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  • Articles  (216)
  • gene expression  (74)
  • Triticum aestivum  (70)
  • Saccharomyces cerevisiae  (57)
  • fish
  • Springer  (216)
  • 1995-1999  (216)
  • 1996  (216)
  • 1
    ISSN: 1572-8773
    Keywords: EPR ; Saccharomyces cerevisiae ; uptake ; vanadate ; vanadyl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO4 3- concentration and the growth was completely arrested at a concentration of 20 mM of VO4 3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A ‘mobile’ and an ‘immobile’ species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time τ r indicated the relative motional freedom at the macromolecular site. A strongly ‘immobilized’ vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO2+ ions with isolated cell walls.
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  • 2
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    Cellular and molecular life sciences 52 (1996), S. 942-949 
    ISSN: 1420-9071
    Keywords: Apoptosis ; transglutaminase ; signalling ; gene expression ; promoter elements ; retinoic acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Clarification of the molecular details of forms of natural cell death, including apoptosis, has become one of the most challenging issues of contemporary biomedical sciences. One of the effector elements of various cell death pathways is the covalent cross-linking of cellular proteins by transglutaminases. This review will discuss the accumulating data related to the induction and regulation of these enzymes, particularly of tissue type transglutaminase, in the molecular program of cell death. A wide range of signalling pathways can lead to the parallel induction of apoptosis and transglutaminase, providing a handle for better understanding the exact molecular interactions responsible for the mechanism of regulated cell death.
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  • 3
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    Cellular and molecular life sciences 52 (1996), S. 1130-1135 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mRNA-specific translational activation ; synthetic genes ; gene regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondrial gene expression in yeast,Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These nuclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.
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  • 4
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    Cellular and molecular life sciences 52 (1996), S. 1033-1041 
    ISSN: 1420-9071
    Keywords: Ubiquitin ; yeast ; Saccharomyces cerevisiae ; Dictyostelium discoideum ; cytoskeleton ; mutants ; endocytosis ; actin ; myosin ; calmodulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Endocytosis is a general term that is used to describe the internalization of external and plasma membrane molecules into the cell interior. In fact, several different mechanisms exist for the internalization step of this process. In this review we emphasize the work on the actin-dependent pathways, in particular in the yeastSaccharomyces cerevisiae, because several components of the molecular machinery are identified. In this yeast, the analysis of endocytosis in various mutants reveals a requirement for actin, calmodulin, a type I myosin, as well as a number of other proteins that affect actin dynamics. Some of these proteins have homology to proteins in animal cells that are believed to be involved in endocytosis. In addition, the demonstration that ubiquitination of some cell surface molecules is required for their efficient internalization is described. We compare the actin, myosin and ubiquitin requirements for endocytosis with recent results found studying these processes usingDictyostelium discoideum and animal cells.
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  • 5
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    Cellular and molecular life sciences 52 (1996), S. 1111-1116 
    ISSN: 1420-9071
    Keywords: Mitochondria ; mitochondrial inheritance ; cytoskeleton ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; membrane proteins ; organelle movement ; mitochondrial morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mechanisms mediating the inheritance of mitochondria are poorly understood, but recent studies with the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe have begun to identify components that facilitate this essential process. These components have been identified through the analysis of conditional yeast mutants that display aberrant mitochondrial distribution at restrictive conditions. The analysis of these mutants has uncovered several novel proteins that are localized either to cytoskeletal structures or to the mitochondria themselves. Many mitochondrial inheritance mutants also show altered mitochondrial morphology and defects in maintenance of the mitochondrial genome. Although some inheritance components and mechanisms appear to function specifically in certain types of cells, other conserved proteins are likely to mediate mitochondrial behavior in all eukaryotic cells.
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  • 6
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondrial ribosomes ; peptidyl transferase ; Varl ribosomal protein ; gene relocation ; posttranscriptional rRNA modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondria posses their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome. In yeast,Saccharomyces cerevisiae, the two ribosomal RNAs and a single ribosomal protein, Varl, are products of mitochondrial genes, and the remaining approximately 80 ribosomal proteins are encoded in the nucleus. The mitochondrial translation system is dispensable in yeast, providing an excellent experimental model for the molecular genetic analysis of the fundamental properties of ribosomes in general as well as adaptations required for the specialized role of ribosomes in mitochondria. Recent studies of the peptidyl transferase center, one of the most highly conserved functional centers of the ribosome, and the Varl protein, an unusual yet essential protein in the small ribosomal subunit, have provided new insight into conserved and divergent features of the mitochondrial ribosome.
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  • 7
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    Biology and fertility of soils 23 (1996), S. 327-331 
    ISSN: 1432-0789
    Keywords: CO2 emission ; Field method ; Soil respiration ; Triticum aestivum ; Soil moisture ; Carbon reservoirs ; Greenhouse effect ; Grey forest soil ; Mineralization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The dynamics of the rate of CO2 evolution from soil in fallow and croplant under spring wheat (Triticum aestivum L.) was studied in a crop rotation in grey forest soil of the Baikal forest-steppe during the growing season and in different years. It was shown that the regional characteristics of soils and hydrothermal conditions in different years affect the rate of CO2 evolution in agroecosystems. The seasonal dynamics of CO2 is characterized by insignificant changes in the autumn to spring period and enhanced emission in hot and dry summers. CO2 evolution is assumed to increase due to enhanced mineralization and partial diffusion from the carbonate horizon at the depth of the seasonal frost. During the growing season the dynamics of CO2 evolution depends on the soil moisture regime. There was a strong correlation between the rate of CO2 emission and soil moisture in the particularly dry year of 1993 (η=0.86) and a moderate correlation in the other years (η=0.38–0.54). The effect of the previous crop and fertilizer application on the rate of CO2 emission was insignificant. In a continuous fallow the total carbon release into the atmosphere varied throughout the years studied from 558 to 1880 kg ha-1. Humus losses varied from 0.9% to 3.1%.
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  • 8
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    Biology and fertility of soils 23 (1996), S. 327-331 
    ISSN: 1432-0789
    Keywords: Key words CO2 emission ; Field method ; Soil respiration ; Triticum aestivum ; Soil moisture ; Carbon reservoirs ; Greenhouse effect ; Grey forest soil ; Mineralization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The dynamics of the rate of CO2 evolution from soil in fallow and cropland under spring wheat (Triticum aestivum L.) was studied in a crop rotation in grey forest soil of the Baikal forest-steppe during the growing season and in different years. It was shown that the regional characteristics of soils and hydrothermal conditions in different years affect the rate of CO2 evolution in agroecosystems. The seasonal dynamics of CO2 is characterized by insignificant changes in the autumn to spring period and enhanced emission in hot and dry summers. CO2 evolution is assumed to increase due to enhanced mineralization and partial diffusion from the carbonate horizon at the depth of the seasonal frost. During the growing season the dynamics of CO2 evolution depends on the soil moisture regime. There was a strong correlation between the rate of CO2 emission and soil moisture in the particularly dry year of 1993 (η=0.86) and a moderate correlation in the other years (η=0.38–0.54). The effect of the previous crop and fertilizer application on the rate of CO2 emission was insignificant. In a continuous fallow the total carbon release into the atmosphere varied throughout the years studied from 558 to 1880 kg ha–1. Humus losses varied from 0.9% to 3.1%.
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  • 9
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    Biology and fertility of soils 23 (1996), S. 273-281 
    ISSN: 1432-0789
    Keywords: Wheat ; Triticum aestivum ; Rhizosphere ; Soil microflora ; Gram-negative bacteria ; API 20 NE ; Flavobacterium spp ; Cytophaga
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract We identified 161 Gram-negative bacterial strains isolated from the root surface of wheat grown under different soil conditions. The strains were divided into seven groups based on major morphological and physiological properties. Taxonomic allocation of the groups was verified by guanine+cytosine contents of DNA. Except for one group, which may be assumed to include bacteria belonging to the genera Flavobacterium and Cytophaga, the various groups were taxonomically united. The distribution of the groups changed with soil improvement. Pseudomonads predominated in unimproved soil, but Flavobacterium and Cytophaga spp. were predominant in the most improved soil. As all the strains were non-fermentative by Hugh and Leifson's test, API 20NE identification was applied. However, many strains were misidentified by this system, especially in the Flavobacterium and Cytophaga spp. group. For ecological studies, the strains were classified to species level by the API 20 NE system and by the results of a combination of guanine+cytosine (mol%) and isoprenoid quinone data. The pattern of distribution of the bacteria on the root surface of wheat varied at species level within one genus depending on soil conditions.
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  • 10
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    Cellular and molecular life sciences 52 (1996), S. 888-891 
    ISSN: 1420-9071
    Keywords: Ageing ; rat ; brain ; gene expression ; differential display
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have used the polymerase chain reaction (PCR)-based technique of differential display to analyse changes in gene expression during ageing of the rat brain. In this approach we have compared three young adult (6 months) with three old adult (20 months) animals. RNA preparations from the homogenised brains were subjected to reverse transcriptase (RT)-PCR using 36 different combinations of primer pairs. Any PCR product which was consistently found to be more prominent in the three young brains compared to the three old brains, and vice versa, was scored as potentially representing a gene which was differentially expressed during the ageing of this tissue. Out of a possible 2000+PCR products we identified 44 that might represent genes that exhibit differential expression during ageing of the rat brain. An initial screen of these fragments, by Southern-blotting the PCR products and hybridising them with cDNA probes derived from either young or old brain RNA preparations, indicated that 40% of them represented genes that were differentially expressed. This approach is likely to prove invaluable for identifying cohorts of genes that show differential expression during the ageing process.
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  • 11
    ISSN: 1432-203X
    Keywords: glyphosate ; gene expression ; gene amplification ; cell culture ; resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The stability and expression of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes was examined in glyphosate resistant tobacco cells grown in glyphosate-free medium, and in plantlets regenerated from resistant cells. Amplified DNA was maintained in resistant cells grown in the absence of glyphosate for three years. Amplified EPSPS genes were retained in regenerated plantlets at levels comparable to those observed in the resistant cells, and EPSPS mRNA was overexpressed (compared to unselected plantlets). However, glyphosate resistance in cell lines grown in glyphosate-free medium declined 7-fold, and in regenerated plantlets approximately 20-fold, compared to resistant cells maintained under glyphosate selection. In plantlets, reduced resistance correlated with lower levels of EPSPS mRNA. Plantlets regenerated from resistant cells exhibited morphological variation, and had an approximate doubling of their nuclear genome size.
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  • 12
    ISSN: 1432-0983
    Keywords: Key words Omnipotent suppression ; Microtubules ; Respiratory deficiency ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  SUP35 and SUP45 genes determine the accuracy of translation at the stage of termination. We present indirect evidence indicating that these genes may also control some cellular process mediated by microtubules. A majority of sup35 and sup45 suppressor mutations confer supersensitivity to benomyl, the drug which de-polymerizes microtubules. In addition, data correlating phenotypic manifestations of sup45 suppressor mutations, involving sensitivity to benomyl, respiratory deficiency and a suppressor effect, are also presented.
