Biomedical subjects

P H Schreier

Publications and source records attributed to P H Schreier.

At least 19 recordsLinked to original sources

PubMed2004

A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (< C4) and produces H2O2. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.

Alcohol Oxidoreductases↗

PubMed2000

The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins.

The nonstructural NSm protein of tomato spotted wilt tospovirus (TSWV) represents a putative viral movement protein involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures. To study the molecular basis of NSm function, we expressed the protein in Escherichia coli and investigated protein-protein and protein-RNA interactions of NSm protein in vitro. NSm specifically interacts with TSWV N protein and binds single-stranded RNA in a sequence-nonspecific manner. Using NSm as a bait in a yeast two-hybrid screen, we identified two homologous NSm-binding proteins of the DnaJ family from Nicotiana tabacum and Arabidopsis thaliana.

Amino Acid Sequence↗

PubMed1999

Homotypic interaction and multimerization of nucleocapsid protein of tomato spotted wilt tospovirus: identification and characterization of two interacting domains.

The nucleocapsid protein (N) of tomato spotted wilt tospovirus (TSWV) plays a central role in the viral life cycle. With the aid of the yeast two-hybrid system and surface plasmon resonance analysis, homotypic interaction and multimerization of the N protein was detected. Analysis of deletion mutants identified two binding regions in the protein, located at the N terminus (amino acids 1-39) and the C terminus (amino acids 233-248), respectively, implying a "head-to-tail" interaction of the N terminus with the C terminus to form a multimeric chain. Further characterization of the binding domains was performed by site-directed mutagenesis. Two phenylalanines (F242 and F246) highly conserved in the N proteins within the Tospovirus genus were shown to play a crucial role in the interaction.

Amino Acid Sequence↗

PubMed1998

Early events of tomato spotted wilt transcription and replication in protoplasts.

A protoplast transfection system for tomato spotted wilt tospovirus (TSWV) was established by PEG-mediated infection of tobacco protoplasts. Analysis of viral RNA synthesis revealed an asymmetric production of viral (v) and viral-complementary (vc) strands of all three genomic RNA segments and a separation in time of subgenomic transcription during the first 72 hr post infection (p.i.). Synthesis of vc-RNA was detectable 8-10 hrs prior to v-RNA production. During the first 72 h of infection the accumulation of S-RNAs exceeded M- and L-RNAs and the amount of vc-RNAs was larger than that of genomic v-RNAs. The subgenomic N-mRNA was first detected 5 hr p.i., followed by vc-S RNA and the mRNA for the nonstructural protein (NSs) 15 hr later. The two subgenomic mRNA species of the S RNA appeared to be regulated independently from each other and from other viral mRNA species. Defective interfering (DI) elements from L RNA associated with the virus inoculum, were not preferentially replicated at the expense of full-sized L-RNA.

Immunohistochemistry↗

PubMed1997

An ozone-responsive region of the grapevine resveratrol synthase promoter differs from the basal pathogen-responsive sequence.

Stilbene synthase (STS) is an enzyme involved in the biosynthesis of stilbenes, which are synthesized in various plants in response to pathogen attack, UV irradiation or exposure to ozone. We describe analysis of an ozone inducible STS transcript and its corresponding promoter (Vst1), combined with the beta-glucuronidase (GUS) reporter gene. A single ozone pulse (0.1 microliter/l, 10 h) resulted in 11-fold GUS expression. Histochemical localization of GUS activity revealed small spots distributed over the whole leaf. Cross-sections of leaf tissue showed that the Vst1 promoter was induced in palisade and spongy parenchyma cells and to a lesser extent in epidermal cells. Deletions at the 5' end showed that a partial promoter sequence between position -430 and -280 constituted the ozone-responsive region, whereas for effective pathogen-inducibility sequences from -280 to -140 have been shown to be necessary.

Acyltransferases↗

PubMed1996

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants.

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.

Amino Acid Sequence↗

PubMed1995

Ribozymes.

