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  • Articles  (66)
  • gene expression  (36)
  • Somatic embryogenesis  (30)
  • Springer  (66)
  • 1995-1999  (66)
  • 1945-1949
  • 1998  (66)
  • Biology  (66)
  • Process Engineering, Biotechnology, Nutrition Technology  (1)
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  • Articles  (66)
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  • 1995-1999  (66)
  • 1945-1949
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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    BioMetals 11 (1998), S. 345-358 
    ISSN: 1572-8773
    Keywords: calcium ; CREB ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Through the evolution of multicellular organisms, calcium has emerged as the preferred ion for intracel-lular signalling. It now occupies a pivotal role in many cell types and nowhere is it more important than in neurons, where it mediates both the relaying and long-term storage of information. The latter is a process that enables learning and memory to be formed and requires the activation of gene expression by calcium signals. Evidence from a number of diverse organisms shows that transcription mediated by the transcrip-tion factor CREB is critical for learning and memory. Here we review the features of CREB activation by calcium signals in mammalian cells. In contrast to other transcription factors, its regulation is dependent on an elevation of nuclear calcium concentration, potentially placing this spatially distinct pool of calcium as an important mediator of information storage.© Kluwer Academic Publishers
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology reporter 16 (1998), S. 323-339 
    ISSN: 1572-9818
    Keywords: Aux/IAA genes ; gene expression ; gene families ; RT-PCR ; tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have developed an improved method for determination of gene expression levels with RT-PCR. The procedure is rapid and does not require extensive optimization or densitometric analysis. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, we were able to rapidly screen nine members of the Aux/IAA family of auxin- responsive genes and identify those genes which vary in message abundance in a tissue- and light-specific manner. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, our method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.
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  • 3
    ISSN: 1432-203X
    Keywords: Key words ABA ; Somatic embryogenesis ; l-glutamine ; Thidiazuron
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l–1 2,4-D and 0.2 mg l–1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l–1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic callus was transferred to MS medium supplemented with 3 mg l–1 ABA and 60 g l–1 sucrose. The addition of 10–30 mM l-glutamine improved embryo development.
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  • 4
    ISSN: 1432-203X
    Keywords: Key words Pigeonpea ; Cajanus cajan ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 µm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters.
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  • 5
    Electronic Resource
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    Plant cell reports 17 (1998), S. 469-475 
    ISSN: 1432-203X
    Keywords: Key wordsAzadirachta indica A. Juss. ; Neem ; Somatic embryogenesis ; Thidiazuron
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1–50 µm). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 µm and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2–3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem.
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  • 6
    ISSN: 1432-203X
    Keywords: Key words Protoplast fusion ; Iodoacetamide ; Somatic embryogenesis ; Morphology ; Chromosome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Somatic hybrids were obtained by polyethylene glycol fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. peruvianum (PI270435) or L. chilense (PI128652). The hybrids were selected by a multiple-step selection procedure relying on the different colors of the fusion partners, IOA treatment of cotyledon protoplasts, and the use of a culture medium which only allowed cotyledon protoplasts to regenerate. The somatic embryos were derived from greenish calli that formed from the fusion mixtures, developed progressively through the globular, heart, and torpedo stages, and finally formed complete plantlets. The excised torpedo-stage embryos could be propagated on a modified medium. The morphology of the somatic hybrids were intermediate to their donor partners, and chromosome observations indicated that the hybrids were tetraploid, hexaploid, and aneuploid.
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  • 7
    Electronic Resource
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    Plant cell reports 17 (1998), S. 251-255 
    ISSN: 1432-203X
    Keywords: Key wordsCymbidium ensifolium var. misericors ; Plant regeneration ; Rhizome ; Somatic embryogenesis ; Totipotent callus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Totipotent calli of Cymbidium ensifolium var. misericors, a locally grown orchid of high commercial value, were induced from sections of pseudobulbs, rhizomes and roots of seed-derived plantlets on 1/2-strength Murashige and Skoog medium plus 10 mg/l 2,4-dichlorophenoxyacetic acid and 0.1 mg/l thiadiazuron. The calli could be maintained by subculturing in the same medium. The calli could be induced to develop further along one of three distinct morphogeneic routes: (1) production of rhizomes, (2) production of shoot buds, or (3) development of granular embryoids. Efficient mass propagation was possible via rhizome proliferation and embryoid formation.
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  • 8
    ISSN: 1432-203X
    Keywords: Key words Gum karaya ; Somatic embryogenesis ; Sterculia urens ; Thidiazuron ; Tree tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nodal explants from selected trees of gum karaya (Sterculia urens Roxb.) in the adult growth phase cultured on Murashige and Skoog (MS) medium supplemented with 6.62 μm N6-benzylaminopurine (BAP) produced an average of six adventitious shoots in 30 days. Shoots were rooted in vitro on 1/4-strength MS medium containing 9.82 μm indole-3-butyric acid. Nodulated callus was produced from hypocotyl explants cultured on MS medium supplemented with 4.52 μm 2,4-dichlorophenoxyacetic acid and 8.90 μm BAP. Somatic embryos developed when the nodulated callus was transferred to MS medium containing 0.45 μm thidiazuron (TDZ). TDZ treatment for 2 days gave the optimum response. Over 30% of the somatic embryos developed into plantlets when transferred to 1/4-strength MS basal medium without any growth regulators. Plantlets produced from adventitious shoots and somatic embryos were acclimatized to ex vitro conditions and established in the field.
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  • 9
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    Plant cell reports 17 (1998), S. 172-176 
    ISSN: 1432-203X
    Keywords: Key words Papaya ; Somatic embryogenesis ; Synthetic seed
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Carica papaya L. (papaya) single somatic embryos (2.0 mm diameter) produced in a high-frequency liquid production system were encapsulated in two different synthetic encapsulation compounds. The frequency of regeneration from encapsulated embryos was significantly affected by (1) the concentration of sodium alginate, (2) the presence or absence of nutrient salts in the capsule, and (3) the duration of exposure to calcium chloride. A 2.5% sodium alginate concentration in a half-strength MS salts base resulted in significantly higher germination frequencies than other treatments. A relatively short (10 min) exposure to CaCl2 provided uniform encapsulation of embryos and the highest frequencies of successful germination (77.5%). Germinated artificial seeds produced normal plantlets.
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  • 10
    Electronic Resource
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    Plant cell reports 18 (1998), S. 59-63 
    ISSN: 1432-203X
    Keywords: Key words Cotton ; Gossypium hirsutum ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Selections were made among individual plants of Gossypium hirsutum cv `Coker 310' for high-frequency in vitro regeneration by somatic embryogenesis. After three generations of selection, a pure line for high-frequency somatic embryogenesis was selected and named Coker 310 FR (FR, fully regenerating). Coker 310 FR could be regenerated by following previously published protocols (see Materials and methods) and a modified protocol developed in this study that reduced the time necessary for in vitro regeneration. Coker 310 FR was crossed with individual plants of major cotton cultivars grown in India, namely `MCU 5', `MCU 7', `Khandwa 2', `Bikaneri Nerma', `F 846' that have been shown to be recalcitrant to in vitro regeneration, to evaluate the regeneration potential of F1s. All the F1s showed regeneration by somatic embryogenesis. However, the F1 of G. barbadense×G. hirsutum Coker 310 FR did not regenerate.
