bluuebunny/crossref_metadata_2025 huggingface dataset:
CD40, CD154, Bax and Bcl‐2 Expression in Sjögren's Syndrome Salivary Glands: a Putative Anti‐Apoptotic Role During its Effector Phases
Authors: Ohlsson M. , Szodoray P. , Loro L. L. , Johannessen A. C. , Jonsson R.
Published: March 2003

Sjögren's syndrome (SS) is an autoimmune rheumatic disorder characterized by chronic lymphocytic infiltration and decreased secretion in the salivary glands (SGs). For some time, apoptosis has been suggested to constitute the major mechanism for acinar epithelial destruction during the effector phases; however, this is still controversial. We have recently demonstrated that despite the expression of Fas and FasL, the incidence of apoptosis is not increased in SS patients compared with control individuals. Our aim was therefore to further evaluate the expression of the pro‐ and anti‐apoptotic Bax and Bcl‐2 proteins. CD40 and CD154 expression was also investigated, as CD40 ligation has been suggested to protect cells from Fas‐mediated apoptosis. Immunohistochemical staining was performed on SG tissue from primary and secondary SS patients, a group of patients with non‐SS‐related degenerative changes as well as on healthy control individuals. The frequency of stained cells in the foci of infiltrating mononuclear cells (IMCs) and in the acinar and ductal epithelium was determined. We found the expression of Bcl‐2 but rarely Bax in SS SG IMCs. Bcl‐2 in epithelial cells was sparse, while Bax expression occurred frequently and with no significant difference between the patient groups. CD40 and CD154 expression was high among SS IMCs, while CD40 levels were slightly decreased in SS epithelium compared with controls. Elevated CD154 expression was found in SS epithelium, being significantly increased in the ducts. In conclusion, our study further supports the hypothesis about apoptosis resistance among SS focal IMCs and suggests a putative protective role of CD40 ligation in SS SG epithelium.
Self‐Peptides with Intermediate Capacity to Bind and Stabilize MHC Class I Molecules may be Immunogenic
Authors: Andersen M. L. M. , Ruhwald M. , Nissen M. H. , Buus S. , Claesson M. H.
Published: March 2003

Thirty self‐peptides were selected on the basis of their predicted binding to H‐2b molecules. The binding of peptides was ascertained experimentally by biochemical (KD measurements) and cellular [major histocompatibility complex class I (MHC‐I) stabilization] assays. A weak, but significant, correlation between KD measurements and MHC‐I stabilization was observed. Mice (n = 99) were immunized with individual peptides. Twenty‐eight peptides were found to induce peptide‐specific cytotoxic activity, and a total of 84 mice developed significant cytotoxic T lymphocyte (CTL) responses after immunization. Only one of the 21 mice immunized with high‐affinity peptides developed a peptide‐specific CTL response of 29 lytic units per 106 splenocytes, whereas 11 of the 42 mice immunized with intermediate‐affinity peptides developed peptide‐specific CTL responses at this level (P < 0.05). These observations suggest the absence of tolerance towards most MHC‐I‐restricted self‐peptides and that strong antiself immunity can be generated preferentially towards self‐peptides with an intermediate affinity for MHC‐I. These data should be considered in the design of tumour vaccines based on MHC‐I‐binding self‐peptides.
Novel Specificity of Anti‐U1A Autoimmune Patient Sera
Authors: Faig O. Z. , Lutz C. S.
Published: March 2003

We have previously described a novel complex of proteins which contains the U1snRNP‐A protein (U1A) but no other small nuclear ribonucleoprotein particle (snRNP) components (O'Connor et al., RNA 1997;3:1444–55). Antibodies to this novel complex inhibit both splicing and polyadenylation in vitro of a test pre‐mRNA (O'Connor et al., RNA 1997;3:1444–55; Lutz et al., RNA 1998;4:1493–9). This novel complex of proteins was identified using an unusual mouse monoclonal antibody (MoAb), called MAb 12E12. We were interested to know if autoimmune patient sera were similar to this MoAb. We have discovered a novel specificity of systemic lupus erythematosus patient sera reminiscent of MAb 12E12 in that the patient serum, like 12E12, (1) does not recognize U1A when bound to U1 RNA, (2) recognizes primarily the epitopes in the amino‐terminal third of the protein, including RNA recognition motif 1 (RRM1) and (3) inhibits in vitro polyadenylation. These findings may lead to the discovery of previously undescribed autoantigens as components of the novel protein complex, and may provide insight into autoimmune diseases.
Downregulation of Mac‐1 Expression in Monocytes by Surface‐Bound IgG
Authors: Fernández‐Calotti P. X. , Salamone G. , Gamberale R. , Trevani A. , Vermeulen M. , Geffner J. , Giordano M.
Published: March 2003

