Plant polyploidy: gene expression and genetic redundancy.
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Evolution, Molecular↗
Biomedical subjects
L D Gottlieb
Publications and source records attributed to L D Gottlieb.
The 5' leader of plant PgiC has an intron: the leader shows both the loss and maintenance of constraints compared with introns and exons in the coding region.
PgiC, a complex gene with 23 coding exons and 22 intervening introns, encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9) in higher plants. Here, we report RNA ligase-mediated rapid amplification of cDNA ends experiments that showed that PgiC in Clarkia (Onagraceae) and Arabidopsis thaliana has an intron in the 5' leader. Comparison of the EMBL accessions of the cDNA and genomic sequences showed that this is also the case in rice (Oryza sativa), suggesting that a leader intron is generally present in higher plant PgiC. The intron is bounded by consensus 5'-GT and AG-3' splice sites but showed alternative splicing in Clarkia, resulting in mature transcripts that differ by 8-19 nt in length. The intron is located 18 or 10 nt upstream of the start codon in Clarkia, 2 nt upstream in Arabidopsis, and 9 nt in rice. PgiC in Clarkia was duplicated before the divergence of the extant species, many of which have two expressed genes PgiC1 and PgiC2. Full-length transcripts of both genes identified the transcription start and made it possible to identify the leader intron and leader exon (between the transcription start and leader intron) from previously obtained genomic sequences of both genes in other Clarkia species. These data permit the comparison of evolution in the leader exon and intron with the exons and introns of the coding region, a topic that has not been studied previously. Both the leader exon and the leader intron resemble introns of the coding region in base substitution rate and accumulation of gaps. But the leader intron splice junctions are not strictly conserved in position as are those of the coding region introns. Also, in base composition, the leader intron resembles the other introns, whereas the leader exon more nearly resembles the coding exons. A difference in base composition between coding exons and flanking introns is known to be important for the recognition of splice sites. Thus, the marked difference in base composition between the leader exon and leader intron is probably maintained by selection despite a high rate of sequence divergence.
5' Untranslated Regions↗
Single mutations silence PGiC2 genes in two very recent allotetraploid species of Clarkia.
Our understanding of how polyploidy influences gene evolution is limited by the fact there have been few molecular descriptions of particular genes and their expression in polyploid plants and their diploid progenitors. Here we use evidence from sequencing of genomic DNA and cDNA obtained by reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends to describe PgiC genes and their expression in two allotetraploid species of the wildflower genus Clarkia, C. delicata and C. similis. PgiC encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9) and was duplicated in the ancestral stock of Clarkia, giving rise to paralogous genes PgiC1 and PgiC2. The active form of the PGIC enzyme is a dimer of like subunits. The electrophoretic patterns in the parent species show three bands of activity, representing two homodimers and a heterodimer of intermediate mobility, and are encoded by two genes. The electrophoretic patterns in the tetraploids also show three bands, but the tetraploids were expected to have multiple PGIC isozymes encoded by four genes. Our molecular studies demonstrated that each tetraploid has two PgiC1 and two PgiC2 genes, as predicted. One gene in each of them has been silenced by a single mutation, and a functional protein is no longer produced. In C. similis, PgiC2(mod) was silenced by a mutation of a single nucleotide in exon 5 that created a stop codon. In C. delicata, a polymorphism exists between a normal allele and a defective allele of PgiC2(epi) that has a deletion of a splice junction in intron 19 that results in the synthesis of a transcript lacking an entire exon, an example of exon skipping. The three-banded PGIC electrophoretic pattern of both tetraploid species arises because isozymes encoded by two or three of the genes comigrate. A very recent origin for both tetraploids is suggested by the near identity of several of their PgiC genes to their corresponding diploid orthologues and the absence of any acceleration in mutation rates. The problem of assessing genetic redundancy in tetraploids is discussed.
Clarkia↗
The status of Clarkia australis (Onagraceae).