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  • 11
    ISSN: 1432-203X
    Keywords: Key wordsPersea americana ; Somatic embryogenesis ; Genetic transformation ; Agrobacterium tumefaciens ; Avocado
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l–1 picloram and 50 mg l–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively.
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  • 12
    ISSN: 1432-203X
    Keywords: Key words Coconut ; Regeneration ; Somatic embryogenesis ; Plumule
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases.
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  • 13
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    Plant cell reports 18 (1998), S. 325-330 
    ISSN: 1432-203X
    Keywords: Key wordsMyrtus communis L. ; Auxins ; Cytokinins ; Morphogenesis ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Immature seeds, as well as hypocotyls and cotyledons excised from seedlings of Myrtus communis L., were cultured on media containing half-strength Murashige and Skoog macronutrients (MS/2) with combinations of auxins and cytokinins, in order to study their morphogenetic competence. Somatic embryogenesis was obtained from cotyledons, hypocotyls and 2-month-old immature seeds with 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The percentage of explants showing this primary somatic embryogenesis ranged from 4% for hypocotyls to 12% for 2-month-old immature seeds. In the latter, somatic embryogenesis was also obtained in media containing 2,4-D plus a cytokinin, and with only a cytokinin. Somatic embryos obtained from hypocotyls, cotyledons or immature seeds were able to develop on MS/2 medium without plant growth regulators. Subculture of primary somatic embryos obtained from immature seeds on MS/2 medium without plant growth regulators gave rise to clusters with secondary somatic embryos and embryogenic calli. These clusters were subcultured every 8 weeks, and they were the source of highly embryogenic cultures. An average of 10% of the secondary somatic embryos developed into plantlets in each subculture. Therefore, the same culture on MS/2 medium without growth regulators yielded both plantlets and de novo secondary embryos.
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  • 14
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; gene expression ; calmodulin ; spontaneous hypertensive rats ; rat kidney cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats ingested with saline was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open reading frame). Rats were freely given saline as drinking water for 7 days. Regucalcin mRNA levels in the kidney cortex were suppressed by saline ingestion. When calcium chloride (10 mg Ca/100 g body weight) was intraperitoneally administered to rats ingested with saline for 7 days, the effect of calcium administration to increase regucalcin mRNA levels was weakened by saline ingestion. Such effect was also seen by the administration of 2.5 and 5 mg Ca/100 g. Regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were not appreciably increased by the administration of calcium (10 mg/100 g). Meanwhile, calcium content in the kidney cortex was significantly elevated by the administration of calcium (10 mg/100 g) to normal rats. This increase was weakened in saline-ingested rats. Moreover, Ca2+/calmodulin-dependent protein kinase activity in the cytosol of kidney cortex was significantly decreased by saline ingestion. These results suggest the possibility that saline ingestion-induced suppression of regucalcin mRNA expression in the kidney cortex is partly involved in the attenuation of Ca2+ signalling.
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  • 15
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    Molecular and cellular biochemistry 178 (1998), S. 157-162 
    ISSN: 1573-4919
    Keywords: protein tyrosine phosphatases ; gene expression ; degenerate deoxyoligonucleotides ; RT-PCR ; Swiss 3T3 fibroblasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.
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  • 16
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    Molecular and cellular biochemistry 186 (1998), S. 43-51 
    ISSN: 1573-4919
    Keywords: myocardial ischemia ; gene expression ; growth factors ; phospholamban ; calsequestrin heat shock proteins ; preconditioning ; stunning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Brief periods of coronary occlusion render the affected myocardium more tolarant to the otherwise devastating effects of long coronary occlusion. Besides this phenomena, called ischemic preconditioning, short periods of ischemia cause a regional dysfunction, namely myocardial stunning. The molecular mechanisms of both syndromes are not very well understood. We therefore investigated the expression of genes which may be involved in cardioprotection or repair processes.Using our porcine model of ischemia and reperfusion we were able to show an induction of genes coding for transcription factors (proto-oncogenes), for proteins involved in repair processes (heat shock genes), for proteins implicated in the calcium homeostasis (calcium-handling genes) and for growth factors. We could show that the increased mRNA levels are due to an enhanced transcriptional activity and not to a prolonged half-life of the transcripts. The angiogenic growth factor vascular endothelial growth factor (VEGF) represents an exception. It exhibits - in addition to a HIF-motif (Hypoxia Inducible Factor) in its promoter/enhancer - a protein binding region in its 3′ UTR which when occupied renders the mRNA more stable. However to what extent the expression of the distinct genes contributes to the cardioprotective effect of ischemic preconditioning or myocardial stunning can only be presumed. Increased mRNA stability can be confered via adenosine which is produced during ischemia by ATP-breakdown. The demasking of unknown genes - via differential display reverse transcription polymerase chain reaction (DDRT-PCR) - should provide a more comprehensive view of the mechanisms underlying both processes.
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  • 17
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    Molecular and cellular biochemistry 178 (1998), S. 283-287 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; gene expression ; fetal development ; rat liver
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, β-actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic β-actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.
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  • 18
    ISSN: 1573-4919
    Keywords: extremely low frequency magnetic fields ; gene expression ; neuron derived orphan receptor-1 ; signal transduction ; Chinese hamster ovary K1 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Enhanced expression of neuron derived orphan receptor (NOR-1) gene was observed by exposure of Chinese hamster ovary K1 (CHO-K1) cells to an extremely low frequency magnetic field (ELFMF) of 50 Hz at 400 mT, but not at 5 mT. The enhanced expression, reaching the maximum at 6 h, was transient and reduced to the control level after exposure to 400 mT ELFMF for 24 h. The NOR-1 expression induced by treatment with forskolin and TPA was further enhanced by the simultaneous treatment with 400 mT ELFMF, in which the maximum response was at 3 h. The NOR-1 expression by these treatments was induced more earlier than that by 400 mT ELFMF alone. When cells were treated with an inhibitor of the protein kinase C (calphostin C or crocetin) and Ca2+ entry blockers (nifedipin and dantrolen) during the 400 mT ELFMF exposure, the enhanced NOR-1 expression was not observed. Exposure of CHO-K1 cells to the high-density 400 mT ELFMF may affect the signal transduction in the cells, resulting in the enhanced NOR-1 gene expression.
