Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli.

cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.     

rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution.

Five genes of the rbcS gene family of Solanum tuberosum (potato) were studied. One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively. The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position. These genes contain two introns, whereas the gene from locus 1 contains three introns. Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbcS could have a special function. A comparison of the rbcS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffling and then were removed stepwise during the evolution of higher plants.     

Lack of association between MnSOD gene polymorphism and sporadic Alzheimer's disease

BACKGROUND AND AIMS: Substantial evidence supports the hypothesis that impairment of mitochondrial function and increased oxidative damage are involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Manganese superoxide dismutase (MnSOD) plays a major role in protecting the mitochondrion from oxidative damage due to superoxide radicals and other excited oxygen species. Recent studies have indicated that MnSOD mRNA levels are significantly increased in the lymphocytes of AD patients, supporting the role of oxidative alterations in the pathogenetic mechanism underlying this neurodegeneration. A potentially functional amino acid polymorphism (Ala-9Val) has been described in the signal sequence of enzymes associated with decreased defense capacity against oxidative stress. The object of this study was to investigate the association between this polymorphism of the MnSOD gene and AD in the Italian population. METHODS: The Ala-9Val polymorphism was genotyped by PCR amplification and SSCP analysis in 227 AD patients and 198 healthy controls. RESULTS: No significant differences in genotype or allele frequencies between cases and controls, even after stratification for APOE carrier status, were observed. CONCLUSIONS: Our data suggest that the Ala-9Val polymorphism in the MnSOD gene is not associated with genetic susceptibility in AD patients.     

The role of manganese superoxide dismutase in health and disease

Manganese superoxide dismutase (MnSOD) is the mitochondrial enzyme that disposes of superoxide generated by respiratory chain activity. Of all electrons passing down the mitochondrial respiratory chain, 1–2% are diverted to form superoxide; thus production of hydrogen peroxide occurs at a constant rate due to MnSOD activity. Mice lacking MnSOD develop cardiomyopathy and basal ganglia lesions, have no lipid peroxidation products, but show destruction of enzymes with 4Fe–4S centres. Patients with complex I (NADH-CoQ oxidoreductase) deficiency show variable hyperinduction of MnSOD that is at least partially dependent on the extent of disturbance of redox state. This in turn appears to result in production of excess hydroxyl radicals, which are damaging to proteins, lipids and DNA. An alternative method of protection from oxygen radicals is employed by complex I-deficient cell types that do not induce MnSOD in that they show induction of the bcl-2 protein.     

Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.     

Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.

A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aap1 mit- mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membrane-associated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily.     

The plastid genome of Cryptomonas phi encodes an hsp70-like protein, a histone-like protein, and an acyl carrier protein.

The plastid genome of Cryptomonas phi, a cryptomonad alga, contains three genes that have not previously been found in any organellar genome. Each of these genes encodes a functional class of organellar gene product not previously reported. The first gene, dnaK, encodes a polypeptide of the hsp70 heat shock protein family. The predicted amino acid sequence of the DnaK protein is 54% identical to that of the Escherichia coli hsp70 protein (DnaK), 50-53% identical to that of two nucleus-encoded mitochondrial hsp70 proteins, and 43-46% identical to that of several eukaryotic cytoplasmic members of the hsp70 protein family. The second gene, hlpA, encodes a polypeptide resembling bacterial histone-like proteins. The predicted amino acid sequence of the HlpA protein is 25-53% identical to that of several bacterial histone-like proteins, and the identity increases to 39-76% over a conserved region corresponding to the long arm that binds DNA. The third gene, acpA, encodes an acyl carrier protein, which is a key cofactor in the synthesis and metabolism of fatty acids. Its predicted amino acid sequence is 36-59% identical to that of eubacterial and plant chloroplast (nucleus-encoded) acyl carrier proteins.     

Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants.

cDNA clones coding for the nuclear-encoded mitochondrial Rieske iron-sulfur protein (RISP) have been isolated from maize and tobacco. Complementation analysis of hybrid proteins consisting of different domains of plant and yeast RISPs showed that the carboxyl two-thirds of the plant protein is functionally equivalent to that of the yeast protein. The amino terminus of the RISP, however, seems to be species specific because this region is not interchangeable between plant and yeast proteins. Complementation analysis of hybrid proteins also identified a structurally conserved domain probably essential for the function of bc1 complex RISPs. A specific domain from the plant RISP was found to cause temperature-sensitive respiratory growth in yeast. We have demonstrated that yeast can serve as a model system to study the structural and functional relationships of plant gene products that are enzymatic components of the mitochondrial respiratory chain.     

Identification and characterization of a mitochondrial thioredoxin system in plants

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.     

Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R2 encoding the Mn-stabilizing protein involved in photosystem II water oxidation.

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amino acid sequence of the mature A. nidulans R2 MSP showed rather low homology (48-49%) to higher plant MSPs, but the conserved amino acid residues appeared to be clustered. Five clusters were tentatively assigned, in which the homology values were in a range of 66-70%. Domains essential for the functioning of MSP are expected to be situated in these clusters. It is of note that the two cysteine residues in MSP were conserved, and the disulfide linkage between them may play an important role in maintaining the tertiary structure of MSP.     