Physical and functional association between the β2‐integrin Mac‐1 (CD11b/CD18) and receptors of immunoglobulin G (IgG) (FcγRs) has been previously reported. In this study, we examined the modulation of Mac‐1 expression by IgG in different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate the expression of Mac‐1 after overnight exposure to surface‐bound IgG. This effect, which requires at least 6 h of incubation, is not associated with a general downmodulation of membrane antigens, and is selectively induced by immobilized IgG (iIgG), as the stimulation of monocytes with N‐formyl‐methionyl‐leucyl‐phenylalanine, lipopolysaccharide, tumour necrosis factor‐α (TNF‐α) or soluble IgG did not modify the Mac‐1 expression after 18 h in culture. The loss of Mac‐1 was completely prevented by blocking antibodies (Abs) directed to FcγRII (CD32) or CD18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac‐1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac‐1. However, there was no inhibition of TNF‐α production induced by zymosan, suggesting that Mac‐1‐dependent responses require different levels of its expression to be fully activated.
Sedimentary patterns and geometries of the Bahamian outer carbonate ramp (Miocene–Lower Pliocene, Great Bahama Bank)
Authors: Betzler Christian , Reijmer John J. G. , Bernet Karin , Eberli Gregor P. , Anselmetti Flavio S.
Published: March 2003

Core, logging and high‐resolution seismic data from ODP Leg 166 were used to analyse deposits of the Neogene (Miocene–Lower Pliocene) Bahamian outer carbonate ramp. Ramp sediments are cyclic alternations of light‐ and dark‐grey wackestones/packstones with interbedded calciturbidite packages and minor slumps. Cyclicity was driven by high‐frequency sea‐level changes. Light‐grey layers containing shallow‐water bioclasts were formed when the ramp exported material, whereas the dark‐grey layers are dominantly pelagic. Calciturbidites are arranged into mounded lobes with feeder channels. Internal bedding of the lobes shows a north‐directed shingling as a result of the asymmetrical growth of these bodies. Calciturbidite packages occur below and above sequence boundaries, indicating that turbidite shedding occurred during third‐order sea‐level highstands and lowstands. Highstand turbidites contain shallow‐water components, such as green algal debris and epiphytic foraminifera, whereas lowstand turbidites are dominated by abraded bioclastic detritus. Gravity flow depocentres shifted from an outer ramp position during the early Miocene to a basin floor setting during the late Miocene to early Pliocene. This change was triggered by an intensification of the strength of bottom currents during the Tortonian, which was also responsible for shaping the convex morphology of the outer ramp. The Miocene and Lower Pliocene of the leeward flank of Great Bahama Bank provides an example of the poorly known depositional setting of the outer part of distally steepened carbonate ramps. The contrast between its sedimentary patterns and the well‐known Upper Pliocene–Quaternary slope facies associations of the flat‐topped Great Bahama Bank shows the strong control that the morphology of a carbonate platform exerts on the depositional architecture of the adjacent slope and base‐of‐slope successions.
Miocene silcretes in argillaceous playa deposits, Madrid Basin, Spain: petrological and geochemical features
Authors: Bustillo M. A. , Bustillo M.
Published: March 2003

Four regressive sequences are present in the opaline rocks and related deposits of the Miocene Intermediate Unit of the Madrid Basin. The sequences consist of silty mudstones and argillaceous opals, separated by transitional facies. The silty mudstone consists mainly of dioctahedral smectites, whereas the argillaceous opal is principally opal‐CT and variable amounts of sepiolite. In the transitional facies, lamina of dioctahedral smectite co‐exist with neoformed opal‐CT and sepiolite. Petrological and geochemical features (major, trace and REE elements) indicate that the opaline levels and the transitional facies are related and are a consequence of silcrete formation in an argillaceous playa deposit. The isocon method was used to calculate changes in element concentration associated with silcrete formation. The geochemical data suggest silicification in an arid environment. The silcrete profile occurs four times, possibly as a result of highstand–lowstand fluctuations of the lake level caused by climatic changes. Structures and cements in the silcretes indicate that, although silicification may have commenced at the top of a groundwater table, it continued in the unsaturated zone above the water table.
Two sibling species of the spruce web‐spinning sawfly Cephalcia fallenii (Hymenoptera: Pamphiliidae) in Europe
Authors: BATTISTI  , BOATO  , ZANOCCO
Published: March 2003

