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D R Gallie

Publications and source records attributed to D R Gallie.

At least 37 records · Page 2Linked to original sources
PubMed1998

The phosphorylation state of the wheat translation initiation factors eIF4B, eIF4A, and eIF2 is differentially regulated during seed development and germination.

The translation initiation factors (eIF) 4B and eIF2 are phosphoproteins whose phosphorylation state differs between mature seed and leaves. We examined the isoforms of eIF4B and the alpha and beta subunits of eIF2 during the development and germination of wheat seed to determine whether the differences in their phosphorylation state are because of tissue-specific regulation or occur concomitant with changes in protein synthetic activity during development. eIF2alpha underwent phosphorylation through several intermediate isoforms that correlated with the increase and subsequent reduction in protein synthetic activity characteristic of seed development. eIF2beta and eIF4B, present as highly phosphorylated isoforms during early seed development, underwent dephosphorylation during late development. eIF4B was rapidly phosphorylated within 20 h of germination, whereas eIF2alpha did not undergo dephosphorylation until 48-60 h of growth. A third factor, eIF4A, was predominantly nonphosphorylated throughout most of seed development and germination. These observations suggest that the phosphorylation state of eIF2alpha, eIF2beta, and eIF4B is developmentally regulated in a way that correlates with the changes in protein synthetic activity but that some differences were also observed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1998

Analysis of translation elongation factors from wheat during development and following heat shock.

Translational activity in plants undergoes rapid changes during developmental stages such as seed formation and germination, and during abiotic stresses such as heat shock, hypoxia and wounding. We examined the protein levels and isoelectric state of two components of the translation machinery, elongation factor (EF) 1 alpha and 2, to determine their roles in the regulation of translation. We found that the apparent protein levels of EF1 alpha increase relative to the EF2 levels which decline slightly during the development of the wheat seed. During germination, high levels of these factors are present in seedling tissues known to be actively engaged in translation; however, no differences in isoelectric state were observed during germination. As an example of abiotic stress, heat shock had little impact on the apparent levels of EF1 alpha or EF2 present in wheat leaves, nor were changes in the number or levels of isoforms observed.

Germination↗
PubMed1998

Controlling gene expression in transgenics.

The repertoire of cis-regulatory elements has increased to a level of sophistication that offers considerable spatial and temporal control over transgene expression. Recent advances made with transgenes have revealed that the control of their expression is also influenced by factors that range from transgene copy number and arrangement to nuclear architecture and chromosomal location. These factors must now be included with the standard considerations of transcriptional and translational enhancers of gene expression during transgene design.

Gene Expression Regulation, Plant↗
PubMed1997

Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity.

The 5'-cap and the poly(A) tail act synergistically to increase the translational efficiency of eukaryotic mRNAs, which suggests that these two mRNA elements communicate during translation. We report here that the cap-associated eukaryotic initiation factors (eIFs), i. e. the two isoforms of the cap-binding complex (eIF-4F and eIF-iso4F) and eIF-4B, bind to the poly(A)-binding protein (PABP) both in the presence and absence of poly(A) RNA. The interactions between PABP and eIF-4F, eIF-iso4F, and eIF-4B were measured in the absence of poly(A) RNA using far Western analysis and confirmed by direct fluorescence titration studies. The functional consequence of the interaction between these initiation factors and PABP was examined using RNA binding assays and RNA mobility shift analysis. eIF-4F, eIF-iso4F, and eIF-4B promoted PABP activity through a shift in its equilibrium affinity for poly(A). eIF-iso4G, the large subunit of eIF-iso4F, was the subunit responsible for the interaction between eIF-iso4F and PABP and was the subunit that promoted PABP RNA binding activity. Truncation analysis of eIF-iso4G indicated that a domain close to its N-terminal end appeared to be involved in binding PABP. These results suggest that the interaction between PABP and eIF-4B and eIF-iso4G may be involved in mediating the functional co-dependence observed between the cap and the poly(A) tail during translation.

Binding Sites↗
PubMed1997

The wheat poly(A)-binding protein functionally complements pab1 in yeast.

Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDS/PAGE and a theoretical mass of 70 823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNP1 and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.

Amino Acid Sequence↗
PubMed1997

The phosphorylation state of translation initiation factors is regulated developmentally and following heat shock in wheat.