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  • 13
    ISSN: 1432-0983
    Keywords: Key words D-ribulose-5-phosphate 3-epimerase ; D-ribose-5-phosphate ketol-isomerase ; Pentose-phosphate pathway ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have cloned and characterized the two remaining unknown genes of the non-oxidative part of the pentose-phosphate pathway of Saccharomyces cerevisiae encoding the enzymes D-ribulose-5-phosphate 3-epimerase (Rpe1p) and D-ribose-5-phosphate ketol-isomerase (Rki1p). Rpe1p has an unexpected high specific activity of 2148 mU × (mg protein)–1 in crude extracts. Deletion mutants of RPE1 show no enzyme activity and are unable to grow on D-xylulose. Unexpectedly, haploid rki1 deletion mutants are not viable. Functional expression of RKI1 was demonstrated following an increase of gene dosage in the haploid rki1 deletion mutant, which restored viability and specific D-ribose-5-phosphate ketol-isomerase activity. Both enzymes show high similarity to the deduced protein sequences of various open reading frames, expressed sequence tags or cDNAs from different organisms.
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  • 14
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    Current genetics 30 (1996), S. 461-468 
    ISSN: 1432-0983
    Keywords: Keywords DNA repair ; Methylation damage ; Epistasis analysis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways.
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  • 15
    ISSN: 1432-0983
    Keywords: Biocontrol ; Secretion ; Chitinase ; Expression cloning ; Saccharomyces cerevisiae ; Trichoderma harzianum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.
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  • 16
    ISSN: 1432-0983
    Keywords: Heat-shock response ; Multidrug resistance ; AP-1 homolog ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have examined whether the stress-induced transcriptional activation ofYDR1/PDR5/STS1 is mediated by yAP-1 and yAP-2. Of the stresses examined, heat shock-induced, rapid and transient PDR5 expression became very low in ayap1 yap2 double-gene disruptant, indicating that the yAP proteins mediate the response. Similar results were obtained withSNQ2, a close homologue ofPDR5. A set of 5′-truncation derivatives of thePDR5 gene identified the region from −484 to −434 as being sufficient for the response. A sequence similar to the yAP-1 recognition element recently identified in the stress-responsive yeast genes was found in this region and in the 5′-flanking sequences ofSNQ2.
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  • 17
    ISSN: 1432-0983
    Keywords: Key words Biocontrol ; Secretion ; Chitinase ; Expression cloning ; Saccharomyces cerevisiae ; Trichoderma harzianum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.
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  • 18
    ISSN: 1432-0983
    Keywords: Key words Cytochrome b ; Mutants ; Mitochondria ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nucleotide changes present in a group of five cytochrome b mit– mutants were analyzed at the sequence level. Two single-base changes were found: one (M10-152) generated a nonsense codon in the first exon while the other (M8-181) created a missense substitution in the second exon. The other mutants all have multiple (three) substitutions that either resulted in a missense mutation in a coding region (M17-162) or else changed nucleotides in the last intron of the gene, so blocking its excision (M6-200 and M8-53). The synthesis of mitochondrial polypeptides and the steady state concentration of the complex-III subunits were examined. The Rieske protein and the core-4 and core-5 subunits were much reduced in all mutants. Consequently the overall stability of complex III is very sensitive even to amino-acid substitutions in the cytochrome b protein. Mutant M8-53 provides direct evidence for the proposed role of the P9.1 stem in the core structure of the group-I type last intron of this gene.
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  • 19
    ISSN: 1432-0983
    Keywords: Key wordsβ-glucosidase ; Candida wickerhamii ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast Candida wickerhamii exports a cell-associated β-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active β-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-µ replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the α-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.
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  • 20
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    Current genetics 29 (1996), S. 227-233 
    ISSN: 1432-0983
    Keywords: Trichoderma reesei ; β-Glucosidase ; Cellulase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An intronless form of thebgl1 gene encoding an extracellularβ-glucosidase fromTrichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL 1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete activeβ-glucosidase into the growth medium. Additionally, active recombinantβ-glucosidase protein was shown to be localized predominantly in the periplasmic space by using ap-nitrophenylβ-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinantβ-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.
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  • 21
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    Current genetics 29 (1996), S. 227-233 
    ISSN: 1432-0983
    Keywords: Key words  Trichoderma reesei ; β-Glucosidase ; Cellulase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract   An intronless form of the bgl1 gene encoding an extracellular β-glucosidase from Trichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete active β-glucosidase into the growth medium. Additionally, active recombinant β-glucosidase protein was shown to be localized predominantly in the periplasmic space by using a p-nitrophenyl β-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinant β-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.
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  • 22
    ISSN: 1432-0983
    Keywords: Key words Adenine biosynthesis ; ade8-18 ; ade2 mutations ; Red/white colony color assay ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.
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  • 23
    ISSN: 1432-072X
    Keywords: Key words Plasma membrane H+-ATPase ; Saccharomyces cerevisiae ; Low pH ; PMA1 gene expression ; PMA2 gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cells of Saccharomyces cerevisiae grown in media with an initial pH of 2.5–6.0, acidified with a strong acid (HCl), exhibited the highest plasma membrane H+-ATPase-specific activity at an initial pH of 6.0. At a lower pH (above pH 2.5) ATPase activity (62–83% of the maximum level) still allowed optimal growth. At pH 2.5, ATPase activity was about 30% of the maximum value and growth was impaired. Quantitative immunoassays showed that the content of ATPase protein in the plasma membrane was similar across the entire pH range tested, although slightly lower at pH 2.5. The decrease of plasma membrane ATPase activity in cells grown at low pH was partially accounted for by its in vitro stability, which decreased sharply at pH below 5.5, although the reduction of activity was far below the values expected from in vitro measurements. Yeast growth under acid stress changed the pattern of gene expression observed at optimal pH. The level of mRNA from the essential plasma-membrane-ATPase-encoding gene PMA1 was reduced by 50% in cells grown at pH 2.5 as compared with cells grown at the optimal pH 5.0, although the content of ATPase in the plasma membrane was only modestly reduced. As observed in response to other kinds of stress, the PMA2 promoter at the optimal pH was up to eightfold more efficient in cells grown at pH 2.5, although it remained several hundred times less efficient than that of the PMA1 gene.
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  • 24
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 102-109 
    ISSN: 1476-5535
    Keywords: Millet ; Pennisetum typhoides ; liquefaction ; saccharification ; baker's yeast ; Saccharomyces cerevisiae ; fermentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4-α-d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca2+, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4-α-d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h−1, biomass yield coefficient of 0.5 g g−1 and feed substrate concentration of 200 g L−1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L−1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g−1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.
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  • 25
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    Molecular and cellular biochemistry 155 (1996), S. 85-90 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; gene expression ; rat hepatoma ; Morris hepatoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Whether the gene expression of hepatic Ca2+-binding protein regucalcin is altered in hepatomas was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). Rat hepatoma was induced by continuous feeding of basal diet containing 0.06% 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB). After 35 weeks feeding, rats were sacrificed, and the non-tumorous and tumorous tissues of the livers were removed. In individual rats, the regucalcin mRNA levels in the tumorous tissues were generally decreased in comparison with that of the non-tumorous tissues of the chemical-fed rats, although the chemical administration might decrease the mRNA expression in normal rat liver, suggesting that the chemical administration causes a suppresive effect on the mRNA expression. When the genomic DNA extracted from the liver tumorous tissues was digested with restriction enzymes (EcoRI, BamHI and HindIII) and analyzed by Southern blotting, no rear-ranged band was found in the regucalcin gene from the hepatoma. Interestingly, in the transplantable Morris hepatoma cells, the regucalcin mRNA was markedly expressed, while the albumin mRNA was expressed only slightly. The present study demonstrates that regucalcin mRNA is clearly expressed in the transformed cells (Morris hepatoma cells).
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  • 26
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    Molecular and cellular biochemistry 155 (1996), S. 105-111 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; aldosterone ; estrogen ; dexamethasone ; gene expression ; rat kidney cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of various steroid hormones on the expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex but not the medulla. Rats received a single subcutaneous administration of steroid; the animals were sacrificed 60 min after the treatment of aldosterone (2.5, 5.0 and 10 μg/100 g body weight) or 6 h after the treatment of estrogen (17β-estradiol; 0.05, 0.1 and 0.2 mg/100 g), hydrocortisone (0.5, 1.0 and 3.0 mg/100 g) and dexamethasone (50, 100 and 150 μg/100 g). Regucalcin mRNA levels in the kidney cortex were clearly diminished by the administration of aldosterone or estrogen, while hydrocortisone administration had no effect. The administration of dexamethasone (100 μg/100 g) caused a remarkable increase of regucalcin mRNA levels in the kidney cortex. The dexamethasone-induced increase in regucalcin mRNA levels was completely blocked by the simultaneous administration of cycloheximide (150 μg/100 g), although the drug administration had no effect on the mRNA levels in control rats. Meanwhile, the dexamethasone administration did not cause an appreciable alteration of calcium content in the kidney cortex. The present study demonstrates that, of the various steroid hormones used, dexamethasone uniquely has a stimulatory effect on regucalcin mRNA expression in the kidney cortex of rats. The steroid effect may be mediated through a newly synthesized protein.
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  • 27
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    Molecular and cellular biochemistry 160-161 (1996), S. 307-313 
    ISSN: 1573-4919
    Keywords: myocardium ; hypertension ; gene expression ; estrogens ; cardiac hypertrophy ; signal transduction ; genetic program
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Gender specific differences in cardiovascular disease are largely mediated by sex hormones. The use of estrogens significantly reduces the overall incidence of heart disease in postmenopausal women. Beneficial effects of estrogens on plasma lipoprotein levels are clearly established. However, these do not explain the magnitude of risk reduction seen in clinical studies. Thus, additional and currently unknown functions of estrogens must be operative. Elucidation of the exact estrogen action in the heart will have important implications in the treatment of cardiovascular disease. It will probably enhance the therapeutic repertoire in treating heart disease, the most common cause of death in industrialized countries. We will review the current understanding of the function of estrogens in the heart and discuss potential strategies on how to apply these data to clinical practice.
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  • 28
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    Molecular and cellular biochemistry 162 (1996), S. 139-144 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; gene expression ; saline ingestion ; hypertensive rats ; kidney cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of adrenalectomy (ADX) or saline ingestion, which is a hypertensive factor, on the expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex but not the medulla. Rats were adrenalectomized, and 48 h later they were sacrificed. ADX caused a reduction of regucalcin mRNA levels in the kidney cortex, suggesting that adrenal glands participate in the regulation of the mRNA expression. This reduction was not restored by the subcutaneous administration of dexamethasone with an effective dose (1 mg/kg body weight), which can stimulate kidney regucalcin mRNA expression. Regucalcin mRNA levels in the kidney cortex of rats were markedly suppressed by the ingestion of saline for 7 days. The ADX-induced decrease of renal cortex regucalcin mRNA levels was not appreciably restored by saline ingestion. Moreover, regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were clearly decreased as compared with that of control (Wistar-Kyoto) rats. Meanwhile, calcium content in the kidney cortex was not significantly decreased by ADX or saline ingestion. The present study suggests that the expression of regucalcin mRNA in the kidney cortex of rats is suppressed by saline administration.
