Biomedical subjects

G W Owttrim

Publications and source records attributed to G W Owttrim.

11 recordsLinked to original sources

PubMed2000

Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase.

We have shown previously that CrhC is a unique member of the DEAD-box family of RNA helicases whose expression occurs specifically under conditions of cold stress. Here we show that recombinant His-tagged CrhC, purified from Escherichia coli, is an ATP-independent RNA binding protein possessing RNA-dependent ATPase activity which is stimulated most efficiently by rRNA and polysome preparations. RNA strand displacement assays indicate that CrhC possesses RNA unwinding activity that is adenosine nucleotide specific. Unwinding of partially duplexed RNA proceeds in the 5'-->3' but not the 3'-->5' direction using standard assay conditions. Immunoprecipitation and far-western analysis indicate that CrhC is a component of a multisubunit complex, interacting specifically with a 37 kDa polypeptide. We propose that CrhC unwinds cold-stabilized secondary structure in the 5'-UTR of RNA during cold stress.

Adenosine Triphosphatases↗

PubMed2000

Redox-regulated RNA helicase expression.

In photosynthetic organisms it is becoming increasingly evident that light-driven shifts in redox potential act as a sensor that initiates alterations in gene expression at both the level of transcription and translation. This report provides evidence that the expression of a cyanobacterial RNA helicase gene, crhR, is controlled at the level of transcription and mRNA stability by a complex series of interacting mechanisms that are redox regulated. Transcript accumulation correlates with reduction of the electron transport chain between Q(A) in photosystem II and Q(O) in cyt b(6)f, when Synechocystis sp. strain PCC 6803 is cultured photoautotrophically or photomixotrophically and subjected to darkness and/or electron transport inhibitors or illumination that preferentially excites photosystem II. crhR mRNA stability is also regulated by a redox responsive mechanism, which differs from that affecting accumulation and does not involve signaling initiated by photoreceptors. The data are most consistent with plastoquinol/cyt b(6)f interaction as the sensor initiating a signal transduction cascade resulting in accumulation of the crhR transcript. Functionally, CrhR RNA unwinding could act as a linker between redox regulated transcription and translation. The potential for translational regulation of redox-induced gene expression through RNA helicase-catalyzed modulation of RNA secondary structure is discussed.

Cyanobacteria↗

PubMed2000

Regulation of cold shock-induced RNA helicase gene expression in the Cyanobacterium anabaena sp. strain PCC 7120.

Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30 degrees C but accumulate at 20 degrees C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for crhC transcript accumulation. Enhanced crhC mRNA stability contributes significantly to the accumulation of crhC transcripts, with the crhC half-life increasing sixfold at 20 degrees C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of crhC transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce crhC expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in Anabaena. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of crhC transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of crhC expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of crhC transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.

Anabaena↗

PubMed1999

A cold shock-induced cyanobacterial RNA helicase.

The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes, crhB and crhC, which are differentially expressed in the cyanobacterium Anabaena sp. strain PCC 7120. crhC transcription is limited specifically to cold shock conditions while crhB is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5' untranslated region of crhC revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.

Amino Acid Sequence↗

PubMed1995

Highly conserved genes coding for eukaryotic translation initiation factor eIF-4A of tobacco have specific alterations in functional motifs.

Eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase that is required for the binding of mRNA to ribosomes. Plant eIF-4A-like proteins are highly homologous to eIF-4As from yeast, mouse and Drosophila melanogaster. The pattern of intron-exon boundaries in eIF-4A-like genes are conserved within tobacco, but are not conserved with other organisms. Fixed spacings between the functionally important sequence motifs, GKT-PTRELA (72 bp), DEAD-SAT (81 bp) and SAT-HRIGR (426 bp), are conserved between plants, mouse, Drosophila and yeast.

Amino Acid Sequence↗

PubMed1994

Characterization of the tobacco eIF-4A gene family.

