendrogram constructed based on simple matching coefficient revealed a distance of 2.257-3.317 between the final clusters. This strengthens the existence of wide genetic variation among the somaclones.     

Nature of the ribosomal RNA transcribed from the X and Y chromosomes of Drosophila melanogaster

In Drosophila melanogaster there is one nucleolar organizer (NO) on each X and Y chromosome. Experiments were carried out to compare the ribosomal RNAs derived from the two nucleolar organizers. ³²PO₄-labelled ribosomal RNA was isolated from two strains of D. melanogaster, one containing only the X chromosome NO, the other containing only the Y chromosome NO. 28 S and 18 S RNA from the two strains were subjected to a variety of “fingerprinting” and sequencing procedures. Fingerprints of 28 S RNA were very different from those of 18 S RNA. Fingerprints of “X” and “Y” 28 S RNA were indistinguishable from each other, as also were fingerprints of “X” and “Y” 18 S RNA. In combined “T₁ plus pancreatic” RNAase fingerprints several distinctive products were characterized and quantitated. Identical products were obtained from X and Y RNA, and the molar yields of the products were indistinguishable. Together these findings imply that the rRNA sequences encoded by the X and Y NOs are closely similar and probably identical to each other.Two further findings were of interest in “T₁ plus pancreatic” RNAase fingerprints: (1) in 28 S (as well as in 18 S) fingerprints several distinctive products were recovered in approximately unimolar yields. This indicates that 28 S RNA does not consist of two identical half molecules, though it does consist of two non-identical half molecules together with a “5.8 S” fragment. (2) Several methylated components in Drosophila rRNA also occur in rRNA from HeLa cells and yeast. This suggests that certain features of rRNA structure involving methylated nucleotides may be highly conserved in eukaryotic evolution.     

Properties of “enkephalinase” from rat kidney: Comparison of dipeptidyl-carboxypeptidase and endopeptidase activities

The “enkephalinase” i.e. the metallopeptidase cleaving the Gly³-Phe⁴ amide bond of enkephalins from rat kidney was studied in its membrane-bound form as well as in a highly purified preparation. It seems identical or very close to three other enzyme activities: “enkephalinase” from cerebral membranes, an endopeptidase from bovine pituitary and the “neutral endopeptidase” from rabbit kidney. Specificity constants of substrates were higher for peptides with a free terminal carboxylate as compared to amidified or typical endopeptidase substrates which were also cleaved. The dipeptidyl carboxypeptidase specificity of “enkephalinase” is attributable to the presence of a critical arginine residue in its active site.     

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.     

Gibberellin like Substances in Parts of Developing Barley Grain

In husks, the activity of gibberellin-like substances extracted with aqueous methanol (M-“free” GAs) showed a maximum on the 9th day after pollination. In developing embryos, M-“free” GAs showed no biological activity, whereas biological active component(s) were obtained when the embryos were extracted with Tris buffer. The “free” GAs found in the buffer homogenates (B-“free” GAs) of developing embryos showed a maximum of activity on the 33rd day after pollination. Bound GAs recovered from the precipitated protein of the buffer homogenate (“Protein-bound” GAs) were found in embryo and endosperm. Developing endosperm generally contains the major amount of the extractable gibberellin-like substances. In this tissue, the amount of all examined fractions (M-“free” GAs, B-“free” GAs and “protein-bound” GAs) increased after pollination to reach a maximum on the 21st day, before decreasing to a minimum at grain maturity. Moreover, the curves for dry weight increase and gibberellin like substances follow a remarkably similar course, with the latter reaching its maximim slightly earlier than the former one. This result indicates that gibberellines may participate in the regulation of the accumulation process in the endosperm of barley grain.     

