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R B Meagher

Publications and source records attributed to R B Meagher.

At least 55 records · Page 3Linked to original sources
PubMed1993

Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants. I. 5'-Phosphoribosyl-5-aminoimidazole synthetase.

We have initiated an investigation of the de novo purine nucleotide biosynthetic pathway in the plant Arabidopsis thaliana. Functional suppression of Escherichia coli auxotrophs allowed the direct isolation of expressed Arabidopsis leaf cDNAs. Using this approach we have successfully suppressed mutants in 4 of the 12 genes in this pathway. One of these cDNA clones, encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase (PUR5) has been characterized in detail. Analysis of genomic DNA suggests that the Arabidopsis genome contains a single AIR synthetase gene. Analysis of the cDNA sequence and mRNA size suggests that this enzyme activity is encoded by a monofunctional polypeptide, similar to that of bacteria and unlike other eukaryotes. The Arabidopsis AIR synthetase contains a basic hydrophobic transit peptide consistent with transport into chloroplasts. Comparison of both the predicted amino acid and nucleotide sequence from Arabidopsis to those of eight other distant organisms suggests that the plant sequence is more similar to the bacterial sequences than to other eukaryotic sequences. This study provides the groundwork for future investigations into the regulation of de novo purine biosynthesis in plants. Additionally, we have demonstrated that functional suppression of bacterial mutants may provide a useful method for cloning a variety of plant genes.

Amino Acid Sequence↗
PubMed1992

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs.

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo. The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.

Base Composition↗
PubMed1992

Expression of foreign genes in transgenic yellow-poplar plants.

Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding beta-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-beta-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-beta-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves.

Journal Article↗
PubMed1992

Degradation products of the mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean and transgenic petunia.

The degradation of a soybean ribulose-1,5-bisphosphate carboxylase small subunit RNA, SRS4, was investigated in soybean seedlings and in petunia plants transformed with an SRS4 gene construct. Polyacrylamide RNA gel blot, primer extension, and S1 nuclease analyses were used to identify and map fragments of the SRS4 mRNA generated in vivo. We showed that SRS4 mRNA is degraded to a characteristic set of fragments in soybean and transgenic petunia and that degradation is not dependent on position of insertion of the gene construct within the genome, on the expression level of the SRS4 mRNA, or on the rbcS promoter. Degradation products lacked poly(A) tails and fractionated with poly(A)-depleted RNA on oligo(dT)-sepharose columns. These products pelleted with polysomes and were released from polysomes prepared with EDTA. Sequences at the 5' end of the SRS4 mRNA were more stable than those at the 3' end of the mRNA. Three models for SRS4 mRNA degradation involving endonucleolytic and exonucleolytic degradation were presented to explain the origin of the 5' proximal fragments.

Base Sequence↗
PubMed1990

Transcriptional and post-transcriptional processes regulate expression of RNA encoding the small subunit of ribulose-1,5-biphosphate carboxylase differently in petunia and in soybean.

The effects of white light, far-red light and darkness on the in vitro transcription and RNA levels of the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) were investigated in petunia and in soybean. In petunia plants treated with 48 hours of darkness the in vitro transcription rate of two of the rbcS subfamilies of petunia, rbcS A and rbcS C, declined 32- and 8-fold respectively, whereas treatment of dark-adapted plants with light caused the in vitro transcription rate of these subfamilies to return to their light-grown levels. Relative RNA levels of rbcS A and rbcS C declined in parallel with in vitro transcription rate changes upon treatment of petunia plants with darkness. However, while relative RNA levels of rbcS C changed in parallel with in vitro transcription rate under all conditions of far-red light and white light tested, there were differences between the changes in rbcS A in vitro transcription rate and RNA levels which were consistent with post-transcriptional regulation of rbcS A RNA. In addition we observed that nuclei isolated from the leaves of plants which were exposed to darkness for periods of 72 hours or longer were transcriptionally inactive. Similar experiments on the in vitro transcription and relative levels of the rbcS RNA in soybean seedlings have lead to the hypothesis that rbcS RNA is less stable in light than in darkness. In contrast, small decreases in rbcS in vitro transcription rate in mature soybean plants treated with darkness were accompanied by large decreases in rbcS RNA, suggesting that rbcS RNA was degraded more rapidly in darkness than in light in these plants. We have shown that differences in the modulation of rbcS RNA levels by post-transcriptional mechanisms exist between plants which belong to different orders, and between different developmental states of the same plant species.

