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  • gene expression  (83)
  • Springer  (83)
  • American Meteorological Society
  • Institute of Physics
  • 1995-1999  (40)
  • 1990-1994  (43)
  • 1940-1944
  • 1998  (40)
  • 1992  (43)
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  • Springer  (83)
  • American Meteorological Society
  • Institute of Physics
  • Wiley-Blackwell  (13)
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  • 1995-1999  (40)
  • 1990-1994  (43)
  • 1940-1944
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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
    Electronic Resource
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    Springer
    Antonie van Leeuwenhoek 62 (1992), S. 131-153 
    ISSN: 1572-9699
    Keywords: mitochondrial DNA ; mutational analysis ; nucleo-mitochondrial interactions ; gene expression ; membrane assembly ; respiratory deficiency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In contrast to most other organisms, the yeastSaccharomyces cerevisiae can survive without functional mitochondria. This ability has been exploited in genetic approaches to the study of mitochondrial biogenesis. In the last two decades, mitochondrial genetics have made major contributions to the identification of genes on the mitochondrial genome, the mapping of these genes and the establishment of structure-function relationships in the products they encode. In parallel, more than 200 complementation groups, corresponding to as many nuclear genes necessary for mitochondrial function or biogenesis have been described. Many of the latter are required for post-transcriptional events in mitochondrial gene expression, including the processing of mitochondrial pre-RNAs, the translation of mitochondrial mRNAs, or the assembly of mitochondrial translation products into the membrane. The aim of this review is to describe the genetic approaches used to unravel the intricacies of mitochondrial biogenesis and to summarize recent insights gained from their application.
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  • 4
    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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  • 5
    ISSN: 1573-4919
    Keywords: mouse ; protein tyrosine phosphatase ; cDNA cloning ; nucleotide sequence ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5′ (61 nucleotides) and 3′ (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain.
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  • 6
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    Springer
    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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  • 7
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    Springer
    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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  • 8
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    Springer
    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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  • 9
    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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  • 10
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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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  • 11
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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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  • 12
    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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  • 13
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    Springer
    Molecular biology reports 16 (1992), S. 277-284 
    ISSN: 1573-4978
    Keywords: translational initiation ; 18S rRNA ; mRNA secondary structure ; gene expression ; initiation mutants ; β-galactosidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To learn if an mRNA·18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5′ to part of the Escherichia coli tuf B-lacZ gene. Deletion of bases potentially complementary to the 18S rRNA highly increased the transient β-galactosidase expressed in transfected CHO cells. Deletion of bases that fostered formation of potential hairpins with the mRNA 5′-terminus or altered the structure of the coding region reduced β-galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation. Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.
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  • 14
    ISSN: 1573-5028
    Keywords: ABA ; Daucus carota ; ECP31 ; gene expression ; LEA clone ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A full-length cDNA for ECP31, an embryogenic cell protein from carrot (Daucus carota L.) with a M r of 31000 (Kiyosue T, Satoh S, Kamada H, Harada H (1991) Plant Physiol 95: 1077–1083), was isolated from a cDNA library prepared from embryogenic cells using PCR-amplified DNA as a probe. The genomic Southern blot analysis revealed that there are two or three genes for ECP31 in the carrot genome. The transcripts of ECP31 accumulated in the peripheral regions of clusters of embryogenic cells and disappeared in the course of somatic embryogenesis that was induced by transfer of the embryogenic cells to auxin-free media. The cDNA encodes a polypeptide of 256 amino acids, and the calculated molecular weight of this polypeptide is 26 111. The deduced amino acid sequence shows a high degree (62.2%) of similarity to that of a protein that is abundant during late embryogenesis of cotton (LEA D34; Baker JC, Steele C, Dure III (1988) Plant Mol Biol 11: 227–291). The mRNAs for ECP31 started to accumulate in zygotic embryos at a late stage of embryogenesis but were undetectable in mature embryos within 24 h after imbibition of seeds. In dry fruits (seeds), the transcripts were detected only in zygotic embryos by in situ hybridization. The level of ECP31 transcripts increased after treatment with abscisic acid (ABA) in torpedo-shaped somatic embryos but not in seven-day-old seedlings. These results suggest that both embryo-specific factor(s) and ABA are involved in the expression of the gene for ECP31.
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  • 15
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    Plant molecular biology 19 (1992), S. 623-630 
    ISSN: 1573-5028
    Keywords: gene expression ; heat shock ; intron ; maize ; pollen ; RNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract While a heat shock treatment of 40 °C or 45 °C induced the vegetative tissues of maize to produce the typical heat shock proteins (HSPs), germinating maize pollen exposed to the same temperatures did not synthesize these characteristic HSPs. Comparison of RNA accumulation in shoot and tassel tissue showed that mRNAs for HSP70 and HSP18 increased several-fold, reaching high levels within 1 or 2 hours. At the higher temperature of 45 °C these vegetative tissues were blocked in removal of an intron from the HSP70 mRNA precursor, which accumulated to a high level in tassel tissue. In germinating pollen exposed to heat shock, mRNAs for these HSPs were induced but accumulated only to low levels. The stressed pollen maintained high levels of RNA for α-tubulin, a representative normal transcript. It is likely that the defective heat shock response of maize pollen is due to inefficient induction of heat shock gene transcription.