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  • 29
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    Molecular and cellular biochemistry 155 (1996), S. 153-162 
    ISSN: 1573-4919
    Keywords: apolipoprotein B and E ; lipid ; gene expression ; rat ; mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The aim of the present investigation was to study the regulation of apolipoprotein E by two dietary nutrients, saturated fat and cholesterol, known to raise plasma cholesterol levels. ApoE is a protein component of several classes of lipoproteins including VLDL and HDL, and dietary lipids may regulate VLDL and apoE-containing HDL particles through their effects on apoE gene. Male rats and mice were fed the following 4 diets: control diet (C); high cholesterol diet with 0.5% cholesterol (HC); high fat diet with 20% hydrogenated coconut oil (HF); and high fat plus high cholesterol diet with 0.5% cholesterol and 20% fat (HF/C). Plasma cholesterol levels remained unchanged on HC diet, but in mice VLDL-cholesterol increased by 31%. HF diet increased VLDL and LDL by 15–17% in rats, and 21% in mice. A combination of fat and cholesterol diet showed pronounced effects on plasma lipoprotein concentrations, raising apoB-containing particles by 21% and 44% in mice and rats, respectively. Plasma apoE levels increased significantly on all diets. The mechanism of regulation of increased plasma apoB and apoE levels was examined. Quantification of hepatic apoB mRNA showed a lack of correlation between plasma apoB and hepatic apoB mRNA levels, suggesting that posttranscriptional regulation increased plasma apoB-containing lipoproteins in animals fed saturated fat diets. Hepatic apoE mRNA levels increased significantly in animals fed cholesterol-rich diets. However, despite increased plasma apoE levels on diet containing only saturated fat, hepatic apoE mRNA did not change. Synthesis of apoE on the liver polysomes increased selectively on cholesterol-rich diets. These results suggest that cholesterol-rich diets altered apoE, in part, by transcriptional mechanism, and saturated fat-rich diets increased plasma apoE levels by posttranscriptional mechanism, possibly decreased receptor-mediated uptake of apoE-containing particles. The regulation of LDL receptor was also studied since plasma apoB and E levels may be altered by LDL receptor-mediated uptake by the hepatocytes. As expected, high cholesterol diet decreased LDL receptor mRNA by 30–40%. However, the LDL receptor protein on liver membranes did not change on any of the test diets in both animal species. Hepatic cholesterol content increased several fold selectively on high cholesterol diets. These findings suggest that: 1) both transcriptional and posttranscriptional mechanisms are important in regulating plasma apoB and E containing lipoproteins; 2) dietary cholesterol regulates apoE gene by a transcriptional mechanism anddietary saturated fat by posttranscriptional mechanism; and 3) changes in the hepatic apoE and LDL receptor mRNA are associated with the changes in intracellular cholesterol concentrations.
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  • 30
    ISSN: 1573-4919
    Keywords: fatty acid transport protein ; gene expression ; subtractive hybridization ; oxidative stress ; ischemia/reperfusion ; ischemic preconditioning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this study, ischemia and oxidative stress-inducible gene expression in heart was examined by subtractive hybridization technique. Total RNA was isolated from ventricular muscle fragments of normal and oxidative stress-induced hearts. Poly (A)+ RNA was purified followed by the construction of a plasmid cDNA library. This was followed by the subtractive screening of oxidative stress-induced cDNA library. The positive colonies were amplified and the plasmid isolated. An aliquot was subjected to restriction cutting with Bam H1 and EcoRl; the fragments corresponding to cDNA insert were separated by electrophoresis, radiolabeled by random-primed DNA synthesis, and used as probes in standard Northern blotting experiments. An aliquot containing the plasmid from the confirmed positives was then subjected to bidirectional partial DNA sequencing (using M13 and T7/T3α primers) by the chain-extension/chain termination method. These sequences were subjected to a computerized search for homologies against all sequences in the updated worldwide Gen Bank and EMBL sequence databases followed by restriction mapping and reading frame identification. Out of 24 putative positive colonies screened, one clone was matched with 〉 97% homology with FAT gene that has been implicated in binding or transport of long chain fatty acids. cDNA probe synthesized from this clone identified two major transcripts of 4.8 and 2.9 kb. Additional experiments were then performed where isolated perfused rat hearts were subjected to the following treatments: (1) 5 min ischemia; (2) 10 min ischemia; (3) 20 min ischemia; (4) 5 min ischemia followed by 10 min reperfusion (ischemic preconditioning); and (5) 5 min ischemia followed by 10 min reperfusion, repeated four times (4 × preconditioning). RNAs were extracted from these hearts and hybridized with the FAT cDNA probe. The results indicated that FAT gene was induced by oxidative stress, ischemic preconditioning, but not by ischemia. (Mol Cell Biochem 160/161: 241–247, 1996)
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  • 31
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    Molecular and cellular biochemistry 163-164 (1996), S. 231-237 
    ISSN: 1573-4919
    Keywords: extracellular matrix ; angiotensin II ; fibrillar collagen ; cardiac fibrosis ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Recent studies suggest that angiotensin II (angiotensin) may be involved in the regulation of metabolism of the cardiac extracellular matrix (ECM). Two major components of ECM are collagen types I and III which play an important role in maintaining the structure and function of the heart. Although the cellular metabolism of collagen is very complex (especially at the posttranslational level), we chose to address events that occur relatively early in the synthesis of cardiac collagen molecules. To gain an understanding of the role of angiotensin in the regulation of cardiac collagen gene expression, we studied the effect of three different doses of angiotensin (12, 24, and 48 μg/kg/h) on adult heart and cultured neonatal cardiac fibroblasts. The steady-state mRNA abundance of collagen types I and III was monitored using Northern blot analysis in both left and right ventricular samples at day 3 of angiotensin infusion and in cultured cardiac fibroblasts stimulated with angiotensin. In all mRNA abundance studies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal was used to normalize the data for possible differences in loading and/or transfer of total RNA. Both collagen types I/GAPDH and III/GAPDH mRNA signal ratios were increased significantly in left ventricle in all dose regimens used for angiotensin infusion. Only the collagen type I/GAPDH mRNA signal ratio was increased in right ventricle with angiotensin infusion. Angiotensin (10−7-10−5 M) had no effect on the steady-state mRNA abundance of collagen genes in cultured neonatal cardiac fibroblasts after 24 h treatment in serum-free conditions. Our results confirm that infusion of angiotensin may upregulate steady-state collagen gene mRNA abundance in the heart. Angiotensin had no observable effect on collagen mRNA abundance in neonatal fibroblast culture. An explanation for the current results may be that angiotensin causes the release of undefined factors from cardiac myocytes, and that these secondary factors may be involved in either the activation of collagen gene transcription or in alteration of stability of collagen mRNA transcripts via a paracrine mechanism. Although our results indicate hemodynamic loading may potentiate the action of angiotensin, this scenario is unlikely as collagen type I gene expression was increased in the normotensive right ventricle.
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    Molecular and cellular biochemistry 162 (1996), S. 51-58 
    ISSN: 1573-4919
    Keywords: metabolism ; glucose transporter ; adipocytes ; gene expression
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We tested the hypothesis that the constitutive glucose transporter (GLUT 1) in 3T3-L 1 adipocytes belongs to the family of glucose-regulated proteins which are transcriptionally regulated by glucose deprivation. Using cDNA probes for both GRP78 (BiP) and GLUT1, we show that the level of GRP78 mRNA increased by 15-fold within 24 h of glucose deprivation with little change in GLUT1 mRNA. The elevated GRP78 mRNA in turn led to a time-dependent increase in GRP78 protein. While glucose deprivation did not alter the expression of the normal glycoform of GLUT 1, a lower molecular weight glycoform accumulated with extended deprivation. Mannose and fructose, but not galactose, prevented the induction of GRP78 and accumulation of the abnormal GLUT1. Because GRP78 acts as a chaperone in other cell systems, we also sought evidence to support this activity in 3T3-L1 adipocytes. Using the technique of co-immunoprecipitation, we demonstrate that GRP78 bound several proteins unique to the glucose-deprived state. No deprivation-specific proteins could be detected in association with GLUT 1. These data lead us to conclude that GLUTl does not display characteristics of the glucose-regulated proteins, at least in 3T3-L1 adipocytes, a widely used model for differentiation, hormone action, and nutrient control. However, the mechanisms for activating traditional members of this family appear intact.
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  • 33
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    Water, air & soil pollution 90 (1996), S. 557-562 
    ISSN: 1573-2932
    Keywords: heavy metal ; accumulation ; fish
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract Levels of cadmium, copper, zinc, lead and iron were determined seasonally in the liver, spleen, kidney, gill and muscle tissues of Mullus barbatus and Sparus aurata from the Iskenderun Gulf, East Mediterranean coast of Turkey. Wet digested tissues were analysed by atomic absorption spectroscopy. Metal levels were higher in liver, spleen and kidney compared with the gill and muscle tissues in both species; the levels of all metals in a given tissue were always higher in Mullus barbatus than in Sparus aurata.
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  • 34
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    Molecular and cellular biochemistry 154 (1996), S. 65-70 
    ISSN: 1573-4919
    Keywords: gene expression ; regulatory elements ; plasmid ; oligonucleotides
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A potentially powerful pharmacological approach to modulating the expression of specific, disease-related genes involves the inhibition of transcription factor binding to promoter or enhancer elements through oligonucleotide-mediated triple-helix formation. In vivo, the typical target for intermolecular triplex formation would most likely be torsionally-strained rather than relaxed duplex DNA. To determine the effects of strained DNA on triplex formation, we investigated the interactions between a G/T rich oligonucleotide and both supercoiled and relaxed plasmid DNA using a restriction endonuclease protection assay. Both the kinetics of formation and dissociation of purine-motif triplexes were unaffected by the conformational state of the duplex DNA. Similarly, the topological state of the plasmid targets was not affected by triplex formation. Taken together, these observations suggest that stable intermolecular triplexes can form in vivo under conditions of moderate torsional strain.
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  • 35
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    Keywords: osmotic stress ; Saccharomyces cerevisiae ; glycerol ; K+/Na+ ions ; osmoregulation
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The intracellular level of Na+ and K+ of S. cerevisiae strain AB1375 revealed that under KCl as well as sorbitol stress, the cationic level was comparable to the level under no stress conditions. On the other hand, there was a sharp drop in the intracellular K+ content and increase in the Na+ content on addition of NaCl to the medium. However, the total cationic level was close to that under control conditions. In addition to changes in the cationic level, an enhanced production and accumulation of glycerol were also observed under osmotic stress. A regulatory mechanism co-ordinating the intracellular concentration of glycerol as well as Na+, K+ content under osmotic stress conditions has been proposed.
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  • 36
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    Molecular biology reports 23 (1996), S. 205-210 
    ISSN: 1573-4978
    Keywords: autoantigen ; cDNA cloning ; gene expression ; ribonucleoprotein Ro ; 5′RACE
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5′ RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kindney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.
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  • 37
    ISSN: 1573-5028
    Keywords: gene expression ; photosynthesis ; protein turnover ; psbA ; tac promoter ; D1 protein
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    Topics: Biology
    Notes: Abstract Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI Q system. Over-expression was induced with 40 μg/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 μmol photons m-2 s-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 μmol photons m-2 s-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.
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  • 38
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; cDNA ; gene expression ; starch biosynthesis ; starch branching enzyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two starch branching enzyme (SBE) cDNAs were identified in an Arabidopsis seedling hypocotyl library using maize Sbe1 and Sbe2 cDNAs as probes. The two cDNAs have diverged 5′ and 3′ ends, but encode proteins which share 90% identity over an extensive region with 70% identity to maize SBE IIb [12]. Genomic Southern blots suggest that the two cDNAs are the products of single, independent genes, and that additional, more distantly related SBE genes may exist in the Arabidopsis genome. The two cDNAs hybridize to transcripts which show similar expression patterns in Arabidopsis vegetative and reproductive tissues, including seedlings, inflorescence rachis, mature leaves, and flowers. This is the first report of the identification of cDNAs encoding two closely related starch branching enzymes from the same species.