The spruce web‐spinning sawfly Cephalcia fallenii is considered to be a minor pest defoliating spruces (Picea spp.) in Europe and northern Asia, and is characterized by a high degree of morphological polymorphism throughout its wide range. A sudden increase in population density observed in Central and southern Europe since 1976, strictly related to the dieback of large areas of spruce forests, made it possible to compare a large number of insects from different sites. The study was performed by combining field observations on epidemics and biological traits with analyses of allozymic, karyological and morphological characters. Two sibling species occurring together in all the study sites were identified by a diagnostic allozyme at the glucose phosphate isomerase (GPI) locus, karyotype differences, emergence times and the colour pattern of adults (light and dark). As the light form matches well with the lectotype of Lyda annulicornis, here designated, this species is redescribed on the basis of a large number of specimens. A new synonymy for the remaining taxa is proposed. Karyotype differences between the two siblings are substantial (C. fallenii, n = 18, 17M + 1A;C. annulicornis, n = 26, 13M + 13A), suggesting that chromosome rearrangements played a role in the speciation process.
The 18.7 ka phreatomagmatic flank eruption on Etna (Italy): relationship between eruptive activity and sedimentary basement setting
Authors: Andronico Daniele , Branca Stefano , Carlo Paola Del
Published: March 2003

This paper documents a phreatomagmatic flank eruption that occurred 18 700 ± 100 a BP, on the lower north‐eastern slope of Etna during the Ellittico volcano activity, which produced fall and surge deposits. This type of eruption is connected to a sedimentary basement ridge at Etna. The interaction between the rising magma and the shallow groundwater hosted in the volcanic pile overlying the impermeable sediments resulted in phreatomagmatic instead of strombolian activity. Three eruptive phases are distinguished based on field and analytical data: (i) an explosive phreatomagmatic opening, (ii) a main phase producing coarse lithic‐rich fallout and a strombolian deposit, and (iii) the final pulsating surge‐forming phase. The discovery of this phreatomagmatic flank eruption, which occurred at lower altitude, raises important issues for previous hazard assessments at Etna.
Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif
Authors: Argüello Gerardo , García‐Hernández Enrique , Sánchez Mireya , Gariglio Patricio , Herrera‐Estrella Luis , Simpson June
Published: March 2003

Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF‐2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF‐2 is present in both green and etiolated tissue and in roots and factors analogous to ABF‐2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes.
The in‐vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts
Authors: Schmid Jürg , Schaller Andreas , Leibinger Urs , Boll Werner , Amrhein Nikolaus
Published: March 2003

Full‐length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in‐vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N‐terminal portion of the deduced amino acid sequence resembles known chloroplast‐specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in‐vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.
Analysis of three tissue‐specific elements from the wheat Cab‐1 enhancer
Authors: Gotor Cecilia , Romero Luis C. , Inouye Keiko , Lam Eric
Published: March 2003

The genes encoding the major light‐harvesting antenna chlorophyll a/b‐binding protein (Cab) of higher plants are regulated by light at the transcriptional level. In addition, their expression is largely restricted to photosynthetically competent organs such as leaves. A 268 bp fragment of the Cab‐1 promoter from wheat functions as a light‐responsive and organ‐specific enhancer in transgenic tobacco. Using DNase I footprinting, four different regions have been mapped (Cab1‐A, Cab1‐B, Cab1‐C and Cab1‐D) in this fragment that bind to protein factors in tobacco nuclear extracts. Three of these regions (A, B and C) coincide with sequences that have been found to be functionally important from previous cis‐element analyses. Synthetic tetramers of these three sites interact with different proteins in gel retardation assays. In addition, cross‐competition analyses demonstrate that Cab1‐C is likely to interact with ASF‐2, a tobacco DNA‐binding activity that binds to a conserved GATA element found in many dicot Cab promoters. In transgenic tobacco, a 95 bp fragment of the Cab‐1 enhancer containing the A, B and C regions can confer leaf expression when fused upstream of a truncated derivative of the cauliflower mosaic virus (CaMV) 35S promoter. However, expression observed with this enhancer fragment in the promoter context of these studies does not appear to be significantly dependent on light. Similar results were obtained with synthetic tetramers of Cab1‐A, ‐B or ‐C. These data thus suggest that the wheat Cab‐1 enhancer contains at least three distinct elements that contribute to leaf‐specific expression in transgenic tobacco. Interaction between factors binding to these positive elements and those that bind elsewhere in the Cab‐1 enhancer may be necessary for light‐responsive transcriptional activation.
Patch‐clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts
Authors: Rück Annegret , Palme Klaus , Venis Michael A. , Napier Richard M. , Felle Hubert H.
Published: March 2003