Clarkia australis and C. virgata grow on the western slope of the central Sierra Nevada of California. Clarkia australis was established to accommodate populations of C. virgata from south of the Tuolumne River that could not be successfully hybridized to populations north of the river. Although the species is maintained in the new Jepson Manual, its validity has been questioned because only two populations were originally tested, and they had no useful morphological traits that distinguished them from C. virgata. We report here the results of a large program of interpopulation hybridizations that show that C. australis is distinct and that its reproductive isolation from C. virgata is complete and absolute and reflects a compatibility block that apparently causes abortion of hybrid seeds in early development. Both species include populations north and south of the Tuolumne River and, in general, those of C. australis occupy higher elevations. Morphologically, the species are extremely similar though the mean values of several dimensions of the petals are different. However, significant variation among their populations has the consequence that, at present, the only certain way to assign particular populations to species is to test their compatibility with previously tested populations.
Journal Article↗
A recently silenced, duplicate PgiC locus in Clarkia.
Previous electrophoretic analysis showed that 17 diploid species of the wildflower Clarkia (Onagraceae) have two cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9), whereas 15 other diploid species have a single PGIC. Molecular studies revealed that the two isozymes in the former species are encoded by duplicate genes, PgiC1 and PgiC2, whereas the single isozyme in the latter is always encoded by PgiC1. Phylogenetic analysis of the nucleotide sequences implied that PgiC2 was silenced four times independently in the genus. Here we describe a psi PgiC2 from C. mildrediae, a species in which only PgiC1 is expressed. The discovery of the psi PgiC2 is significant because it confirms a formal prediction of the phylogenetic analysis. The psi PgiC2 includes 5,039 nucleotides corresponding to 18 of the 23 exons of PgiC, as well as the intervening introns and 3' nontranslated region. The absence of an increase of nucleotide substitutions in its "exons" suggests that the gene was silenced recently. The present study appears to be the first to establish that a specific duplicate gene locus regularly expressed in a group of related plant species has been silenced in one of them. The multiple independent silencings of PgiC2 suggest that it remained functional but inessential in ancestral lineages. We discuss the possibility that PgiC2 may have been preserved in these lineages by selection against mutants causing defective PGIC1-PGIC2 heterodimers.
Base Sequence↗
A collaborative project in Connecticut to improve the care of patients with acute myocardial infarction.
BACKGROUND: State-based peer review organizations (PROs) and individual hospitals are challenged to achieve their quality improvement (QI) goals with shrinking resources. In 1993-1994 the Connecticut PRO and 15 local hospitals generated a comparative QI database on acute myocardial infarction (AMI) care for 1,202 Medicare and non-Medicare patients discharged in 1992 and 1993. METHODS: A steering committee composed of hospital and PRO representatives was assembled to provide oversight. PRO staff developed a chart abstraction tool and trained hospital abstracters who collected and submitted data to the PRO for comparative analyses. Written feedback was provided to all hospitals and supplemented with onsite presentations when requested. Each hospital prepared a written QI plan based on its unique data profile. RESULTS: Opportunities for improvement were identified at all hospitals. The most commonly targeted areas for improvement included the use of thrombolytics at presentation, aspirin at presentation and at discharge, and beta blockers at discharge. Improvement interventions included staff education sessions, development of AMI critical paths and standing orders, and storage of appropriate medications in emergency departments. Self-report data from the hospitals indicate improvements in care. DISCUSSION: PROs and hospitals can augment their individual QI activities by working together to share data, resources, and lessons learned. Twenty-three hospitals are now collaborating with the Connecticut PRO on a similarly designed QI project aimed at improving the care of patients hospitalized with atrial fibrillation. This project includes a more formal means of communicating QI interventions.
Aged↗
A collaborative project in Connecticut to improve the care of patients with acute myocardial infarction.
BACKGROUND: State-based peer review organizations (PROs) and individual hospitals are challenged to achieve their quality improvement (QI) goals with shrinking resources. In 1993-1994 the Connecticut PRO and 15 local hospitals generated a comparative QI database on acute myocardial infarction (AMI) care for 1,202 Medicare and non-Medicare patients discharged in 1992 and 1993. METHODS: A steering committee composed of hospital and PRO representatives was assembled to provide oversight. PRO staff developed a chart abstraction tool and trained hospital abstractors who collected and submitted data to the PRO for comparative analyses. Written feedback was provided to all hospitals and supplemented with onsite presentations when requested. Each hospital prepared a written QI plan based on its unique data profile. RESULTS: Opportunities for improvement were identified at all hospitals. The most commonly targeted areas for improvement included the use of thrombolytics at presentation, aspirin at presentation and at discharge, and beta blockers at discharge. Improvement interventions included staff education sessions, development of AMI critical paths and standing orders, and storage of appropriate medications in emergency departments. Self-report data from the hospitals indicate improvements in care. DISCUSSION: PROs and hospitals can augment their individual QI activities by working together to share data, resources, and lessons learned. Twenty-three hospitals are now collaborating with the Connecticut PRO on a similarly designed QI project aimed at improving the care of patients hospitalized with atrial fibrillation. This project includes a more formal means of communicating QI interventions.