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  • 39
    ISSN: 1573-5028
    Keywords: Agrobacterium rhizogenes ; gene expression ; GUS staining ; in situ hybridization ; rolA gene ; tobacco
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    Topics: Biology
    Notes: Abstract The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the β-glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elengation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B+C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.
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  • 40
    ISSN: 1573-5028
    Keywords: Arabidopsis ; gene expression ; hypersensitive response ; plant-pathogen interactions ; cell suspensions ; sulfotransferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotrans-ferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hyper-sensitive reaction, as compared to virulent pathogens, which lead to disease.
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  • 41
    ISSN: 1573-5028
    Keywords: cDNAs ; Douglas fir ; gene expression ; germination ; low-molecular-weight heat shock proteins ; methyl jasmonate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated and sequenced two cDNA clones (PM 18.2A;PM 18.2B) from Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) which encode for the low-molecular-weight heat shock proteins (LMW HSPs) of 18.2 kDa. The predicted amino acid sequences of the two Douglas fir proteins are 97.5% identical. A phylogenetic tree of class I LMW HSPs showed that the PM LMW HSPs are found within a subgroup consisting exclusively of dicot species indicating that class I LMW HSPs evolved from a common ancestor predating the divergence of gymnosperms and angiosperms. Northern blots of RNA from dry, imbibed, stratified and germinated seeds revealed a notable induction of LMW HSP transcripts during post-germination and early seedling growth. Unlike previous reports, the expression of these HSPs appears to be primarily restricted to seedlings as mRNA transcripts were detected at very low levels during seed development and desiccation. Maximum induction of LMW HSPs in seedlings occurred during heat shock treatment at 38–40°C, whereas cold shock or wounding failed to induce HSP transcripts. The transcription of HSP genes is up regulated by GA, MeJA and auxin and is down regulated by ABA. Methyl jasmonate treatment induced expression of these genes in dormant seeds of Douglas fir. The expression of class I cytoplasmic LMW HSPs in seedlings and their regulation by plant growth regulators suggests specific roles in plant development other than desiccation tolerance.
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  • 42
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    Plant molecular biology 30 (1996), S. 711-722 
    ISSN: 1573-5028
    Keywords: developmental regulation ; gene expression ; gene family ; phenylalanine ammonia-lyase ; tobacco ; wound response
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the general phenylpropanoid pathway leading to the production of phenolic compounds with a significant range of biological functions. A PAL gene we designated gPAL1, cloned from tobacco, consists of two exons separated by an intron of 1932 bp. Exon I, 398 bp, and exon II, 1747 bp, together encode a polypeptide of 715 amino acids. A putative TATA box and polyadenylation signal are found 144 bp upstream of the initiation codon and 193 bp downstream from the stop codon, respectively. Using various parts of gPAL1 as probes, genomic Southern blots indicated the presence of a small family of PAL genes in the tobacco genome that can be divided into two distinct subfamilies, one consisting of pal1 and pal2 and another of pal3 and pal4. Comparative genomic blot analysis of progenitor species (Nicotiana tomentosiformis and N. sylvestris) indicated that each species contains one PAL gene from each of the subfamilies, suggesting that pal1 and pal3 (or pal2 and pal4) diverged prior to the evolution of N. tabacum. Expression of the PAL gene family was examined using RNA gel blots. PAL transcript levels were significantly higher in flowers and roots than in leaves and stems of mature plants. PAL transcripts accumulate differentially during flower and leaf maturation in that mRNA levels decline during flower maturation but increase during leaf maturation. In leaves, PAL transcripts rapidly accumulated after wounding.
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  • 43
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; auxin ; gene expression ; glutathione S-transferase
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    Topics: Biology
    Notes: Abstract Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a λ clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.
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  • 44
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    Plant molecular biology 30 (1996), S. 863-872 
    ISSN: 1573-5028
    Keywords: gene expression ; sucrose synthase ; sugar beet (Beta vulgaris L.)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To investigate the expression pattern of sucrose synthase, a cDNA from tap roots of sugar beet (Beta vulgaris L.) was isolated using a heterologous sucrose synthase cDNA from potato. The 2762 bp long cDNA clone designated SBSS 1 encodes for a 822 amino acid polypeptide of a predicted molecular mass of 93.7 kDa. The deduced amino acid sequence of sugar beet sucrose synthase has homologies of 65–70% when compared to predicted amino acid sequences of sucrose synthases from other species. RNA blot analysis shows that SBSS1 is expressed most predominant in tap root under normal growth conditions. Cold treatment and anaerobiosis lead to an increase in the steady-state levels of SBSS 1 mRNA in leaf and root tissue. In tap root slices, sugars in various concentrations had no influence on the SBSS 1 transcript level. On the other hand, wounding resulted in a decreased transcript level.
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  • 45
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    Plant molecular biology 30 (1996), S. 913-924 
    ISSN: 1573-5028
    Keywords: catalase ; gene expression ; gene structure ; isozyme ; reactive oxygen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The maize Cat2 gene was isolated by direct cloning and PCR. The clones were mapped and sequenced. The start site of transcription was determined by primer extension. Computer analysis of the 1.6 kb Cat2 promoter sequence has revealed an obvious TATA box, two GC boxes, a putative GA response element, and several ACGT core sequences known to have diverse regulatory functions in plants. Several other protein binding motifs were also identified within 800 bp upstream from the transcriptional start site. Five introns were identified in the Cat2 coding region. All five Cat2 introns are located in exactly the same position as five of the six introns in Cat1. Two of the Cat2 introns are located in the same position as the two Cat3 introns. The identical positioning of these introns suggests an evolutionary link between all three maize catalase genes. The response of the Cat2 gene to plant growth regulators was examined. Results clearly showed that the response of Cat2 to several environmental factors are developmental stage-dependent. Thus, complex regulatory mechanisms appear to be involved in the regulation of Cat2 expression in maize.
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  • 46
    ISSN: 1573-5028
    Keywords: ethylene ; ethylene receptor ; signal transduction ; ETR1 ; tomato ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Dominant mutations in the Arabidopsis ETR1 gene block the ethylene signal transduction pathway. The ETR1 gene has been cloned and sequenced. Using the ETR1 cDNA as a probe, we identified a cDNA homologue (eTAE1) from tomato. eTAE1 contains an open reading frame encoding a polypeptide of 754 amino acid residues. The nucleic acid sequence for the coding sequence in eTAE1 is 74% identical to that for ETR1, and the deduced amino acid sequence is 81% identical and 90% similar. Genomic Southern blot analysis indicates that three or more ETR1 homologues exist in tomato. RNA blots show that eTAE1 mRNA is constitutively expressed in all the tissues examined, and its accumulation in leaf abscission zones was unaffected by ethylene, silver ions (an inhibitor of ethylene action) or auxin.
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  • 47
    ISSN: 1573-5028
    Keywords: elongation growth ; gene expression ; Hordeum vulgare ; protochlorophyllide oxidoreductase ; slender mutant ; tonoplast intrinsic protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three cDNA clones have been isolated on the basis of altered patterns of expression in the leaf extension zone of the developmental mutant,slender barley, compared with the wild type. mRNAs corresponding to two of the cDNAs, 7s and 8s, are increased inslender compared with normal. 7s encodes a putative γ-TIP and is expressed throughout the elongation zone. γ-TIPs form transmembrane channels which allow the passive transfer of water. Although expression of 7s was increased inslender leaf tissue, the increase was much less extreme than that shown by Phillips and Huttly (1994) following the application of GA to an extreme dwarf ofArabidopsis. 8s is maximally expressed in the region of early cell elongation and has 66% encoded protein identity with MFS18, a cDNA encoding a putative cell wall structural protein isolated from male flowers of maize. Both 8s and MFS18 encode small (128 amino acids) basic proteins rich in glycine, alanine, proline and serine. mRNA corresponding to the third cDNA, 24n, is present at a greatly reduced level inslender compared with normal and encodes protochlorophyllide oxidoreductase (POR). POR catalyses the conversion of protochlorophyllide into chlorophyllide. The reduced level of POR mRNA is not correlated with a similar reduction in expanded leaf blade chlorophyll levels. Western analysis identified two POR proteins present in light-grown seedlings. Whilst the larger of the proteins is present throughout most of the leaf, the smaller protein mimics the mRNA results, being both maximally present in the elongation tissue and present at a reduced level inslender. An antagonistic relationship between chlorophyll biosynthesis and extension growth is suggested.
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  • 48
    ISSN: 1573-5028
    Keywords: ACC synthase ; differential expression ; ethylene ; root-specific expression ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two partial 1-aminocyclopropane-1-carboxylic acid (ACC) synthase cDNA clones (pWAS1, 1089 bp; andpWAS3, 779 bp) were isolated by polymerase chain reaction (PCR) using cDNA to total mRNA purified from etiolated wheat seedlings as template and degenerate oligonucleotides synthesized based on the regions of the ACC synthase amino acid sequence that are highly conserved among different plants. Northern analysis showed that the expression of the corresponding genes are differentially regulated. While the transcripts ofpWAS1 were found in all the tissues of wheat that were tested with a maximum level at the early stages of spike development,pWAS3 mRNA was present almost exclusively in the root. A 5590-bp genomic clone,TA-ACS2, corresponding topWAS3 cDNA has been isolated. TheTA-ACS2 sequence consists of a 589-bp 5′-upstream region, 2743 bp of transcribed region with four exons and three introns and a 3′-downstream region of 2257 bp. Expression inEscherichia coli confirmed the ACC synthase activity of TA-ACS2 polypeptide. Sequence comparisons show that the two wheat ACC synthases are more similar to each other and to the rice ACC synthase,OS-ACS1, at the nucleotide level than at the amino acid level. The amino acid sequence of TA-ACS2 is most similar (66.1% identity) to that of broccoli. The chromosomal location of both wheat ACC synthase genes have been determined by aneuploid analysis.TA-ACS1 is located on the short arm of chromosomes 7A and 7D and on the long arm of chromosome 4A.TA-ACS2 is located on the long arm of homoeologous group 2 chromosomes.
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  • 49
    ISSN: 1573-5028
    Keywords: Chlamydomonas reinhardtii ; chlorophyll a/b-binding protein gene ; circadian rhythms ; gene expression ; light-regulated genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cabI/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.
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  • 50
    ISSN: 1573-5028
    Keywords: gene expression ; Glycine max. L. ; nodule development ; purN ; purD ; GAR synthetase ; GAR transformylase
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    Topics: Biology
    Notes: Abstract Soybean nodule cDNA clones encoding glycinamide ribonucleotide (GAR) synthetase (GMpurD) and GAR transformylase (GMpurN) were isolated by complementation of corresponding Escherichia coli mutants. GAR synthetase and GAR transformylase catalyse the second and the third steps in the de novo purine biosynthesis pathway, respectively. One class of GAR synthetase and three classes of GAR transformylase cDNA clones were identified. Northern blot analysis clearly shows that these purine biosynthetic genes are highly expressed in young and mature nodules but weakly expressed in roots and leaves. Expression levels of GMpurD and GMpurN mRNAs were not enhanced when ammonia was provided to non-nodulated roots.
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  • 51
    ISSN: 1573-5028
    Keywords: abscisic acid ; early cysteine-labeled protein ; fluridone ; metallothioneins ; pollen embryogenesis ; Triticum aestivum
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    Notes: Abstract A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.