The electrical response of Zea mays protoplasts to different auxins and to antibodies raised against an ER‐located auxin binding protein from maize (Zm‐ERabp1), was investigated using the patch‐clamp technique (whole‐cell configuration). Following a lag‐phase of 30–40 seconds, indole‐3‐acetic acid and 1‐naphthylacetic acid induced an outwardly directed current of positive charge in a concentration‐dependent manner. This current was further increased by the fungal toxin fusicoccin (FC). The current was observed only in the presence of Mg2+‐ATP in the patch‐pipette and was abolished after addition of erythrosin B, an inhibitor of H+‐ATPase, to the protoplasts indicating that the plasma membrane H+‐ATPase is activated by auxins and fusicoccin. Addition of antibodies directed against Zm‐ERabp1 abolished the current induced by auxins, without affecting the response of protoplasts to fusicoccin. Antibodies directed against a peptide representing part of the putative auxin binding domain of Zm‐ERabp1 showed auxin agonist activity, stimulating an outwardly directed membrane current in the absence of auxin. These results suggest that (i) Zm‐ERabp1 or antigenically related proteins represent a site for auxin perception through which the plasma membrane H+‐ATPase is activated, and (ii) that the activation of the H+‐ATPase by such proteins is initiated from outside the plasma membrane.
Isolation and characterization of two related Arabidopsis ocs‐element bZIP binding proteins
Authors: Zhang Bei , Foley Rhonda C. , Singh Karam B.
Published: March 2003

Ocs‐elements are a group of related, bipartite promoter elements which have been exploited by two distinct groups of plant pathogens, Agrobacterium and certain viruses to express genes in plants. The genes for two Arabidopsis bZIP (basic region‐leucine zipper) proteins that bind to ocs‐elements have been isolated and characterized. The genes, called OBF4 and OBF5, were isolated by screening an Arabidopsis genomic library with degenerate oligonucleotides complementary to the DNA‐binding domains of other plant ocs‐element‐binding proteins. The OBF4 and OBF5 proteins show 53% amino acid identity but low DNA homology. Southern blot analysis demonstrated that each of the OBF genes is a member of a small family. OBF4 is more similar to the tobacco TGA1a and Arabidopsis TGA1 proteins, while OBF5 is more similar to the maize OBF3.1, wheat HBP1b and Arabidopsis aHBP1b proteins. The DNA‐binding properties of OBF4 and OBF5 were similar although OBF5 was able to bind simultaneously to both halves of the ocs‐element more efficiently than OBF4. This difference in binding to the ocs‐element between two closely related proteins from the same species is potentially significant since binding to both halves of the ocs‐element is a pre‐requisite for in vivo transcriptional activity.
Analysis of a maize α‐tubulin gene promoter by transient expression and in transgenic tobacco plants
Authors: Rigau Joan , Capellades Montserrat , Montoliu Lluis , Torres Miguel Angel , Romera Carme , Martínez‐Izquierdo José Antonio , Tagu Denis , Puigdomènech Pere
Published: March 2003