Differential induction of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes in Arabidopsis thaliana by wounding and pathogenic attack.

We have isolated cDNAs from two distinct genes encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) in Arabidopsis thaliana. Predicted protein sequences from both genes, DHS1 and DHS2, and a potato DAHP synthase gene are highly related, but none shows significant sequence similarity to conserved microbial DAHP synthase proteins. Despite this structural difference, the DHS1 cDNA complements mutations in a yeast strain lacking DAHP synthase activity. DHS1 RNA levels increase in Arabidopsis leaves subjected either to physical wounding or to infiltration with pathogenic Pseudomonas syringae strains. DHS2 RNA levels are not increased by these treatments, suggesting that the DHS1 and DHS2 proteins fulfill different physiological functions. Other enzymes in the Arabidopsis aromatic pathway are also encoded by duplicated genes, an arrangement that may allow independent regulation of aromatic amino acid biosynthesis by distinct physiological requirements such as protein synthesis and secondary metabolism. The presence of amino-terminal extensions characteristic of chloroplast transit peptides on DHS1 and DHS2 suggests that both proteins may be targeted to the chloroplast.     

Nucleotide Sequence and Genomic Structure of the Chicken Insulin-like Growth Factor-II (IGF-II) Coding Region

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide with structural similarity to insulin that appears to be involved in regulating embryonic growth. While mammalian IGF-II proteins and IGF-II genes have been well characterized, little is known about avian IGF-II proteins and the genomic sequences that encode them. In this paper we report the isolation and characterization of chicken IGF-II cDNA and genomic clones. The predicted chicken prepro-IGF-II protein contains 187 residues and comprises a signal peptide (24 residues), IGF-II peptide (67 residues), and C-terminal peptide (96 residues), which share 33, 82, and 25/28% amino acid sequence identity, respectively, with their rat/human counterparts. Like the human and rat IGF-II genes, the chicken IGF-II gene has three coding exons. These are interrupted by introns at similar positions to those found in the human and rat IGF-II genes.     

Multivariate analysis of maize disease resistances suggests a pleiotropic genetic basis and implicates a GST gene

Plants are attacked by pathogens representing diverse taxonomic groups, such that genes providing multiple disease resistance (MDR) are expected to be under positive selection pressure. To address the hypothesis that naturally occurring allelic variation conditions MDR, we extended the framework of structured association mapping to allow for the analysis of correlated complex traits and the identification of pleiotropic genes. The multivariate analytical approach used here is directly applicable to any species and set of traits exhibiting correlation. From our analysis of a diverse panel of maize inbred lines, we discovered high positive genetic correlations between resistances to three globally threatening fungal diseases. The maize panel studied exhibits rapidly decaying linkage disequilibrium that generally occurs within 1 or 2 kb, which is less than the average length of a maize gene. The positive correlations therefore suggested that functional allelic variation at specific genes for MDR exists in maize. Using a multivariate test statistic, a glutathione S-transferase (GST) gene was found to be associated with modest levels of resistance to all three diseases. Resequencing analysis pinpointed the association to a histidine (basic amino acid) for aspartic acid (acidic amino acid) substitution in the encoded protein domain that defines GST substrate specificity and biochemical activity. The known functions of GSTs suggested that variability in detoxification pathways underlie natural variation in maize MDR.     

Intron conservation across the prokaryote-eukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.

The nuclear gene encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from maize has been cloned and sequenced. The gene is G + C rich in its coding sequences and, in addition, contains a CpG-rich region surrounding the promoter. Further upstream several enhancer-like repetitions have been identified that may control the light- and phytochrome-mediated expression of this gene. The gene is interrupted by three introns. Introns 1 and 2 are located within the sequence encoding the transit peptide, dividing it into three parts, each containing one of the three major homology blocks typical for transit peptides of nucleus-encoded chloroplast proteins. Intron 3 is located at codon 166 (glycine) at the same nucleotide position as intron 1 in the GAPDH gene from the nematode Caenorhabditis elegans, suggesting that this intron was present in the parental GAPDH gene from which these two modern descendants originated. Intron 3 divides the GAPDH protein into its two constituent domains, the NAD-binding and the catalytic domain, immediately after helix alpha 1 at a position homologous to that of intron 9 in the gene for maize alcohol dehydrogenase, thereby confirming the prediction of Br?ndén et al. on the basis of gene-protein structure correlations in maize alcohol dehydrogenase for the placement of introns in the GAPDH gene [Br?ndén, C.I., Eklund, H., Cambillau, C. & Pryor, A.J. (1984) EMBO J. 3, 1307-1310]. These results suggest that intron 3 is an archetypical relic of early GAPDH and alcohol dehydrogenase evolution, whereas introns 1 and 2 were implicated in the evolution of chloroplast transit peptides.