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O A Olsen

Publications and source records attributed to O A Olsen.

At least 19 recordsLinked to original sources
PubMed1999

Silencing of an aleurone-specific gene in transgenic rice is caused by a rearranged transgene.

In rice, silencing of the aleurone-specific Ltp2-gus transgene, causing easily detectable staining patterns on the grain surface, offers a convenient tool to study quantitative aspects of gene silencing in monocots. In this paper we analyzed phenotypes, occurrence, inheritance and environmental effects on the silencing. We also report on the cloning of transgenes, determination of their structure and analysis of transcripts from the transgene loci. The results show that various patterns of silencing appeared in the R2 generation at which most of the transgenes became homozygous and that they were inherited for five generations. In addition, silencing independently occurred in three generations and reversion to full expression was also found. Cloning of transgenes from a silenced L3.3 line demonstrated that this line carried two transgene loci: one carried an intact Ltp2-gus gene and the other carried a rearranged transgene in which part of the gus gene was in the antisense orientation. Analysis of gus transcripts indicated that partial antisense RNA derived from the rearranged transgene was present in silenced lines and was polyadenylated but that it was absent in non-silenced lines. RNA analyses suggested that the Ltp2-gus silencing in the aleurone layer was post-transcriptional and that it may be caused by interaction of partial antisense gus transcripts with normal sense transcripts. Possible involvement of antisense transcripts in post-transcriptional silencing is discussed.

Base Sequence↗
PubMed1998

Nucellain, a barley homolog of the dicot vacuolar-processing protease, is localized in nucellar cell walls.

The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.

Amino Acid Sequence↗
PubMed1997

A (His)6-tagged recombinant barley (Hordeum vulgare L.) endosperm ADP-glucose pyrophosphorylase expressed in the baculovirus-insect cell system is insensitive to allosteric regulation by 3-phosphoglycerate and inorganic phosphate.

ADP-glucose pyrophosphorylase from photosynthetic tissue is allosterically regulated by 3-phosphoglycerate and inorganic phosphate. In contrast, data from our laboratory indicated that the major AGPase from barley seeds is insensitive to these regulators. Verification of this conclusion has, however, been hindered by the proteolytic degradation of the enzyme from seeds. This report characterizes the barley seed AGPase expressed in the baculovirus-insect cell system, confirming that lack of allosteric regulation by 3-PGA/Pi is an intrinsic property of the enzyme. Purification of the enzyme was by Ni2+-NTA agarose chromatography using a (His)6 tag attached to the N-terminus of the small AGPase subunit.

Allosteric Regulation↗
PubMed1997

Cell wall (1-->3)- and (1-->3, 1-->4)-beta-glucans during early grain development in rice (Oryza sativa L.).

Immunogold labeling was used to study the distribution of (1-->3)-beta-glucans and (1-->3, 1-->4)-beta-glucans in the rice grain during cellularization of the endosperm. At approximately 3-5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1-->3)-beta-glucans but not (1-->3, 1-->4)-beta-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1-->3, 1-->4)-beta-glucans with (1-->3)-beta-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state.

Cell Wall↗
PubMed1997

Barley elongation factor 1 alpha: genomic organization, DNA sequence, and phylogenetic implications.

A full length cDNA clone encoding the 447 amino acid long barley (Hordeum vulgare cv. Bomi) endosperm elongation factor 1 alpha (eF-1 alpha) was isolated by a differential screening procedure. RFLP mapping of eF-1 alpha showed that the barley genome contains a small eF-1 alpha gene family of 4 copies, with 1 copy of the gene being located on each of chromosomes 2, 4, 6, and 7. Analysis of barley endosperm total proteins by Western blot with antibodies directed towards wheat eF-1 alpha and the sea urchin 51 kDa proteins gave a single band of the expected molecular weight. Amino acid sequence comparison with other plant eF-1 alpha sequences showed that the isolated barley endosperm eF-1 alpha is more similar to the published wheat eF-1 alpha sequence than to eF-1 alpha sequences previously published for the barley cultivars Igri and Dicktoo. The phylogenetic analysis suggests that the barley eF-1 alpha gene family can be divided into two subfamilies and that two ancestral genes existed before the divergence of monocotyledonous and dicotyledonous plants.

Amino Acid Sequence↗
PubMed1996

A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.

We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans.

Amino Acid Sequence↗
PubMed1996

A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare).

ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5' ends. In blps14, the unique 5' end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5' end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5' ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5'-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing.

Alternative Splicing↗
PubMed1996

Isolation of molecular markers from the barley endosperm coenocyte and the surrounding nucellus cell layers.

The cereal endosperm develops from a coenocyte to a cellular storage organ through formation of nucleo-cytoplasmic domains and cell wall deposition in the interzones between these domains. During its early stages, the endosperm develops in close contact with nucellus, the sporophytic tissue which gives rise to the megagametophyte. Owing to the positioning of the two tissues deeply within the ovary, neither cell types have been easily accessible for molecular studies. In this paper we report for the first time the cloning of molecular markers for the barley endosperm coenocyte and the nucellus. The novel END1 and NUC1 cDNAs were isolated by differential screening of a cDNA library from 5 DAP (days after pollination) ovaries using a positive probe from hand-dissected embryo sacs with adhering nucellus and testa cell layers, and a negative probe from pericarp. In situ and northern blot hybridization data show that END1 transcripts are asymmetrically distributed in the endosperm coenocyte limited to an area over the nucellar projection. In the cellular endosperm, END1 transcripts are present in modified aleurone cells and a few layers of ventral starchy endosperm cells. The second clone, NUC1, hybridizes to transcripts in the nucellus before fertilization and in autolyzing nucellus cells after fertilization. At later stages, after the disappearance of nucellus, NUC1 transcripts are present in the nucellar epidermis and in the lateral cells of the nucellar projection. This work provide tools for future elucidation of the genes specifying endosperm histogenesis.