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  • 16
    ISSN: 1573-5028
    Keywords: sunflower ; gene expression ; zygotic embryogenesis ; Lea proteins ; heat-shock proteins ; abscisic acid ; osmotic stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have cloned and sequenced three different cDNAs from sunflower seed-stored mRNA. Sequence similarities and response to heat-shock identified one of the cDNAs as a low-molecular-weight heat-shock protein (lmw-HSP). The other two clones showed significant sequence similarity to the cotton and carrot late-embryogenesis-abundant (Lea) proteins D-113 and Emb-1, respectively. The three cDNAs showed similar expression patterns during zygotic embryo development, as well as in vegetative tissues of 3-day-old seedlings in response to stress. Maximal accumulation of all three mRNAs was detected in dry seeds and during embryo mid-maturation stage, in the absence of exogenous stress. In seedlings, mRNAs accumulated to lower levels in response to osmotic stress and exogenous abscisic acid (ABA) treatments. A differential time course of response to osmotic stress was observed: lmw-HSP mRNA accumulation was induced earlier than that of Lea mRNAs. The coordinate accumulation of Lea and lmw-HSP transcripts during embryo development and in response to stress and ABA suggests the existence of common regulatory elements for Lea and lmw-HSP genes, and supports the notion that HSPs might have alternative functions in the plant cell.
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  • 17
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    Plant molecular biology 19 (1992), S. 1049-1055 
    ISSN: 1573-5028
    Keywords: Brassica napus ; rapeseed ; gene expression ; nucleotide sequence ; storage proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated a five-member gene subfamily which encodes cruciferin, a legumin-like 12S storage protein of Brassica napus L., and have analyzed the structure and expression of the family members in developing embryos. Sequence analysis has shown that the coding regions of all five genes are highly similar, with the two most divergent members of the family retaining 89% sequence identity. The analysis of this cruciferin gene family's expression indicates that the developmental pattern of expression of each gene is similar, and the steady-state mRNA levels of each gene are approximately equivalent to each other at all developmental stages.
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  • 18
    ISSN: 1573-5028
    Keywords: gene expression ; microsporogenesis ; Nicotiana tabacum ; pectate lyase ; pollen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A genomic clone has been isolated which contains an open reading frame of 1191 bp interrupted by two small introns. The ORF has been sequenced and the transcriptional start determined. The predicted amino acid sequence shows homology to the deduced amino acid sequences of two pollen-specific pectate lyase genes identified in tomato. The genomic clone was isolated using a partial cDNA clone, TP10, which had been isolated from a Nicotiana tabacum pollen cDNA library by means of differential screening. TP10 has been fully sequenced and contains an open reading frame of 792 bp which shows 96% homology to the ORF in the genomic clone. The transcript corresponding to TP10 is maximally expressed late in pollen development, and has not been detected in vegetative tissues.
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  • 19
    ISSN: 1573-5028
    Keywords: β-1,3-glucanase ; gene expression ; pathogenesis-related proteins ; plant-fungus interaction ; protein P14
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a β-1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa β-1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic β-1,3-glucanase and a basic 35 kDa β-1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic β-1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa β-1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.
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  • 20
    ISSN: 1573-5028
    Keywords: aleurone ; Avena fatua ; cDNA nucleotide sequence ; gene expression ; gibberellin ; polyubiquitin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A λgt11 cDNA library, constructed from poly(A)+ mRNA isolated from Avena fatua aleurone layers incubated with 1 μM gibberellin A1 (GA1) for 4 days, was screened with an anti-idiotypic antiserum raised against the GA-specific monoclonal antibody MAC 182. One positive clone was isolated, sequenced and shown to encode a tetraubiquitin based on the deduced amino acid sequence. This polyubiquitin cDNA exhibited a high degree of homology to a cloned wheat hexaubiquitin in its 3′-non-coding region. Analysis of total RNA isolated from A. fatua aleurone layers, treated without or with a range of concentrations of GA1 from 10-11 to 10-6 M, by northern blotting using the cDNA probe revealed 8 different ubiquitin-containing transcript classes all of which are constitutively expressed in aleurone and are regulated by GA1.
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  • 21
    ISSN: 1573-5028
    Keywords: Cell division ; gene expression ; Nicotiana sylvestris ; protoplast ; stress ; ubiquitin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris. Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed. cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized. The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5- mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids. The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation. The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.
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  • 22
    ISSN: 1573-5028
    Keywords: β-D-glucuronidase ; mannopine synthase promoter ; Agrobacterium ; gene expression ; initiation of translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcriptional and translational fusions between the reading frame of the β-D-glucuronidase gene (gusA) and the 2′ as well as the 1′ promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level. In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves. In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.
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  • 23
    ISSN: 1573-5028
    Keywords: Agrobacterium ; binary vector ; CaMV 35S ; gene expression ; β-glucuronidase ; Nicotiana plumbaginifolia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for β-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5′ to 3′ model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ′ region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.
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  • 24
    ISSN: 1573-5028
    Keywords: Chlamydomonas eugametos ; chlorophyll a/b-binding proteins ; circadian oscillator ; gene expression ; light-regulated genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)+ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40 000 clones in each library revealed 44 clones which hybridize preferentially with a [32P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [32P] cDNA probe synthesized from poly(A)+ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.
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  • 25
    ISSN: 1573-5028
    Keywords: Tomato ; gene expression ; wounding ; ethylene ; glycine-rich protein ; rRNA ; polyamines
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.
