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R B Meagher

Publications and source records attributed to R B Meagher.

At least 37 records · Page 2Linked to original sources
PubMed1997

The Arabidopsis ACT11 actin gene is strongly expressed in tissues of the emerging inflorescence, pollen, and developing ovules.

ACT11 represents a unique and ancient actin subclass in the complex Arabidopsis actin gene family. We have isolated and characterized the Arabidopsis ACT11 actin gene and examined its expression. Southern blotting with a 5' gene-specific probe showed that ACT11 was a single-copy gene in the genome. Northern analysis with a 3' gene-specific probe and reverse transcriptase-mediated PCR (RT-PCR) using gene-specific primers detected ACT11 mRNA at low levels in seedling, root, leaf, and silique tissue; at moderate levels in the inflorescence stem and flower; and at very high levels in pollen. The 5' region of the ACT11 gene, including the promoter region, the 5'-untranslated leader, the intron within the leader, and the first 19 actin codons, was fused to a beta-glucuronidase (GUS) reporter gene. The expression of the ACT11/GUS fusion was examined histochemically in numerous independent transgenic Arabidopsis plants. Strong ACT11/GUS activity was detected in rapidly elongating tissues and organs (e.g., etiolated hypocotyls, expanding leaves, stems) and in floral organ primordia. As the floral buds developed into mature flowers, strong GUS activity was gradually restricted to mature pollen and developing ovules. ACT11 appears to be the only Arabidopsis actin gene expressed at significant levels in ovule, embryo, and endosperm. The unique expression patterns in reproductive organs and the sequence divergence of the ACT11 actin gene suggest that the ACT11 isovariant plays distinct and required roles during Arabidopsis development.

Actins↗
PubMed1996

Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution.

Amino Acid Sequence↗
PubMed1996

Sequence, function, and phylogenetic analysis of an ascovirus DNA polymerase gene.

We have sequenced a 5.5-kb region of the DNA genome of the Spodoptera Ascovirus (SAV) containing a DNA polymerase gene. The gene codes for a 1104-amino-acid polypeptide with seven motifs characteristic of DNA polymerases and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The SAV DNA polymerase gene was able to functionally substitute for a baculovirus DNA polymerase gene in a transient assay that relies on origin-specific reporter plasmid DNA replication. Analysis of the predicted DNA polymerase sequence using neighbor-joining and protein parsimony algorithms indicated that this gene was only distantly related to other known viral and cellular DNA polymerases. The SAV DNA polymerase gene is the first ascovirus gene to be identified and sequenced. The molecular phylogenetic analyses of this gene supports the placement of insect ascoviruses in a separate virus family.

Amino Acid Sequence↗
PubMed1996

Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues.

Arabidopsis has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by ACT2 and ACT8, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene-specific RNA gel blot hybridization and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays showed that ACT2 and/or ACT8mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most Arabidopsis organs. The 5' flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a beta-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The ACT2/GUS construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the ACT8/GUS construct was weaker. It was observed only in a subset of the organs and tissues expressing ACT2/GUS and was not significantly expressed in the flower. ACT2, ACT8, and ACT8/GUS mRNAs were present at moderate to high levels in pollen, and yet neither ACT2/GUS nor ACT8/GUS enzyme expression could be detected in pollen. This suggested a mechanism of translational control affecting ACT2 and ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.

Actins↗
PubMed1996

The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development.

Plants contain complex actin gene families composed of several diverse and ancient subclasses of genes. One Arabidopsis actin gene subclass represented by the ACT4 and ACT12 genes has been isolated and characterized. Both actin genes have typical plant actin gene structures, including three small introns interrupting the coding region and an intron within the mRNA leader. Their encoded proteins differ from each other in only one amino acid, whereas they differ in 3-10% of their amino acids from the other five Arabidopsis actin subclasses. They also share a few small blocks of DNA sequence homology in the 5' flanking region near their TATA boxs, but not in their introns, 3' flanking regions, or degenerate positions within codons. Southern analysis with gene-specific probes from 5' flanking sequences showed that both were single copy genes in the genome. Both RNA gel blot analysis with 3' gene-specific probes and reverse transcriptase-mediated polymerase chain reactions (RT-PCR) with gene-specific primers detected low levels of ACT4 and ACT12 mRNAs in flowers and very high levels in pollen. The RT-PCR detected very low levels of these mRNAs in the vegetative organs. The 5' region from both genes, including the promoter region, TATA box, the sequence for the mRNA leader and its intron, and the first 19 actin codons, was fused to a beta-glucuronidase (GUS) reporter gene. Expression of the GUS fusions were examined histochemically in 40 independent transgenic Arabidopsis plants. Expression of the ACT4/GUS fusion was restricted to young vascular tissues, tapetum, and developing and mature pollen. Similar expression patterns in these tissues and cell types were observed for ACT12/GUS fusion, yet unlike ACT4, ACT12 was also strongly expressed in the root cap and in a ring of pericycle tissues during lateral root initiation and early development. The unique expression patterns of the ACT4/ACT12 actin gene subclass are discussed in light of recent data on the other expressed members of the Arabidopsis actin gene family.