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Base Sequence↗

PubMed1994

Expression of a reporter gene is reduced by a ribozyme in transgenic plants.

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.

Drug Resistance↗

PubMed1994

A stable hammerhead structure is not required for endonucleolytic activity of a ribozyme in vivo.

Cleavage of a specific target, the mRNA encoding the bacterial neomycin phosphotransferase, by mutant satellite RNA of subterranean clover mottle virus (sSCMoV) ribozymes (Rz) was used to study the role of the hammerhead (Hh) structure in Rz activity in cis and in trans. The bimolecular Rz-target RNA interaction was predicted by computer secondary structure analysis. In vivo, endonucleolytic cleavage was determined in plant protoplasts and compared with in vitro results. Two point mutations within the Hh were studied in detail. A Rz mutant with a point mutation in the most distal nucleotide of the catalytic domain (A14G) showed no endonucleolytic activity in vivo. A second point mutation inside helix II (G11.3C) which destabilizes the helix and, according to thermodynamic calculations, should disrupt the conserved Hh structure, unexpectedly displayed Rz activity in trans in vivo. In vitro, this mutant exhibited an activity similar to the wild-type Rz in cis, but no significant activity in trans. It therefore appears that helix II within the Rz Hh structure is not required in vivo for endonucleolytic activity, nor for stability of the Rz transcript, and that in vitro results are inadequate to predict Rz activity in living cells.

Amino Acid Sequence↗

PubMed1993

The gene family encoding the ribulose-(1,5)-bisphosphate carboxylase/oxygenase (Rubisco) small subunit of potato.

We determined the nucleotide sequences of five members of the rbcS gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of potato. The genomic organization, structure and expression of the genes is compared to the features of the rbcS genes in tomato. Within the two species, Lycopersicon esculentum and Solanum tuberosum, both members of the Solanaceae, the rbcS genes share more interspecific sequence identity, especially in the 5'- and 3'-untranslated regions and the intron sequences, than within one species. However, the expression data of orthologous rbcS genes containing highly identical regulatory cis-acting elements were found to be different, suggesting that the simple finding of these motifs does not automatically imply similar transcriptional and/or post-transcriptional gene regulation.

Amino Acid Sequence↗

PubMed1993

Disease resistance results from foreign phytoalexin expression in a novel plant.

Although phytoalexins have long been inferred to be important in the defence of plants against fungal infection, there are few reports showing that they provide resistance to infection. Several plants, including grapevine, synthesize the stilbene-type phytoalexin resveratrol when attacked by pathogens. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Stilbene biosynthesis only specifically requires the presence of stilbene synthase. Furthermore, the precursor molecules for the formation of hydroxy-stilbenes are malonyl-CoA and p-coumaroyl-CoA, both present in plants. To investigate the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance, we isolated stilbene synthase genes from grapevine, where they are expressed at a high level, and transferred them into tobacco. We report here that regenerated tobacco plants containing these genes are more resistant to infection by Botrytis cinerea. This is, to our knowledge, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.

Acyltransferases↗

PubMed1992

Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo.

The subclass of catalytic RNAs termed ribozymes cleave specific target RNA sequences in vitro. Only circumstantial evidence supports the idea that ribozymes may also act in vivo. In this study, ribozymes with a hammerhead motif directed against a target sequence within the mRNA of the neomycin phosphotransferase gene (npt) were embedded into a functional chimeric gene. Two genes, one containing the ribozyme and the other producing the target, were cotransfected into plant protoplasts. Following in vivo expression, a predefined cleavage product of the target mRNA was detected by ribonuclease protection. Expression of both the ribozyme gene and the target gene was driven by the CaMV 35S promoter. Concomitant with the endonucleolytic cleavage of the target mRNA, a complete reduction of NPT activity was observed. An A to G substitution within the ribozyme domain completely inactivates ribozyme-mediated hydrolysis but still shows a reduction in NPT activity, albeit less pronounced. Therefore, the reduction of NPT activity produced by the active ribozyme is best explained by both hydrolytic cleavage and an antisense effect. However, the mutant ribozyme--target complex was more stable than the wildtype ribozyme--target complex. This may result in an overestimation of the antisense effect contributing to the overall reduction of gene expression.