The pattern of expression directed by the promoter of the maize Tubα1 gene was investigated by analysis of chloramphenicol acetyl transferase (CAT) and β‐glucuronidase (GUS) activities in transient expression experiments of maize and tobacco protoplasts. The same promoter was also investigated by histochemical GUS analysis in transgenic tobacco plants containing promoter gene fusions. As determined by histochemical tests, the Tubα1 promoter gene preferentially directs GUS expression in regenerating root tip meristems and pollen. This pattern corresponds to the distinctive features of natural expression of the gene in maize as determined by Northern analysis. However, no expression is observed in other meristematic tissues of the transgenic tobacco plants, as in shoot apex or in coleoptiles, which is weakly detected in maize. Analysis of the regulatory properties of 5′ promoter deletions showed that the proximal region of the promoter, from positions −1410 or −449 to 15 bp upstream of the ATG, is sufficient to establish the qualitative pattern of expression in transgenic tobacco plants. Deletions to positions −352 or −117 abolished the expression in roots, but not in pollen, suggesting that upstream of these positions there are elements responsible for the pattern in root. Further deletions abolished all the promoter activity, suggesting that this promoter region contains the elements essential for expression in pollen. The different patterns and levels of transient and stable expression are discussed.
Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†
Authors: Nehra Narender S. , Chibbar Ravindra N. , Leung Nick , Caswell Karen , Mallard Cliff , Steinhauer Lee , Baga Monica , Kartha Kutty K.
Published: March 2003

A system for enhanced induction of somatic embryo‐genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self‐fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long‐term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L‐phosphinothricin (L‐PPT) to obtain herbicide‐resistant self‐fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L‐PPT whereas none of the plants showed β‐glucuronidase (GUS) activity. Molecular analysis of PAT‐positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho‐transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny.
Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana
Authors: Bauwens Serge , Katsanis Katerina , Van Montagu Marc , Van Oostveldt Patrick , Engler Gilbert
Published: March 2003

A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal‐to‐noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.
A laccase‐like phenoloxidase is correlated with lignin biosynthesis in Zinnia elegans stem tissues
Authors: Liu Lan , Dean Jeffrey F.D. , Friedman William E. , Eriksson Karl‐Erik L.
Published: March 2003

Stem tissues from different internodes of 4–6 week‐old Zinnia elegans cv. Envy plants were sectioned and stained with chromogenic substrates previously used in studies of laccases (p‐diphenol:O2 oxidoreductases) isolated from tree tissues. The pattern of color development found when stem sections were stained in the presence and absence of H2O2 suggested that p‐diphenol:O2 oxidoreductase activity was tightly correlated spatially and temporally with the lignification of secondary cell walls in developing primary xylem. The correlation between this laccase‐like phenoloxidase activity and lignification appeared tighter than that between lignification and peroxidases stained using the same substrates. Zymogram analysis of the phenoloxidase activities catalyzed by enzymes that were not boiled prior to separation by SDS—PAGE suggested that a single enzyme was predominantly responsible for the laccase‐like phenoloxidase activity in Zinnia stems. Some of this enzyme was released from cell wall residue by washing with high ionic strength buffer; however, substantial amounts of the enzyme could only be recovered after treatment of the residue with cell wall‐degrading enzymes. This phenoloxidase appears to share significant characteristics with the coniferyl alcohol oxidase isolated from developing secondary xylem in pines, which suggests that such enzymes may be widespread in vascular plants.
Petunia hybrida homologues of shaggy/zeste‐white 3 expressed in female and male reproductive organs
Authors: Decroocq‐Ferrant Véronique , Van Went Jac , Bianchi Michele W. , de Vries Sacco C. , Kreis Martin
Published: March 2003

Petunia hybrida ovule‐specific cDNA libraries were screened for genes encoding homologues of protein kinases known to be involved in developmental processes. Two cDNAs, PSK4 and PSK6 (Petunia hybridashaggy related protein kinase), homologous to GSK‐3 from rat, MDS1 from yeast and to the segment polarity gene shaggy/zeste‐white 3 (sgg/zw3) from Drosophila were characterized in detail. Southern blot analysis showed that the PSK4 and PSK6 genes belong to a small multigene family in P. hybrida. The PSK4 and PSK6 proteins, which are 70% identical to sgg/zw3 and its functional homologue GSK‐3 over the protein kinase catalytic domain and 55% identical to MDS1, represent two different subgroups of protein kinases. RNA gel blot and RT‐PCR analyses revealed that the PSK4 gene is expressed during the vegetative phase and the female reproductive phase of development and the PSK6 gene predominantly during the male and female reproductive phases. In situ hybridization on developing flower buds confirmed that PSK4 is expressed throughout the different developmental stages from ovule primordium formation until embryo sac maturation, while PSK6 showed expression during anther cell differentiation and at pollen maturity, and an expression pattern similar to PSK4 in the female reproductive organs. The results also revealed transcripts of a PSK gene in embryos at the globular stage.
Biochemical and molecular characterization of a novel starch synthase from potato tubers
Authors: Edwards Anne , Marshall Jacqueline , Sidebottom Christopher , Visser Richard G.F. , Smith Alison M. , Martin Cathie
Published: March 2003

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N‐terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule‐bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity.