Aged↗
Molecular characterization of duplicate cytosolic phosphoglucose isomerase genes in Clarkia and comparison to the single gene in Arabidopsis.
The nucleotide sequence of PgiC1-a which encodes a cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5.
Amino Acid Sequence↗
Molecular analysis of the plant gene encoding cytosolic phosphoglucose isomerase.
The gene encoding a cytosolic isozyme of phosphoglucose isomerase (PGI, EC 5.3.1.9) was isolated from Clarkia lewisii, a wild flower native to California, and the structure and sequence of the entire coding region determined. PGI catalyzes an essential step in glycolysis and carbohydrate biosynthesis in plants. Spanning about 6 kb, the gene has 23 exons and 22 introns, the highest number yet reported in plants. The exons range in size from 43 to 156 nt and encode a protein of 569 amino acids. The protein is about 44-46% identical to the inferred protein sequences of pig, Escherichia coli and Saccharomyces cerevisiae. All of the introns are bordered with the consensus 5'-GT...AG-3' dinucleotides. The longest intron includes a large stem-loop structure bounded by a perfect 9 nt direct repeat. We cloned the PGI gene from a genomic library prepared from a single plant of known PGI genotype. The locus and allele of the clone were identified by matching restriction fragments to fragments from genetically defined genomic DNAs by Southern hybridization.
Amino Acid Sequence↗
Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.
1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.
Chloroplasts↗
Isolation and characterization of the phosphoglucose isomerase gene from Escherichia coli.
The nucleotide sequence of the gene encoding the glycolytic enzyme phosphoglucose isomerase (PGI) from Escherichia coli is presented. The gene encodes a polypeptide of 549 amino acids. The transcriptional start point of the gene was determined and found to lie within a consensus promoter region. The amino acid sequence derived from the E. coli PGI gene can be aligned without insertions or deletions to the predicted amino acid sequence of a nuclear-encoded chloroplast isozyme of PGI from a higher plant, and the two sequences have a similarity of 87.6%. The amino acid sequence similarity between E. coli and that predicted from cDNA sequences for mouse and pig PGI is approximately 65%.
Amino Acid Sequence↗
The efficacy of atenolol in the outpatient management of the alcohol withdrawal syndrome. Results of a randomized clinical trial.
We conducted a randomized, double-blind, clinical trial of atenolol compared with placebo in the outpatient management of patients with the alcohol withdrawal syndrome. In addition to receiving customary therapy, 88 patients were randomly assigned to receive atenolol and 92 to receive placebo. Outcome during the next 14 days was assessed using two main measures: the patient's clinical course as assessed by an alcohol withdrawal severity index; and the occurrence of treatment failure (composite measure including return to drinking, dropouts, and withdrawal lasting longer than 5 days). In addition, levels of craving for alcohol were assessed as an associated response variable. Overall, treatment failure occurred for 37% of the patients receiving atenolol and 52% of those receiving placebo. Among patients who had withdrawal symptoms at baseline, vital signs became normal more rapidly in the patients receiving atenolol, and their abnormal behavioral characteristics also resolved more rapidly. Levels of craving for alcohol were strongly associated with treatment failure; the group of patients who received atenolol included significantly fewer who reported high levels of craving 24 hours after treatment began (7% of patients receiving atenolol and 20% of those receiving placebo). We conclude that the outpatient management and treatment outcomes of the alcohol withdrawal syndrome are improved in patients who receive atenolol, and that the beneficial effects are associated with reduced levels of craving for alcohol. If these results are confirmed by other investigators, atenolol may prove to play an important role in the outpatient management of the alcohol withdrawal syndrome.
Adult↗
The role of beta blockers in alcohol withdrawal syndrome.
