PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2α(PGF2α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA (CHO-PGF2α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2α-induced PLD activation in CHO-PGF2α·R cells. PLD activation was examined by measuring the production of [³H]phosphatidylbutanol ([³H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2α-induced [³H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2α. The maximal [³H]PBut formation was observed at 2 min after addition of PGF2α. Depletion of extracellular Ca²⁺ with EGTA suppressed PGF2α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2α-induced [³H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2α-induced PLD activation. A combination of maximal effective concentrations of PGF2α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKCα and decreased PGF2α-induced PLD activation. These results suggest that PLD activation by PGF2α is mediated by both PKC-dependent and -independent pathways and that PKCα is involved in the former pathway.     

La procréation médicalement assistée avec spermatozoïdes immatures: Impératifs cliniques et techniques chirurgicales

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.     

THE RELATIONSHIP BETWEEN A FLOWER GALL MIDGE PSEUDAS-PHONDYLIA SP. (DIPTERA: CECIDOMYIIDAE) AND ITS HOST PLANT ACTINIDIA VALVATA*

Abstract Kiwifruit plants, Actinidia sp., are native to subtropical China. The flower-bud gall of A. valvata, which is induced by an undescribed gall midge in the genus Pseud as phond ylia, is valued by the pharmaceutical industry. When studying the biology of the Actinid ia/Pseud as phond ylia interaction in Central-south China we found evidence suggesting that under certain circumstances the gall insect modifies the reproductive mode of the dioecious host plant. Surveys and field experiments in the National Hupingshan Natural Reserve showed a high frequency of galled trees. The density of galled trees varied among valleys and among trees within the valleys. In two valleys, 92% and 75%, respectively, of all trees were attacked, while in a third valley no trees were attacked. When infested, staminate tree only produced galls, whereas pistillate plants produced normal fruits as well as galls. Gall shape differed between male and female trees. Trees with galls tended to produce more fruits than treea without galls. We speculate that this is one of a few documented examples of an insect that induces androdioecy in an otherwise functionally dioecious plant.     

Polymerization of Actin from Maize Pollen

Here we describe the in vitro polymerization of actin from maize (Zea mays) pollen. The purified actin from maize pollen reported in our previous paper (X. Liu, L.F. Yen [1992] Plant Physiol 99: 1151-1155) is biologically active. In the presence of ATP, KCl, and MgCl2 the purified pollen actin polymerized into filaments. During polymerization the spectra of absorbance at 232 nm increased gradually. Polymerization of pollen actin was evidently accompanied by an increase in viscosity of the pollen actin solution. Also, the specific viscosity of pollen F-actin increased in a concentration-dependent manner. The ultraviolet difference spectrum of pollen actin is very similar to that of rabbit muscle actin. The activity of myosin ATPase from rabbit muscle was activated 7-fold by the polymerized pollen actin (F-actin). The actin filaments were visualized under the electron microscope as doubly wound strands of 7 nm diameter. If cytochalasin B was added before staining, no actin filaments were observed. When actin filaments were treated with rabbit heavy meromyosin, the actin filaments were decorated with an arrowhead structure. These results imply that there is much similarity between pollen and muscle actin.     

Galactinol synthase from kidney bean cotyledon and zucchini leaf. Purification and N-terminal sequences.

Galactinol synthase (GS) was purified 1591-fold with a 3.9% recovery from the cotyledon of kidney bean (Phaseolus vulgaris) by a novel scheme consisting of ammonium sulfate fractionation followed by diethylaminoethyl, Affi-Gel Blue, and UDP-hexanolamine affinity chromatography. The purified enzyme had a specific activity of 8.75 mumol mg-1 min-1, a pH optimum of 7.0, and requirements for manganese ion and DTT. The enzyme exhibited a Km = 0.4 mM for UDP-galactose and a Km = 4.5 mM for myo-inositol. It was identified as a 38-kD peptide that co-purified with a 41- and a 43-kD peptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purification to homogeneity was achieved by isolating the 38-kD peptide from the SDS-PAGE gel. To clarify conflicting reports in the literature about the relative molecular mass of purified GS from zucchini leaf (Cucurbita pepo), a similar scheme with modified eluting conditions was used to purify GS from this source. Zucchini leaf GS was purified to homogeneity and identified as a 36-kD peptide on SDS-PAGE. Partial N-terminal sequences of the 38-kD peptide from kidney bean cotyledon and the 36-kD peptide from zucchini leaf were obtained. To facilitate identification of GS during the purification, an assay utilizing thin-layer chromatography and an isotopic analytic imaging scanner was developed.     