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  • 19
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    Molecular and cellular biochemistry 188 (1998), S. 41-48 
    ISSN: 1573-4919
    Keywords: zinc ; transcription factors ; gene expression ; organogenesis ; Xenopus laevis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Zinc regulates the gene expression machinery. It affects the structure of chromatin, the template function of its DNA, the activity of numerous transcription factors and of RNA polymerases. Hence, it determines both the types of mRNA transcripts synthesized and the rate of transcription itself. Alterations in one or more of these zinc dependent processes have been proposed to account for the proliferative arrest and teratology induced by zinc deficiency. To examine this proposal, studies of zinc during X. laevis development have been initiated. The kinetics of X. laevis oocyte zinc uptake and storage and of zinc utilization during embryogenesis have been examined first. Vitellogenin carries zinc into the oocyte. Ten % of the total zinc (10 ng/egg) remains within the cytosol while 90% (90 ng/egg) is stored in the yolk platelets associated with lipovitellin. The cytosolic pool is the source of the zinc for all newly formed metalloproteins involved in embryo development. The yolk platelet zinc pool is stored for later use during early metamorphosis. It is now possible to examine zinc transfer to molecules, such as e.g. transcription factors, and the role of the metal in their function in development and organogenesis.
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  • 20
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    Molecular and cellular biochemistry 189 (1998), S. 107-111 
    ISSN: 1573-4919
    Keywords: gene expression ; electromagnetic fields ; superinduction ; anisomycin ; immediate early gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Rat pheochromocytoma PC12 cells have been treated with nerve growth factor (NGF) at final concentrations of 2, 4, 8, and 16 ng/ml, and then were exposed to 60-Hz, sinusoidal magnetic fields (MF) of 12.5, 25, 50, and 100 μT (rms) for 30 min. Transcript levels for both c-fos and glyceraldehyde-3 -phosphate dehydrogenase were determined by Northern blot analysis using 32P-labeled cDNA probes. No change in c-fos expression was measured at any condition employed. Treatment of PC12 cells with a combination of agents (NGF, forskolin, and tetradecanoylphorbol acetate [TPA]) increased c-fos expression over that detected with NGF alone. MF exposure of cells treated with the three-agent regimen produced two outcomes, either no change or a doubling of c-fos expression. In subsequent experiments, cells were treated with NGF, NGF + forskolin + TPA, or pre-treated with anisomycin and then treated with NGF + forskolin + TPA. It was determined that MF exposure, like superinduction with anisomycin, increased c-fos expression only in cultures which were not yet exhibiting maximal c-fos expression. It is hypothesized that MF exposure, like anisomycin, may alter the activity of key intracellular protein kinases.
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  • 21
    ISSN: 1573-4978
    Keywords: gene expression ; nuclear matrix proteins ; ocular lens epithelial cells ; transcription factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Association of transcription factors with the nuclear matrix represents a mechanism by which nuclear architecture may influence transcriptional control of gene expression. This investigation examines nuclear matrix associated proteins (NMP's) isolated from ocular lens epithelial cells by monitoring DNA binding activities using consensus oligonucleotides recognized by the transcription factors YY1, AML-1, AP-1, SP-1 and ATF. The nuclear matrix fractions tested included an immortilized human lens epithelial cell line containing the SV40 large T-antigen, and two mouse lens epithelial cell lines derived from either a normal mouse or a cataract mouse. A rabbit epidermal epithelial cell line and HeLa cells were also included in this study for comparison. The data from these experiments reveal that ubiquitously represented and tissue restricted regulatory proteins are associated with nuclear matrix of lens epithelial cells. The functional significance of the nuclear matrix association of these transcription factors remains to be determined. However, our findings raise the possibility that the transcription factors associated with the nuclear matrix could have specific roles in gene regulation and eye tissue development.
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  • 22
    ISSN: 1432-2048
    Keywords: Key words: Arabinogalactan protein ; Daucus (arabinogalactan proteins) ; β-Glucosyl Yariv reagent ; Cell suspension culture ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The arabinogalactan protein-binding β-d-glucosyl Yariv reagent (βGlcY) was applied to the various developmental stages of embryogenic carrot (Daucus carota L. cv. Early Nantes) cell-suspension cultures. Roots without shoot structures were produced in cultures grown under embryo-inducing conditions in medium containing βGlcY. Only low concentrations of βGlcY permitted the subsequent production of embryos in these cultures. When early stage embryos were transferred to medium containing βGlcY, the roots elongated greatly while the shoot apices expanded radially. These embryos did not progress to the next developmental stage. Torpedo embryos and plantlets, however, showed an overall inhibition of growth in the presence of βGlcY. Developmental stage therefore appears to determine how cultures and embryos respond to βGlcY, root growth being promoted in the early stages, and overall growth reduced in the late stages.
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  • 23
    ISSN: 1432-2048
    Keywords: Key words: Acetylesterase ; Cardenolide ; Cell wall ; Digitalis ; Lanatoside 15′-O-acetylesterase ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Lanatoside 15′-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 μmol · s−1 · (g protein)−1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4–24 nmol · s−1 · (g protein)−1.
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  • 24
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    Plant cell reports 17 (1998), S. 866-869 
    ISSN: 1432-203X
    Keywords: Key words Conservation ; Heracleum candicans ; Plant regeneration ; Somatic embryogenesis ; Xanthotoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol has been developed for achieving somatic embryogenesis and plant regeneration from petiole-derived callus of Heracleum candicans Wall. Callus was initiated on MS medium supplemented with 0.5 mg l–1 2,4-D and 0.5 mg l–1 BAP and subcultured on a medium containing double strength MS macrosalts, 1 mg l–12,4-D and 0.25 mg l–1 Kn. Numerous globular embryos were formed on the surface of the callus upon transfer to auxin-rich MS medium that lacked cytokinins. The globular embryos differentiated into mature embryos only when 2,4-D was removed from the medium. Mature embryo formation was significantly influenced by the pH of the medium and the addition of AgNO3 and ABA. Eighty-five percent of the somatic embryos were converted into plantlets when transferred to a medium supplemented with 0.01 mg l–1 BAP and 0.01 mg l–1 IBA. The regenerated plants have been established in soil and appear to be identical to the parent plants in morphology and chromosome number.
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  • 25
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    Plant cell reports 17 (1998), S. 902-906 
    ISSN: 1432-203X
    Keywords: Key words Barley ; Somatic embryogenesis ; Plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation.
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  • 26
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    Plant cell reports 17 (1998), S. 765-772 
    ISSN: 1432-203X
    Keywords: Key words Microprojectile bombardment ; Mitotic index ; Somatic embryogenesis ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic cultures of soybean [Glycine max (L.) Merr. cv. Jack and Asgrow A2872] were established in liquid Finer and Nagasawa medium, maintained by transfer to fresh medium at biweekly intervals, and subjected to microprojectile bombardment over time. Cultures were not amenable to transformation until they were at least 6 months old. Over time, different cell lines of the same genotype acquired very different culture phenotypes. Histological analysis of cell lines differing in transformation ability showed that the most transformable cultures had cytoplasmic-rich cells in the outermost layers of the tissue. In contrast, the outer layers of less transformable cultures contained cells with prominent vacuoles. Although fresh weight accumulation of the cultures was curvilinear during the 2-week subculture period, a burst of mitotic activity was evident shortly after transfer to fresh medium. This activity usually lasted from the 2nd to the 6th day following subculture, and peaked on the 4th day. Tissues at or near this stage always produced more transient expression of a reporter gene than did bombardments at other times. In addition, the cell lines most amenable to transformation also exhibited the highest mitotic index. Thus any treatment to increase the mitotic index, especially when the cell lines are less than 6 months old, may facilitate the transformation of cell lines from which efficient recovery of transgenic plants is still possible.