Characterization of cDNAs encoding eukaryotic translation initiation factor 4A (eIF-4A) indicates the expression of a minimum of ten related genes in tobacco leaf cells. The ten groups fall into two gene families, NeIF-4A2 and NeIF-4A3. The majority of the cDNAs exhibit significant sequence similarity to the NeIF-4A2 family at both the DNA and deduced amino acid levels. Northern analysis using specific probes indicates variable expression of four family members in various tobacco organs. Western analysis, using an anti-tobacco eIF-4A polyclonal antibody, reveals a complex pattern of immunologically related polypeptides of approximately 46 kDa. Subcellular fractionation suggests that at least one eIF-4A-related polypeptide is located in the chloroplast where it is ribosome-associated.

Amino Acid Sequence↗

PubMed1991

Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco.

Three cDNA clones coding for eukaryotic translation initiation factor 4A, eIF-4A, were isolated from a Nicotiana plumbaginifolia root cDNA library by heterologous screening. The clones comprise two distinct gene classes as two clones are highly similar while the third is divergent. The genes belong to a highly conserved gene family, the DEAD box supergene family, although the divergent clone contains a DESD box rather than the characteristic DEAD box. The two clones are representatives of separate small multigene families in both N. plumbaginifolia and N. tabacum. Representatives of each family are coordinately expressed in all plant organs examined. The 47 kD polypeptide product of one clone, overexpressed in E. coli, crossreacts immunologically with a rabbit reticulocyte eIF-4A polyclonal antibody. Taken together the data suggest that the two Nicotiana eIF-4A genes encode translation initiation factors. The sequence divergence and the coordinate expression of the two Nicotiana eIF-4A families provide an excellent system to determine if functionally distinct eIF-4A polypeptides are required for translation initiation in plants.

Amino Acid Sequence↗

PubMed1989

Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene.

The expression of the cyanobacterial recA gene, isolated from Anabaena variabilis, has been examined at the levels of transcript and protein abundance. Exposure of the cyanobacterium to a variety of DNA-damaging agents, including mitomycin C, methyl methanesulfonate, and UV irradiation, results in a rapid increase in the abundance of the recA transcript above basal levels as determined by Northern (RNA) blot analysis. A concomitant increase in the abundance of a 37- to 38-kilodalton polypeptide was also detected by Western (immuno-) blot analysis of soluble cyanobacterial polypeptides using polyclonal antiserum directed against the Escherichia coli recA protein. The cyanobacterial polypeptide is of the same molecular mass as that synthesized by an in vitro, DNA-directed procaryotic transcription-translation system primed with an A. variabilis genomic fragment containing the recA gene. Nucleotide sequence analysis of the cyanobacterial gene revealed a protein of 358 amino acids with a molecular weight of 38,403 daltons. The A. variabilis and E. coli recA genes share similarity at 58% of the amino acid residues; however, an E. coli-like lexA repressor-binding site is not present in the A. variabilis promoter region. The similarities of A. variabilis and E. coli recA expression and gene sequence are discussed.

Amino Acid Sequence↗

PubMed1987

Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis.

A recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system.

Cloning, Molecular↗

PubMed1986

Inorganic Carbon Uptake during Photosynthesis : II. Uptake by Isolated Asparagus Mesophyll Cells during Isotopic Disequilibrium.

The species of inorganic carbon (CO(2) or HCO(3) (-)) taken up a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO(2) or HCO(3) (-) transport, during steady state photosynthesis, is achieved by monitoring the changes (by (14)C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO(2) or HCO(3) (-) transport) and experimental time-courses of (14)C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO(2), rather than HCO(3) (-), is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO(2) is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO(3) (-) transport, as the incorporation of (14)C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO(2) uptake alone. The contribution of HCO(3) (-) to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO(3) (-) concentration. The evidence for direct HCO(3) (-) transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of (14)C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO(2), which is partially alleviated by a high extracellular concentration of HCO(3) (-).

Journal Article↗

PubMed1986

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.

Carboxy-Lyases↗