Several translation initiation factors in mammals and yeast are regulated by phosphorylation. The phosphorylation state of these factors is subject to alteration during development, environmental stress (heat shock, starvation, or heme deprivation), or viral infection. The phosphorylation state and the effect of changes in phosphorylation of the translation initiation factors of higher plants have not been previously investigated. We have determined the isoelectric states for the wheat translation initiation factors eIF-4A, eIF-4B, eIF-4F, eIF-iso4F, and eIF-2 and the poly(A)-binding protein in the seed, during germination, and following heat shock of wheat seedlings using two-dimensional gel electrophoresis and Western analysis. We found that the developmentally induced changes in isoelectric state observed during germination or the stress-induced changes were consistent with changes in phosphorylation. Treatment of the phosphorylated forms of the factors with phosphatases confirmed that the nature of the modification was due to phosphorylation. The isoelectric states of eIF-4B, eIF-4F (eIF-4E, p26), eIF-iso4F (eIF-iso4E, p28), and eIF-2alpha (p42) were altered during germination, suggesting that phosphorylation of these factors is developmentally regulated and correlates with the resumption of protein synthesis that occurs during germination. The phosphorylation of eIF-2beta (p38) or poly(A)-binding protein did not change either during germination or following a thermal stress. Only the phosphorylation state of two factors, eIF-4A and eIF-4B, changed following a heat shock, suggesting that plants may differ significantly from animals in the way in which their translational machinery is modified in response to a thermal stress.

Blotting, Western↗
PubMed1997

Visualization of poly(A)-binding protein complex formation with poly(A) RNA using atomic force microscopy.

Poly(A)-binding protein (PABP) is an RNA-binding protein that binds specifically to the poly(A) tail of messenger RNAs in eukaryotes. The PABP/poly(A) tail complex has been implicated as being important in promoting the efficient initiation of translation as well as in maintaining the integrity of the mRNA. PABP binds poly(A) cooperatively with a packing density of one PABP molecule per 25 adenosine residues. We have investigated the complexes formed between purified PABP and poly(A) RNA using atomic force microscopy (AFM). PABP alone was observed to be primarily in a monomer form with a height of 1.0 +/- 0.2 nm. Following binding to poly(A), PABP appeared to be present in variable size complexes that bound lengthwise along the RNA. This size of the PABP/poly(A) complex appeared to be maximal, suggesting that PABP binding to poly(A) may be self-limiting. Poly(A) RNA alone appeared to contain a knob-like structure that largely disappeared once PABP was bound. The use of AFM has therefore provided potential new insights into the complexes formed by this RNA-binding protein.

Aluminum Silicates↗
PubMed1997

Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources.

Adaptation, Physiological↗
PubMed1996

Translational control during recovery from heat shock in the absence of heat shock proteins.

Heat shock results in a reduction in translational efficiency and an increase in mRNA stability in plants that is proportional to the severity of the stress. To determine whether the absence of heat shock proteins may affect the observed changes during translation or mRNA turnover, the effect that inhibiting transcription during a heat shock has on the subsequent translation of a reporter mRNA was examined. The presence of actinomycin D repressed heat shock protein synthesis in response to the application of a heat shock by more than 80%. The translational efficiency of the reporter mRNA was subject to a greater degree of repression following a heat shock when transcription was inhibited than it was in heat-shocked cells in which transcription was not inhibited. In contrast, inhibiting transcription during a heat shock did not prevent the heat-mediated increase in mRNA stability. These data suggest that ongoing transcription is needed during a heat shock to support a basal level of translational activity in the subsequent recovery from the stress but does not appear to be required for the heat-mediated increase in mRNA stability.

Animals↗
PubMed1996

The effect of the length of the 3'-untranslated region on expression in plants.

The effect of increasing the length of the 3'-untranslated region (UTR) on expression of luciferase mRNA was examined in transiently transfected carrot protoplasts. The effect of the 3'-UTR on both poly(A)- and poly(A)+ mRNA was examined. A nested set of constructs in which the 3'-UTR increased from 4 to 104 bases was generated by the introduction and reiteration of a 20 base sequence downstream of the luc stop codon. For poly(A)- mRNA, there was a consistent increase in expression when the length of the 3'-UTR was increased from 4 to 104 bases. For poly(A)+ mRNA, expression increased 18-fold when the length of the 3'-UTR was increased from 7 to 27 bases. Further increases in the length of the 3'-UTR did not affect expression. The increase in expression was largely due to an increase in translational efficiency. These data suggest that the length of the 3'-UTR plays an important role in determining the extent to which a poly(A) tail can stimulate the translation of an mRNA.

