Darwinian evolution across the disciplines.
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Animals↗
Biomedical subjects
C R McClung
Publications and source records attributed to C R McClung.
Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase.
The photorespiratory pathway is comprised of enzymes localized within three distinct cellular compartments: chloroplasts, peroxisomes, and mitochondria. Photorespiratory enzymes are encoded by nuclear genes, translated in the cytosol, and targeted into these distinct subcellular compartments. One likely means by which to regulate the expression of the genes encoding photorespiratory enzymes is coordinated temporal control. We have previously shown in Arabidopsis that a circadian clock regulates the expression of the nuclear genes encoding both chloroplastic (Rubisco small subunit and Rubisco activase) and peroxisomal (catalase) components of the photorespiratory pathway. To determine whether a circadian clock also regulates the expression of genes encoding mitochondrial components of the photorespiratory pathway, we characterized a family of Arabidopsis serine hydroxymethyltransferase (SHM) genes. We examined mRNA accumulation for two of these family members, including one probable photorespiratory gene (SHM1) and a second gene expressed maximally in roots (SHM4), and show that both exhibit circadian oscillations in mRNA abundance that are in phase with those described for other photorespiratory genes. In addition, we show that SHM1 mRNA accumulates in light-grown seedlings, although this response is probably an indirect consequence of the induction of photosynthesis and photorespiration by illumination.
Amino Acid Sequence↗
Intron loss and gain during evolution of the catalase gene family in angiosperms.
Angiosperms (flowering plants), including both monocots and dicots, contain small catalase gene families. In the dicot, Arabidopsis thaliana, two catalase (CAT) genes, CAT1 and CAT3, are tightly linked on chromosome 1 and a third, CAT2, which is more similar to CAT1 than to CAT3, is unlinked on chromosome 4. Comparison of positions and numbers of introns among 13 angiosperm catalase genomic sequences indicates that intron positions are conserved, and suggests that an ancestral catalase gene common to monocots and dicots contained seven introns. Arabidopsis CAT2 has seven introns; both CAT1 and CAT3 have six introns in positions conserved with CAT2, but each has lost a different intron. We suggest the following sequence of events during the evolution of the Arabidopsis catalase gene family. An initial duplication of an ancestral catalase gene gave rise to CAT3 and CAT1. CAT1 then served as the template for a second duplication, yielding CAT2. Intron losses from CAT1 and CAT3 followed these duplications. One subclade of monocot catalases has lost all but the 5'-most and 3'-most introns, which is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a fully spliced mRNA. Following this event of concerted intron loss, the Oryza sativa (rice, a monocot) CAT1 lineage acquired an intron in a novel position, consistent with a mechanism of intron gain at proto-splice sites.
Arabidopsis Proteins↗
Imbibition, but not release from stratification, sets the circadian clock in Arabidopsis seedlings.
Circadian rhythms in the abundance of the CAT2 catalase mRNA were not seen in etiolated seedlings but developed upon illumination. These circadian oscillations were preceded by a rapid and transient induction of CAT2 mRNA abundance that varied strikingly according to the timing (circadian phase) of the onset of illumination. This variation oscillated with a circadian periodicity of approximately 28 hr, indicating that the circadian oscillator is running in etiolated seedlings and regulates (gates) the induction of CAT2 by light. Moreover, because we assayed populations of seedlings, we infer that the individual clocks among populations of etiolated seedlings were synchronized before the onset of illumination. What developmental or environmental signals synchronized the clocks among seedlings? Varying the phase of the onset of illumination relative to release from stratification failed to affect the acute induction of CAT2, indicating that the temperature step from 4 to 22 degrees C associated with release from stratification did not reset the circadian clock. However, the acute induction of CAT2 mRNA varied with time after imbibition, demonstrating that imbibition provides a signal capable of resetting the circadian clock and of synchronizing the clocks among populations of seedlings.
Arabidopsis↗
Regulation of catalases in Arabidopsis.
The catalase multi-gene family in Arabidopsis includes three genes encoding individual subunits which associate to form at least six isozymes that are readily resolved by non-denaturing gel electrophoresis. CAT1 and CAT3 map to chromosome 1, and CAT2 maps to chromosome 4. The nucleotide and deduced amino acids sequences of the three coding regions are highly related to each other and to other catalases. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. All three mRNAs are highly expressed in inflorescences, and CAT2 and CAT3 are highly expressed in leaves. All three mRNAs are detectable in freshly imbibed seeds, although the pattern of mRNA relative abundance varies among the three genes during early germination. CAT1 and CAT2 mRNA abundance is induced by light. In contrast, CAT3 is negatively light-responsive. CAT2 and CAT3 mRNA abundance is controlled by the circadian clock. Interestingly, the peak in CAT3 mRNA abundance occurs in the subjective evening, which is out of phase with expression of the Arabidopsis CAT2 catalase gene that shows clock-regulated expression gated to the subjective early morning. CAT1 mRNA abundance is not clock-regulated.