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  • 52
    ISSN: 1573-5028
    Keywords: cell division cycle ; gene expression ; mitotic cyclin ; plant suc1 homologue ; Ntcdc2-1 ; Nicotiana tabacum
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    Topics: Biology
    Notes: Abstract We have previously reported the isolation of a cDNA encoding a mitotic cyclin, NTCYC1, from a tobacco cell suspension library. Here we describe the expression patterns of NTCYC1 and of Ntsuc1, a suc1 plant homologue, in synchronized tobacco cell suspensions. Furthermore, the expression pattern of this cyclin is compared to that of Ntcdc2-1, a Nicotiana tabacum homologue of cdc2. While no NTCYC1 transcript was detected in cells synchronized in the G1 and S phases, NTCYC1 expression was observed in late G2 and early M phases, disappearing in the G1′ of a new cell cycle. On the other hand, Ntsuc1 and Ntcdc2-1 exhibited a constitutive expression during the cell cycle. A functional analysis performed by microinjecting NTCYC1 mRNA into immature Xenopus oocytes, indicates that NTCYC1 could participate in the control of the G2/M transition in plant cells. Subsequently NTCYC1 expression was used to assess the status of mesophyll cells in expanded leaves of N. tabacum. Depending on leaf position along the shoot axis, a large population of mesophyll cells appeared with a 4C DNA content, suggesting a G2 arrest. It was found that leaves with such a population also contained high levels of NTCYC1 transcripts. With respect to these results concerning a naturally occurring G2-arrested cell population, the regulation of NTCYC1 expression in planta is discussed.
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  • 53
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    Molecular biology reports 23 (1996), S. 119-121 
    ISSN: 1573-4978
    Keywords: gene expression ; Portulaca grandiflora ; protein profiles ; rosette ; ratooning ; SDS-PAGE
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    Topics: Biology
    Notes: Abstract Rosetteness is either developmentally controlled or induced by various biotic and abiotic stresses. Prolonged vegetative growth and/or pruning seems to be inducing rosetteness in Portulaca grandiflora. Analysis of cellular extracts from rosette stems by SDS-PAGE, revealed induction of a polypeptide of high molecular mass (≃ 58 kDa) and over-expression of a few lower molecular weight polypeptides. However, leaves showed no differences in the protein profiles.
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  • 54
    ISSN: 1573-5028
    Keywords: abscisic acid ; cold acclimation ; frost tolerance ; gene expression ; PCR
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    Notes: Abstract A DNA fragment corresponding to a low-temperature- and ABA-responsive gene (Scdhn1) was amplified by PCR from genomic DNA of a wild, frost-resistant potato species, Solanum commersonii. A homologous gene (Stdhn1) was identified in Solanum tuberosum cv. Bintje, a frost-sensitive domesticated potato cultivar. The expression of the gene was studied during low temperature and ABA treatments in both Solanum species. The analysis revealed that both low temperature and ABA lead to the accumulation of a 1 kb transcript that corresponded to the PCR fragment. The induction of the gene was relatively rapid and maximum amounts of the transcripts were detected already after 1 day and 7 h of treatment with low temperature and ABA, respectively. Previous results have shown that there is no increase in the amount of endogenous ABA in S. tuberosum during low-temperature treatment, which indicates that two independent signalling pathways lead to the induction of this gene.
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  • 55
    ISSN: 1573-5028
    Keywords: Chinese cabbage (Brassica campestris L. spp. pekinensis) ; cDNA ; cystatin ; inhibitor ; gene expression
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    Notes: Abstract A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.
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  • 56
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    Plant molecular biology 30 (1996), S. 673-677 
    ISSN: 1573-5028
    Keywords: cDNA sequence ; chymotrypsin inhibitor ; histidine-tag ; protein Z ; serpin ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27–33% identical with human serpins such as α1-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with α-chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.
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  • 57
    ISSN: 1573-5028
    Keywords: pathogenesis-related protein ; gene expression ; flowering ; plant reproduction ; transgenic plants
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    Notes: Abstract A gene (pMON9617; Chi2;1) identified by screening a tomato pistil cDNA library has been found to encode a protein similar in sequence to class II chitinases. Using a baculovirus expression system we show that the Chi2;1 protein is an active endochitinase. The Chi2;1 protein is most similar in sequence to a previously described stylar chitinase (SK2) isolated from potato. Both proteins lack the diagnostic N-terminal cysteine-rich regions and the C-terminal vacuolar targeting signals of class I chitinases and appear to define a novel type of class II endochitinases associated with flowers. Chi2;1 is expressed predominantly in floral organs and its expression within these organs is temporally regulated. The highest level of expression is found in the transmitting tissue of the style where Chi2;1 mRNA begins to accumulate just prior to anthesis. In vegetative tissue, low levels of Chi2;1 mRNA were detected, and these levels did not increase in response to wounding or treatment with ethephon. mRNA from Chi2;1 orthologs was not detected in most other angiosperms tested, even including some members of the Solanaceae, and it is therefore unlikely that Chi2;1 is essential for stylar function. A fragment containing 1.4 kilobase pairs of 5′-flanking DNA from the Chi2;1 gene was shown to drive high-level expression of an attached reporter gene in the styles of transgenic tomatoes. This fragment has utility for engineering expression of other coding regions in styles.
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  • 58
    ISSN: 1573-5028
    Keywords: MAP kinase ; osmotic stress ; Pisum sativum ; Saccharomyces cerevisiae ; signal transduction
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    Topics: Biology
    Notes: Abstract Previous analysis of the MAP kinase homologue from Pisum sativum (PsMAPK) revealed a potential MAP kinase motif homologous to that found in eukaryotic cdc2 kinases. Sequence comparison showed a 47% identity on amino acid sequence basis to the Saccharomyces cerevisiae Hog 1p MAP kinase involved in the osmoregulatory pathway. Under conditions of salt-stress aberrant morphology of a hog1 deletion mutant was completely restored and growth was partially restored by expression of the PsMAPK. This shows that PsMAPK is functionally active as a MAP kinase in S. cerevisiae. Comparison of PsMAPK with other kinases involved in osmosensitivity, showed a high degree of homology and implicates a possible role for PsMAPK in a P. sativum osmosensing signal transduction pathway.
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  • 59
    ISSN: 1573-5028
    Keywords: oleosin ; gene expression ; seed development ; Arabidopsis thaliana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Oleosins are proteins associated with lipid bodies mainly synthesised during seed development. Using a subtractive hybridisation approach two new members of the oleosin gene family of Arabidopsis thaliana have been isolated. The quantitative and temporal expression patterns of both genes are found to be affected in the fus3 mutant defective in late embryogenesis. This pattern is interpreted as a molecular marker for a mutant specific developmental change from a seed maturation toa germination pathway.
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  • 60
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    Plant molecular biology 31 (1996), S. 565-573 
    ISSN: 1573-5028
    Keywords: gene expression ; Nicotiana tabacum ; peroxidase ; tobacco ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The tobacco anionic peroxidase gene encodes the predominant peroxidase isoenzyme in the aerial portions of tobacco. Three kb of the peroxidase promoter was joined to the coding region of theEscherichia coli β-glucuronidase gene (GUS), and transiently expressed in tobacco mesophyll protoplasts in the presence or absence of plant growth regulators. Benzyladenine, ethylene, and gibberellic acid did not affect peroxidase gene expression. Abscisic acid slightly inhibited expression at high concentrations. The auxins indole-3-acetic acid (IAA) and naphthaleneacetic acid strongly suppressed peroxidase expression. We observed half maximal suppression at 30 μM IAA. An antiauxin,p-chlorophenoxyisobutyric acid (PCIB), enhanced expression from the peroxidase promoter above that of untreated controls or restored activity when used in combination with IAA. Sequencing 3 kb of the peroxidase promoter revealed many potential regulatory elements based on sequence homology to previously characterized genes. This includes several consensus transcription factor binding sites found in auxin-regulated promoters. 5′ deletions of the peroxidase promoter/GUS fusion revealed several positive and negative regulatory elements. An upstream enhancer element was found between −3146 and −638 from the start of transcription. A strong silencer element was observed between −638 and −220. Removal of this silencer resulted in a truncated promoter (−220) with 100% activity of the full-length promoter (−3146). Inhibition by auxin was observed with all 5′ deletions.
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  • 61
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    Plant molecular biology 31 (1996), S. 295-306 
    ISSN: 1573-5028
    Keywords: Barley ; gene expression ; Hordeum vulgare ; promoter ; retrotransposon ; transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The BARE-1 retrotransposon occurs in more than 104 copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.
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  • 62
    ISSN: 1573-5028
    Keywords: ubiquitin ; proteolysis ; ubiquitin-conjugating enzymes ; gene families ; gene expression ; Arabidopsis thaliana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the β-glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.
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  • 63
    ISSN: 1573-5028
    Keywords: chaperone ; cyanobacteria ; gene expression ; heat shock ; protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Clp family consists of large, ubiquitous proteins that function as molecular chaperones and/or regulators of ATP-dependent proteolysis. A single copy gene coding for one of these proteins, ClpC, was cloned from the unicellular cyanobacterium Synechococcus sp. PCC 7942. The predicted polypeptide is most similar (ca. 88%) to the chloroplast-localized ClpC protein from higher plants. Using degenerate PCR primers specific for the two distinct ATP-binding domains characteristic of all ClpA-C proteins, partial sequences homologous to clpC from Synechococcus were also identified in five other cyanobacterial strains. The Synechococcus clpC gene is transcribed under standard growth conditions as a monocistronic message of around 2.7 kb. The level of this message, however, decreases slightly after a shift from 37 to 47.5°C for 2 h, similar to expression previously observed for clpC mRNA from heat-shocked higher plants. At the protein level, the amount of ClpC remains relatively unchanged during the high temperature shift, while that of the known heat shock protein GroEL rises considerably. In contrast, the constitutive level of ClpC in Synechococcus increases considerably under conditions of rapid growth, both with increasing light intensities or CO2 concentrations. This, and the fact that attempts to inactivate clpC expression fail to produce a viable phenotype, suggest that ClpC activity is essential for growth in this obligate photoautotrophic cyanobacterium.
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  • 64
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    Plant molecular biology 32 (1996), S. 393-405 
    ISSN: 1573-5028
    Keywords: gene expression ; endotoxin ; untranslated leader ; intron ; splicing ; synthetic genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Introducing a foreign gene into a new plant host does not always result in a high level of expression of the incoming gene. Numerous promoters have been used to express foreign genes in different plant tissues, but there are sometime various features of the new gene which are deleterious to expression in the new host. There are a number of posttranscriptional steps in the expression of a gene and sometimes sequences present in a particular coding region can resemble the signals which initiate these processing steps. When aberrantly carried out, these steps diminish the level of expression. By removing such fortuitous signals, one can dramatically increase expression of a transgene in plants. Ensuring proper protein folding and/or targeting the protein product to a particular cellular compartment can also be used to increase the level of protein obtained. The various methods used to optimize expression of a foreign gene in plants by concentrating on post-transcriptional events are discussed.
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  • 65
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    Plant molecular biology 32 (1996), S. 447-452 
    ISSN: 1573-5028
    Keywords: pentose-phosphate pathway ; Solanum tuberosum ; lignin ; wound ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.
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  • 66
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    Plant molecular biology 32 (1996), S. 565-569 
    ISSN: 1573-5028
    Keywords: anthocyanin ; flavonoid ; gene expression ; grape ; proanthocyanidin ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.