The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.
Temporal and spatial expression patterns of PHYA and PHYB genes in Arabidopsis
Authors: Somers David E. , Quail Peter H.
Published: March 2003

Phytochromes A and B have discrete photosensory functions in Arabidopsis. To determine whether differential temporal or spatial expression patterns of the PHYA and PHYB genes contribute to this phenomenon the expression of PHYA—GUS and PHYB—GUS reporter genes has been examined in transgenic Arabidopsis. Histochemical and quantitative biochemical analyses indicate that both transgenes are expressed extensively throughout the plant, including roots, shoots and flowers, during the entire life cycle, but with strong differences between the two in expression level and photoregulation, and more limited differences in spatial expression patterns. The data indicate that regulation is at the transcriptional level. In dry seed, PHYB—GUS is expressed throughout the embryo at three‐fold higher levels than PHYA—GUS, which is confined primarily to the embryonic root tip. By contrast, PHYA promoter activity, despite strong negative regulation in shoots by light, is consistently higher than PHYB (two‐ to 20‐fold) in both the light and dark in most tissues during all subsequent developmental phases, from seedling to mature adult. At the tissue level, most cells appear to express both transgenes at some level at all stages examined, with highest apparent activity in vascular tissue and root tips. With the notable exception of pollen, where high PHYB—GUS but not PHYA—GUS expression occurs, few major differences are observed in the quantitative spatial distribution pattern between the two transgenes. The strongly similar spatial and temporal expression patterns of PHYA—GUS and PHYB—GUS transgenes suggest that the differential photosensory activity of these two phytochromes occurs largely through differences in their (i) intrinsic biochemical activities, (ii) relative abundances, and/or (iii) independent and separate reaction partners, rather than through discrete, developmentally controlled expression patterns.
A general method to isolate genes tagged by a high copy number transposable element
Authors: Souer Erik , Quattrocchio Francesca , De Vetten Nick , Mol Joseph , Koes Ronald
Published: March 2003

The Petunia hybrida line W138 contains more than 200 copies of the transposable element dTph1. In W138 progeny these elements give rise to new unstable mutations at high frequency. With the aim of isolating these mutated genes a method was developed to isolate dTph1 flanking sequences unique for mutant plants. This method is based on differential screening of cloned inverse polymerase chain reaction (IPCR) products originating from the mutated plant. It directly yields a probe for the mutated gene which can be used to screen pre‐existing cDNA and genomic libraries. This method may be generally applicable to isolate genes tagged by other high copy number transposable elements, like Mutator (Mu) or Dissociation (Ds) in Zea mays.
Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity
Authors: Park Y.‐D. , Papp I. , Moscone E.A. , Iglesias V.A. , Vaucheret H. , Matzke A.J.M. , Matzke M.A.
Published: March 2003

The promoter homology‐dependent inactivation of a 35Spro‐hygromycin phosphotransferase (hpt) gene, which is present at the H2 locus, by the multipurpose 271 silencing locus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro‐RiN). All F1 progeny of a cross between homozygous H2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were accompanied by a reduction in the steady‐state levels of Hyg and NiR transcripts. Transcriptional run‐on experiments indicated, however, that while NiR silencing occurred post‐transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with increased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild‐type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygromycin resistance was only partially and non‐uniformly regained among adult BC1 plants. Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 locus was correlated with cytosine methylation primarily at CpG and CpNpG residues. Despite this residual methylation, H2 loci weakened by an association with 271 did not acquire the ability to silence a ‘naive’H2 locus. Fluorescence in situ hybridization revealed that the 271 locus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a post‐transcriptional process that is meiotically reversible; the latter is due to transcriptional inactivation and is associated with increased promoter methylation, which can lead to meiotically heritable reductions in target gene activity. The relevance of these data for the meiotic heritability of silencing, the non‐transferability of silencing activity, and the basis of 271 silencing effects is discussed.
A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular‐arbuscular (VA) mycorrhizal associations
Authors: Harrison Maria J.
Published: March 2003