Gene Expression Regulation↗
PubMed1990

A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit in soybean.

Post-transcriptional regulation of the genes encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase was examined in soybean seedlings. Substantial discrepancies were detected between relative in vitro transcription rates and steady-state RNA levels in light- and dark-grown seedling leaves, indicating that rbcS RNA may be degraded more rapidly in light than in darkness. Additional data imply that the turnover mechanism is rapidly induced by light, maintained for some time in darkness, and that it may be negatively controlled by far-red light. The proposed RNA turnover system does not affect all RNAs equally since a soybean actin gene showed equivalent in vitro transcription rates and RNA levels in light and darkness. Soybean rbcS genes may be subject to a novel mode of control in which light-induced expression is accompanied by an increased rate of RNA degradation. Models for the specific regulation of rbcS RNA stability in response to light are presented.

Blotting, Western↗
PubMed1990

Plants contain highly divergent actin isovariants.

Actin protein isovariants have been identified in animals with distinct cytoplasmic or muscle specific patterns of expression. Analysis of vascular plant actin gene sequences suggests that an even greater diversity should exist within the plant actin protein families, but previous studies on plant proteins have not demonstrated the presence of multiple actin isovariants. Antibodies recognizing a conserved amino-terminal plant actin peptide, a family of plant actin peptides from a variable region, and two monoclonal antibodies to conserved epitopes within animal actins were used to identify isovariants of soybean actin resolved by two-dimensional isoelectric focusing (IEF) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately six to eight actin isovariants with pI values ranging from 5.1 to 5.8 have been identified from soybean hypocotyls, stems, leaves, and roots with varying amounts of most isovariants present in all four organs. Acidic isovariants were present in much higher levels in leaves and stems. Antisera with lambda-class actin specificity detected a subset of three isovariants in all organs examined. One monoclonal and one antipeptide antisera are shown to react well with a wide variety of plant actin isovariants. Similar patterns of actin isovariants were detected in the distant angiosperms, Arabidopsis, petunia, and maize. It is likely that many of these diverse classes of isovariants have been preserved throughout vascular plant evolution and reflect the ancient diversity within plant actin gene families. The extreme difference among isovariants implies the presence of a complex actin-based cytoskeletal system in plants.

Actins↗
PubMed1990

Comparison of the expression of two highly homologous members of the soybean ribulose-1,5-bisphosphate carboxylase small subunit gene family.

Two soybean ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are highly homologous over a region that includes 4 kb of 5' and 1 kb of 3' flanking sequences. The expression of these genes was compared using synthetic oligonucleotide probes. Analysis of a soybean leaf cDNA library indicates that SRS1 and SRS4 are the most highly expressed members of the soybean SSU gene family. Similar changes were observed in the RNA levels for these genes in response to white light, far-red light and darkness, although SRS1 was expressed at a four-fold higher level in total RNA than SRS4 under all conditions. However, nuclear run-on assays indicate that SRS1 is transcribed at a lower rate than SRS4, which suggests that SRS1 RNA is more stable. S1 nuclease analysis and oligonucleotide directed RNase H cleavage indicate that transcripts from both genes are polyadenylated within two principle regions separated by 35 nt. Sequence analysis of 16 independent cDNA clones identified seven different polyadenylation sites, and six of these sites lie within these two regions. Although SRS1 RNA was poorly recovered during poly(A)+ fractionation, RNase H cleavage experiments showed that transcripts from SRS1 and SRS4 had similar poly (A) tail lengths ranging from 0 to 220 nt. In addition, and despite differences in the untranslated leader sequences, SRS1 and SRS4 RNAs are assembled into polysomes with equal efficiencies. The overall similarity in expression patterns for these two genes further illustrates the coordinate evolution of individual members of a SSU gene family and is consistent with the proposal that gene conversion homogenizes both the coding and regulatory regions of these genes.