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  • 67
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    Plant molecular biology 32 (1996), S. 559-564 
    ISSN: 1573-5028
    Keywords: gene expression ; Trollius ledebouri ; germination ; glucose/ribitol dehydrogenase ; glutathione S-transferase ; late embryogenesis abundant ; seed dormancy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA library was generated from seeds of Trollius ledebouri cv. Golden Queen after GA3 treament. Five clones encoded mRNAs which were down-regulated during dormancy breaking and the initial stages of germination. Two of these showed homology to storage proteins (pPCB3 and pPCB4) and one each to the late-embryogenesis-abundant (LEA) group 2 dehydrin proteins (pPCB2), a barley glucose dehydrogenase (pPCB6) and the glutathione S-transferase (GST) superfamily (pPCB7). Transcript levels declined over 8 days in GA3-treated seeds. In dormant imbibed seeds transcript levels were relatively unchanged over the same period except for the PCB3 transcript, the level of which increased.
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  • 68
    ISSN: 1573-5028
    Keywords: rice seed storage protein ; albumin ; gene expression ; glutelin ; prolamin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of rice seed storage-protein genes is dramatically regulated over a short period of seed maturation. To characterize the expression mechanism of the rice seed storage protein genes, their expression of major storage protein genes (16 kDa albumin, 13 kDa prolamin and type II glutelin) were compared by RNA blot analysis. Their coordinate expression suggests that the transcriptional regulatory machinery is shared among the glutelin, prolamin and albumin-genes. We isolated two novel genomic genes for prolamins (PG5a and PG5b) and obtained the promoter region of the glutelin gene by PCR. The 5′-flanking regions of these three rice seed storage protein genes were found to contain some similar conserved sequences. Nuclear extract partially purified from maturing rice seeds was used for the gel shift assay of the 5′ region of the RA gene. We identified two DNA sequences of RA gene which were recognized by independent DNA-binding proteins. The complexes of these DNA sequences and DNA-binding proteins were inhibited by the fragments containing the 5′ regions of the prolamin and glutelin genes, suggesting that these three genes share transcription factors.
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  • 69
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    Plant molecular biology 32 (1996), S. 745-749 
    ISSN: 1573-5028
    Keywords: circadian rhythm ; CO2 fixation ; gene expression ; green alga
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have investigated whether the expression of carbonic anhydrase genes (Cah1 and Cah2) is regulated by a circadian clock in Chlamydomonas. When cells were grown in ordinary air under 12 h light/12 h dark (LD) cycles, the levels of the Cah2 mRNA hardly altered during the cycles, while the Cah1 mRNA showed a strong diurnal rhythm. The rhythm of about 24 h continued at least 3 days even under continuous light. Temperature compensation of the rhythm was demonstrated, using cultures maintained at 16, 22, and 28°C. These results indicate that the abundance of the Cah1 transcript is controlled by a circadian clock.
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  • 70
    ISSN: 1573-5028
    Keywords: Chlamydomonas ; chlorophyll biosynthesis ; gene expression ; protochlorophyllide reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The pc-1 mutant of Chlamydomonas reinhardtii has been shown to be incapable of protochlorophyllide photoconversion in vivo and is thought to be defective in light-dependent NADPH:protochlorophyllide oxidoreductase activity. We have isolated and characterized the nuclear genes encoding this enzyme from wild-type and pc-1 mutant Chlamydomonas cells. The wild-type CRlpcr-1 gene encodes a 397 amino acid polypeptide of which the N-terminal 57 residues comprise the chloroplast transit sequence. The Chlamydomonas protochlorophyllide reductase has 66–70% identity (79–82% similarity) to the higher plant enzymes. Transcripts encoding protochlorophyllide reductase are abundant in dark-grown wild-type cells, but absent or at very low levels in cells grown in the light. Similarily, immunoreactive protochlorophyllide reductase protein is also present to a greater extent in dark-versus light-grown wild-type cells. Both pc-1 and pc-1 y-7 cells lack CRlpcr-1 mRNA and the major (36 kDa) immunodetectable form of protochlorophyllide reductase consistent with their inability to photoreduce protochlorophyllide. DNA sequence analysis revealed that the lpcr gene in pc-1 y-7 cells contains a two-nucleotide deletion within the fourth and fifth codons of the protochlorophyllide reductase precursor that causes a shift in the reading frame and results in premature termination of translation. The absence of protochlorophyllide reductase message in pc-1 and pc-1 y-7 cells is likely the consequence of this frameshift mutation in the lpcr gene. Introduction of the CRlpcr-1 gene into pc-1 y-7 cells by nuclear transformation was sufficient to restore the wild-type phenotype. Transformants contained both protochlorophyllide reductase mRNA and immunodetectable enzyme protein. These studies demonstrate that pc-1 was in fact a defect in protochlorophyllide reductase activity and provide the first in vivo molecular evidence that the lpcr gene product is essential for light-dependent protochlorophyllide reduction.
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  • 71
    ISSN: 1573-5028
    Keywords: 2-oxoglutarate/malate translocator ; C4 plant ; gene expression ; mitochondria ; Panicum miliaceum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C4 plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane α-helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.
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  • 72
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    Plant molecular biology 30 (1996), S. 359-366 
    ISSN: 1573-5028
    Keywords: anti-fungal killer toxin ; double-stranded RNA ; gene expression ; transgenic plant ; Ustilago maydis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Ustilago maydis killer toxins are small polypeptides (7–14 kDa) whichkill susceptible cells of closely related fungal species. The KP4 toxin is a single polypeptide subunit with a molecular weight of 11.1 kDa. In this work, a transgenic tobacco plant was constructed which secretes the KP4 toxin at a high level. The KP4 toxin expressed in this transgenic plant was of the same size and specificity as the authentic Ustilago KP4 toxin. The expression level was at least 500 times higher than that of the KP6 toxin expressed in plants. Transgenic crop plants producing the KP4 toxin could be rendered resistant to KP4-susceptible fungal pathogens.
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  • 73
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    Plant molecular biology 30 (1996), S. 439-453 
    ISSN: 1573-5028
    Keywords: Zea mays ; homeobox gene family ; gene expression ; DNA-binding
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    Topics: Biology
    Notes: Abstract Clones of two highly related genes, ZmHox2a and ZmHox2b (Zea mays homeobox), were isolated from maize embryo cDNA libraries by screening with the ZmHox1a homeobox sequence. The genes map to chromosomes 3 and 8, respectively, and encode mRNA transcripts of 6 kb. The encoded proteins, ZmHox2a and b, share 84% sequence identity and exhibit a modular structure with several novel plant-specific protein domains. Interestingly, each ZmHox2 gene product contains two complete homeodomains which, for Zmhox2a, were both shown to be functional DNA-binding motifs in vitro. Not only probes encoding the homeobox but also DNA fragments corresponding to other ZmHox2 domains hybridize to multiple bands in genomic Southern blots, indicating that related protein domains may be conserved in other maize genes. The ZmHox2a/b genes, therefore, are members of a novel and large class of maize genes, some of which can be expected to encode new transcription factors.
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  • 74
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    Plant molecular biology 30 (1996), S. 611-623 
    ISSN: 1573-5028
    Keywords: aleurone layer ; embryo ; gene expression ; gibberellin ; Hordeum vulgare ; thionin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Gibberellins are noted for their ability to induce expression of genes, such as α-amylase, in the aleurone layers of cereals. However, a number of mRNA species in the mature imbibed aleurone cell of barley, such as a storage globulin (Heck et al., Mol Gen Genet 239: 209–218 1993), are simultaneously and specifically repressed by gibberellin. In a continuing effort to understand this effect, we report cloning and characterization of two additional cDNAs from barley designated pHvGS-1 and pcHth3 that have high corresponding mRNA levels in the mature imbibed aleurone but are repressed 10-fold or more within 24 h of treatment with gibberellic acid (GA3). The extent of repression was concentration dependent and maximally effective at 10-6 M GA3. Repression was also noted in the constitutive gibberellin response mutant, slender, in the absence of exogenous GA3. The antagonistic phytohormone, abscisic acid, had no effect or was weakly inductive of the steady-state levels of these mRNAs. During development of the seed, repressible mRNAs are present to different degrees in the maturing aleurone layer and embryo, but not in the starchy endosperm. Some repressible mRNA persists in the mature dry aleurone layer, but is degraded during imbibition, replenished by de novo transcription, and maintained at high steady-state levels until GA3 is perceived. Preliminary investigation suggests that repression is at least partly due to destabilization of the mRNAs which have estimated half-lives of 12 h or greater in the absence of GA3. pcHth3 encodes a member of the γ-thionin gene family located on chromosome 7. pHvGS-1 corresponds to a gene on chromosome 3 of unknown function.
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  • 75
    ISSN: 1573-5028
    Keywords: differential display ; HSP70 gene family ; 3′ non-coding region ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have modified the differential display technique to isolate 3′ regions from different members of the wheat HSP70 gene family. An HSP70 gene family-specific degenerate primer was used as a 5′ primer in place of the arbitrary primer used in the original technique. We cloned and sequenced three cDNA fragments that were differentially expressed in heat stressed wheat seedlings. Based on the high similarity between predicted translation products of these three sequences and known members of the HSP70 family from plants, these cDNAs were identified as members of the HSP70 gene family. Two of these members appeared distinct in the 3′ non-coding region with only 48% identity. Therefore differential display could successfully be used to isolate 3′ regions of different members of a multigene family in a relatively short period, even if the members had highly similar protein-coding regions.
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  • 76
    ISSN: 1573-5028
    Keywords: gene evolution ; gene expression ; Zea mays L. ; starch biosynthesis ; starch branching enzyme
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    Notes: Abstract Expression of the maize (Zea mays L.) starch branching enzyme (SBE) genes Sbe1 and Sbe2 were characterized during kernel development and in vegetative tissues. The onset of Sbe1 and Sbe2 expression during endosperm development was similar to that of other genes involved in starch biosynthesis (Wx, Sh2 and Bt2). However, the expression of Sbe2 peaked earlier than that of Sbe1 in developing endosperm and embryos resulting in a shift in the ratio of Sbe1 to Sbe2 relative message levels during kernel and embryo development. Transcripts hybridizing to the Sbe2 probe were not detectable in leaves or roots which nonetheless have SBEII enzymatic activity, suggesting that there may be another divergent SBEII-like gene(s) in maize. A similar expression pattern is shared between the maize genes and related genes in pea, which together with their evolutionary conservation, suggests that the SBE isoforms may play unique roles in starch biosynthesis during plant development.
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  • 77
    ISSN: 1573-5028
    Keywords: cryptic splice sites ; intron-enhancement ; gene expression ; nitA cDNA ; Volvox ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Use of the nitrate reductase encoding gene (nitA) as selection marker has facilitated the successful nuclear transformation of Volvox carteri. The Volvox nitA gene contains 10 introns. A stable nitA mutation in the Volvox recipient strain 153–81 resides in a G-to-A transition of the first nucleotide in the 5′ splice site of nitA intron 2. This mutation resulted in at least three non-functional splice variants, namely: (1) intron 2 was not spliced at all; (2) a cryptic 5′ splice site 60 nt upstream or (3) a cryptic 5′ splice site 16 nt downstream of the mutation were activated and used for splicing. When we used nitA cDNA (pVcNR13) for transformation of V. carteri 153–81, a low efficiency of about 5×10-5 transformants per reproductive cell was observed. Re-integration of either intron 1 (pVcNR15) or introns 9 and 10 (pVcNR16) in the transforming cDNA increased transformation rates to 5×10-4. In parallel, pVcNR15-transformed Volvox exhibited growth rates that were 100-fold increased over the pVcNR13-transformed alga. This intron-enhancement of nitA gene expression appears to be associated with post-transcriptional processing and ‘channelling’ of the message. These data suggest an important role of splicing for gene expression in V. carteri.