A cDNA clone encoding a hexose transporter has been isolated from a library prepared from Medicago truncatula roots colonized by the mycorrhizal fungus Glomus versiforme. The clone (Mtst1) represents a M. truncatula gene and expression studies in yeast indicate that the encoded protein transports glucose and fructose but not sucrose. Transcripts corresponding to Mtst1 are expressed in leaves, stems and roots of M. truncatula, with the highest levels of expression in roots. In the roots, Mtst1 transcripts were detected in two distinct locations; the phloem fiber cells of the vascular tissue, and the cells of the root tip. Mtst1 expression in the roots is regulated in response to colonization by G. versiforme; transcript levels increased two‐ to fourfold in both M. truncatula and M. sativa following colonization by G. versiforme but did not increase during the unsuccessful interaction between G. versiforme and a M. sativa myc− mutant, suggesting that the increase in Mtst1 transcripts in the successful mycorrhizal interaction is correlated with internal growth of the fungus and potentially with a functioning symbiosis. Mtst1 transcripts were also detected in the cortical cells of the mycorrhizal root, specifically in areas of the root that were highly colonized by the mycorrhizal fungus. Thus, the formation of a symbiotic association with a VA mycorrhizal fungus is acompanied by a change in the cell type‐specific expression of a transporter that potentially functions to supply sugars to root cells critically involved in the symbiotic association.
Differential expression of the 1‐aminocyclopropane‐1‐carboxylate oxidase gene family of tomato
Authors: Barry Cornelius S. , Blume Beatrix , Bouzayen Mondher , Cooper Wendy , Hamilton Andrew J. , Grierson Donald
Published: March 2003

The tomato ACC oxidase gene family is comprised of three members designated ACO1, ACO2 and ACO3. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3′ untranslated regions. These regions have been cloned and used as gene‐specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both ACO1 and ACO3 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that ACO1 is wound‐inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; ACO1 is predominantly expressed in the petals and the stigma and style, ACO2 expression is mainly restricted to tissues associated with the anther cone whereas ACO3 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed.
Coordinate transcriptional induction of myo‐inositol metabolism during environmental stress
Authors: Ishitani Manabu , Majumder Arun Lahiri , Bornhouser Angela , Michalowski Christine B. , Jensen Richard G. , Bohnert Hans J.
Published: March 2003

The pathway from glucose 6‐phosphate (G 6‐P) to myo‐inositol 1‐phosphate (Ins 1‐P) and myo‐inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo‐inositol O‐methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, d‐ononitol and d‐pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo‐inositol 1‐phosphate synthase (INPS1), from the ice plant. Two Inps‐like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87–90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10‐fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt‐stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress‐induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.
Functional analysis of yeast‐derived phytochrome A and B phycocyanobilin adducts
Authors: Kunkel Tim , Neuhaus Gunther , Batschauer Alfred , Chua Nam‐Hai , Schäfer Eberhard
Published: March 2003

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase (chs) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast‐derived phytochrome‐phycocyanobilin adducts (phyA*, phyB*) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as β‐glucuronidase (GUS) expression mediated by light‐regulated promoters (chs, chlorophyll a/b binding protein (lhcb1) and ferredoxin NADP+ oxidoreductase (fnr)). Microinjection of phyA* under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs—GUS, lhcb1—GUS and fnr—GUS expression. Microinjection of phyB* under identical conditions induced chlorophyll accumulation and mediated lhcb1—GUS and fnr—GUS expression but neither anthocyanin accumulation nor chs—GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far‐red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB* injections under red light conditions indicated that phyB* needs interactions with other photoreceptors to mediate a rapid and efficient de‐etiolation signal.
The pef mutants of Arabidopsis thaliana define lesions early in the phytochrome signaling pathway
Authors: Ahmad Margaret , Cashmore Anthony R.
Published: March 2003

In a screen for early‐flowering mutants, a number of mutants that were early flowering under both short and long days were isolated. One such mutant, pef1, was selectively insensitive to both red and far‐red light in the inhibition of hypocotyl elongation response; a classic phytochrome phenotype mediated by both PHYA and PHYB. The pef1 mutant seedlings could not be phenotypically rescued by biliverdin, a precursor of the phytochrome chromophore, nor did they fail to complement any previously identified elongated hypocotyl (hy) mutants. Difference spectra and Western blot analysis showed normal concentrations of PHYA photoreceptor apoprotein, which appeared photochemically active. It was concluded that the pef1 mutant is defective in both PHYA‐ and PHYB‐ mediated signaling pathways, and may represent a lesion in an early step of the phytochrome signal transduction pathway. Additional pef mutants deficient specifically in PHYB‐mediated responses were also identified by this screen.
The plant ER: a dynamic organelle composed of a large number of discrete functional domains
Authors: Staehelin L. Andrew
Published: March 2003