Base Sequence↗
PubMed1990

Diverse soybean actin transcripts contain a large intron in the 5' untranslated leader: structural similarity to vertebrate muscle actin genes.

Plant actins are encoded by complex and highly divergent multigene families. Despite the general lack of intron conservation in animal, fungal and protist actin genes, evidence is presented which indicates that higher plant actin genes have an untranslated leader exon with structural similarity to that found in vertebrate actin genes. All functional higher plant actin genes sequenced to date contain a potential intron acceptor site in the 5' untranslated region 10 to 13 nucleotides upstream of the initiator ATG. A leader specific cDNA probe hybridized to sequences over 1.0 kbp upstream from the coding region confirming the presence of an upstream exon. Primer extension of mRNA with gene-specific oligonucleotides was used to analyze the 5' untranslated exon and leader intron from four divergent soybean actin genes, SAc3, 4, 6 and 7. The 5' ends of all four mRNAs are heterogeneous. The consensus promoter elements of the SAc7 actin promoter were identified. Gene specific primer extension sequencing of actin mRNAs indicated that splicing of the 5' leader intron occurred at the predicted acceptor site in SAc6 and SAc7. The SAc6 and SAc7 5' untranslated exons are small (88-111 nt) and the leader introns are relatively large (844-1496 nt). The presence of an intron within the 5' RNA leader and an intron which splits a glycine codon at position 152 in all plant actin genes and all vertebrate muscle actin genes suggests that these structures may have been conserved due to a functional role in actin expression. The 5' regions of these two soybean actin genes contain many unusual features including (CT) repeats and long stretches of pyrimidine-rich DNA. The possible roles of the upstream exon/intron and the C + T-rich regions are discussed.

Actins↗
PubMed1990

Characterization of the expression of the petunia glycine-rich protein-1 gene product.

We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels.

Journal Article↗
PubMed1990

Tissue-specific expression of divergent actins in soybean root.

It has been proposed that the evolution of distinct classes of genes encoding the kappa-, lambda-, and mu-actins in soybean is the result of an ancient divergence in patterns of actin gene expression. In this study, antisera against a family of synthetic actin peptides from a divergent region within the predicted actin polypeptide sequences have been used to explore the differential expression of plant actins. Antiserum elicited against a 16-residue synthetic lambda-actin peptide SAc4:257 reacted with a 46-kilodalton protein in soybean extracts, showed specificity for the lambda-peptide over the divergent kappa- and mu-actin peptides in enzyme-linked immunosorbent assays, and reacted strongly and preferentially with root protoderm in apical roots and in lateral root primordia. Antiserum elicited against the synthetic kappa-actin peptide SAc1:257 reacted with 46-kilodalton protein on protein gel blots, showed partial specificity toward the immunogenic kappa-peptide over the divergent lambda- and mu-peptides, and reacted strongly with all root tissues with the exception of root cap. These data support the hypothesis that ancient classes of plant actin genes may have been preserved because of their role in developmentally controlled differences in tissue-specific actin expression and/or function. The possibility that other diverse actin classes have unique patterns of regulation is discussed.

Actins↗
PubMed1989

Molecular evolution of the small subunit of ribulose bisphosphate carboxylase: nucleotide substitution and gene conversion.

The nucleotide sequences encoding the mature portion of 31 ribulose 1.5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is approximately 6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species.

Amino Acid Sequence↗
PubMed1988

Recombination within a subclass of restriction fragment length polymorphisms may help link classical and molecular genetics.