Actins↗
PubMed1996

Structure and evolution of the actin gene family in Arabidopsis thaliana.

Higher plants contain families of actin-encoding genes that are divergent and differentially expressed. Progress in understanding the functions and evolution of plant actins has been hindered by the large size of the actin gene families. In this study, we characterized the structure and evolution of the actin gene family in Arabidopsis thaliana. DNA blot analyses with gene-specific probes suggested that all 10 of the Arabidopsis actin gene family members have been isolated and established that Arabidopsis has a much simpler actin gene family than other plants that have been examined. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least two ancient classes of genes that diverged early in land plant evolution and may have separated vegetative from reproductive actins. Subsequent divergence produced a total of six distinct subclasses of actin, and five showed a distinct pattern of tissue specific expression. The concordance of expression patterns with the phylogenetic structure is discussed. These subclasses appear to be evolving independently, as no evidence of gene conversion was found. The Arabidopsis actin proteins have an unusually large number of nonconservative amino acid substitutions, which mapped to the surface of the actin molecule, and should effect protein-protein interactions.

Actins↗
PubMed1996

The Arabidopsis profilin gene family. Evidence for an ancient split between constitutive and pollen-specific profilin genes.

Profilin is a ubiquitous eukaryotic protein that regulates the actin cytoskeleton and recently has been identified as a potent allergen in pollen. We examined the profilin gene family in the model plant, Arabidopsis thaliana, and found that it contained approximately 8 to 10 members. Four distinct profilin sequences, three cDNAs, PRF1, PRF2, and PRF3, and two genomic clones, PRF1 and PRF4, were isolated and characterized. These genes encoded four distinct profilin isoforms of 131 to 134 amino acids. Northern and reverse-transcriptase polymerase chain reaction analyses demonstrated that Arabidopsis PRF1 was expressed in all major plant organs, whereas PRF4 was specifically expressed in mature pollen. Gene trees constructed from amino acid sequence data revealed the presence of two ancient, distinct profilin gene classes in plants. PRF4 was in a class with previously identified pollen-specific profilins from monocot and dicot species. PRF1, PRF2, PRF3, and a distant dicot sequence formed a separate novel class, suggesting an ancient separation of plant profilins based on regulation and perhaps function. The coevolution of plant actin and profilin classes with similar patterns of expression is discussed. The similarity of plant, fungal, protist, insect, and nematode profilins and their extreme divergence from the vertebrate profilins has striking implications for the evolution of fungal-spore- and plant-pollen-profilins as allergens.

Allergens↗
PubMed1996

The arabidopsis ACT7 actin gene is expressed in rapidly developing tissues and responds to several external stimuli.

ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of aCT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5' end of ACT 7 (1.9 kb) joined to the beta-glucuronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no beta-glucuronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements.

Actins↗
PubMed1996

De novo purine synthesis in Arabidopsis thaliana. II. The PUR7 gene encoding 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole synthetase is expressed in rapidly dividing tissues.