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  • 27
    ISSN: 1432-203X
    Keywords: Key words Conifer ; Somatic embryogenesis ; Tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Low initiation frequency is one of the main barriers in applying somatic embryogenesis to the clonal production of Pinus species. Factors affecting initiation, including basal medium, plant growth regulators, and Phytagel concentration, have been investigated in loblolly pine (Pinus taeda L.). BM1 basal medium proved superior to DCR1 and LP (LP basal salts plus BM1 organic nutrients). No extrusion from megagametophytes was exhibited on LP medium. The combination of 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) resulted in a higher extrusion frequency than that of 11 mg/l 2,4-D, 4.5 mg/l BA and 4.3 mg/l kinetin. Phytagel at 1 g/l resulted in the highest explant browning, but the lowest extrusion frequency, while 4 g/l Phytagel induced some dry embryogenic extrusions. Phytagel at 2 g/l was regarded as the best level for initiation of embryogenic cultures.
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  • 28
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    Plant cell reports 17 (1998), S. 446-450 
    ISSN: 1432-203X
    Keywords: Key wordsPhalaenopsis ; Protocorm-like bodies ; Callus induction ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l–1. Medium containing 200 ml ⋅ l–1 coconut water together with 40 g ⋅ l–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis.
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  • 29
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    Plant cell reports 17 (1998), S. 522-525 
    ISSN: 1432-203X
    Keywords: Key wordsArachis hypogaea L. ; Somatic embryogenesis ; Genotypes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets could be a genotype-dependent characteristic.
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  • 30
    ISSN: 1432-203X
    Keywords: Key wordsBacopa monniera ; Brahmi ; Shoot regeneration ; Somatic embryogenesis
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    Topics: Biology
    Notes: Abstract The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil.
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  • 31
    ISSN: 1432-203X
    Keywords: Key words Carrot ; Somatic embryogenesis ; Conditioned medium ; High-cell-density culture
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    Topics: Biology
    Notes: Abstract Carrot somatic embryogenesis was strongly inhibited in high-cell-density cultures. This inhibition was not caused by depletion of nutrients or physical damage but by factor(s) released into the culture medium from cells during culture. A conditioned medium prepared by eliminating cells after high-cell-density culture inhibited somatic embryogenesis. The degree of inhibition increased with the amount of conditioned medium. A dialysis experiment revealed that the molecular weight of the inhibiting factor(s) was below 3,500. We also found that the conditioned medium contained a high-molecular-weight factor(s), which stimulated somatic embryogenesis.
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  • 32
    ISSN: 1432-203X
    Keywords: Key words Genetic transformation ; Agrobacterium rhizogenes ; Ri T-DNA ; Colt ; Somatic embryogenesis
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    Topics: Biology
    Notes: Abstract Hairy roots were obtained after inoculation with Agrobacterium rhizogenes strain NCPPB 1855 of the in-vitro-grown shoots of the cherry rootstocks Colt (Prunus avium×P. pseudocerasus) and Mazzard F12/1 (P. avium L.). Not all putatively transgenic roots were able to grow in hormone-free medium. Mazzard F12/1 roots, induced with A. rhizogenes, did not differentiate any shoot or embryo, while both somatic embryos and shoots differentiated from the transgenic roots of Colt in medium containing 1 mg/l 6-benzylaminopurine and 1 mg/l 1-naphthaleneacetic acid. Somatic embryos were capable of secondary embryogenesis, but few developed into whole plants. DNA hybridization showed both a different number of bands and signal intensity in each of the five transgenic shoot clones and embryos examined. In a morphogenetic in vitro test, leaf explants of the transgenic shoot clones showed an increased capacity to differentiate roots, although clones differed in their sensitivity to the hormone ratio. Clones from the transgenic shoots had not only an increased rooting ability when grown in vitro but also exhibited various hairy root phenotypes when cultured in vitro and when transferred into the greenhouse.
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  • 33
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    Plant cell reports 17 (1998), S. 665-669 
    ISSN: 1432-203X
    Keywords: Key words Sweet potato ; Ipomoea batatas ; Protoplast ; Somatic embryogenesis ; Plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×106 were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 105 protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.
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    Plant cell reports 18 (1998), S. 111-116 
    ISSN: 1432-203X
    Keywords: Key wordsAllium cepa ; Somatic embryogenesis
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    Notes: Abstract Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5 mg/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The production of somatic embryos was significantly affected by the addition of auxin, embryo size and cultivar. The potential of somatic embryogenic cultures for plantlet regeneration has been maintained for over 1 year in some lines. Three types of immature-embryo-derived cultures were characterized by histology. Some cultures were morphologically similar to immature-embryo-derived embryogenic cultures of other monocotyledonous species. Cultures such as these have proven to be useful target tissues in transformation studies.
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  • 35
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    Plant cell reports 18 (1998), S. 148-153 
    ISSN: 1432-203X
    Keywords: Key words Carnation ; Somatic embryogenesis ; Regeneration
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    Topics: Biology
    Notes: Abstract Conditions for efficient direct somatic embryogenesis and plant regeneration of leaf explants from carnation cultivars Lena (SIM group) and Bulgarian spray cultivars Nasslada, Yanita, Regina and Line 84 were established. Murashige and Skoog (MS) liquid medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-benzylaminopurine was used for direct induction of embryoids without an additional callus phase. The first globular structures were observed after 20 days of cultivation. Their further development to the torpedo stage was correlated with the presence of polyethylene glycol (PEG 6000). Somatic embryo maturation was promoted by casein hydrolysate (1000 mg/l) in MS liquid media. The percentage conversion of embryos and polyembryos to whole plants varied between 10 and 75% among studied cultivars. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in a greenhouse.
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  • 36
    ISSN: 1432-2242
    Keywords: Key words Bird’s-foot treefoil ; Lotus corniculatus ; Root culture ; Somatic embryogenesis ; Super-growth
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    Topics: Biology
    Notes: Abstract  Root cultures, displaying vigorous growth and high embryogenic capacity, were established in the legume forage species Lotus corniculatus (bird’s-foot trefoil). Root cloning as well as plant regeneration was achieved on hormone-free medium, in agitated culture in the dark or under stationary conditions in the light, respectively. These qualities of vigorous growth and regeneration faded with time in hormone-free culture, with slow-growing roots turning brown in color. Addition of the synthetic cytokinin-like hormone benzylaminopurine to the culture medium, however, re-established the aging tissue’s capacity for somatic embryogenesis and plant formation. During continuous initiation of new cultures, it was possible to obtain one root culture (selected from 11 960 seeds at a 65% germination rate) which did not show the typical decline of qualities after prolonged proliferation but distinguished itself by displaying even faster growth and more vigorous embryogenic plant production on hormone-free medium. There was no decline since its initiation 9 months earlier. This super-growing root culture produces plants that show no morphological differences as compared to wild-type regenerants or seedlings. Roots, dissected from plantlets derived from super-root embryogenesis, expressed all the super-root qualities again when cultured in vitro. This is the first report on somatic embryogenesis from sustained root cultures without exogenous hormone application. Such a hormone-free, continuous root culture should provide a superior experimental system for genetic or developmental studies that might be sensitive to exogenous hormones, such as somaclonal variation in transgenesis or, since introduced in a legume species, nodulation in vitro.