Daucus carota↗
PubMed1996

Isolation and characterization of the 102-kilodalton RNA-binding protein that binds to the 5' and 3' translational enhancers of tobacco mosaic virus RNA.

Tobacco mosaic virus (TMV) is a positive-sense, single-stranded RNA virus the genome of which acts as a mRNA in the cytoplasm. On infection, TMV mRNA is efficiently and selectively translated by the host translation machinery despite the lack of a poly(A) tail, which is normally required for efficient translation. Both the 68-base 5' leader (Omega) and the 205-base 3' untranslated region of TMV promote efficient translation. A 25-base poly(CAA) region within Omega and the upstream pseudoknot domain, a 72-base region composed of three RNA pseudoknots, are responsible for the translational regulation. We have identified, purified, and characterized a 102-kDa RNA-binding protein (p102) from wheat that binds specifically to the poly(CAA) region within Omega and the upstream pseudoknot domain within the TMV 3' untranslated region. Polyclonal antibodies raised against wheat p102 were used to demonstrate that p102 is widely conserved in plant species. Moreover, specific RNA binding activity was detected in all plant species tested. Addition of anti-p102 antibodies to an in vitro translation lysate derived from wheat germ repressed translation, which was subsequently reversed by supplementing the lysate with p102. These findings suggest that this protein may play an important role in determining translational efficiency in plants.

Antibodies↗
PubMed1996

The histone 3'-terminal stem-loop is necessary for translation in Chinese hamster ovary cells.

The metazoan cell cycle-regulated histone mRNAs are the only known cellular mRNAs that do not terminate in a poly(A) tall. Instead, mammalian histone mRNAs terminate in a highly conserved stem-loop structure which is required for 3'-end processing and regulates mRNA stability. The poly(A) tail not only regulates translational efficiency and mRNA stability but is required for the function of the cap in translation (m(7)GpppN). We show that the histone terminal stem-loop is functionally similar to a poly(A) tail in that it enhances translational efficiency and is co-dependent on a cap in order to establish an efficient level of translation. The histone stem-loop is sufficient and necessary to increase the translation of reporter mRNA in transfected Chinese hamster ovary cells but must be positioned at the 3'-terminus in order to function optimally. Mutations within the conserved stem or loop regions reduced its ability to facilitate translation. All histone mRNAs in higher plants are polyadenylated. The histone stem-loop did not function to influence translational efficiency or mRNA stability in plant protoplasts. These data demonstrate that the histone stem/loop directs efficient translation and that it is functionally analogous to a poly(A) tail.

Animals↗
PubMed1996

Translational control of cellular and viral mRNAs.

We are becoming increasingly aware of the role that translational control plays in regulating gene expression in plants. There are now many examples in which specific mechanisms have evolved at the translational level that directly impact the amount of protein produced from an mRNA. All regions of an mRNA, i.e., the 5' leader, the coding region, and the 3'-untranslated region, have the potential to influence translation. The 5'-terminal cap structure and the poly(A) tail at the 3' terminus serve as additional elements controlling translation. Many viral mRNAs have evolved alternatives to the cap and poly(A) tail that are functionally equivalent. Nevertheless, for both cellular and viral mRNAs, a co-dependent interaction between the terminal controlling elements appears to be the universal basis for efficient translation.

Nucleic Acid Conformation↗
PubMed1996

Translational efficiency is regulated by the length of the 3' untranslated region.