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  • 26
    ISSN: 1573-5028
    Keywords: adventitious buds ; cDNA cloning ; cytokinin ; gene expression ; germination ; Norway spruce
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A pulse treatment of embryos of Norway spruce with cytokinin suppresses germinative development and induces the coordinate formation of adventitious buds from subepidermal cell layers. To analyse the patterns of gene expression associated with germination and the alterations induced by the bud induction treatment, we have isolated cDNA clones corresponding to genes that are differentially expressed in cytokinin-treated and untreatedin vitro germinating embryos. One category of 14 clones hybridized to transcripts that were abundant specifically during germination. The expression of 8 of these genes was reduced by the bud induction treatment. Four clones, including one identified as a histone H2A gene, recognized transcripts that showed an increased abundance in bud-induced versusin vitro germinating embryos. A second category of 13 clones hybridized to transcripts that increased in abundance during post-germinative development of the seedling. Among these a subset of 8 clones, including an α-tubulin clone, corresponds to genes suppressed by the bud induction treatment, whereas 5 clones, including a gene with sequence similarity to polyubiquitin, were unaffected by the treatment. One clone hybridized to a message abundant in the seed, during early germination as well as in the vegetative bud, and showed 60% partial sequence identity to a barley (1→3)-β-glucanase gene. Genes expressed exclusively in bud-induced orin vitro germinating embryos were not found. The results show that a major difference in gene expression between treated and untreated embryos is related to the shift from extensive cell proliferation to elongation and differentiation that occurs at the transition from germination to post-germinative development, and which is suppressed in the bud-induced embryos.
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  • 27
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    Plant molecular biology 18 (1992), S. 749-757 
    ISSN: 1573-5028
    Keywords: salt stress ; stem-specific expression ; lipid transfer protein ; cDNA sequence ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A full-length tomato cDNA clone, TSW12, which is developmentally and environmentally regulated, has been isolated and characterized. TSW12 mRNA is accumulated during tomato seed germination and its level increases after NaCl treatment or heat shock. In mature plants, TSW12 mRNA is only detected upon treatment with NaCl, mannitol or ABA and its expression mainly occurs in stems. The nucleotide sequence of TSW12 includes an open reading frame coding for a basic protein of 114 amino acids; the first 23 amino acids exhibit the sequence characteristic of a signal peptide. The high similarity between the TSW12-deduced amino acid sequence and reported lipid transfer proteins suggests that TSW12 encodes a lipid transfer protein.
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  • 28
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    Plant molecular biology 18 (1992), S. 815-818 
    ISSN: 1573-5028
    Keywords: expression cassette ; gene expression ; protoplasts ; translation initiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the β-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.
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  • 29
    ISSN: 1573-5028
    Keywords: glucanase ; gene expression ; pathogenesis response ; stress response ; plant growth regulators ; gene family
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A rice β-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots expressGns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogenSclerotium oryzae or from the non-pathogenSaccharomyces cereviseae. Shoots also expressGns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The β-glucanase shows 82% amino acid similarity to the barley 1,3;1,4-β-D-glucanases, and from hybridization studies it is the β-glucanase gene in the rice genome closest to the barley 1,3;1,4-β-glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G+C in the third position of the codon in the mature peptide coding region, but only 61% G+C in the signal peptide region.
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  • 30
    ISSN: 1573-5028
    Keywords: cDNA sequence ; gene expression ; glutamine synthetase ; phytochrome ; Solanaceae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A full-length cDNA encoding glutamine synthetase (GS) was cloned from a λgt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C-and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression during greening in tomato.
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  • 31
    ISSN: 1573-5028
    Keywords: cab gene ; chlorophyll a/b-binding protein ; gymnosperm ; gene expression ; pine (Pinus thunbergii)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A gene for chlorophyll a/b-binding protein (cab) of pine (Pinus thunbergii) was isolated and sequenced. The gene (cab-6) contains an intron at a position equivalent to the type II cab genes of angiosperms. Transcript mapping analyses show that the amount of the mRNA in the dark is about half of that in the light. The cab-6 gene is expressed in dark-grown seedlings at a very high level, differing from angiosperm cab genes which are induced by light. The cab-6 gene typifies the coniferous plant cab genes in light-independent gene expression.
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  • 32
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    Plant molecular biology 19 (1992), S. 1031-1044 
    ISSN: 1573-5028
    Keywords: ABA ; dehydrin ; gene expression ; pea (Pisum sativum L.) ; seed development ; water stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species. The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum. A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins. B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water. During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.
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  • 33
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    Plant molecular biology 18 (1992), S. 1-11 
    ISSN: 1573-5028
    Keywords: betaine aldehyde dehydrogenase ; gene expression ; glycine betaine ; osmotic stress ; salt tolerance ; sugar beet
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a λgt10 libary representing poly(A)+ RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome.
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  • 34
    ISSN: 1573-5028
    Keywords: ribosomal protein S15a ; cDNA clones ; rapessed ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated two cDNA clones which appear to encode the 40S ribosomal subunit protein S15a from Brassica napus (oilseed rape). The open reading frame in both clones contains 390 bases, encoding a deduced polypeptide sequence of 130 amino acids (100% homology between clones) with 76% sequence identity to the N-terminal 37 amino acids of the rat ribosomal protein S15a and 80% identity to the S24 polypeptide of yeast. Both the yeast and rapeseed proteins have a net positive charge of +9 and the rapeseed S15a protein has a molecular mass of 14778 Da compared to 14762 Da for the yeast protein. The rapeseed ribosomal protein S15a is encoded by a small multi-gene family with at least two actively transcribed members. A single transcript of ca. 1.0 kb, corresponding to ribosomal protein S15a, is abundant in actively dividing tissues such as apical meristem, flower buds and young leaves and less abundant in mature stem and fully expanded leaves.
