Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens

Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.     

Nucleolar transformation in plants grown on clinostats

Summary Cells of carrot calli (Daucus carota L.) grown on clinostats (simulated weightlessness) exhibit increases in nucleolar number and volume. In clinostat-grown whole barley plants (Hordeum vulgare L. cv. Steptoe), nucleoli in 70% of root meristem and root cortical cells in the 1 mm root apex exhibit multiple nodulations after one day of growth. The nucleolar nodules (1.1 m mean diameter) are densely and finely fibrous, distinctly different from the nucleolus in which the content is so compact that the granular component is masked. Control nucleoli (from vertically rotated and stationary seedlings) rarely exhibit nodule-like protrusions, are not compact, and contain a well defined granular component. Proteins that are heat soluble, characteristic of many stress responses, rapidly increase in barley grown on clinostats. Barley growth on clinostats is slowly and steadily inhibited. There is no difference between vertically rotated and stationary controls for any of the parameters measured, indicating that clinostat motion per se does not affect significantly barley development. The evidence taken together suggests that barley plants germinated and grown on clinostats are stressed, the effects of which are expressed sequentially by alteration of nucleolar morphology, increased production of heat-soluble proteins, and decreased plant growth. Similar stress-related changes may be expected to occur in plants subjected to weightlessness during space flight. It is therefore of interest that nucleoli in wheat roots (Triticum aestivum L. cv. Broom) obtained from the space flight IML-1 mission show irregularity that is not observed in any of the ground controls for the flight experiment.Abbreviations Act D actinomycin D - C clinostat rotation - EM electron microscopy - LM light microscopy - R vertical rotation - rDNA ribosomal DNA - S stationary     

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for -glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the -glucuronidase (GUS) gene, under the control of the doubled enhancer element (the –208 to –46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to –389 bp from ATG) promoter of wheat, -amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 10⁶ protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.     

Agrobacterium-mediated transient transformation of annatto (Bixa orellana) hypocotyls with the GUS reporter gene

Hypocotyls from annatto seedlings, were inoculated with Agrobacterium tumefaciens harboring a binary vector, pBI.121 or pCAMBIA2301, containing the -glucuronidase (gus) gene. Histochemical GUS assay of infected hypocotyls from two annatto varieties showed transient gus gene expression between 3 and 12 days after inoculation.These authors contributed equally to this work.)     

Transgenic haploid plants ofNicotiana rustica produced by bombardment-mediated transformation of pollen

Immature pollen fromNicotiana rustica was bombarded with gold particles coated with plasmid DNA encoding neomycin phosphotransferase II (NPTII) and -glucuronidase (GUS) genes which, respectively, are under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator in the plasmid. Kanamycin-resistant pollen embryoids were selected from the bombarded pollen cells and two independent lines of transgenic plants were regenerated. Enzyme assays showed that one has both NPTII and GUS activities and the other only weak NPTII activity. Southern blot analyses indicated that the former has a DNA fragment corresponding to the intact expression cassettes for both genes in its genome; whereas the latter lacks intact expression cassettes for both genes and has only the intactnptII coding sequence in its genome. The transgenic plants of both lines have 24 chromosomes, confirming haploidy, and they are infertile. These results indicate that transgenic haploid plants can be produced directly by the bombardment-mediated transformation of immature pollen.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Using Pair-Coupled Amino Acid Composition to Predict Protein Secondary Structure Content

The pair-coupled amino acid composition is introduced to predict the secondary structure contents of a protein. Compared with the existing methods all based on singlewise amino acid composition as defined in a 20D (dimensional) space, this represents a step forward to the consideration of the sequence coupling effect. The test results indicate that the introduction of the pair-coupled amino acid composition can significantly improve the prediction quality. It is anticipated that the concept of the pair-coupled amino acid composition can be used to simplify the formulation of sequence coupling (or sequence order) effects and to study many other features of proteins as well.     