Arabidopsis↗
The circadian clock gates expression of two Arabidopsis catalase genes to distinct and opposite circadian phases.
In Arabidopsis thaliana, catalase is encoded by a small gene family. We have characterized cDNA and genomic clones containing the Arabidopsis catalase gene CAT3, present as a single copy in the nuclear genome. Six introns were identified in the CAT3 coding region and two transcription start sites have been been mapped by primer extension. The deduced amino acid sequence of CAT3 is highly similar to other catalases. CAT3 expression is similar in seedlings germinated and grown either in continuous light or in continuous dark, suggesting that CAT3 expression in seedlings is not light responsive. CAT3 expression is controlled by the circadian clock; in 5-week-old plants grown on a light-dark cycle and then transferred to continuous light, robust oscillations in CAT3 mRNA abundance with circadian period persist for at least five circadian cycles. Interestingly, the peak in CAT3 mRNA abundance occurs in the subjective evening, which is out of phase with expression of the Arabidopsis CAT2 catalase gene, which shows clock-regulated expression gated to the subjective early morning.
Amino Acid Sequence↗
Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh.
The catalase multigene family in Arabidopsis includes three genes encoding individual subunits that associate to form at least six isozymes that are readily resolved by nondenaturing gel electrophoresis. CAT1 and CAT3 map to chromosome 1, and CAT2 maps to chromosome 4. The nucleotide sequences of the three coding regions are 70 to 72% identical. The amino acid sequences of the three catalase subunits are 75 to 84% identical and 87 to 94% similar, considering conservative substitutions. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. Similarly, mRNA abundance of the three genes varies among organs. All three mRNAs are highly expressed in bolts, and CAT2 and CAT3 are highly expressed in leaves.
Amino Acid Sequence↗
Identification of an Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA) minimal promoter regulated by light and the circadian clock.
Transcription of the Arabidopsis thaliana gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is organ-specific, light-responsive, and regulated by the circadian clock. RCA is transcribed throughout the green parts of the plant, but not in roots and petals. Responses elicited by short pulses of light indicate that the light response is mediated, at least in part, by phytochrome. Analysis of transgenic tobacco and Arabidopsis carrying RCA 5' untranscribed regions fused to reporter genes (uidA, encoding beta-glucuronidase, or cat, encoding chloramphenicol acetyltransferase) indicate that elements sufficient to confer organ-specific, light-responsive, and clock-regulated transcription are localized within 317 base pairs upstream of the site of transcription initiation. A clock-responsive element sufficient to confer a low-amplitude (approximately 2-fold) circadian oscillation lies within 317 base pairs of the trascription start, but other elements necessary for high-amplitude (approximately 10-fold) circadian oscillation lie upstream of -317 and are removed by deletion from -970 to -317.
Arabidopsis↗
Molecular characterization of the proline-1 (pro-1) locus of Neurospora crassa, which encodes delta 1-pyrroline-5-carboxylate reductase.
delta 1-pyrroline-5-carboxylate reductase (P5CR; [L-proline: NAD(P+) 5-oxidoreductase]; EC 1.5.1.2) catalyzes the final step in proline biosynthesis. We have shown that the proline-1 (pro-1) locus of Neurospora crassa encodes P5CR. The pro-1 gene was localized to a 3.2 kb region by complementation of (restoration of proline-independent growth to) a proline auxotroph carrying a recessive mutation at the pro-1 locus. The nucleotide sequence of this 3.2 kb region contains an open reading frame with coding capacity of 311 amino acids. The deduced polypeptide shows significant similarity to P5CR amino acid sequences. Similarity of N. crassa P5CR is greatest to that of the yeast, Saccharomyces cerevisiae, but is also strong to P5CR sequences from archaea, eubacteria, plants, and humans. In N. crassa, amino acid imbalance, including deficiency or excess of a single amino acid, such as histidine, induces expression of many amino acid biosynthetic genes that are under cross-pathway control, a general regulatory system analogous to general amino acid control in Saccharomyces. Although P5CR catalyzes the only committed step in proline biosynthesis, pro-1 expression was unaltered by histidine starvation and independent of CPC1, a positively acting transcription factor that mediates cross-pathway control in N. crassa.
Alleles↗
Correlation of the physical and genetic maps of the centromeric region of the right arm of linkage group III of Neurospora crassa.