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  • 35
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    Plant molecular biology 19 (1992), S. 959-971 
    ISSN: 1573-5028
    Keywords: gene expression ; gene variants ; pre-mRNA splicing ; pseudogenes ; U1 small nuclear RNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract U1 small nuclear RNAs (U1snRNAs) occur in the nucleus of plants and animals where, complexed with several proteins in the form of U1 small nuclear ribonucleoprotein particles (U1snRNPs), they play an important role in precursor messenger RNA (pre-mRNA) splicing. Ten potato U1snRNA genes have been isolated on two genomic clones illustrating the clustering of this multigene family on the potato genome. Based on both the sequence of their coding regions and upstream regulatory elements, seven of the genes are potentially functional. The other three genes were pseudogenes with defective promoter or coding region sequences. Analysis of expression of individual cloned U1snRNA genes in transfected tobacco protoplasts was impossible due to the similarity of U1snRNA sequences in tobacco. However, by marking the coding regions with oligonucleotides or constructing chimaeric genes consisting of a potato U1snRNA promoter region and maize U5snRNA coding region, three of the U1 promoter regions were shown to be transcriptionally active.
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  • 36
    ISSN: 1573-5028
    Keywords: gene expression ; promoter specificity ; snRNA gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3′-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to α-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.
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  • 37
    ISSN: 1573-5028
    Keywords: ADP-glucose pyrophosphorylase ; Anabaena 7120 ; Escherichia coli ; gene cloning ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.
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  • 38
    ISSN: 1573-5028
    Keywords: gene expression ; desaturation ; oil synthesis ; embryogenesis ; stearoyl-acyl carrier protein desaturase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nucleotide sequence is reported for a cDNA containing the entire coding region of a stearoyl-ACP desaturase (EC 1.14.99.6) fromBrassica napus L. cv. Jet neuf. The cDNA was obtained from a library constructed from poly(A)+ RNA purified from embryo tissue. The derived amino acid sequence demonstrates substantial similarity with those from other plant Δ9-desaturases. Comparative RNA-dot blot analyses using the Δ9-desaturase cDNA and a rapessed oleosin cDNA as probes showed that although both these transcripts were seed-specific, they exhibited distinct patterns of temporal regulation. The desaturase message was induced by 25 days after anthesis (DAA), peaking at 45 DAA but decreasing considerably thereafter. In contrast, the oleosin transcript did not increase until 45–50 DAA, reaching a peak much later at about 70 DAA.
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  • 39
    ISSN: 1573-5028
    Keywords: barley (Hordeum vulgare) ; zygotic embryogenesis ; plant development ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The temporal and spatial pattern of expression of a novel barley gene is described. The gene has been identified through the differential screening of a cDNA library constructed to poly(A)+ RNA of zygotic embryos. Transcripts corresponding to the cDNA, pZE40, become abundant in the non-axial tissues of the developing embryo within 8–10 days after anthesis, when steady-state levels are high in the scutellum, coleoptile and coleorhiza, with the exception of the scutellar epithelium. This expression pattern is maintained throughout maturation of the embryo until levels eventually decline as the grain desiccates. On germination, there is a transient re-appearance of mRNA to pZE40, with accumulation specifically restricted to the scutellum of the seedling. In situ hybridization has enabled the detection of transcripts elsewhere in the barley plant, in highly localized groups of cells. The timing and cell specificity of expression suggests the gene product is involved in the synthesis and/or transport of metabolites.
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  • 40
    ISSN: 1573-5028
    Keywords: S-adenosylmethionine decarboxylase ; differential screening ; gene expression ; Solanum tuberosum ; tuberisation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.
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  • 41
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    Plant molecular biology 20 (1992), S. 663-671 
    ISSN: 1573-5028
    Keywords: PEP carboxylase ; housekeeping gene ; gene expression ; gene structure ; light-mediated activation ; Saccharum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A gene (SCPEPCD1) encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from the C-4 monocot sugarcane (Saccharum hybrid var. H32-8560). SCPEPCD1 is ca. 6800 bp long, with 10 exons. The entire gene sequence from −1561 to 262 bp downstream of the putative poly(A) addition signal is reported. A low-level, essentially constitutive pattern of expression, amino acid sequence similarities to other ‘housekeeping’ PEPC enzymes, and the absence of DNA sequence elements conserved in the upstream region of maize and sorghum C-4-specific PEPC genes indicate that SCPEPCD1 encodes a housekeeping PEPC. Despite this, a motif proposed to act as a phosphorylation site in light-mediated activation of photosynthetic PEPC enzymes [10] is present in the SCPEPCD1 protein; evidence is presented for the presence of this site in other housekeeping PEPC proteins.
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  • 42
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    Plant molecular biology 20 (1992), S. 705-713 
    ISSN: 1573-5028
    Keywords: alanine aminotransferase ; C4 photosynthesis ; gene expression ; nucleotide sequencing ; Panicum miliaceum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.