Aluminum borate whisker-mediated production of transgenic tobacco plants

An aluminum borate whiskers-mediated transformation system for calluses of tobacco (Nicotiana tabacum, cv. SR-1) has been developed. A total of 50 small pieces of calluses were vigorously agitated in a liquid medium containing aluminum borate whiskers, pBI221 plasmid carrying the -glucuronidase (GUS) gene, and pBI222 plasmid carrying the hygromycin phosphotransferase (HPT) gene. After treatment, calluses were cultured to select for hygromycin resistance, and three resistant calluses were obtained. Adventitious shoots were produced from each hygromycin-resistant callus and were transferred to rooting medium. A total of three plantlets obtained from each hygromycin-resistant callus were acclimatized and established in soil. Polymerase chain reaction analysis revealed that all the plantlets were cotransformed with both the GUS and HPT genes. Detached leaves of transgenic individuals showed clear hygromycin resistance when cultured in liquid medium. Histochemical assay for GUS revealed that one of these transgenic plants expressed the GUS gene, indicating coexpression of foreign genes.     

Infertility in mice induced by the rhesus monkey chorionic gonadotropin β-subunit glycoprotein (rmCGβ) using DNA immunization

The recombinant eukaryotic expression vector pCMV4-rmCG, inserted full-length cDNA of the -subunit of rhesus monkey chorionic gonadotropin (rmCG), as DNA immuno-contraceptive against CG glycoprotein, has previously demonstrated the biological expression of rmCG in vitro and in vivo. The plasmid DNA of pCMV4-rmCG was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCG DNA delivery method for BALB/c mice, and the dose of 10 g should be enough to elicit immune response. With different doses from 10–50 g, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCG DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 g of pCMV4-rmCG DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCG DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of -subunit of mouse chorionic gonadotropin (muCG) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCG shares 99.6% homology with rmCG and 90.6% with hCG respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCG contraceptive should be further studied as a CG DNA contraceptive.     

Identity of esterase-22 and egasyn,the protein which complexes with microsomal β-glucuronidase

Recent experiments have demonstrated that egasyn not only sequesters -glucuronidase in microsomes by forming high molecular weight complexes with -glucuronidase, but also has carboxyl esterase activity. We have found several new phenotypes of egasyn-esterase after electrophoresis and isoelectric focusing of liver homogenates and purified egasyn of inbred and wild mouse strains. Several phenotypes corresponded in relative mobility and relative isoelectric point among inbred strains to that recently reported for esterase-22 by Eisenhardt and von Deimling [(1982). Comp. Biochem. Physiol. 73B:719]. This genetic evidence, plus a wide variety of comparative biochemical and physiological data, indicates that egasyn is identical to esterase-22. Both parental types of egasyn isozymes are expressed in heterozygous F₁ progeny, suggesting that alterations in the egasyn structural gene are responsible for the altered isoelectric points. Also, egasyn is a monomer since no new esterase bands appear in F₁ progeny. The variants in isoelectric point of egasyn map at or near the egasyn (Eg) gene within the esterases of cluster 1 near Es-9 on chromosome 8.This work was supported by Grant GM-33559 from the National Institutes of Health.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants

Summary A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the -glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Carrot (Daucus carota) hypocotyl transformation usingAgrobacterium tumefaciens

Summary Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, -glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.Abbreviations NPT II neomycin phosphotransferase II - Nos nopaline synthase - GUS -glucuronidase - CaMV cauliflower mosaic virus - 2,4-D 2,4 dichlorophenoxyacetic acid - PMSF phenylmethylsulfonyl fluoride     

Conversion of free β-amylase to bound β-amylase on starch granules in the barley endosperm during desiccation phase of seed development

Summary Association of -amylase with starch granules in the starchy endosperm of barley (Hordeum vulgare L. cv. Menuet) grains was characterized biochemically. In whole homogenates of dry seeds, two forms of -amylase were detected: one is free -amylase extractable with saline solution and the other is bound -amylase extractable with saline solution containing a reducing agent. The two forms of -amylase were shown to be identical in terms of mobility on disc gels, antigenicity, and molecular specific activity, indicating that the -amylase molecules of the two forms are identical. The starch granules were isolated from either dry seeds or mature seeds harvested before the desiccation phase. Both starch granule preparations were morphologically identical by microscopic inspection. The bound -amylase was predominantly associated with starch granules isolated from dry seeds, whereas it was not associated with starch granules from mature seeds harvested before desiccation. Overall results show that the periphery of starch granules is the major site of deposition for bound -amylase in dry seeds. The association of -amylase with starch granules occurs during the desiccation phase of seed development, resulting in the conversion of free -amylase into a bound form.Recipient of an award from the Union Générale de la Brasserie Française (I. H.-N.) and from the Centre National de la Recherche Scientifique and the Japan Society for the Promotion of Science under the France-Japan Cooperative Science Programme, 1985 (M.N.).