A gene encoding a truncated large subunit of Rubisco is transcribed and salt-inducible in rice

Using the rice salt-tolerant mutant 20 as material, a cDNA library was constructed and two salt-inducible clones, SIR5.5 and SIR8.1, were isolated by differential screening. Homology analysis revealed that the two clones together constituted a chimeric rbcL which encoded a truncated large subunit of Rubisco with 337 amino-acids, plus 64 amino-acids of unknown origin. The expressions of both the normal and the chimeric locus appeared to be developmentally regulated and salt-inducible in shoots of the salt-tolerant mutant 20 and its original variety 77–170. In roots, their expressions were salt-inducible in the salt-tolerant mutant 20 whereas no, or only premature, forms were present in the salt-treated original variety 77–170. Higher concentrations of salt reduced the expressions of both normal rbcL and the chimeric locus. ABA showed no effect on their expression.     

Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz)

A size-fractionated TaqI genomic library of seashore paspalum (Paspalum vaginatum Swartz) was screened for the presence of (GA) _n and (CA) _n simple sequence repeats (SSRs). A total of 54 clones with a positive signal were detected among 13,000 clones screened. Forty-seven clones having repeats of n 3 were identified, of which 85% were perfect, 13% were imperfect and 2% were compound repeat sequences. Five of ten primer pairs synthesized to amplify selected loci resulted in a product in the expected size range and were subsequently used to examine SSR polymorphisms among 46 ecotypes of P. vaginatum. The number of alleles resolved on agarose or polyacrylamide gels were similar and ranged from 6 to 16 with an average of 14 per locus. Phenetic analysis of SSR polymorphisms revealed genetic relationships among the P. vaginatum ecotypes that were in general agreement with relationships determined previously by RAPD analysis of the same plant materials. Further screening of the genomic library did not identify (AT) _n , trimeric or tetrameric repeats. Hybridization of an (ATT)₈ oligonucleotide probe to genomic DNA isolated from I. batatas, E. coli, Citrullis lanatus and P. vaginatum suggested that the P. vaginatum genome contained significantly fewer ATT repeats than either the I. batatas or C. lanatus genome.     

Isolation,characterization and application of a species-specific repeated sequence from Haynaldia villosa

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.     

Mapping quantitative trait loci for downy mildew resistance in pearl millet

Quantitative trait loci (QTLs) for resistance to pathogen populations of Scelerospora graminicola from India, Nigeria, Niger and Senegal were mapped using a resistant x susceptible pearl millet cross. An RFLP map constructed using F₂ plants was used to map QTLs for traits scored on F₄ families. QTL analysis was carried out using the interval mapping programme Mapmaker/QTL. Independent inheritance of resistance to pathogen populations from India, Senegal, and populations from Niger and Nigeria was shown. These results demonstrate the existence of differing virulences in the pathogen populations from within Africa and between Africa and India. QTLs of large effect, contributing towards a large porportion of the variation in resistance, were consistently detected in repeated screens. QTLs of smaller and more variable effect were also detected. There was no QTLs that were effective against all four pathogen populations, demonstrating that pathotype-specific resistance is a major mechanism of downy mildew resistance in this cross. For all but one of the QTLs, resistance was inherited from the resistant parent and the inheritance of resistance tended to be the result of dominance or over-dominance. The implications of this research for pearl millet breeding are discussed.