Previous studies have suggested that beta blockers might be useful in the treatment of alcohol withdrawal syndrome. A randomized, double-blind clinical trial was therefore conducted to compare results with atenolol versus those with placebo in patients hospitalized with alcohol withdrawal syndrome. In addition to receiving customary therapy, patients were randomly assigned to receive atenolol (61 patients) or placebo (59 patients). Outcome was assessed daily by the measurement of nine features in three categories: vital signs, clinical signs (eg, tremor), and behavioral signs (eg, agitation). Among patients who had withdrawal symptoms at baseline, vital signs became normal more rapidly in the patients receiving atenolol; abnormal behavior and clinical characteristics also resolved more rapidly. On each treatment day, significantly fewer patients receiving atenolol required concomitant oxazepam therapy for agitation. Patients receiving placebo, however, required a significantly higher mean daily dose of oxazepam. The results indicate that atenolol is helpful in the treatment of patients with alcohol withdrawal syndrome.
Adolescent↗
Craving and the social context: a new interaction model for enhancing recovery from alcoholism.
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Alcohol Drinking↗
Chloroplast DNA evidence for the origin of the genus Heterogaura from a species of Clarkia (Onagraceae).
Restriction-site variation in chloroplast DNA was examined in the morphologically distinct and monotypic genus Heterogaura and the related speciose genus Clarkia (Onagraceae), both native to California. Of the 605 restriction sites surveyed, a total of 119 mutations were identified. Of these, 55 were shared by at least two species and were used to construct a most parsimonious phylogenetic tree. This analysis, as well as one based on a distance metric, provided evidence that Heterogaura and Clarkia dudleyana, a member of a phylogenetically advanced section, share a more recent common ancestor than either does with any other species. The two species are more closely related than nearly all paris of Clarkia tested. The origin of Heterogaura from within another genus raises important questions about the adequacy of morphological data and suggests that the relationships of other well-known monotypic plant genera should be reinvestigated.
Journal Article↗
Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase.
Ethyl methane sulfonate treatment was used to induce a mutation in the nuclear gene encoding the chloroplast isozyme of phosphoglucose isomerase in Clarkia xantiana. The mutation, which proved allelic to wild type activity, was backcrossed to wild type for five generations so that the two could be compared in a near isogenic background. An immunological analysis showed that the mutant, when homozygous, reduced the activity of the isozyme by about 50%. In contrast to wild type, the mutant showed little change in leaf starch level over a diurnal period or following a 72-hour continuous light treatment. By the end of the diurnal light period, the mutant accumulated only about 60% as much starch as wild type. However, mutant leaves had an increased sucrose level presumably because photosynthate was directly exported from the chloroplasts. The mutant also exhibited reduced leaf weight. These changes in metabolism and growth suggest that the wild type level of plastid phosphoglucose isomerase activity is necessary to achieve wild type carbohydrate status.
Journal Article↗
Randomized clinical trial of atenolol in patients with alcohol withdrawal.
We conducted a randomized, double-blind clinical trial of atenolol as compared with placebo in the treatment of patients hospitalized with the alcohol withdrawal syndrome. In addition to receiving customary therapy, 61 patients were randomly assigned to receive atenolol, and 59 to receive placebo. Outcome was assessed daily by the measurement of nine features in three categories: vital signs, clinical signs (e.g., tremor), and behavioral signs (e.g., agitation and anxiety). Compared with placebo patients, atenolol patients had a significant reduction in the mean length of hospital stay (four as compared with five days, P less than 0.02). On each treatment day, significantly fewer patients receiving atenolol required concomitant benzodiazepines, and patients receiving placebo required a significantly higher mean daily dose of benzodiazepines. Among patients who had withdrawal symptoms at base line, vital signs became normal more rapidly in the patients receiving atenolol, and their abnormal behavior and clinical characteristics also resolved more rapidly. We conclude that atenolol is helpful in the treatment of patients with the alcohol withdrawal syndrome.
Adult↗
Nucleotide sequence of the triose phosphate isomerase gene of Escherichia coli.
We report here the complete nucleotide sequence of the E. coli triose phosphate isomerase gene. The gene encodes a polypeptide of 255 amino acids which is approximately 46% homologous to eukaryotic triose phosphate isomerases, and approximately 38% homologous to the enzyme from a thermophilic bacterium, Bacillus stearothermophilus. The nucleotide sequence is 55% homologous to that of the corresponding gene in the yeast Saccharomyces cerevisiae. To our knowledge, this is the first report of the sequence of a gene coding a glycolytic enzyme from a prokaryotic organism.
Amino Acid Sequence↗