The small genome size and excellent genetics of Arabidopsis, as well as the ease with which it is transformed, make it a superb candidate for molecular genetic studies of the purine biosynthetic pathway. Herein we report the isolation, physical characterization, and dissection of the expression patterns of the single gene encoding 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole synthetase. This enzyme, encoded by the PUR7 gene, catalyzes aspartate addition at the alpha-amino group to the growing purine backbone. The expression of the PUR7 as directed by the 5' region, containing the promoter, mRNA leader, and leader intron, was examined in Arabidopsis using a transgenic reporter system. Our analysis demonstrates that the highest level of purine biosynthesis occurs in mitotically active tissues of the plant. Furthermore, purine biosynthesis appears to be under developmental and hormonal regulation. Inhibition of purine biosynthesis using substrate analogs results in arrested plant development and induction of purine gene expression. Purine nucleotides and their derivatives provide multiple cofactors for a variety of metabolic processes. Our findings begin to identify some of the regulatory mechanisms that affect the production of purine nucleotides in Arabidopsis and may give important insights into nitrogen metabolism in general.

Amino Acid Sequence↗
PubMed1996

Conserved expression of the Arabidopsis ACT1 and ACT 3 actin subclass in organ primordia and mature pollen.

We have proposed that ancient and divergent classes of plant actin genes have been preserved throughout vascular plant evolution, because they have distinct patterns of gene regulation. The hypothesis was explored for ACT1 and ACT3, which represent one of the six ancient subclasses in the Arabidopsis actin gene family. Comparison of ACT1 and ACT3 cDNA and genomic sequences revealed highly divergent flaking and intron sequences, whereas they encoded nearly identical proteins. Quantification of their level of divergence suggests that they have not shared a common ancestor for 30 to 60 million years. Gene-specific RNA gel blot hybridization and reverse transcriptase-polymerase chain reaction analyses demonstrated that the distribution of ACT1 and ACT3 mRNAs was very similar: both preferentially accumulated at high levels in mature pollen and at very low levels in the other major organs. The 5' flanking regions of both genes, including the promoter, leader exon and intron, and the first 19 condons, were fused to the beta-glucuronidase (GUS) reporter gene. The expression of these reporter fusions was examined in a large number of transgenic Arabidopsis plants. Histochemical assays demonstrated that both ACT1-GUS and ACT3-GUS constructs were expressed preferentially in pollen, pollen tubes, and in all organ primordia, including those in roots shoots, and the inflorescence. Comparison of the 5' flanking regions of ACT1 and ACT3 revealed a number of short conserved sequences, which may direct their common transcriptional and post-transcriptional regulation. The expression patterns observed were distinct from those of any other other Arabidopsis actin subclass. The conservation of their expression pattern and amino acid sequences suggests that this actin subclass plays a distinct and required role in the plant cytoskeleton.

Actins↗
PubMed1996

A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast.

Poly(A) tails of eukaryotic mRNAs serve as targets for regulatory proteins affecting mRNA stability and translation. Differential mRNA polyadenylation and deadenylation during gametogenesis and early development are now widely recognized as mechanisms of translational regulation in animals, but they have not been observed in plants. Here, we report that the expression of the PAB5 gene encoding one of the poly(A) binding proteins (PABPs) in Arabidopsis is restricted to pollen and ovule development and early embryogenesis. Furthermore, PAB5 is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP. However, PAB5 did not restore the linkage of deadenylation and decapping, thus demonstrating that this function of PABP is not essential for viability. Also, like endogenous PABP, PAB5 expressed in yeast demonstrated genetic interaction with a recently characterized yeast protein SIS1, which is also involved in translational initiation. We propose that PAB5 encodes a post-transcriptional regulatory factor acting through molecular mechanisms similar to those reported for yeast PABP. This factor may have evolved further to post-transcriptionally regulate plant sexual reproduction and early development.

Arabidopsis↗
PubMed1995

Sequence-based identification of T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 and act4-1.

A method is presented to facilitate the isolation of mutations in plant genes, which requires knowledge of the target gene or protein sequence, and is independent of mutant phenotype. The polymerase chain reaction was used to amplify the junctions between a T-DNA insert and the gene of interest from pools of mutant plant lines. The approach was used to identify mutations in Arabidopsis thaliana actin genes. The Arabidopsis genome encodes 10 actins in six ancient subclasses each with distinct expression patterns. Primers in the T-DNA border and highly degenerate actin primers, designed from conserved amino acid motifs, were used to prime the amplification. The PCR products were transferred to filters and probed for actin at low stringency. Thus, mutations in all 10 actin genes were screened for simultaneously. Mutations in the vegetative constitutive actin gene, ACT2, and the pollen-specific actin gene, ACT4, were identified in a population of 5300 lines containing approximately 1.5 T-DNA insertions per line. The screen was sensitive enough that actin insertion alleles were easily distinguished among pools of 100 plant lines. PCR techniques were used which accelerated the purification of mutant lines, and segregation, physical mapping, and sequencing of the act2-1 and act4-1 mutations. This strategy should be generally useful in screening mutant libraries made with a variety of plant insertion elements for mutations in any known sequence.