Base Sequence↗
PubMed1994

The promoter of the barley aleurone-specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell-specific expression in transgenic rice.

This paper describes the aleurone-specific gene Ltp2 from barley, which encodes a putative 7 kDa non-specific lipid transfer protein. As shown by Northern and in situ hybridization analyses, the Ltp2 transcript is present in barley aleurone cells shortly after the initiation of aleurone cell differentiation. The expression of Ltp2 increases until grain mid-maturity, but the mRNA is absent from mature grains. The Ltp2 transcript is undetectable in the embryo and vegetative tissues, confirming the aleurone specificity of the Ltp2 gene. The ability of the isolated 801 bp Ltp2 promoter to direct aleurone-specific expression in immature barley grains is demonstrated by particle bombardment experiments. In these experiments, the activity of the Ltp2 promoter is 5% of the activity of the strong constitutive Actin1 promoter from rice, as quantified by GUS activity measurements. In stably transformed rice plants containing the Ltp2 promoter-Gus construct, the specificity of the Ltp2 promoter is confirmed in vivo by the presence of GUS activity exclusively in the aleurone layer. This study demonstrates the conserved nature of the regulatory signals involved in aleurone-specific gene transcription in cereal grains.

Amino Acid Sequence↗
PubMed1994

Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds.

In cereal seeds, the aleurone layer and the embryo share several characteristics, including synthesis and accumulation of lipid bodies, desiccation tolerance and dormancy. A number of Balem transcripts present in both the barley aleurone layer and the embryo have been cloned by differential screening of a cDNA library from aleurone layers of immature barley grains. The Balem clones constitute two subgroups, one for which the transcripts are detectable in aleurone layers and embryos of developing seeds only (B23D and B15C), and another for which transcripts are present also in germinating embryos and in maternal tissues (B12D, B14E and B31E). Sequence analysis identified B23D and B15C as the barley homologues of the 18 kDa oleosin of maize embryos (72% amino acid identity) and the dormancy-associated transcript pBS128 from Bromus secalinus (95% identity), respectively. In situ hybridization experiments demonstrate that in the embryo, the B23D transcript is mainly present in the scutellum, whereas the B15C transcript is predominantly present in shoot and root apices. Using anther-derived embryos and embryogenic cell suspensions, it is demonstrated that the B23D and B15C transcripts can be used as molecular markers for somatic embryogenesis. The functions of the transcripts in the second Balem subgroup remain unknown. Further studies on the Balem transcripts may shed light on the molecular basis for the extensive similarities between the embryo and the aleurone layer of the endosperm in the grass family.

Amino Acid Sequence↗
PubMed1993

Kinetic mechanism and regulation of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves.

Barley (Hordeum vulgare, cv. Bomi) leaf ADP-glucose pyrophosphorylase (AGP) was purified to near-homogeneity, using ammonium sulfate fractionation and heat treatment as well as ion exchange, hydrophobic, and dye-ligand chromatography. The enzyme was found to be composed of two subunit types of 51 and 54 kDa, which were recognized by the respective rabbit antibodies against the small and/or large subunit of spinach leaf AGP. Substrate kinetics and product inhibition studies, carried out in the direction of ADP-glucose synthesis, suggested a sequential Iso Ordered Bi Bi kinetic mechanism for the enzyme. In addition, inhibition patterns with CrATP, a dead-end inhibitor of ATP-utilizing enzymes, were consistent with ATP (synthesis reaction) and ADP-glucose (pyrophosphorolysis reaction) binding first to different free forms of the enzyme. The AGP was potently activated by 3-phosphoglycerate (PGA) (K alpha of 5 microM) and inhibited by orthophosphate (P(i)), with the ratio of the two effectors playing a major role in modulating AGP activity. At low [PGA]/[P(i)] ratios, P(i) could reverse the activating effect of PGA. However, at the [PGA]/[Pi] ratios of 1.4, or higher, some activating effect of P(i) in the presence of PGA was observed. PGA decreased the Km values for all substrates of AGP (in both the synthesis and pyrophosphorolysis directions). Properties of the barley leaf AGP are compared to those of AGP from barley seed endosperm, which is insensitive to PGA/P(i) regulation.

Adenosine Triphosphate↗
PubMed1993

Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

PCR amplification of cDNA prepared from poly(A)+ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis.

Arabidopsis↗
PubMed1993

Insensitivity of barley endosperm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation.

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 mumol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4 degrees C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

Adenosine Diphosphate Glucose↗
PubMed1992

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Amino Acid Sequence↗
PubMed1992

Structured modeling of fish physiology.

The use of models in simulation and state estimation has proved useful in diverse applications, especially in industrial process control. The project presented here looked into the modeling of fish physiology for applications in fish physiology research and aquaculture. The models deal with gastric evacuation, metabolism, kidneys, gills, the cardiovascular system, and feeding behavior and are based on data from the literature. Model responses are mostly in accord with real responses in principle, but, as with most models of complex biological systems, the numerical accuracy is low in several cases. However, these structured models enable researchers to test hypotheses by altering the submodels and parameters.

Animals↗
PubMed1991

Primary structure of a novel barley gene differentially expressed in immature aleurone layers.

As a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridies to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were the nearly full-length aleurone/pericarp clone pB22E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene designated B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcript and contains two introns of 145 and 125 bp. Particle bombardment of barley aleurone with a B22EL8 promoter-GUS (beta-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.

Amino Acid Sequence↗