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.     

Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins

Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.     

Mouse genes encoding DNA topoisomerase I

We have initiated a genetic analysis of the physiologically important enzyme type I DNA topoisomerase in mouse. The exon-intron structures of the 5 part and the 3 part of the active gene, Top-1, were determined and shown to be quite similar to those of the previously determined human gene TOP1. The active mouse gene was mapped to the distal Chromosome (Chr) 2. In addition, the mouse genome contains one truncated processed topoisomerase-I-related pseudogene (retroposon), Top-1ps, on Chr 16. The Top-1ps locus, together with the immunoglobulin-lambda-light-chain locus, defines and additional conserved linkage group common to murine Chr 16 and human Chr 22, the site of the human pseudogene TOP1P2. The mapping data suggest that the pseudogene was established before mammalian radiation. Structural features, shared by the mouse and the human pseudogene, support this possibility.     

Analysis of Arabidopsis genes encoding putative class III lipases

Triacylglycerol lipases are class of enzymes which catalyze the hydrolysis of long-chain triglycerides. They are widely found in the plant kingdom, numerous genes putatively encoding triacylglycerol lipases are sequenced but only a few of them have been characterized. Here we systematically analyzed Arabidopsis gene sequences deposited in public databases, and identified 38 putative class III lipase proteins, all of which contain a highly conserved lipase_3 domain (Pfam ID: PF01764). These 38 genes are randomly distributed on all chromosomes, and their genomic sequences consist of variable numbers of introns from zero to 13. They can be divided into four groups based on homology of protein sequences, and their potential subcellular localization is predicted to cytosol, chloroplast, mitochondria or endoplasmic reticulum. Furthermore, ten typical genes are selected to investigate their expression patterns. Most of them show weak tissue- or organ-specificity expression pattern. Several of them significantly accumulates in some tissues or organs in addition to germinated seedlings. Some of them are specifically transcribed during seed germination while others are not detected during stages of normal growth which are probably induced by stresses. In conclusion, putative Arabidopsis class III lipases display polymorphism in their sequences, gene structures and expression patterns.     

Differential expression of two paralogous genes of Bacillus subtilis encoding single-stranded DNA binding protein

The Bacillus subtilis genome comprises two paralogous single-stranded DNA binding protein (SSB) genes, ssb and ywpH, which show distinct expression patterns. The main ssb gene is strongly expressed during exponential growth and is coregulated with genes encoding the ribosomal proteins S6 and S18. The gene organization rpsF-ssb-rpsR as observed in B. subtilis is found in many gram-positive as well as some gram-negative bacteria, but not in Escherichia coli. The ssb gene is essential for cell viability, and like other SSBs its expression is elevated during SOS response. In contrast, the paralogous ywpH gene is transcribed from its own promoter at the onset of stationary phase in minimal medium only. Its expression is ComK dependent and its gene product is required for optimal natural transformation.     

Arabidopsis genes encoding components of the chloroplastic protein import apparatus

The process of protein import into plastids has been studied extensively using isolated pea (Pisum sativum) chloroplasts. As a consequence, virtually all of the known components of the proteinaceous apparatus that mediates import were originally cloned from pea. With the recent completion of the Arabidopsis genome sequencing project, it is now possible to identify putative homologs of the import components in this species. Our analysis has revealed that Arabidopsis homologs with high sequence similarity exist for all of the pea import complex subunits, making Arabidopsis a valid model for further study of this system. Multiple homologs can be identified for over one-half of the components. In all but one case it is known that more than one of the putative isoforms for a particular subunit are expressed. Thus, it is possible that multiple types of import complexes are present within the same cell, each having a unique affinity for different chloroplastic precursor proteins, depending upon the exact mix of isoforms it contains. Sequence analysis of the putative Arabidopsis homologs for the chloroplast protein import apparatus has revealed many questions concerning subunit function and evolution. It should now be possible to use the genetic tools available in Arabidopsis, including the generation of knockout mutants and antisense technology, to address these questions and learn more about the molecular functions of each of the components during the import process.     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

Characterization of two genes encoding ferritin-like protein in Actinobacillus actinomycetemcomitans.

Two genes encoding ferritin-like protein, designated afnA and afnB, were identified in the upstream region of actX on the Actinobacillus actinomycetemcomitans chromosomal DNA. The actX has been reported to be a regulatory gene homologous to the Escherichia coli fnr, which controls the growth and virulence of A. actinomycetemcomitans under anaerobic conditions. The afnB located 340 bp-upstream from the actX, and the afnA located just 15 bp-upstream from afnB. The afnA and afnB encoded 161 and 165 amino acid residues, respectively, which were similar to ferritin-like proteins of other microorganisms. Western immunoblotting using rabbit antiserum against E. coli ferritin showed these two proteins, which are reactive with the serum with 19-kDa molecular masses, are produced from A. actinomycetemcomitans. The N-terminal amino acid sequences of the two proteins were consequent with those deduced from afnA and afnB. Northern hybridization revealed that the afnA and afnB constituted a bicistronic operon and the accumulation of afnA and afnB mRNA was upregulated under aerobic conditions. These findings suggested that the operon was regulated by the presence of oxygen. The two ferritin-like proteins may have important roles in the adaptation of A. actinomycetemcomitans to oxidative environmental changes.     

The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent

Many eukaryotic genomes have experienced ancient whole-genome duplication (WGD) followed by massive gene loss. These eliminations were not random since some gene families were preferentially retained as duplicates. The gene balance hypothesis suggests that those genes with dosage reduction can imbalance their interacting partners or complex, resulting in decreased fitness. In Arabidopsis, the cytoplasmic ribosomal proteins (RP) are encoded by gene families with at least two members. We have focused our study on the two RPS6 genes in an attempt to understand why they have been retained as duplicates. We demonstrate that RPS6 function is vital for the plant. We also show that reducing the level of RPS6 accumulation (in the knock-out rps6a or rps6b single mutants, or in the double heterozygous RPS6A/rps6a,RPS6B/rps6b), confers a slow growth phenotype (haplodeficiency). Importantly, we demonstrate that the functions of two RPS6 genes are redundant and interchangeable. Finally, like in most other described Arabidopsis rp mutants, we observed that a reduced RPS6 level slightly alters the dorsoventral leaf patterning. Our results support the idea that the Arabidopsis RPS6 gene duplicates were evolutionarily retained in order to maintain an expression level necessary to sustain the translational demand of the cell, in agreement with the gene balance hypothesis.