Actins↗
PubMed1995

Degradation of the soybean ribulose-1,5-bisphosphate carboxylase small-subunit mRNA, SRS4, initiates with endonucleolytic cleavage.

The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3' ends of the proximal products and the 5' ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5' ends of the proximal products and the 3' ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m7G cap(+) and m7G cap(-) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5' cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.

Base Sequence↗
PubMed1994

Structure, expression and phylogenetic analysis of the gene encoding actin I in Pneumocystis carinii.

Actin is a major component of the cytoskeleton and one of the most abundant proteins found in eukaryotic cells. Comparative sequence analysis shows that this essential gene has been highly conserved throughout eukaryotic evolution making it useful for phylogenetic analysis. Complete cDNA clones for the actin-encoding gene were isolated and characterized from Pneumocystis carinii purified from immunosuppressed rat lungs. The nucleotide sequence encodes a protein of 376 amino acids. The predicted actin protein of P. carinii shares a high degree of conservation to other known actins. Only one major actin gene was found in P. carinii. The P. carinii actin sequence was compared with 30 other actin sequences. Gene phylogenies constructed using both neighbor-joining and protein parsimony methods places the P. carinii actin sequence closest to the majority of the fungi. Since the phylogenetic relationship of P. carinii to fungi and protists has been questioned, these data on the actin gene phylogeny support the grouping of P. carinii with the fungi.

Actins↗
PubMed1994

Faithful degradation of soybean rbcS mRNA in vitro.

The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.

Base Sequence↗
PubMed1993

Identification of profilin as an actin-binding protein in higher plants.

Profilin is a low molecular weight protein involved in the organization of the mammalian and protozoan cytoskeleton as well as in signal transduction. In this study, profilin is identified as an actin-binding protein in higher plants which is present in monocot and dicot angiosperms. Birch pollen profilin and actin can be copurified as a complex, and purified recombinant birch profilin can be used as an affinity matrix to obtain birch pollen actin. The binding of 125I-labeled recombinant birch pollen profilin to plant and animal actins can be blocked by profilin-specific antibodies that react with different epitopes of birch profilin. One of the blocking antibodies was raised against the 25 COOH-terminal amino acids indicating the importance of this region in the profilactin complex formation.

Actins↗
PubMed1993

Differential organ-specific expression of three poly(A)-binding-protein genes from Arabidopsis thaliana.

Poly(A)-binding protein (PABP) is considered an essential component of a eukaryotic cell; deletion of the PABP-coding gene in yeast leads to a lethal phenotype. PABP is implicated in numerous aspects of posttranscriptional regulation, including mRNA turnover and translational initiation. A nested set of degenerate PCR primers designed from regions conserved among yeast, Xenopus, and human PABP sequences was used to amplify genomic DNA fragments from Arabidopsis thaliana. Hybridization screening of genomic and cDNA libraries with a genomic PCR probe led to the isolation of three diverse Arabidopsis genes encoding PABPs, PAB1, PAB3, and PAB5. All three sequences contain the expected four RNA-recognition motifs. Sequence diversity between these genes equals or exceeds the diversity among animal and fungal sequences. One of the genes, PAB5, and its cDNA were completely sequenced. Its open reading frame encodes a 73.2-kDa protein containing a number of amino acid motifs characteristic of PABPs from different species. Moreover, in vitro synthesized PAB5 protein bound to poly(A)-Sepharose with high specificity. All three genes isolated showed organ-specific patterns of expression. PAB5 and PAB3 RNAs were detected only in floral organs, with the highest level of expression in immature flowers. PAB1 RNA was observed predominantly in roots, was less abundant in immature flowers, and was not detected in any other organ examined (stems, leaves, mature flowers, siliques). This suggests a potentially unique role for PABPs in organ-specific posttranscriptional regulation in plants.

Amino Acid Sequence↗