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  • 78
    ISSN: 1573-5028
    Keywords: developmental regulation ; elongation factor ; evolution ; gene expression ; rhodophyte, sporophyte
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    Notes: Abstract The life cycle of the red alga Porphyra purpurea alternates between two morphologically distinct phases: a shell-boring, filamentous sporophyte and a free-living, foliose gametophyte. From a subtracted cDNA library enriched for sporophyte-specific sequences, we isolated a cDNA encoding an unusual elongation factor 1α (EF-1α) that is expressed only in the sporophyte. A second EF-1α gene that is expressed equally in the sporophyte and the gametophyte was isolated from a genomic library. These are the only EF-1α genes detectable in P. purpurea. The constitutively expressed gene encodes and EF-1α very similar to those of most eukaryotes. However, the sporophyte-specific EF-1α is one of the most divergent yet described, with nine insertions or deletions ranging in size from 1 to 26 amino acids. This is the first report of a developmental stage-specific EF-1α outside of the animal kingdom and suggests a fundamental role for EF-1α in the developmental process.
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  • 79
    ISSN: 1573-5028
    Keywords: cotton ; gene expression ; Gossypium hirsutum ; metallothionein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have characterized cotton (Gossypium hirsutum L.) genes encoding type 1 metallothionein-like proteins that are highly expressed in roots. Little or no expression of these genes was detected in other organs and tissues. The deduced amino acid sequences have a high degree of similarity with type 1 metallothionein-like proteins from other plants, including a central hydrophobic domain flanked by conserved cysteine-rich motifs. The type 1 metallothionein-like genes of cotton are encoded by a small gene family. One gene (MT1-A) was analyzed in detail and found to have three exons which are 52, 83 and 397 bp long, and two introns 130 and 1042 bp in length. Three of the type 1 metallothionein-like genes are organized in a tandom array, and the 5′-flanking regions of these genes share a high degree of sequence similarity. Two of the clustered genes (MT1-A andMT1-B) are expressed at about equal levels in roots and use the same transcription start site. A 640 bp promoter fragment from theMT1-A gene was sufficient to direct expression of beta-glucuronidase (GUS) in transformed cotton roots. The expression was highest near the root tip.
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  • 80
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    Plant molecular biology 31 (1996), S. 905-910 
    ISSN: 1573-5028
    Keywords: cytoskeleton ; Eucalyptus globulus ; gene expression ; mycorrhiza ; Pisolithus tinctorius ; tubulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Because symbionts are experiencing major morphological changes during ectomycorrhiza development, the expression of genes encoding cytoskeletal proteins is likely altered. To test this contention, we have cloned and characterized in a α-tubulin cDNA (EgTubA1) from Eucalyptus globulus. A poorly-aggressive isolate (No. 270) of the ectomycorrhizal basidiomycete Pisolithus tinctorius caused no changes in root transcript levels of EgTubA1, whereas a drastic up-regulation in its expression was observed between 3 to 4 days after contact with the aggressive isolate 441. This enhanced α-tubulin expression coincided with the increase lateral root formation induced by fungal colonisation. The changes in α-tubulin expression support a role for cytoskeleton components in ectomycorrhiza development.
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  • 81
    ISSN: 1573-5028
    Keywords: chitinase ; cotton ; gene expression ; 1,3-β-glucanase ; Gossypium hirsutum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3-β-glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3-β-glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3-β-glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3-β-glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5′-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.
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  • 82
    ISSN: 1573-5028
    Keywords: leghemoglobin ; gene expression ; site-directed mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5′-Srglb3-uidA-3′nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.
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  • 83
    ISSN: 1573-5028
    Keywords: cDNA cloning ; gene expression ; glutamine synthetase ; grapevine ; nitrogen ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity. Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity. Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized. Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa. Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5′-and 3′-untranslated sequences showed only 40% similarity. The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS. Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome. In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues. Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1-and pGS1;2-related genes.
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  • 84
    ISSN: 1573-5028
    Keywords: Generative cells ; gene expression ; Lilium longiflorum ; pollen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Metabolic labelling with [35S]-methionine demonstrated that generative cells ofLilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [35S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.
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  • 85
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    Plant molecular biology 31 (1996), S. 1173-1184 
    ISSN: 1573-5028
    Keywords: CABII ; Chlamydomonas reinhardtii ; circadian rhythm ; gene expression ; lhcb ; transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The small gene family encoding the chlorophylla/b-binding proteins of photosystem II (CABII orlhcb) is known to exhibit circadian rhythms of mRNA abundance inChlamydomonas reinhardtii. In this study we investigated the role of transcription in the phenomenon. We used as reportersChlamydomonas genes that encode nitrate reductase (NITI) and arylsulfatase (ARS2) transcriptionally fused to sequences upstream of one of theCABII genes (calledCABII-1). We found that both reporters exhibited the same circadian rhythm of mRNA abundance in phase, period, and amplitude as does the endogenousCABII-1 gene. We also evaluated the efficacy of arylsulfatase enzymatic activity as a reporter and found that its half-life is too long to make it a useful reporter of rhythmic transcription during a circadian or diurnal cycle. The amount of mRNA synthesis from theCABII-1 gene was examined byin vivo labeling experiments and a circadian rhythm in transcription rate was demonstrated.In vivo labeling also revealed a circadian rhythm of mRNA synthesis for theCABII gene family as a whole. The results from the transcriptional reporter assays together with thein vivo labeling experiments strongly support the conclusion that the biological clock regulates the transcriptional activity of theCABII-1 gene, and moreover that regulation at the transcriptional level is the predominant mode by which the clock regulates this gene.
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  • 86
    ISSN: 1573-5028
    Keywords: cell wall ; gene expression ; pectin methylesterase ; Phaseolus vulgaris ; polygalacturonase ; texture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tomato fruit maturation is accompanied by a depolymerization of cell wall pectins which is due to the action of endopolygalacturonase (endoPG) preceded by pectin methylesterase (PE) activity. To investigate the role of endoPG and PE in determining the structure of green bean (Phaseolus vulgaris L.) pectins, these pectinases were studied during pod development. Early developmental stages displayed low endoPG or exoPG activities while PE activities were measurable during all stages of pod and seed development. These results do not favour a possible synergistic action of PE and PG. For seeds, the relatively high PE activities concurred with relatively low levels of pectin methyl esterification. At a molecular level, one partial chromosomal clone of 210 pb (PE1V), two partial PE cDNA clones of 660 bp (PE2V and PE3V) from cv. verona and one full-length PE cDNA clone of 1990 bp (PE3M), from cv. Masai were isolated. The identity of the CDNA clones was confirmed by expression inEscherichia coli and immunodetection with antibodies directed towards a tomato fruit PE. Transcripts corresponding with the genomic clone PE1V were not detected but both PE2 and PE3 cDNAs corresponded with mRNAs 1.8 kb in length. In contrast to PE2, PE3 gene expression levels varied significantly in pods from different cultivars suggesting an involvement in determining pod morphology.
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  • 87
    ISSN: 1573-5028
    Keywords: ATP synthase subunit γ ; gene expression ; leader sequences ; plastocyanin ; Spinacia oleracea ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of chimeric uidA gene fusions (for bacterial β-glucuronidase) with 5′-flanking sequences of the spinach AtpC and PetE genes (encoding the subunit γ of the chloroplast ATP synthase and plastocyanin, respectively) requires sequences for the 5′-untranslated leaders. The sequence for the PetE leader does not exhibit significant similarities to those of other leader sequences. Closer inspection of PetE uncovered that the crucial region is located in the vicinity of the transcription start site (+5/+15, TTGTCATTTCT). In contrast, 3′ deletions of sequences for the AtpC leader revealed that the region in the vicinity of the translation initiation codon is essential for uidA gene expression (+103/+176). This segment contains a CT-rich sequence (TTCTCTCTCCT), which is found identically or in a slightly modified form in sequences for 85 plant leaders deposited in the EMBL data bank. Site-directed mutagenesis of the CT-rich sequence resulted in a three-fold reduction of the transcription of the transgene. It is concluded (1) that different elements in the sequences for the spinach PetE and AtpC leaders control the expression of the uidA gene, (2) that these elements operate transcriptionally rather than post-transcriptionally and (3) that a CT-rich sequence represents a crucial cis element for the transcription of the AtpC::uidA gene fusion.
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  • 88
    ISSN: 1573-5028
    Keywords: β-tubulin ; gene expression ; light regulation ; Lupinus albus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We present here the characterization of a Lupinus albus β-tubulin gene, TubB2, which is closely related to TubB1 [22]. Both TubB1 and TubB2 transcripts are present in the embryonic axis of lupin dry seeds. The patterns of developmental expression of TubB1 and TubB2 β-tubulin genes are strongly correlated. Both genes are expressed at higher levels in hypocotyls of 7-day-old etiolated plants compared to hypocotyls from plants grown under light/dark cycles. When etiolated plants are exposed to continuous white light, differential changes in the steady state levels of the TubB1 and TubB2 mRNAs from hypocotyls are observed. These changes are accompanied by an inhibition of hypocotyl extension.
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  • 89
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    Plant molecular biology 32 (1996), S. 923-936 
    ISSN: 1573-5028
    Keywords: PEPC ; C3 metabolism ; gene expression ; evolution ; gymnosperm ; Picea abies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Phosphoenolpyruvate carboxylase (PEPC) genes and cDNA sequences have so far been isolated from a broad range of angiosperm but not from gymnosperm species. We constructed a cDNA library from seedlings of Norway spruce (Picea abies) and identified cDNAs coding for PEPC. A full-length PEPC cDNA was sequenced. It consists of 3522 nucleotides and has an open reading frame (ORF) that encodes a polypeptide (963 amino acids) with a molecular mass of 109 551. The deduced amino acid sequence revealed a higher similarity to the C3-form PEPC of angiosperm species (86–88%) than to the CAM and C4 forms (76–84%). The putative motif (Lys/Arg-X-X-Ser) for serine kinase, which is conserved in all angiosperm PEPCs analysed so far, is also present in this gymnosperm sequence. Southern blot analysis of spruce genomic DNA under low-stringency conditions using the PEPC cDNA as a hybridization probe showed a complex hybridization pattern, indicating the presence of additional PEPC-related sequences in the genome of the spruce. In contrast, the probe hybridized to only a few bands under high-stringency conditions. Whereas this PEPC gene is highly expressed in roots of seedlings, a low-level expression can be detected in cotyledons and adult needles. A molecular phyiogeny of plant PEPC including the spruce PEPC sequence revealed that the spruce PEPC sequence is clustered with monocot and dicot C3-form PEPCs including the only dicot C4 form characterized so far.