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  • 43
    ISSN: 1573-5028
    Keywords: chloroplast ; gene expression ; photosystem 2 ; transcription ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The time course of the accumulation of the transcripts from 13 psb genes encoding a major part of the proteins composing photosystem II during light-induced greening of dark-grown wheat seedlings was examined focusing on early stages of plastid development (0.5 h through 72 h). The 13 genes can be divided into three groups. (1) The psbA gene is transcribed as a single transcript of 1.3 kb in the dark-grown seedlings, but its level increases 5- to 7-fold in response to light due to selective increase in RNA stability as well as in transcription activity. (2) The psbE-F-L-J operon, psbM and psbN genes are transcribed as a single transcript of 1.1 kb, two transcripts of 0.5 and 0.7 kb and a single transcript of 0.3 kb, respectively, in the dark-grown seedlings. The levels of accumulation of every transcript remain unchanged or rather decrease during plastid development under illumination. (3) The psbK-I-D-C gene cluster and psbB-H operon exhibit fairly complicated northern hybridization patterns during the greening process. When a psbC or psbD gene probe was used for northern hybridization, five transcripts differing in length were detected in the etioplasts from 5-day old dark-grown seedlings. After 2 h illumination, two new transcripts of different length appeared. Light induction of new transcripts was also observed in the psbB-H operon.
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  • 44
    ISSN: 1573-9368
    Keywords: transgenic mice ; gene expression ; mammary gland ; co-injection ; milk protein
    Source: Springer Online Journal Archives 1860-2000
    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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  • 45
    ISSN: 1573-9368
    Keywords: transgenic rabbits ; gene expression ; mammary gland ; erythropoietin ; WAP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    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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  • 46
    ISSN: 1572-9788
    Keywords: differential display ; gene expression ; hybrid vigor ; molecular marker heterozygosity ; Oryza sativa L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Using differential display analysis, we assessed the patterns of differential gene expression in hybrids relative to their parents in a diallel cross involving 8 elite rice lines. The analysis revealed several patterns of differential expression including: (1) bands present in one parent and F1 but absent in the other parent, (2) bands observed in both parents but not in the F1, (3) bands occurring in only one parent but not in the F1 or the other parent, and, (4) bands detected only in the F1 but in neither of the parents. Relationships between differential gene expression and heterosis and marker heterozygosity were evaluated using data for RFLPs, SSRs and a number of agronomic characters. The analysis showed that there was very little correlation between patterns of differential expression and the F1 means for all six agronomic traits. Differentially expressed fragments that occurred only in one parent but not in the other parent or in F1 in each of the respective crosses were positively correlated with heterosis and heterozygosity. And conversely, fragments that were detected in F1s but in neither of the respective parents were negatively correlated with heterosis and heterozygosity. The remaining patterns of differential expression were not correlated with heterosis or heterozygosity. The relationships between the patterns of differential expression and heterosis observed in this study were not consistent with expectations based on dominance or overdominance hypotheses.
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  • 47
    ISSN: 1573-0778
    Keywords: Drosophila cell culture ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE),Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1α promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 μg FK2P/106 cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 μg FK2P/106 cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominately as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.
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  • 48
    ISSN: 1573-8469
    Keywords: quorum-sensing ; gene expression ; autoinducer ; secondary metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract N-acyl homoserine lactones (AHLs) function as cell density (quorum) sensing signals and regulate diverse metabolic processes in several gram negative bacteria. We report that strains of Pseudomonas syringae pvs. syringae (Pss), tabaci and tomato as well as P. corrugata and P. savastanoi produce difussible AHLs that activate the lux operons of Vibrio fischeri or the tra::lacZ fusion of Agrobacterium tumefaciens. In Pss strain B3A, AHL production occurs in cell density dependent manner. Nucleotide sequence and genetic complementation data revealed the presence of ahlIPss, a luxI homolog within the Ahl+ DNA of Pss strain B3A. The $$ahlI_{Pss}^ + $$ DNA expresses in AHL-deficient strains of P. fluorescens and E. carotovora subsp. carotovora (Ecc), and restores extracellular enzyme production and pathogenicity in the Ecc strain. The derivatives of Pss strains B3A and 301D carrying chromosomal ahlI::lacZ do not produce AHL, but like their wild type parents, produce extracellular protease and the phytotoxin syringomycin as well as elicit the hypersensitive reaction in tobacco leaves. While these strains also produce a basal level of β-galactosidase activity, the expression of ahlI::lacZ is substantially stimulated in the presence of multiple copies of the $$ahlI_{Pss}^ + $$ DNA or by the addition of cell-free spent cultures containing AHL. The activation of β-galactosidase production occurs with spent cultures of some, but not all Pseudomonas strains which produce AHL as indicated by the Lux and tra::lacZ assays. Pss strains deficient in the global regulatory genes, gacA or lemA, produce very low levels of AHL. Since inactivation of ahlIPss eliminates AHL production and since Ahl+ Pseudomonas strains carry the homolog of ahlIPss, we conclude that ahlIPss specifies a key step in AHL biosynthesis and it has been conserved in many plant pathogenic pseudomonads.