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  • 37
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    Theoretical and applied genetics 97 (1998), S. 557-562 
    ISSN: 1432-2242
    Keywords: Key words Red clover ; Trifolium pratense ; Somatic embryogenesis ; Inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Red clover genotypes capable of regenerating plantlets in vitro from non-meristematic tissue-derived callus are rare. Selection for genotypes capable of somatic embryogenesis identified a clone comprised of a group of plantlets regenerated from a hypocotyl-derived callus culture on L2-based media and another group of plantlets originating from crown divisions of the epicotyl-derived plant. The callus-derived plants of this clone were highly regenerative when reintroduced to callus culture, but the epicotyl-derived plants produced nonregenerative callus cultures. F1, F2 and BC1 populations were evaluated to determine the mode of inheritance of the regeneration trait. Reciprocal crosses did not differ, indicating a lack of maternal effects. Results were compatible with genetic control of regeneration by two complementary genes. We propose the genotype Rn1-Rn2- for regenerative plants. Three petiole segment explants were sufficient to evaluate regenerative ability in seedlings. Regenerative ability was often associated with abnormal leaf morphology in a few to several leaves.
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  • 38
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    Theoretical and applied genetics 96 (1998), S. 823-831 
    ISSN: 1432-2242
    Keywords: Key words Mangifera indica L. ; Anthracnose ; Somatic embryogenesis ; Dual culture ; RAPD
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Embryogenic nucellar cultures of two polyembryonic mango cultivars, ‘Hindi’ and ‘Carabao’, were selected for resistance to the culture filtrate and phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated from mango leaves. The cultures were recurrently selected either with progressively increasing concentrations of culture filtrate or by continuous challenge with the same concentration of either culture filtrate phytotoxin. Mycelium growth was inhibited when the pathogen was cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth medium containing macerated resistant embryogenic cultures did not inhibit mycelium growth, confirming that extracellular antifungal compounds were involved in the defense response. Enhanced secretion of chitinase and glucanase was observed in the plant growth medium in which resistant embryogenic cultures and regenerated somatic embryos were grown in comparison with the controls.
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  • 39
    ISSN: 1573-9368
    Keywords: transgenic mice ; gene expression ; mammary gland ; co-injection ; milk protein
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    Topics: Biology
    Notes: Abstract In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine β-lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus
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  • 40
    ISSN: 1573-9368
    Keywords: transgenic rabbits ; gene expression ; mammary gland ; erythropoietin ; WAP
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    Notes: Abstract An understanding of the expression of transgenes in the mammary gland during gestation and lactation is crucial for the use of transgenic mammals as bioreactors. Here we describe the temporal pattern of expression of the endogenous rabbit WAP gene and human erythropoietin (hEPO) transgenes under the control of rabbit WAP promoter and 3′ flanking sequences. The endogenous rabbit WAP gene was expressed throughout gestation including the day of mating, as well as during lactation in transgenic rabbits bearing a minigene construct. In non-pregnant cycling females, WAP expression was found independent of transgenic status; however, WAP expression was not detected in non-cycling females. The significance of this new finding is not clear at present. hEPO mRNA was detected in mammary gland biopsies from pregnant transgenic rabbits only on day 28 of gestation. During lactation, transcripts were present in mammary gland biopsy samples taken on days 0, 7, 14 and 21. A sharp decline in the levels ...
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  • 41
    ISSN: 1615-6102
    Keywords: Abscisic acid ; Daucus carota ; Desiccation tolerance ; Low-temperature scanning electron microscopy ; Membrane permeability ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Carrot (Daucus carota) somatic embryos that were treated with abscisic acid during their development have been shown to acquire complete desiccation tolerance when slowly dried, but fail to do so when rapidly dried. We studied plasma membrane permeability by a spin probe technique. On rehydration, the plasma membranes of rapidly dried somatic embryos became permanently leaky, whereas those of slowly dried embryos retained permeability levels as low as for fresh embryos. The leakage was associated with extensive deesterification of the phospholipids. Using low-temperature scanning electron microscopy (LTSEM), we examined (re)hydrated and dry somatic and zygotic embryos. No differences in surface morphology were detected between dry tolerant and intolerant somatic embryos that were obtained by slow and rapid drying, respectively. However, on rehydration, the nonviable somatic embryos had lost turgor and intracellular organization, as revealed by LTSEM images of fractured embryos, whereas the viable somatic embryos had turgescent cells with intact cell structure. In 3-day-imbibed, viable embryos, cell extension was visible. These findings are consistent with the electron paramagnetic resonance data. Somatic embryos always had reduced and abnormal cotyledons, mostly fused, and the surface was irregular. The surface of dry somatic embryo cells was considerably more wrinkled than that of dry zygotic embryo cells. Maturation in polyethylene glycol-containing medium increased the percent dry matter in the fresh somatic embryos and reduced the extent of wrinkling after drying. Wrinkling of the dried embryo cells apparently depends on the filling with dry matter in the hydrated state. We suggest that wrinkling per se does not limit desiccation tolerance of somatic embryos.
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  • 42
    ISSN: 1615-6102
    Keywords: 2,4-Dichlorophenoxyacetic acid ; Auxin ; Carya illinoinensis ; Embryo induction ; Naphthaleneacetic acid ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.
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  • 43
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    Plant molecular biology 36 (1998), S. 439-449 
    ISSN: 1573-5028
    Keywords: ethylene ; gene expression ; leaf senescence ; RNase ; tomato ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A main feature of leaf senescence is the hydrolysis of macromolecules by hydrolases of various types, and redistribution of released materials. We have initiated a study for the characterization of RNases involved in nucleic acid catabolism during senescence. Using a PCR-based cloning approach we isolated from tomato two senescence-induced RNase cDNA clones. Each of these cDNAs hybridized to a senescence-induced transcript in northern analysis. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both LX and LE RNase genes had originally been demonstrated to be induced after phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. We observed that the expression of the LX and LE genes is induced in leaves during an advanced stage of senescence with the LX transcript level being much more induced than that of LE. Low-level expression of the RNase genes was observed in flowers and artificially senescing detached leaves while no expression could be detected in stems, roots, or fruits at different ripening stages. Ethylene activated the LX gene expression in detached young leaves while LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may function during wounding as a plant defense protein.