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  • 90
    ISSN: 1432-2242
    Keywords: Key wordsSuppressor gene ; Powdery mildew resistance ; Gene location ; Triticum aestivum ; Secale cereale ; Monosomic analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The chromosomal location of a suppressor for the powdery mildew resistance genes Pm8 and Pm17 was determined by a monosomic set of the wheat cultivar Caribo. This cultivar carries a suppressor gene inhibiting the expression of Pm8 in cv Disponent and of Pm17 in line Helami-105. In disease resistance assessments, monosomic F1 hybrids (2n=41) of Caribo × Disponent and Caribo × Helami-105 lacking chromosome 7D were resistant, whereas monosomic F1 hybrids involving the other 20 chromosomes, as well as disomic F1 hybrids (2n=42) of all cross combinations, were susceptible revealing that the suppressor gene for Pm8 and Pm17 is localized on chromosome 7D. It is suggested that genotypes without the suppressor gene be used for the exploitation of genes Pm8 and Pm17 in enhancing powdery mildew resistance in common wheat.
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  • 91
    ISSN: 1432-2242
    Keywords: Haynaldia villosa ; Triticum aestivum ; C-banding ; Genomic in situ hybridization ; Alien chromosome addition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F3 plants from the amphiploid Triticum aestivum-H. villosa x ‘Yangmai 158’ hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.
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  • 92
    ISSN: 1432-2242
    Keywords: Triticum timopheevii ; Triticum aestivum ; Chromosome substitution ; C-banding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Whether the two tetraploid wheat species, the well known Triticum turgidum L. (macaroni wheat, AABB genomes) and the obscure T. timopheevii Zhuk. (AtAtGG), have monophyletic or diphyletic origin from the same or different diploid species presents an interesting evolutionary problem. Moreover, T. timopheevii and its wild form T. araraticum are an important genetic resource for macaroni and bread-wheat improvement. To study these objectives, the substitution and genetic compensation abilities of individual T. timopheevii chromosomes for missing chromosomes of T. aestivum ‘Chinese Spring’ (AABBDD) were analyzed. ‘Chinese Spring’ aneuploids (nullisomic-tetrasomics) were crossed with a T. timopheevii x Aegilops tauschii amphiploid to isolate T. timopheevii chromosomes in a monosomic condition. The F1 hybrids were backcrossed one to four times to Chinese Spring aneuploids without selection for the T. timopheevii chromosome of interest. While spontaneous substitutions involving all At- and G-genome chromosomes were identified, the targeted T. timopheevii chromosome was not always recovered. Lines with spontaneous substitutions from T. timopheevii were chosen for further backcrossing. Six T. timopheevii chromosome substitutions were isolated: 6At (6A), 2G (2B), 3G (3B), 4G (4B), 5G (5B) and 6G (6B). The substitution lines had normal morphology and fertility. The 6At of T. timopheevii was involved in a translocation with chromosome 1G, resulting in the transfer of the group-1 gliadin locus to 6At. Chromosome 2G substituted for 2B at a frequency higher than expected and may carry putative homoeoalleles of gametocidal genes present on group-2 chromosomes of several alien species. Our data indicate a common origin for tetraploid wheat species, but from separate hybridization events because of the presence of a different spectrum of intergenomic translocations.
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  • 93
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    Theoretical and applied genetics 92 (1996), S. 666-672 
    ISSN: 1432-2242
    Keywords: Triticum aestivum ; Puccinia striiformis ; Diversity ; Competition ; Path analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Four club wheat cultivars and three two-component cultivar mixtures, planted at five frequencies, were grown in three environments in both the presence and absence of stripe rust. The effect of stripe rust on wheat yield was through the yield components, with weight of individual seed being the component most affected by rust. In some cases, yield component compensation was indicated by the presence of negative correlations among the yield components. Path analysis of the yield components revealed that components with the highest correlations to yield also had the largest direct effects on yield. Of the yield components, number of heads per unit area exerted the largest direct influence on yield. The direct effects of number of seeds per head and weight of individual seed were similar, although number of seeds per head was more important in the absence of rust than in its presence. The pure stands and mixtures differed considerably with respect to correlation coefficients, but were very similar for direct effects of yield components on yield. Most of these discrepancies were due to opposing indirect effects, which were not evident from correlation coefficients alone.
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  • 94
    ISSN: 1432-2242
    Keywords: Suppressor gene ; Powdery mildew resistance ; Gene location ; Triticum aestivum ; Secale cereale ; Monosomic analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The chromosomal location of a suppressor for the powdery mildew resistance genes Pm8 and Pm17 was determined by a monosomic set of the wheat cultivar Caribo. This cultivar carries a suppressor gene inhibiting the expression of Pm8 in cv Disponent and of Pm17 in line Helami-105. In disease resistance assessments, monosomic F1 hybrids (2n=41) of Caribo x Disponent and Caribo x Helami-105 lacking chromosome 7D were resistant, whereas monosomic F1 hybrids involving the other 20 chromosomes, as well as disomic F1 hybrids (2n=42) of all cross combinations, were susceptible revealing that the suppressor gene for Pm8 and Pm17 is localized on chromosome 7D. It is suggested that genotypes without the suppressor gene be used for the exploitation of genes Pm8 and Pm17 in enhancing powdery mildew resistance in common wheat.
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  • 95
    ISSN: 1432-2242
    Keywords: Triticum aestivum ; HMW glutenin genes ; Unequal crossing-over ; PCR ; Glu-D1 locus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.
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  • 96
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    Theoretical and applied genetics 93 (1996), S. 1066-1070 
    ISSN: 1432-2242
    Keywords: Amylose synthesis ; Aneuploid ; Granule-bound starch synthase ; Triticum aestivum ; Wx gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Amylose synthesis in wheat endosperm is mainly controlled by the granule-bound starch synthase of about 60 kDa, the so-called waxy (Wx) protein. The Wx proteins are the product of the Wx genes at a triplicate set of single-copy homoeoloci located on chromosomes 7A (Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). Using ‘Chinese Spring’ and its aneuploid lines, including nullisomic-tetrasomics, tetrasomics, ditelosomics and deletion stocks, together with single-chromosome substitution lines for these chromosomes, the effects of varying the dosage of whole chromosomes and chromosome arms, as well as the effects of null alleles, upon amylose synthesis were investigated. Nullisomic 4A and the deletion of chromosome segments carrying the Wx-B1 gene reduced the amylose content by more than 3%. A reasonable agreement was found in the substitution lines. This confirms that the absence of the Wx-B1 gene, or else substitution of this gene by its null allele, has the most striking effect on decreasing amylose synthesis. The removal of chromosomes carrying either the Wx-A1 or the Wx-D1 gene reduces the amylose content by less than 2%. A similar reduction was revealed by substitution of these two genes by the null alleles. Double dosages of chromosomes 7A, 4A and 7D did not increase amylose content, while the tetrasomic chromosomes produced more of the respective Wx proteins. This suggests that a certain level of Wx gene activity or of the Wx proteins led to the maximum amount of amylose.
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  • 97
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    Theoretical and applied genetics 93 (1996), S. 1112-1118 
    ISSN: 1432-2242
    Keywords: Genetic map ; RFLP ; RAPD ; Secale cereale ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A genetic map of six chromosomes of rye, (all of the rye chromosomes except for 2R), was constructed using 77 RFLP and 12 RAPD markers. The map was developed using an F2 population of 54 plants from a cross between two inbred lines. A rye genomic library was constructed as a source of clones for RFLP mapping. Comparisons were made between the rye map and other rye and wheat maps by including additional probes previously mapped in those species. These comparisons allowed (1) chromosome arm orientation to the linkage groups to be given, (2) the corroboration of several evolutionary translocations between rye chromosomes and homoeologous chromosomes of wheat; (3) an increase in the number of available markers for target regions of rye that show colinearity with wheat. Inconsistencies in the location of markers between the wheat and rye maps were mostly detected by multi-copy probes.
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  • 98
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    Theoretical and applied genetics 93 (1996), S. 1078-1082 
    ISSN: 1432-2242
    Keywords: Allele-specific PCR (AS-PCR) ; Powdery mildew ; Hordeum vulgare ; Triticum aestivum ; Sequence-tagged site (STS)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Primers for the polymerase chain reaction (PCR) were tailored to selectively amplify RFLP marker alleles associated with resistance and susceptibility for powdery mildew in cereals. The differentiation between marker alleles for susceptible and resistant genotypes is based on the discrimination of a single nucleotide by using allele-specific oligonucleotides as PCR primers. The PCR assays developed are diagnostic for RFLP alleles at the loci MWG097 in the barley genome and Whs350 in the wheat genome. The first marker locus is closely linked to MlLa resistance in barley, while the latter is linked to Pm2 resistance locus in wheat. PCR analysis of 31 barley and 30 wheat cultivars, with some exceptions, verified the presence or absence of the resistance loci investigated. These rapid PCR-based approaches are proposed as an efficient alternative to conventional procedures for selecting powdery mildew-resistant genotypes in breeding programs.
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  • 99
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    Theoretical and applied genetics 92 (1996), S. 163-169 
    ISSN: 1432-2242
    Keywords: Bread wheat ; Triticum aestivum ; Culture medium ; Embryogenic callus ; Plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Forty-eight bread wheat (Triticum aestivum L.) released cultivars and elite advanced lines were evaluated for their ability to produce embryogenic callus using three different media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,4-D (E3) or MS medium containing 2,4-D plus different amino acids (E5) were used for callus initiation and maintenance. Plant regeneration was achieved on basal MS medium with indole-3-acetic acid (IAA) and 6-benzylamino purine (BAP) and rooting on MS with 1-naphthaleneacetic acid (NAA). Percentage regeneration varied widely with both genotype and initiation medium, with values ranging from 2% to 94%. The number of plantlets produced per embryo ranged from 6 to 42. Thirteen genotypes showed at least 50% regeneration after culture on E5 medium; 3 genotypes after culture on E3 initiation medium and 1 after initiation on E1. After four subcultures, over a 16-week period, 41 genotypes (85%) lost their ability to regenerate plants while the remaining 7 lines (15%) retained plant regeneration potential but at reduced levels. E3 medium was found to be the best for maintaining regeneration potential after four subcultures.
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  • 100
    ISSN: 1432-2242
    Keywords: Wheat ; Triticum aestivum ; Triticum peregrinum ; Chromosome addition lines ; RFLPs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Analyses of RFLPs, isozymes, morphological markers and chromosome pairing were used to isolate 12 Triticum aestivum cv Chinese Spring (genomes A, B, and D)-T. peregrinum (genomes Sv and Uv) disomic chromosome addition lines. The evidence obtained indicates that each of the 12 lines contains an intact pair of T. peregrinum chromosomes. One monosomic addition line, believed to contain an intact 6Sv chromosome, was also isolated. A CS-7Uv chromosome addition line was not obtained. Syntenic relationships in common with the standard Triticeae arrangement were found for five of the seven Sv genome chromosomes. The exceptions were 4Sv and 7Sv. A reciprocal translocation exists between 4S1 and 7S1 in T. longissimum and evidence was obtained that the same translocation exists in T. peregrinum. In contrast, evidence for syntenic relationships in common with the standard Triticeae arrangements were found for only one Uv chromosome of T. peregrinum.; namely, chromosome 2Uv. All other Uv genome chromosomes are involved in at least one translocation, and the same translocations were found in the U genome of T. umbellulatum. Evidence was also obtained indicating that the centromeric regions of 4U and 4Uv are homoeologous to the centromeric regions of Triticeae homoeologous group-6 chromosomes, that the centromeric regions of 6U and 6Uv are homoeologous to the centromeric regions of group-4 chromosomes, and that 4U and 4Uv are more closely related overall to Triticeae homoeologous group-6 chromosomes than they are to group-4 chromosomes.
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