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  • 49
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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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  • 50
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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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  • 51
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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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  • 52
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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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  • 53
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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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  • 54
    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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  • 55
    ISSN: 1573-5028
    Keywords: rice ; waxy ; splicing ; gene expression ; intron
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    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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  • 56
    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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  • 57
    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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  • 58
    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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  • 59
    ISSN: 1573-5079
    Keywords: environmental stress ; Mesembryanthemum crystallinum ; phosphoribulokinase ; gene expression ; protein expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of PRK (phosphoribulokinase, E.C.2.7.1.19) in ice plant (Mesembryanthemum crystallinum) during development and under environmental stress was studied. cDNA clones were isolated and full-length cDNAs were characterized. Ice plant PRK is contained in a 1520 nucleotide transcript including a 126 nucleotide leader sequence, a 175 nucleotide 3′-end and a 20–30 nucleotide polyA+-stretch. The coding region, 397 codons, specifies a protein of Mr 44 064. The mature sequence is preceded by a transit peptide of approximately 46 amino acids. The mature portion of ice plant PRK is 86.4% identical to that of spinach and, e.g., 16.2% identical to PRK from Xanthomonas flavus. Under salt stress or cold adaptation conditions, the amount of mRNA declined by a factor of approximately three within days, followed by an increase to approximately pre-stress levels. The fluctuation in mRNA amount is not reflected on the level of transcription of the gene, suggesting post-transcriptional control, nor is PRK protein amount affected significantly over the short stress period. The recovery of transcript levels for photosynthesis-related proteins after stress appears to be a general response to environmental stresses that affect water status in ice plant. We suggest that the photosynthetic machinery in this facultative halophyte is effectively buffered from damage caused by such environmental stress.
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  • 60
    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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  • 61
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    Plant and soil 146 (1992), S. 145-151 
    ISSN: 1573-5036
    Keywords: barley ; gene expression ; ice plant ; rice ; salt stress ; tobacco ; tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Soil salinity is an important agricultural problem, particularly since the majority of crop plants have low salt tolerance. The identification of genes whose expression enables plants to adapt to or tolerate salt stress is essential for breeding programs, but little is known about the genetic mechanisms for salt tolerance. Recent research demonstrates that salt stress modulates the levels of a number of gene products. Although the detection of gene products that respons specifically to salt stress is a significant finding, they must be identified, functions assigned, and their relation to salt tolerance determined. This article focuses on a few of the salt-responsive proteins and mRNAs that have been discovered and the methods employed to identify and characterize them.
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  • 62
    ISSN: 1573-5087
    Keywords: desiccation tolerance ; dehydrin ; gene expression ; glyoxalase ; grass ; Lea ; protease ; Sporobolus stapfianus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract In order to identify genes involved in expression of desiccation tolerance in the foliage of the grass Sporobolus stapfianus, a cDNA library was constructed from desiccated leaf tissue of S. stapfianus. Differential screening resulted in the isolation of a number of clones which detect transcripts whose abundance alters during drought stress and the associated induction of desiccation tolerance. The characteristics of 6 of these cDNA clones are presented here. Transcripts represented by three of the cDNA clones accumulate during drying and following treatment of leaves with abscisic acid (ABA). A fourth cDNA clone detects a transcript which also accumulates following application of ABA but the transcript level fluctuates during drying. The remaining two cDNA clones are not responsive to ABA but transcript levels are present throughout the drying process. Characterisation of these cDNAs has led to the identification of the encoded proteins. Some have similarity to proteins which are known to be involved in the protection of cellular organelles and detoxification processes and they include dehydrin, LEA group 3 and thiol proteases which have been identified in other systems and shown to be induced by water stress. In addition one clone showed similarity to glyoxalase I, an enzyme involved in the removal of toxic byproducts produced during glycolysis, which has been shown to be induced by salt stress in tomato.
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  • 63
    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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  • 64
    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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  • 65
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    Cellular and molecular neurobiology 12 (1992), S. 45-58 
    ISSN: 1573-6830
    Keywords: retinoid ; thymosin ; neuroblastoma ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. Investigations have demonstrated that the gene encoding thymosinβ 10 (a 43-amino acid member of a family of related proteins originally described in the rat immune system) is a target for morphogenic retinoids in both human and rat neuroblastoma cells. 2. Structure-activity studies revealed that the stimulatory actions of retinoids upon the thymosinβ 10 gene reflect the differing affinities of retinoid analogues for a retinoic acid receptor. 3. To examine further the possibility that the trophic actions of retinoic acid upon expression of the thymosinβ 10 gene involved retinoid receptors, neuroblastoma cells were transiently transfected with an expression vector encoding the nuclear retinoic acid receptor (α) protein. 4. Northern blot and slot-blot analyses revealed that neuronal cells overexpressing RARα-mRNA exhibited an enhanced sensitivity to exogenous and endogeneous retinoic acid in terms of thymosinβ 10 mRNA. Although the RAR-α gene was expressed (at low levels) a priori in these neuroblastoma cells, retinoic acid (2 × 10−7 M for 3 days) slightly stimulated RAR-α-mRNA accumulation. 5. Collectively, these findings indicate the the retinoic acid receptor (α) is regulated by retinoid acid and that the developmentally regulated, retinoidresponsive thymosinβ 10 gene is a target for this nuclear transcription factor in cells derived from the neural crest.