We have cloned three linked genes serine-1 (ser-1), proline-1 (pro-1) and acetate-2 (ace-2) that lie near the centromere on the right arm of linkage group III (LGIIIR) of Neurospora crassa. The ser-1 gene was cloned by sib selection. A chromosomal walk that spans 205 kilobases (kb) was initiated from ser-1. Complementation analysis with clones isolated during the walk allowed identification of the pro-1 and ace-2 genes. Restriction fragment length polymorphism analysis has confirmed the localization of ser-1, pro-1 and ace-2 to the centromeric region of LGIIIR. Genetically, we measured 1% recombination between ser-1 and pro-1 and 2% recombination between pro-1 and ace-2. Physical distances for these intervals were 114 kb from ser-1 to pro-1 and 36 kb from pro-1 to ace-2. Thus, for the pro-1 to ace-2 interval we calculate a physical/genetic correlation of 18 kb/map unit (mu) whereas, in the immediately adjacent, centromere-proximal interval from ser-1 to pro-1, we calculate 114 kb/mu. This provides evidence for a centromere effect, a decrease in recombination frequency as one approaches the centromere.
Centromere↗
Circadian and light-regulated expression of nitrate reductase in Arabidopsis.
The expression of a number of plant genes is regulated by an endogenous circadian clock. We report that the Arabidopsis NIA2 (nitrate reductase) gene shows robust circadian oscillations in mRNA accumulation which persist for at least 5 days in plants that have been grown in a light-dark (LD) cycle and then transformed to continuous light (LL). We further show that NIA2 mRNA accumulation oscillates in a circadian fashion in plants that have been grown in LD and then transferred to continuous darkness (DD). Results from nuclear run-on transcriptional analysis suggest that the oscillations in steady-state levels of NIA2 mRNA abundance are not primarily due to changes in transcription but, instead, reflect post-transcriptional regulation. The circadian oscillations in NIA2 mRNA abundance are paralleled by circadian oscillations in nitrate reductase enzyme activity (NR activity) in Arabidopsis plants that have been grown in LD and then transferred either to DD or to LL. Etiolated Arabidopsis seedlings express neither NIA2 mRNA nor NR activity. However, both NIA2 mRNA accumulation and NR activity are induced by exposure to white light. The inductive effects of light on NIA2 mRNA accumulation are due, at least in part, to a very low fluence phytochrome-mediated response. However, the persistence of circadian oscillations in NIA2 mRNA abundance for at least 5 days in LL demonstrates that the circadian clock is capable of overriding or gating the inductive effects of light on NIA2 mRNA accumulation in Arabidopsis for an extended, continuous period of time.
Arabidopsis↗
Molecular basis of the ribulose-1,5-bisphosphate carboxylase/oxygenase activase mutation in Arabidopsis thaliana is a guanine-to-adenine transition at the 5'-splice junction of intron 3.
Analysis of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene and gene products from Arabidopsis thaliana wild-type plants and the Rubisco activase-deficient mutant strain showed that the rca mutation caused GT to be changed to AT at the 5'-splice junction of intron 3 in the six-intron pre-mRNA. Northern blot analysis, genomic and cDNA sequencing, and primer extension analysis indicated that the mutation causes inefficient and incomplete splicing of the pre-mRNA, resulting in the accumulation of three aberrant mRNAs. One mutant mRNA was identical with wild-type mRNA except that it included intron 3, a second mRNA comprised intron 3 and exons 4 through 7, and the third mRNA contained exons 1 through 3. The G-to-A transition is consistent with the known mechanism of mutagenesis by ethyl methanesulfonate, the mutagen used to create the Rubisco activase-deficient strain.
Amino Acid Sequence↗
Characterization of the formate (for) locus, which encodes the cytosolic serine hydroxymethyltransferase of Neurospora crassa.
Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine. We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT. The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate. The for+ gene encodes a polypeptide of 479 amino acids which shows significant similarity to amino acid sequences of SHMT from bacterial and mammalian sources (47 and 60% amino acid identity, respectively). The for+ mRNA has several different start and stop sites. The abundance of for+ mRNA increased in response to amino acid imbalance induced by glycine supplementation, suggesting regulation by the N. crassa cross-pathway control system, which is analogous to general amino acid control in Saccharomyces cerevisiae. This was confirmed by documenting that for+ expression increased in response to histidine limitation (induced by 3-amino-1,2,4-triazole) and that this response was dependent on the presence of a functional cross-pathway control-1 (cpc-1) gene, which encodes CPC1, a positively acting transcription factor. There are at least five potential CPC1 binding sites upstream of the for+ transcriptional start, as well as one that exactly matches the consensus CPC1 binding site in the first intron of the for+ gene.