Amino Acid Sequence↗

PubMed1991

Reduced steady-state levels of rbcS mRNA in plants kept in the dark are due to differential degradation.

When plants are placed in the dark, the level of the abundant mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) declines rapidly. We present evidence demonstrating an active degradation of rbcS mRNA in the dark. Detailed analysis shows that transcripts originating from different members of the rbcS gene family are differentially affected by this degradation. This phenomenon is not common to all light-regulated plant genes since the mRNA for ST-LS1, another leaf-specific and light-induced gene, is not degraded in the dark within the same time scale.

Base Sequence↗

PubMed1991

Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco.

A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. The ldcgene from Hafnia alvei was therefore (a) placed under the control of the 1' promoter of the bidirectional Tr promoter from Agrobacterium tumefaciens Ti-plasmid, and (b) cloned behind the rbcS promoter from potato fused to the coding region of the rbcS transit peptide. Both ldc constructs, introduced into Nicotiana tabacum with the aid of A. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of the rbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3-1% of dry mass.

Biological Transport↗

PubMed1990

Elicitor-specific induction of one member of the chitinase gene family in Arachis hypogaea.

Chitinases are believed to play an important role in plant defence against bacterial and fungal attack. In peanut (Arachis hypogaea) chitinase genes form a small multigene family. Four chitinase cDNAs (chit 1-4) were isolated from cultured peanut cells. Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP). UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Pmg) elicitor or yeast extract were used to induce expression of chit genes. The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit 1 gene is activated by cell culture dilution and by yeast extract treatment. The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperma but not by the other stimuli. These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.

Arachis↗

PubMed1989

A functional analysis of T-DNA gene 6b: the fine tuning of cytokinin effects on shoot development.

The physiological function in planta of T-DNA gene 6b was studied under various experimental conditions. For this purpose the coding region of gene 6b was cloned behind the 1'-promoter of the TR-DNA to enhance expression of the gene product in transformed plant cells. Expression of the recombinant gene in leaf discs of Nicotiana tabacum altered the capacity for shoot formation of the discs, induced by exogenous (i.e. BAP in the growth medium or agrobacterial trans-zeatin produced under control of gene tzs) or endogenous cytokinins (i.e. isopentenyladenosine produced under control of T-DNA gene 4). The data obtained indicate a reduction of cytokinin activity within the plant cells by the product of T-DNA gene 6b.

Base Sequence↗

PubMed1989

Rubber dam usage among practicing dentists.

This study reports on a survey administered to graduates of the State University of New York at Buffalo School of Dental Medicine concerning usage of and attitudes toward the rubber dam. Differences in both reported usage and attitudes were found between graduates who had received minimal rubber dam training and those who had received more intensive training. Those with graduate training reported more frequent use of the dam than those with no graduate training; however, overall reported usage of the rubber dam was quite low. Comments provided by some respondents suggest that in the educational process a greater emphasis should be put on the reasons for using the dam rather than placement technique.

Dentistry, Operative↗

PubMed1988

Point mutations in the 23 S rRNA genes of four lincomycin resistant Nicotiana plumbaginifolia mutants could provide new selectable markers for chloroplast transformation.

Experiments designed to establish stable chloroplast transformation require selectable marker genes encoded by the chloroplast genome. The antibiotic lincomycin is a specific inhibitor of chloroplast ribosomal activity and is known to bind to the large ribosomal subunit. We have investigated a defined region of the chloroplast 23 S rRNA genes from four lincomycin resistant Nicotiana plumbaginifolia mutants and from wild-type N. plumbaginifolia. The mutants LR415, LR421 and LR446 have A to G transitions at positions equivalent to the nucleotides 2058 and 2059 in the Escherichia coli 23 S rRNA. The mutant, LR400, possesses a G to A transition at a position corresponding to nucleotide 2032 of the E. coli 23 S rRNA.

Base Sequence↗