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  • 66
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    Antonie van Leeuwenhoek 62 (1992), S. 63-78 
    ISSN: 1572-9699
    Keywords: biogenesis ; gene expression ; mutants ; peroxisomes ; yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The observation that peroxisomes ofSaccharomyces cerevisiae can be induced by oleic acid has opened the possibility to investigate the biogenesis of these organelles in a biochemically and genetically well characterized organism. Only few enzymes have been identified as peroxisomal proteins inSaccharomyces cerevisiae so far; the three enzymes involved in β-oxidation of fatty acids, enzymes of the glyoxylate cycle, catalase A and the PAS3 gene product have been unequivocally assigned to the peroxisomal compartment. However, more proteins are expected to be constituents of the peroxisomes inSaccharomyces cerevisiae. Mutagenesis ofSaccharomyces cerevisiae cells gave rise to mutants unable to use oleic acid as sole carbon source. These mutants could be divided in two groups: those with defects in structural genes of β-oxidation enzymes (fox-mutants) and those with defects in peroxisomal assembly (pas-mutants). All fox-mutants possess morphologically normal peroxisomes and can be assigned to one of three complementation groups (FOX1, 2, 3). All three FOX genes have been cloned and characterized. The pas-mutants isolated are distributed among 13 complementation groups and represent 3 different classes: peroxisomes are either morphologically not detectable (type I) or present but non-proliferating (type II). Mislocalization concerns all peroxisomal proteins in cells of these two classes. The third class of mutants contains peroxisomes normal in size and number, however, distinct peroxisomal matrix proteins are mislocalized (type III). Five additional complementation groups were found in the laboratory of H.F. Tabak. Not all PAS genes have been cloned and characterized so far, and only for few of them the function could be deduced from sequence comparisons. Proliferation of microbodies is repressed by glucose, derepressed by non-fermentable carbon sources and fully induced by oleic acid. The regulation of four genes encoding peroxisomal proteins (PAS1, CTA1, FOX2, FOX3) occurs on the transcriptional level and reflects the morphological observations: repression by glucose and induction by oleic acid. Moreover, trans-acting factors like ADR1, SNF1 and SNF4, all involved in derepression of various cellular processes, have been demonstrated to affect transcriptional regulation of genes encoding peroxisomal proteins. The peroxisomal import machinery seems to be conserved between different organisms as indicated by import of heterologous proteins into microbodies of different host cells. In addition, many peroxisomal proteins contain C-terminal targeting signals. However, more than one import route into peroxisomes does exist. Dissection of the import mechanism in a genetically well suited organism likeSaccharomyces cerevisiae together with further characterization and functional assignment of the PAS gene products will provide insight into the biogenesis of peroxisomes. Moreover, these studies will lead to a good model system for elucidation of the mechanisms underlying human peroxisomal disorders.
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  • 67
    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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  • 68
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    Biodegradation 3 (1992), S. 219-238 
    ISSN: 1572-9729
    Keywords: cellulase ; gene expression ; lignin ; Phanerochaete chrysosporium ; Streptomyces cyaneus ; xylanase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Lignocellulose is the predominant renewable resource. It uses include fuel, as the feedstock for the pulp and paper industry, and for animal nutrition. It also constitutes a large proportion of agricultural and urban waste. Biotechnology has roles in its efficient production and utilisation. The types of lignin substrates available for study of lignin biodegradation are described. The white rot fungus Phanerochaete chrysosporium is the archetypal system for the study of lignocellulose degradation, since it mineralises lignin and degrades both cellulose and hemicellulose. The salient features of the P. chrysosporium system are described. The lignin peroxidases are a family of proteins, and it is shown that expression of their genes is differential. P. chrysosporium is heterokaryotic with two gene equivalents that have abundant RFLPs. A set of basidiospore-derived strains with genetic compositions defined by such RFLPs provided the potential basis for a strain improvement programme for lignin degradation. However, analysis of this system using radiolabelled synthetic lignin (DHP) as the substrate confirmed previous evidence that both the substrate and the fungal cultures displayed much variation, so that it was difficult to quantify performance for this property. The cellobiohydrolase I enzymes are also coded for by a family of genes, and evidence is also presented for allelic variants, for differential expression and for differential splicing. In contrast, the cellobiohydrolase II function is encoded at a unique genetic locus. Approaches to an homologous integrative transformation system are discussed. Some actinomycete bacteria represent an alternative system for lignin solubilisation in which strains differ in their spectra of activities on lignocellulose substrates. The xylanase system of Streptomyces cyaneus is shown to include three enzymes, two of which are inducible by xylan. A novel assay method was developed and used to demonstrate that the third is constitutive and also non-repressible by glucose. It is proposed that this acts as a sensor for xylans in the environment that can yield breakdown products that are taken up and can then act as inducers of the other two enzymes. The studies on microbial lignocellulose degradation from different laboratories have allowed the formulation of specific biotechnological goals, and some of the problems and opportunities in this area are identified.
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  • 69
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    Biodegradation 3 (1992), S. 125-135 
    ISSN: 1572-9729
    Keywords: natural evolution ; directed evolution ; biodegradation ; environmental pollutants ; environmental signal transduction ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Microorganisms in nature are largely responsible for the biodegradation and removal of toxic and non-toxic chemicals. Many organisms are also known to have specific ecological niches for proliferation and colonization. The nature of the environment dictates to a large extent the biodegradability of synthetic compounds by modulating the evolutionary processes in microorganisms for new degradative genes. Similarly, environmental factors often determine the extent of microbial gene expression by activating or repressing specific gene or sets of genes through a sensory signal transduction process. Understanding how the environment modulates microbial activity is critical for successful bioremediative applications.