Restriction fragment length polymorphisms (RFLPs) are being used to construct complete linkage maps for many eukaryotic genomes. These RFLP maps can be used to predict the inheritance of important phenotypic loci and will assist in the molecular cloning of linked gene(s) which affect phenotypes of scientific, medical and agronomic importance. However, genetic linkage implies very little about the actual physical distances between loci. An assay is described which uses genetic recombinants to measure physical distance from a DNA probe to linked phenotypic loci. We have defined the subset of all RFLPs which have polymorphic restriction sites at both ends as class II RFLPs. The frequency of class II RFLPs is computed as a function of sequence divergence and total RFLP frequency for highly divergent genomes. Useful frequencies exist between organisms which differ by more than 7% in DNA sequence. Recombination within class II RFLPs will produce fragments of novel sizes which can be assayed by pulsed field electrophoresis to estimate physical distance in kilobase pairs between linked RFLP and phenotypic loci. This proposed assay should have particular applications to crop plants where highly divergent and polymorphic species are often genetically compatible and thus, where class II RFLPs will be most frequent.

Chromosome Mapping↗
PubMed1987

5' proximal sequences of a soybean ribulose-1,5-bisphosphate carboxylase small subunit gene direct light and phytochrome controlled transcription.

Two closely related ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are transcribed at high levels in soybean plants in response to light. Transgenic petunia plants containing 5' sequences from SRS1 or SRS4 fused to the polypeptide encoding region of a neomycin phosphotransferase (NPTII) gene exhibit selectable kanamycin resistance. Deletion of three ATG codons from the region preceding the normal NPTII translation start site has little effect on the levels of kanamycin resistance in transformed plants. Run-on transcription assays in isolated nuclei demonstrate that transcription of the SRS1/NPTII chimera and the native petunia SSU11A gene subfamily is light regulated and under phytochrome control in leaves of transgenic plants. In young expanding leaves of fully light grown plants, transcription of these genes is markedly reduced within minutes of far-red treatment, while ribosomal DNA and actin gene transcription remains unchanged. This is analogous to the transcriptional response we observed for SRS1 and SRS4 in soybean seedlings. These data suggest (1) that transcription of SSU genes in both soybean and petunia require the continued presence or synthesis of phytochrome in the Pfr form and (2) that 5' sequences are sufficient to direct the phytochrome controlled transcriptional response of the SRS1 gene. In fully expanded mature leaves we found the transcription rates of the native SSU11A gene subfamily, the chimeric SRS1/NPTII gene, the rDNA genes, and several other control genes to be reduced markedly after far-red treatment or after extended periods of darkness. The contrast between results in young and mature leaves is discussed.

Chlorophyll↗
PubMed1987

A complex gene superfamily encodes actin in petunia.

We have shown by several independent criteria that actin is encoded by a very large and complex superfamily of genes in Petunia. Several cDNA and genomic probes encoding actins from diverse organisms (Dictyostelium, Drosophila, chicken and soybean) hybridize to hundreds of restriction fragments in the petunia genome. Actin-hybridizing sequences were isolated from a petunia genomic library at a rate of at least 200 per genome equivalent. Twenty randomly selected actin-hybridizing clones were characterized in more detail. DNA sequence data from four representative and highly divergent clones, PAc2, PAc3, PAc4 and PAc7, demonstrate that these actin-like sequences are related to functional actin genes. Intron positions typical of other known plant actin genes are conserved in these clones. Four of six clones analyzed (PAc1, PAc2, PAc3, PAc4) hybridize to leaf mRNA of the same size (1.7 kb) as that reported for other plant actin mRNAs and to a slightly smaller mRNA species (1.5 kb). Five distinct subfamilies of actin-related genes were characterized which varied in size from a few members to several dozen members. It is clear from our data that other actin gene subfamilies must also exist within the genome. Possible mechanisms of actin gene amplification and genome turnover are discussed.

Actins↗