The endoplasmic reticulum (ER) of plants is comprised of a three‐dimensional network of continuous tubules and sheets that underlies the plasma membrane, courses through the cytoplasm, and links up with the nuclear envelope. Aside from discussing the dynamic properties of this versatile and adaptable organelle, the review highlights the structure and the functional properties of 16 types of morphologically defined ER membrane domains. Owing to their lablie or transient nature, several of these domains can only be visualized reliably through the use of ultrarapid freezing techniques. The ER domains discussed are: the lamin receptor domain; the nuclear pores; the nuclear envelope‐ER gates, the microtubule nucleation domains; the protein and oil body‐forming domains; the vacuole‐forming ER; the actin‐binding, the plasma membrane‐anchoring and the vacuole and mitochondrion‐attachment domains; the lipid recycling ER cisternae and the plasmodesmata. Preliminary evidence suggests that this list will have to be expanded in the near future. Understanding the assembly, the functional roles, and the developmental regulation of these domains has implications both for understanding cell structure and function, and for exploiting plants for agricultural and biotechnological purposes.
Reversible protein phosphorylation regulates the activity of the slow‐vacuolar ion channel
Authors: Bethke Paul C. , Jones Russell L.
Published: March 2003

Protein storage vacuoles (PSVs) within barley (Hordeum vulgare) aleurone cells contain abundant K, Ca, Mg and P reserves. These minerals are transported from the PSV and are used to support growth of the embryo. In this study, the regulation of transport through slow‐vacuolar (SV) ion channels in the tonoplast of barley aleurone PSVs was examined using the patch—clamp technique. Okadaic acid (OA), an inhibitor of protein phosphatase types 1 and 2A, reduced whole‐vacuole SV currents by 60%. This inhibition by OA was overcome by exogenous calcineurin. Adding ATP (200 µM) to the bath solution as a substrate for kinase activity decreased SV channel activity by 70%. This reduction in activity was prevented by the kinase inhibitor H‐7. From these data, it is concluded that protein phosphorylation can inhibit SV channel activity, and that both the protein kinase and protein phosphatase involved in this regulation are present at the PSV tonoplast. Whole‐vacuolar SV currents were significantly higher when 2 mM ATP was used to bathe PSVs than with 200 µM ATP. Calmodulin‐like domain protein kinase (CDPK) at either ATP concentration increased SV channel activity by ∼ 150%, implying that protein phosphorylation can also stimulate SV channel activity. When PSVs were treated with the ATP analog AMP‐PNP, SV channel activity was not reduced. Hence, ATP hydrolysis is not essential for sustained SV channel activity. A model in which SV channel activity is regulated by protein phosphorylation at two sites is presented.
Monoclonal antibodies to barley aleurain and homologs from other plants
Authors: Rogers Sally W. , Burks Marlyn , Rogers John C.
Published: March 2003

Barley aleurain is contained within a specific type of vacuole characterized by acidic pH and the presence of other hydrolytic enzymes. The aleurain‐containing vacuole is distinct from protein storage vacuoles, and anti‐aleurain antibodies serve as markers for this organelle in barley cells. Aleurain is a unique type of cysteine protease, and other plant species have genes for homologs whose sequences are highly conserved, but little is known about these homologs at the protein level. Seven monoclonal antibodies to barley aleurain were isolated, which bind to and define aleurain homologues in Arabidopsis, Petunia, and tobacco cell extracts. Interestingly, in addition to 29–32 kDa aleurain homologs, Petunia extracts contain a protein of ∼50 kDa and tobacco extracts a protein of ∼40 kDa that are recognized by multiple different mono‐clonal antibodies, indicating an unexpected diversity to the aleurain protein family. Among the group of antibodies are some that efficiently immunoprecipitate metabolically labeled aleurain from barley cell extracts, and some that efficiently label aleurain in immunofluorescence assays using root tip cells. These antibodies should be useful for plant cell biologists who study vacuole biogenesis and function and sorting of proteins to specific vacuolar compartments, in barley as well as other plant species.