Amino Acid Sequence↗
The Neurospora clock gene frequency shares a sequence element with the Drosophila clock gene period.
The isolation and characterization of single gene mutations affecting the circadian biological clocks of several organisms has left little doubt that circadian rhythms can be subjected to classical genetical analysis. Many of these mutations occur at the same few genetic loci (frequency (frq) in the fungus Neurospora, and period (per) in fruit fly Drosophila); these loci represent the best studied clock-affecting genes known. Mutant strains are usually affected in more than one basic clock property, suggesting an inter-relatedness at the molecular level among these basic properties that would not have been predicted a priori. The extensive background information available concerning the frq locus provides a basis for the molecular dissection of the Neurospora circadian clock--the most minimal circadian system thus far described. We report here the cloning and analysis of the frq locus and show it to be larger and more complex than would have been predicted from the available genetic data. Complete rescue of all of the pleiotropic mutant phenotypes of the recessive frq allele requires transformation with a 7.7-kilobase (kb) region of DNA encoding at least two transcripts. Sequence analysis of this region has allowed the identification of a common element between frq and per which, given the background similarities in their classical genetic characteristics, suggests the possibility of a common element in the clock mechanisms of these two organisms.
Animals↗
Structure of the Bradyrhizobium japonicum gene hemA encoding 5-aminolevulinic acid synthase.
The nucleotide (nt) sequence of the hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS) from the bacterium Bradyrhizobium japonicum, is presented. This sequence predicts a protein of 408 amino acids (aa) with an Mr of 44,599. This predicted amino acid sequence is highly homologous to that of the chicken embryonic liver ALAS, exhibiting a 48.8% identical amino acid sequence over the entire length of the bacterial protein. A single mRNA start point was demonstrated by S1 protection analysis for the B. japonicum hemA. The 5' end of the transcript is 100 nt upstream from the start codon. The sequence of the promoter region has some sequence homology to the -35 nt region of the Escherichia coli consensus promoter sequence but not to the -10 nt region. There is also one block of 9 nt found in both the B. japonicum glnA and hemA promoter regions.
5-Aminolevulinate Synthetase↗
A genetic locus essential for formate-dependent growth of Bradyrhizobium japonicum.
A genetic locus essential for the formate-dependent growth of Bradyrhizobium japonicum was isolated by complementation of ethyl methanesulfonate-induced mutants with a cosmid gene library of B. japonicum DNA. Three related cosmids containing 18.7 kilobase pairs of B. japonicum DNA in common were identified as being able to restore formate-dependent growth capability to mutants lacking either ribulosebisphosphate carboxylase or both ribulosebisphosphate carboxylase and phosphoribulokinase activities. To further localize the complementing gene(s), a series of four deletions spanning a total of 16.1 kilobase pairs were introduced into the B. japonicum chromosome. Each resulting deletion mutant lacked formate dehydrogenase activity and lacked ribulosebisphosphate carboxylase activity and immunologically detectable protein. Three of the four also lacked phosphoribulokinase activity. Two other mutants in which the deletion-bearing recombinant plasmid had integrated into the chromosome also lacked ribulosebisphosphate carboxylase activity and protein and phosphoribulokinase activities. The genetic locus defined by these mutants could contain the structural genes for these enzymes or a regulatory gene(s) controlling their expression or both.
Cloning, Molecular↗
Physical organization of the Bradyrhizobium japonicum nitrogenase gene region.
In Bradyrhizobium japonicum USDA 110 the three genes that encode the nitrogenase enzyme complex are separated into two transcription units, nifH and nifDK. We have physically mapped a 33-kilobase-pair region of the B. japonicum genome that contains both nifH and nifDK. The nifDK operon is located transcriptionally upstream from nifH, and all three genes are transcribed in the same direction. Within the 20-kilobase-pair region that separates the promoters for these two transcription units, we have identified a region homologous to the Klebsiella pneumoniae nifA gene. This nifA homology is situated about 6 kilobase pairs upstream from the nifH transcriptional initiation signal, in an analogous position to the nifA-like locus previously described for Rhizobium meliloti. In addition, we have characterized a second distinct nifA homology in B. japonicum that is not directly linked to the nitrogenase structural gene region.
Base Sequence↗
Enumeration and Localization of N(2)-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel.
Numbers and possible locations of N(2)-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N(2)-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N(2)-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N(2)-fixing bacteria recoverable from the rhizoplane, although high numbers of N(2)-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N(2) fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.
Journal Article↗