All polyadenylated mRNAs contain sequence of variable length between the coding region and the poly(A) tail. Little has been done to establish what role the length of the 3' untranslated region (3'UTR) plays in posttranscriptional regulation. Using firefly luciferase (luc) reporter mRNA in transiently transfected Chinese hamster ovary (CHO) cells, we observed that the addition of a poly(A) tail increased expression 97-fold when the length of the 3'UTR was 19 bases but that its stimulatory effect was only 2.3-fold when the length of the 3'UTR was increased to 156 bases. The effect of the luc 3'UTR on poly(A) tail function was orientation independent, suggesting that its length and not its primary sequence was the important factor. Increasing the length of the 3'UTR increased expression from poly(A)- mRNA but had little effect on poly(A)+ mRNA. To examine the effect of length on translational efficiency and mRNA stability, a 20-base sequence was introduced and reiterated downstream of the luc stop codon to generate a nested set of constructs in which the length of the 3'UTR increased from 4 to 104 bases. For poly(A)- reporter mRNA, translational efficiency in CHO cells increased 38-fold as the length of the 3'UTR increased from 4 to 104 bases. Increasing the length of the 3'UTR beyond 104 bases increased expression even further. Increasing the length of the 3'UTR also resulted in a 2.5-fold stabilization of the reporter mRNA. For poly(A)+ mRNA, the translational efficiency and mRNA half-life increased only marginally as the length of the 3'UTR increased from 27 to 161 bases. However, positioning the poly(A) tail only 7 bases downstream of the stop codon resulted in a 39-fold reduction in the rate of translation relative to a construct with a 27-base 3'UTR, which may be a consequence of the poly(A) tail-poly(A)-binding protein complex functioning as a steric block to translocating ribosomes as they approached the termination codon. The optimal length of the 3' noncoding region for maximal poly(A) tail-mediated stimulation of translation is approximately 27 bases. These data suggest that the length of the 3'UTR plays an important role in determining both the translational efficiency and the stability of an mRNA.

Animals↗
PubMed1995

The tobacco etch viral 5' leader and poly(A) tail are functionally synergistic regulators of translation.

The 5' cap (m7GpppN) and the poly(A) tail of eukaryotic mRNAs work in concert to establish an efficient level of translation in vivo. Nevertheless, several mRNAs naturally lack a cap or a poly(A) tail. Determining how these messages effectively compete for the translational machinery not only reveals alternative mechanisms for translational competence, but can also underscore similarities between alternative mechanisms and the standard cap/poly(A) tail interaction. The genomic RNA of tobacco etch virus (TEV), a potyvirus, is a polyadenylated mRNA that naturally lacks a cap (m7GpppN) at the 5'-terminus and yet is a highly competitive mRNA during translation. The 144-nt 5'-leader is largely responsible for directing efficient translation and can greatly increase the translational competence of reporter mRNAs. We have examined the synergy between the TEV 5'-leader and the poly(A) tail in transfected plant and animal cells. The TEV 5'-leader functioned optimally as a regulator of reporter mRNA translation only when a poly(A) tail was present. The effect of the TEV 5'-leader on the translation of capped transcripts was significantly less than that for uncapped mRNAs, suggesting that the TEV 5'-leader and the cap may promote similar steps in translation.

Animals↗
PubMed1995

m7GpppG cap dependence for efficient translation of Drosophila 70-kDa heat-shock-protein (Hsp70) mRNA.

To investigate whether preferential translation of the heat-shock mRNAs occurs via cap-independent translation, the requirement for the m7GpppG cap structure for efficient translation of 70-kDa heat-shock-protein (Hsp70) mRNA was quantified by in vitro translation and by in vivo translation following electroporation. Hsp70 mRNA was transcribed in vitro with and without a cap structure. Translation in the rabbit reticulocyte or wheat germ lysate was reduced about 70% when the cap was absent. For comparison, translation of uncapped encephalomyocarditis virus 5'-untranslated-region-containing mRNA was equal to or greater than capped mRNA, whereas translation of several non-heat-shock mRNAs was reduced by 85-95% when capping was omitted. Cap-dependent translational stimulation of Hsp70 is not due to increased stability, is not a kinetic effect, and requires the methylated GpppG. To confirm the in vitro analyses, capped and uncapped mRNA were introduced into Drosophila tissue culture cells by electroporation, followed by heat shock. Paralleling the in vitro results, uncapped Hsp70 mRNA translation was 70-80% reduced relative to the capped form. Complementary experiments in which eIF-4 was inactivated in vitro using either m7GTP cap analogue or foot-and-mouth-disease virus L protease expression likewise indicated that the cap-dependent translation pathway is required for optimal Hsp mRNA translation. Since cellular Hsp70 mRNA translation during heat shock is very efficient, it is unlikely that translation via a cap-independent pathway is the principal basis for preferential translation.

Animals↗