Molecular divergence of alfalfa somaclones

Summary Plantlets were regenerated from alfalfa callus following passage through a tissue culture medium which contained gibberellic acid. A proportion of these plantlets showed obvious morphological variations. Leaflet, stem and petiole tissue of these plants were extracted to yield a soluble protein homogenate which was characterized by polyacrylamide gel electrophoresis. Over 18 individual protein bands were resolved and visualized by staining with coomassie blue G250. Electrophoretic gels from regenerated plantlets and from the parent plant were scanned spectrophotometrically and analyzed. The relative quantity of each of the proteins resolved from plants was correlated with proteins of other plants via the Pearson's product-moment correlation. Cluster analysis was then performed using these correlation coefficients to judge relatedness among somaclones and the parent plant. Two of 22 somaclones (9.1%) differed significantly from the parent and from the other somaclones judged by quantitative protein pattern variations. Three distinctive lineages through tissue culture produced plantlets. Using a discriminant analysis strategy somaclones could be grouped according to lineage with 80.8% accuracy based upon distinctions between protein electrophoretic patterns. Two of the somaclone lineage groupings showed no overlap with the parental grouping which indicated significant molecular divergence of these plantlets as judged by quantitative protein differences.     

EXPECTATIONS AND STABILITY OF PREFERENCE CHOICE

When identical milk samples are presented, only 30% of participants respond with a “no preference” rating. Stability of the “no preference” rating was studied under a variety of conditions, having consumer panelists rate both identical and different pairs of milk samples with varying fat content. The proportion of participants choosing the “no preference” option, when the samples in the pair were identical, was largely consistent despite manipulation of pretest conditions and changes in test questions and answer formats. However, when the milk preference test was preceded by a same/different test, and those responding “same” were assumed to have no preference, the percent of “no preference” was two to three times larger for identical test samples (60–69%). Thus by branching the question, the “false preference” choice for identical milks was lowered. Among those responding “different” to identical milks, the false‐alarm rate increased to 91%, suggesting that perception of (spurious or momentary) differences is driving at least part of the preference choice.     

A structural basis for CD8+ T cell-dependent recognition of non-homologous peptide ligands: implications for molecular mimicry in autoreactivity

Molecular mimicry of self-epitopes by viral antigens is one possible pathogenic mechanism underlying induction of autoimmunity. A self-epitope, mDBM, derived from mouse dopamine beta-mono-oxygenase (KALYDYAPI) sharing 44% sequence identity with the lymphocytic choriomeningitis virus-derived immunodominant epitope gp33 (KAVYNFATC/M), has previously been identified as a cross-reactive self-ligand, presentation of which results in autoimmunity. A rat peptide homologue, rDBM (KALYNYAPI, 56% identity to gp33), which displayed similar properties to mDBM, has also been identified. We herein report the crystal structure of H-2Db.rDBM and a comparison with the crystal structures of the cross-reactive H-2Db.gp33 and non-cross-reactive H-2Db.gp33 (V3L) escape variant (KALYNFATM, 88% identity to gp33). Despite the large sequence disparity, rDBM and gp33 peptides are presented in nearly identical manners by H-2Db, with a striking juxtaposition of the central sections of both peptides from residues p3 to p7. The structural similarity provides H-2Db in complex with either a virus-derived or a dopamine beta-mono-oxygenase-derived peptide with a shared antigenic identity that conserves the positioning of the heavy chain and peptide residues that interact with the T cell receptor (TCR). This stands in contrast to the structure of H-2Db.gp33 (V3L), in which a single conserved mutation, also present in rDBM, induces large movements of both the peptide backbone and the side chains that interact with the TCR. The TCR-interacting surfaces of the H-2Db.rDBM and H-2Db.gp33 major histocompatibility complexes are very similar with regard to shape, topology, and charge distribution, providing a structural basis for CD8 T cell activation by molecular mimicry and potential subsequent development of autoreactivity.     

Serological Comparison of Antigens Related to Local Virus and Bacterial Infections and Aspecific Stresses in Tobacco Leaves

Two “new” precipitin bands (antigens) detected by the immunodiffusion test were demon strated in leaf extracts of tobacco inoculated with tobacco mosaic virus (TMV), Pseudomonas tabaci or treated with mercuric chloride, sodium azide or sodium hypochlorite. One of the precipitin bands was stronger, than the other, These antigens were also detected in the upper, non-infected leaves of tobacco plants when the lower leaves were locally stressed (necrotized) either by TMV or by chemical injury. The “new” antigens formed in the upper leaves were detected even if the TMV-inoculated lower leaves were removed one day after inoculation. The “new” antigens were identical both in the lower and upper leaves and their induction was independent from the stress whether pathogenic or chemical. A coincidence exists between the appearance of “new” antigens and acquired resistance, but this does not mean necessarily a cause-and-effect relationship between the two phenomena. Our experiments indicate that the induction of the synthesis of “new” stress proteins in tobacco is aspecific and the proteins formed are related to the aspecific stress itself rather than to pathogenesis.     

NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa

The complete 1H- and 13C-NMR assignments of the major Cannabis constituents, delta9-tetrahydrocannabinol, tetrahydrocannabinolic acid, delta8-tetrahydrocannabinol, cannabigerol, cannabinol, cannabidiol, cannabidiolic acid, cannflavin A and cannflavin B have been determined on the basis of one- and two-dimensional NMR spectra including 1H- and 13C-NMR, 1H-1H-COSY, HMQC and HMBC. The substitution of carboxylic acid on the cannabinoid nucleus (as in tetrahydrocannabinolic acid and cannabidiolic acid) has a large effect on the chemical shift of H-1" of the C5 side chain and 2'-OH. It was also observed that carboxylic acid substitution reduces intermolecular hydrogen bonding resulting in a sharpening of the H-5' signal in cannabinolic acid in deuterated chloroform. The additional aromaticity of cannabinol causes the two angular methyl groups (H-8 and H-9) to show identical 1H-NMR shifts, which indicates that the two aromatic rings are in one plane in contrast to the other cannabinoids. For the cannabiflavonoids, the unambiguous assignments of C-3' and C-4' of cannflavin A and B were determined by HMBC spectra.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

普通小麦F_1杂种Glu-1基因表达过程中的共显性,基因组互作和剂量效应

普通小麦F_1杂种Glu-1基因表达过程中的共显性,基因组互作和剂量效应@潘幸来$山西农业科学院棉花研究所!运城044000小麦;;基因表达;;基因组     

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2K ^b mutants analyzed.Abbreviations used in this paper B6 C57BL/6Kh - bm5 B6-H-2^bm5 - bm6 B6-H-2 ^bm6 - bm7 B6.C-H-2 ^bm7 - bm9 B6.C-H-2 ^bm9 - bm16 B6-H-2 ^bm16 - D H-2D - K H-2K - MHC major histocompatibility complex     

Contamination of exosome preparations,isolated from biological fluids

The aim of this study was to attract attention of researchers to the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 (JEOL, Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: blood plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and conditioned cell culture medium (MDCK, MDA-MB, and MCF-7 cells). All examined preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations were characterized by the presence of contaminating structures: low electron density particles without limiting membrane and therefore could not be attributed to exosomes (“non-vesicles”). Two main types of the “non-vesicles” were found in the exosome preparations: particles of 20–40 nm in size, representing 10–40% of all structures in the exosome preparations; and particles of 40–100 nm in size (identical to exosomes by size). Morphology of the “non-vesicles” corresponded to that of intermediate and low density lipoproteins (20–40 nm), and very low density lipoproteins (40–100 nm), which were identical to exosomes in their size. The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparations by electron microscopy and to take into consideration the presence of contaminating structures in the analysis of experimental data.     

CONSUMER ACCEPTANCE OF FOOD BARS

Increased use of conjoint analysis during the concept testing stage of food product development raises the question of whether conjoint analysis results translate into successful product development. Five food bar concepts from a previous conjoint analysis study targeted for the “Overall” panel, “Female” segment, “Male” segment, “Label Readers” segment and “Calorie Health Nuts” segment, were used for consumer testing. Standardized packaging was also developed to test elements not easily perceived from the product itself, i.e., low fat, low calorie and other nutritional information. The “Female #1 – commercial (Fiber One)” bar had the highest overall acceptance, 6.9 on a 9-point hedonic scale. Packaging significantly increased all ratings. Cluster analysis results were compared with segmentation from conjoint analysis. Similar results were found, showing there were clusters of consumers specifically interested in prototypes developed for specific conjoint analysis segments, which supports that conjoint analysis results translate into actual products.     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。