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  • 44
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    Plant molecular biology 36 (1998), S. 699-707 
    ISSN: 1573-5028
    Keywords: actin cytoskeleton ; in situ hybridization ; gene expression ; profilin ; RT-PCR ; tomato pollen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The actin cytoskeleton plays an important role in the growth of pollen tube. The actin-binding protein profilin could play a role in regulating the organization of the actin filaments. Using the RT-PCR technique, we isolated a cDNA clone (designated LePro1) encoding profilin from pollen grains of tomato (Lycopersicon esculentum Mill. cv. Moneymaker). Sequence analysis of the insert shows 87% similarity to tobacco ntPro2, 78% to timothy grass profilin, 77% to Arabidopsis AthPRF4, 77% to maize ZmPro3, and 73% to birch profilin. Both quantitative PCR and RNA gel blot analyses demonstrated that LePro1 is expressed in a tissue- or cell-type specific manner in the tomato plant. In situ hybridization of 2 µm thick anther sections using a non-radioactive labeling method reveals that LePro1 is expressed only in pollen grains, with undetectable transcription in other parts of the anther or other organs. Phylogenetic analysis of amino acid sequences of 18 plant profilins indicates that two distinct profilin gene classes are present in higher plants. One is pollen-specific, another is constitutive. LePro1 belongs to the former class.
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  • 45
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    Plant molecular biology 36 (1998), S. 733-739 
    ISSN: 1573-5028
    Keywords: AGAMOUS ; tomato ; ripening ; calyx ; gene expression ; sepals ; in vitro
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In vitro culture of VFNT Cherry tomato sepals (calyx) at 16–21 °C results in developmental changes that are similar to those that occur in fruit tissue [10]. Sepals become swollen, red, and succulent, produce ethylene, and have increased levels of polygalacturonase RNA. They also produce many flavor volatiles characteristic of ripe tomato fruit and undergo similar changes in sugar content [11]. We examined the expression of the tomato AGAMOUS gene, TAG1, in ripening, in vitro sepal cultures and other tissues from the plant and found that TAG1 RNA accumulates to higher levels than expected from data from other plants. Contrary to reports on the absence of AGAMOUS in sepals, TAG1 RNA levels in green sepals from greenhouse-grown plants is detectable, its concentration increasing with in vitro ripening to levels that were even higher than in red, ripe fruit. Sepals of fruit on transgenic tomato plants that expressed TAG1 ectopically were induced by low temperature to ripen in vivo, producing lycopene and undergoing cell wall softening as is characteristic of pericarpic tissue. We therefore propose that the induction of elevated TAG1 gene expression plays a key role in developmental changes that result in sepal ripening.
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  • 46
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    Plant molecular biology 37 (1998), S. 735-748 
    ISSN: 1573-5028
    Keywords: AMP-activated protein kinase ; carbohydrates ; gene expression ; gene family ; HMG-CoA reductase ; intracellular signalling ; isoprenoids ; nitrate reductase ; phosphorylation ; SNF1 ; starch ; sucrose phosphate synthase ; sucrose synthase ; sugar sensing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 47
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    Plant molecular biology 37 (1998), S. 1023-1033 
    ISSN: 1573-5028
    Keywords: gene expression ; psbA gene ; truncated psbA messages ; DNA-binding protein ; D1 protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of the psbA genes, which in the cyanobacterium Synechococcus sp. PCC 7942 encode two different forms of the reaction centre D1 protein of photosystem II (D1:1 and D1:2), was studied under different light and temperature conditions. In addition to the mature 1200 nt psbA messages, three shorter mRNA fragments of 220, 320 and 900 nt were also found. All three mRNA fragments could be recognized by using different gene probes from the coding region of the psbAI gene, whereas the corresponding psbAII/III gene probes recognized only the 220 nt mRNA fragment. The 5' 320 nt mRNA fragment from the psbAI gene probably represents a degradation product, since the corresponding 3' 900 nt psbAI mRNA fragment was also detected. By contrast, the 5' 220 nt mRNA fragment of all psbA messages is suggested to be a truncated psbA transcript, since no corresponding 3' fragment was ever found. Inhibition of translation either by a protein synthesis inhibitor or by a shift of cells to lower temperature, increased the number of 1200 nt psbAII/III messages but the number of 5' 220 nt psbAII/III mRNA fragment increased even more dramatically. The first 66 bp after ATG, where the psbAI and psbAII/III genes mostly differ from each other, also appeared important in determining the amount of produced truncated psbA transcripts, as evidenced by the expression of different tac-psbA constructs in the presence of protein synthesis inhibitor. We suggest that both the psbAI and the psbAII/III genes have a latent intragenic termination site and truncated psbA transcripts are produced at high levels under stress conditions when transcription becomes uncoupled from translation.This is to prevent wasting metabolic energy in the production of unused transcripts.
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  • 48
    ISSN: 1573-5028
    Keywords: gene expression ; Candida albicans ; dihydrofolate reductase ; dominant selectable marker ; Nicotiana tabacum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.
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  • 49
    ISSN: 1573-5028
    Keywords: rice ; waxy ; splicing ; gene expression ; intron
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AGTTATA at the putative leader intron 5′ splice site, while all cultivars with a higher proportion of amylose had AGGTATA. Here we demonstrate that this single-base mutation reduces the efficiency of GBSS pre-mRNA processing and results in alternate splicing at three cryptic sites. The predominant 5′ splice site in CT18 low-amylose varieties is 93 bp upstream of the splice site used in intermediate and high amylose varieties and is immediately 5′ to the CT microsatellite that we previously demonstrated to be tightly correlated with amylose content. Use of the leader intron 5′ splice site at either -93 or -1 in conjunction with the predominant 3′ splice site results in formation of a small open reading frame 38 bp upstream of the normal ATG and out of frame with it. This open reading frame is not produced when any of the 5′ leader intron splice sites are used in conjunction with an alternate 3′ splice site five bases further downstream which was observed in all rice varieties tested.
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  • 50
    ISSN: 1573-5028
    Keywords: antisense RNA ; chalcone synthase ; flavonoid ; gene expression ; rooting ; walnut
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Walnut somatic embryos (Juglans nigra × Juglans regia) were transformed with a vector containing a neomycin phosphotransferase II, a β-glucuronidase and an antisense chalcone synthase (chs) gene. This antisense construct included a 400 bp cDNA fragment of a walnut chs gene under the control of the duplicated CaMV-35S promoter. Molecular, biochemical and biological characterizations were performed both on transformed embryos propagated by secondary somatic embryogenesis and on microshoots developed by in vitro culture of embryonic epicotyls from somatic embryos. Thirteen transformed lines with the vector containing the antisense chs gene, one line with only the gus and nptII genes and one untransformed line were maintained in tissue culture. Six of the antisense lines were shown to be flavonoid-deficient. They exhibited a strongly reduced expression of chs genes, very low chalcone synthase activity and no detectable amounts of quercitrin, myricitrin, flavane-3-ols and proanthocyanidins in stems. Rooting tests showed that decreased flavonoid content in stems of antisense chs transformed lines was associated with enhanced adventitious root formation. Free auxin and conjugated auxin contents were determined during the latter phase of the micropropagation, and no variations were detected between control and antisense chs transformed lines. The in vitro plants developed a large basal callus and apical necrosis upon auxinic induction and the transformed lines highly deficient in flavonoids were more sensitive to exogenous application of indolebutyric acid (IBA).
