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Biomedical subjects

O D Anderson

Publications and source records attributed to O D Anderson.

32 records · Page 2Linked to original sources
PubMed1996

Construction and expression of a synthetic wheat storage protein gene.

A synthetic wheat high-molecular-weight (HMW) glutenin storage protein gene analog was constructed for expression in E. coli. This first synthetic HMW-glutenin gene and future modifications are intended to allow systematic dissection of the molecular basis of HMW-glutenin role in the visco-elastic properties critical for wheat product processing and utilization. The design of the gene included four features: different construction strategies for the separate assembly of major polypeptide domains, the inclusion of convenient restriction sites for modifications, use of a codon selection similar to E. coli highly expressed genes, and the ability to produce repetitive sequence domains of exact numbers of defined repeats. The complete synthetic HMW-glutenin construct was 1908 bp, and contained 32 identical copies of one of the HMW-glutenin repetitive domain motifs. The gene expressed the novel HMW-glutenin protein to relatively high levels in bacterial cultures and the protein exhibited the known anomalous behavior of HMW-glutenins in SDS-PAGE.

Amino Acid Sequence↗
PubMed1996

Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat.

High-molecular-weight glutenin subunits (HMW-GS), one class of seed storage proteins, are important determinants of the bread-making quality of wheat flour. To change the amount and composition of these proteins via genetic engineering, a gene encoding a novel hybrid subunit under the control of native HMW-GS regulatory sequences was inserted into wheat. Of 26 independent transgenic lines identified by bialaphos selection, 18 expressed the cotransformed hybrid HMW-GS gene in their seed. The hybrid subunit accumulated to levels comparable to those of the native HMW-GS. These results show that a native HMW-GS gene promoter can be used to obtain high levels of expression of seed storage and, potentially, other proteins in transgenic wheat endosperm. Transgene expression was stable for at least three seed generations in the majority of lines. These experiments demonstrate the feasibility of constructing wheat plants with novel seed protein compositions.

Gene Expression Regulation, Plant↗
PubMed1993

Activity of a maize ubiquitin promoter in transgenic rice.

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.

Cloning, Molecular↗
PubMed1992

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits.

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.

Journal Article↗
PubMed1987

Expression of a wheat alpha-gliadin gene in Saccharomyces cerevisiae.

A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae. This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S. cerevisiae. The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene. A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site. A CYC1::alpha-gliadin transcript of approx. 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region. The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence. The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx. 0.1% of the total cellular protein. The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin. This is consistent with recognition and cleavage of the signal peptide by yeast. Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast.

Codon↗
PubMed1986

Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos.

The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages.

Animals↗
PubMed1985

The nucleotide sequence of a HMW glutenin subunit gene located on chromosome 1A of wheat (Triticum aestivum L.).

A cloned 8.2 kb EcoRI fragment has been isolated from a genomic library of DNA derived from Triticum aestivum L. cv. Cheyenne. This fragment contains sequences related to the high molecular weight (HMW) subunits of glutenin, proteins considered to be important in determining the elastic properties of gluten. The cloned HMW subunit gene appears to be derived from chromosome 1A. The nucleotide sequence of this gene has provided new information on the structure and evolution of the HMW subunits. However, hybrid-selection translation experiments suggest that this gene is silent.

Amino Acid Sequence↗
PubMed1984

Nucleic acid sequence and chromosome assignment of a wheat storage protein gene.

A cloned gliadin gene was isolated from a wheat genomic library, and 2.4 kb of its primary sequence determined. The gene, alpha-1Y, was found by Southern analysis to be located on chromosome 6A, and its derived amino acid sequence identifies it as a member of the A-gliadin subgroup of alpha-gliadins located on the short arm of that chromosome. alpha-1Y is apparently functional, and contains consensus TATA and CAAT boxes, and polyadenylation signals. This gliadin gene has no introns, and its noncoding flanking regions contain several short repeats and inverted sequences. The gene is contained in a 6.2 kb EcoRI genomic fragment whose apparent copy number varies in different wheat cultivars.

Amino Acid Sequence↗