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  • 70
    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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  • 71
    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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  • 72
    ISSN: 1573-5060
    Keywords: Triticum aestivum ; Triticum durum ; bread wheat ; durum wheat Secale cereale ; rye ; gene expression ; alien introduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Expression of 17 rye traits in 24 bread wheat x rye and 8 durum wheat x rye crosses was studied, using a self-compatible, homozygous, dwarf rye. Rye showed epistasis for hairiness on the peduncle in all the crosses of Triticum aestivum and T. durum wheats with rye. Dark greenness of leaves of rye was expressed in all the durum wheat x rye and in some of the bread wheat x rye crosses. Similarly, absence of auricle pubescence, a rye trait, was expressed in most of the durum wheat x rye crosses but not in the bread wheat x rye crosses, indicating the presence of inhibitors for these traits frequently on the D genome and rarely on the A and/or B genome of wheat. Most of the wide hybrids resembled rye fully or partially for intense waxy bloom on the leaf-sheath and for the absence of basal underdeveloped spikelets. Similarly, most of the amphihaploids resembled rye for the anthocyanin in the coleoptile, stem and node. The presence of some inhibitors on A and/or B genome of wheat was indicated in some of the wheat genotypes for the expression of rye traits viz. intense waxy bloom, anthocyanin in node and absence of basal underdeveloped spikelets. Enhancement in the level of expression of the intensity and length of bristles on the mid-rib of the glume of the hybrids might be due to wheat-rye interaction. Less number of florets/spikelet as in rye showed variable expression in different wheat backgrounds. Some other rye traits like absence of auricles, terminal spikelet and glume-awn were not expressed in the wheat background. The expression of some of the rye genes might have been influenced by their interaction with Triticum cytoplasm and/or the environment.
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  • 73
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    Plant growth regulation 11 (1992), S. 211-224 
    ISSN: 1573-5087
    Keywords: Germination ; energy metabolism ; gene expression ; regulation ; respiration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Oxygen uptake and carbon dioxide release rapidly increase in seeds during imbibition. The oxygen uptake is associated with oxidative phosphorylation through cytochrome oxidase. During the early stage of germination substrate level phosphorylation may also contribute to ATP production. All indications suggest that this route of ATP production is insignificant during aerobic germination. However, during oxygen stress, substrate level phosphorylation does significantly contribute to ATP production in some species. Carbohydrate oxidation plays a significant role in the germination process. Up to two thirds of the carbon from carbohydrate breakdown enters the tricarboxylic acid cycle through the phosphoenolpyruvate carboxylase reaction. This anapleurotic input into the Krebs cycle most probably reflects the high demand on intermediates from the cycle for biosynthesis. The extent to which other substrates are utilized for respiration is uncertain. Information regarding the levels of key metabolites and enzymes, as well as their cellular distribution is limited. The involvement of gene expression in the regulation of respiratory metabolism is poorly characterised. Several genes which have been cloned are only expressed during germination. With the exception of the early methionine labeled polypeptide, little is known about the function of these genes.
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  • 74
    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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  • 75
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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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  • 76
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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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  • 77
    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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  • 78
    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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  • 79
    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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  • 80
    ISSN: 1573-904X
    Keywords: gene expression ; hPepTl ; Caco-2 cells ; adenovirus ; drug screening
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligo-peptide transporter (hPepTl). This study characterizes the expression of hPepTl in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction. Methods. A recombinant replication-deficient adenovirus carrying the hPepTl gene was made and used as a vector for the expression of hPepTl. The increase in the uptake permeability of cephalexin and Gly-Sar was determined. The effects of time, dose, apical pH, and substrate specificity were evaluated. Results. A significant increase in the uptake permeability of Gly-Sar and cephalexin was found in all three cell lines after viral transduction. The increase of Gly-Sar permeability in Hela, IEC-18, and Caco-2 cells was 85-, 46-, and 15-fold respectively. Immunoblotting using an antibody against hPepTl detected high levels of a 85-98-kDa protein in all three infected cell lines. Substrate permeability was dependent on time of infection, inward pH gradients, and multiplicity of infection (MOI). Decreased infectivity and lower hPepTl expression were observed in differentiated Caco-2 cells. The uptake was inhibited by dipeptides and β-lactam antibiotics but not amino acids. Conclusions. Adenoviral infected Hela cells displayed a pronounced level of hPepTl expression with a low background and high specificity to dipeptides. These features make this system a useful tool for screening of potential substrates. The success of overexpression of hPepTl in Caco-2 and IEC-18 cells may lead to a novel approach in improving oral absorption of peptides and peptidomimetic drugs.
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  • 81
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    European journal of plant pathology 98 (1992), S. 175-181 
    ISSN: 1573-8469
    Keywords: Nematodes ; Globodera ; plant pathogen ; infection ; monoclonal antibodies ; PCR ; cDNA libraries ; gene expression ; modified plant cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Current control of nematodes is inadequate and this justifies work towards the design of novel bases for plant defence. Our approach for cyst nematodes began by improving understanding of critical events in the establishment of these biotrophic pathogens. The first step involved development of an experimental system for achieving synchronous infection and establishment of cyst-nematodes in roots. Monoclonal antibodies have been raised against these nematodes, their specificity defined and those of particular interest used to define events in the establishment of the animals within plants. A similar approach has been explored for host responses using antibodies raised to plant tissue containing feeding sites. Changes in translatable mRNA populations at the feeding site have been described.
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  • 82
    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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    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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