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  • 51
    ISSN: 1573-5028
    Keywords: ribonucleotide reductase ; gene expression ; cell cycle ; S-phase ; tobacco BY2 cell suspension
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Eukaryotic ribonucleotide reductase (RNR), the enzyme involved in the synthesis of the deoxyribonucleotides, consists of two R1 and R2 subunits whose activities and gene expression are differentially regulated during the cell cycle and are preferentially induced at the G1/S transition. We have isolated three cDNA clones from a tobacco S phase library, two encoding the large R1 subunit, the first cloned in plants, and one encoding the small R2 subunit. From Southern blot hybridization we deduce that RNR2 is encoded by a single-copy gene whereas RNR1 is encoded by a small multigene family. The level of RNR mRNA is cell-cycle regulated showing a maximum in S phase. In mid-S phase, RNR2 transcripts show a higher maximum level than RNR1 transcripts. Analysis of the effects of various cell cycle inhibitors added to freshly subcultured stationary phase cells leads to the conclusion that RNR gene induction at the entry of the cells into the cell cycle takes place in late G1-early S phase. Addition of DNA synthesis-blocking agents to cycling cells synchronized in mid-S phase resulted in an enhancement of RNR transcript level, thus suggesting that RNR gene expression may be linked to the DNA synthesis rate by a feedback-like regulatory mechanism.
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  • 52
    ISSN: 1573-5028
    Keywords: Nicotiana tabacum ; tobacco BY-2 cells ; gene expression ; jasmonic acid ; methyl jasmonate ; ornithine decarboxylase ; polyamine ; nicotine ; SAM synthase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.p〉
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  • 53
    ISSN: 1573-5028
    Keywords: apoplast ; cDNA cloning ; conifer embryogenesis ; gene expression ; germin-like proteins ; Pinus caribaea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Germin-like proteins (GLPs) ionically bound to the walls of preglobular somatic embryos of Pinus caribaea Morelet are markers of this early developmental stage. In order to reveal the physiological implications of such markers during early embryo development, we isolated a cDNA clone from somatic embryos predicted to encode a protein with sequence similarity to GLPs. PcGER1 has an open reading frame corresponding to a 220 amino acid polypeptide with a putative N-glycosylation site on Asn-69. The presence of a 24 amino acid putative signal peptide supports the hypothesis of an apoplastic location. The N-terminal 20 amino acid sequence of the predicted mature protein is identical to the amino terminal sequence of GP111, one of the extracellular pine GLPs previously identified. Southern blot hybridizations indicate that PcGER1 is probably unique in the pine genome. Transcripts homologous to PcGER1 are abundant in all embryogenic lines, absent from nonembryogenic lines, and present in quiescent zygotic embryos but not in the female gametophyte, the haploid storage tissue of conifers. Their abundance sharply decreases during germination. Isolation of gf-0.8, a genomic fragment identical to PcGER1 cDNA sequence, confirms that no introns disrupt the coding region as it has been already described for wheat gf-2.8 and gf-3.8 genomic clones. Recombinant PcGER1, produced in Escherichia coli, is recognized by antibodies raised against the GP111 N-terminal nonapeptide and the unglycosylated wheat germin monomer. The implications of GLPs in pine embryogenesis are discussed.
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  • 54
    ISSN: 1573-5028
    Keywords: sedoheptulose-1,7-bisphosphatase ; Chlamydomonas reinhardtii ; light regulation ; Calvin cycle ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have studied the light-dependent expression of the Chlamydomonas reinhardtii csbp gene encoding sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme of the pentose-phosphate pathway. Expression studies using light/dark-synchronized cultures revealed that csbp mRNA abundance increases significantly during illumination. We have used a 1.4 kb region upstream of the csbp gene in transcriptional fusions to the homologous arylsulfatase-encoding reporter gene (ars). In transformants carrying the chimeric csbp/ars reporter gene, arylsulfatase activity is detected in the absence of sulfate, a condition under which the endogenous ars gene is repressed. Moreover, ars mRNA accumulation is dramatically stimulated by light, indicating that 1.4 kb of the csbp 5′-untranslated region are sufficient to confer light-dependent expression on the ars reporter gene.
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  • 55
    ISSN: 1573-5028
    Keywords: blue/UV-A-light ; gene expression ; glutamine synthetase ; phytochrome ; tomato aurea mutant ; UV-B-light
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition.
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  • 56
    ISSN: 1572-9699
    Keywords: Quorum sensing ; N-acylhomoserine lactones ; gene expression ; virulence ; secondary metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Although it has been clear for some time that individual bacterial cells employ intra-cellular signalling systems to sense, integrate and process information from their surroundings, their widespread capacity to perceive information from other bacterial cells is only just beginning to be recognised. Recent work has established that diverse bacteria exploit a cell-cell communication device to regulate the transcription of multiple target genes. This communication device termed ‘quorum sensing’, depends on the production of one or more diffusible signal molecules termed ‘autoinducers’ or ‘pheromones’ which enable a bacterium to monitor its own cell population density. Quorum sensing is thus an example of multicellular behaviour in prokaryotes and regulates diverse physiological processes including bioluminescence, swarming, antibiotic biosynthesis, plasmid conjugal transfer and the production of virulence determinants in animal, fish and plant pathogens. In Gram-negative bacteria, the best understood family of signal molecules are the N-acylhomoserine lactones (AHLs) which vary predominantly in the presence or absence of an acyl chain C3 substituent (oxo- or hydroxy-) and length of the N-acyl side chain. However not all quorum sensing signal molecules are AHLs; in Gram-positive bacteria, they are often post-translationally modified peptides. Irrespective of the chemical ‘language’ employed, interference with either the synthesis or transmission of a quorum sensing signal molecule in pathogenic bacteria offers an exciting new strategy for controlling infection.
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  • 57
    ISSN: 1573-5028
    Keywords: aspen ; Populus tremuloides ; xylem ; lignin ; caffeoyl-CoA ; caffeate ; O-methyltransferase ; gene expression ; enzyme activity ; recombinant bacterial overexpression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68). Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season. These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season. To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid. Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates. The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms. We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution. Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively. This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms.
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  • 58
    ISSN: 1573-5028
    Keywords: binding site selection ; gene expression ; homeobox-leucine zipper genes ; protein-DNA interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report the isolation and characterization of two Arabidopsis homeobox genes highly related to the Athb-8 gene. The full-length cDNAs encode proteins of 841 and 852 amino acids which we have designated Athb-9 and -14, respectively. Athb-8, -9 and -14 are members of a small family of HD-Zip proteins (HD-ZIP III) characterized by a HD-Zip motif confined to the N-terminus of the polypeptide. The spatial organization of the HD-Zip domain of Athb-8, -9 and -14 is different from that of the Athb-1 (a member of the HD-ZIP I family) and Athb-2 (a member of the HD-ZIP II family) HD-Zip domains. DNA binding analysis performed with random-sequence DNA templates showed that the Athb-9 HD-Zip (HD-Zip-9) domain, but not the Athb-9 HD alone, binds to DNA. The HD-Zip-9 domain recognizes a 11 bp pseudopalindromic sequence (GTAAT(G/C)ATTAC), as determined by selecting high-affinity binding sites from random-sequence DNA. Moreover, gel retardation assays demonstrated that the HD-Zip-9 domain binds to DNA as a dimer. These data support the notion that the HD-ZIP III domain interacts with DNA recognition elements in a fashion similar to the HD-ZIP I and II domains.
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  • 59
    ISSN: 1573-5028
    Keywords: gene expression ; nodules ; phosphoribosyl aminoimidazole synthetase ; purine biosynthesis ; N assimilation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules [7]. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.
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  • 60
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    Plant molecular biology 37 (1998), S. 425-435 
    ISSN: 1573-5028
    Keywords: gene expression ; protein-DNA interaction ; plant ; stress ; seed development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The phytohormone abscisic acid is probably present in all higher plants. This hormone is necessary for regulation of several events during seed development and for the response to environmental stresses such as desiccation, salt and cold. An important part of the physiological response to abscisic acid is achieved through gene expression. Here, we summarize the current knowledge of regulation of abscisic acid-induced transcription. The main focus is on a description of the known abscisic acid-responsive cis-elements, their properties and the possible transacting factors binding to the elements. Results have shown that cooperative action of cis-elements and the promoter configuraton is crucial for regulation by abscisic acid. Furthermore, several elements are organ- and species-specific. Recent studies of the chromatin structure of abscisic acid-responsive genes point to the importance of induction of transcription by coactivators or by phosphorylation/dephosphorylation of transcription factors. An interesting example of activation by a cofactor is the cooperative action between abscisic acid-signaling and the regulatory protein Viviparous 1 through the abscisic acid responsive element.
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  • 61
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    Plant molecular biology 37 (1998), S. 587-596 
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; gene expression ; leucine-rich repeat ; PCR ; receptor-like kinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated three receptor-like kinase cDNAs from an Arabidopsis flower cDNA library by PCR using degenerate oligonucleotide primers for conserved domains of protein kinases. Cloning and sequencing of the full-length cDNAs, designated RKF1 to 3 (receptor-like kinase in flowers), showed that the putative extracellular domain of the RKF1 protein contains 13 tandem repeats of leucine-rich sequences and those of RKF2 and RKF3 have no significant homology with other plant sequences. RNA blot analysis revealed that the RKF1 mRNA is highly expressed in stamens while RKF2 and RKF3 mRNAs are present at low levels in all organs examined. In situ localization experiments indicated that the RKF1 mRNA is detectable in early flower primordia and during stamen development. In addition, when fused to a GUS reporter gene, the RKF1 promoter directed high GUS expression in pollen grains. Recombinant RKF1, produced in Escherichia coli, was found to have kinase activity with serine/threonine specificity in vitro.
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  • 62
    ISSN: 1573-5028
    Keywords: gene expression ; phytohormone ; Lea ; leaf senescence ; senescence-associated gene ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of several Arabidopsis thaliana senescence-associated genes (SAGs) in attached and/or detached leaves was compared in response to age, dehydration, darkness, abscisic acid, cytokinin, and ethylene treatments. Most of the SAGs responded to most of the treatments in a similar fashion. Detachment in darkness and ethylene were the strongest inducers of both SAGs and visible yellowing. Detachment in light was also a strong inducer of SAGs, but not of visible yellowing. The other treatments varied more in their effects on individual SAGs. Responses were examined in both older and younger leaves, and generally were much stronger in the older ones. Individual SAGs differed from the norms in different ways, however, suggesting that their gene products play a role in overlapping but not identical circumstances. Some SAGs responded quickly to treatments, which may indicate a direct response. Others responded more slowly, which may indicate an indirect response via treatment-induced senescence. Four new SAGs were isolated as part of this work, one of which shows strong similarity to late embryogenesis-abundant (Lea) genes.
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  • 63
    ISSN: 1573-5117
    Keywords: Planarian ; Hox ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Seven Hox cluster-related genes (Dthox-A to -G) have been isolated from the freshwater triclad Dugesia (G.) tigrina, their sequence compared to other Hox genes and their expression in intact and regenerating organisms analyzed by whole mount in situ hybridization. Sequence comparison analyses show high similarities of D. tigrina Hox genes to anterior and medial groups of coelomate Hox genes. Expression analyses show very early, synchronous, and overlapping expression of Dthox -A, -E, -G and -F in anterior, posterior and lateral regenerative tissues. At one hour of regeneration all Dthox genes studied showed a neat, clear expression at the wound boundary. Later, as the blastema grows, the expression area expands to more proximal regions covering the blastema and the distal postblastema regions. Blastemas formed by intercalary regeneration also show a synchronous expression of the same Hox genes though the onset of activation is much delayed. The finding that the same set of Hox genes is synchronously activated in anterior, posterior, intercalary and lateral regeneration is in sharp contrast to its well established role in specifying antero-posterior pattern during embryonic development. The implications of these results as regards ancestral versus co-opted roles of Hox genes in development and regeneration are discussed.
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  • 64
    ISSN: 1573-0778
    Keywords: CHO ; DHFR ; gene expression ; growth factor ; recombinant protein ; serum-free
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.
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  • 65
    ISSN: 1573-9368
    Keywords: luciferase ; scaffold attachment regions ; heat shock ; blastocyst ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 °C, 15 min) and 4–6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.
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  • 66
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    Transgenic research 7 (1998), S. 403-411 
    ISSN: 1573-9368
    Keywords: transgenic organism ; gene expression ; promoter ; plant ; gene regulation ; heterologous system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The regulation of gene expression represents a specific process which has different structural and functional requirements in different groups of organisms. It is thus assumed that regulatory sequences of eucaryotes cannot be recognized in procaryotes. This assumption is of interest for risk assessments of the environmental impact of deliberate release experiments with genetically modified organisms. In order to analyse the extent of heterologous gene expression caused by the transfer of plant-specific regulatory sequences into bacteria, we constructed fusions between plant-specific regulatory sequences and the coding regions of the luxAB genes for the luciferase of the bioluminescent bacterium Vibrio harveyi, transferred the fusions into different bacterial species and measured the luminescence to quantify the expression of the luciferase genes. The regulatory sequences investigated included (a) the 35S promoter of the Cauliflower mosaic virus, (b) the B33 promoter of a class I patatin gene of potatoes, (c) the promoter of the ST-LS1 gene of potatoes and (d) the promoter of the rolC gene of Agrobacterium rhizogenes. We could show that in addition to the 35S promoter, which has already been described as being recognized in Escherichia coli, the sequences containing the B33 and the ST-LS1 promoters are recognized in bacteria. Luciferase gene expression promoted by the sequence with the ST-LS1 promoter could be observed in E. coli, Yersinia enterocolitica and Agrobacterium tumefaciens. Comparison of the luminescence caused by fusions between luxAB and different promoters on the chromosome and on an endogenous plasmid of Y. enterocolitica demonstrated that the level of the heterologous gene expression caused by the fragment with the ST-LS1 promoter was within the range of gene expression levels caused by endogenous promoters of Y. enterocolitica.
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