Ionising radiation induces the expression of PARP-1 and PARP-2 genes in Arabidopsis.

By screening for Arabidopsis genes activated by ionising radiation (IR)-induced DNA damage, we have isolated a cDNA hybridising with a 3.2-kb mRNA that accumulates rapidly and strongly in irradiated cell suspensions or whole plants. The cDNA codes for a 110-kDa protein that is highly homologous to the 116-kDa vertebrate poly(ADP-ribose) polymerase (PARP-1). It is recognised by a human anti-PARP-1 antibody, binds efficiently to DNA strand interruptions in vitro, and catalyses DNA damage-dependent (ADP-ribose) polymer synthesis. We have named this protein AtPARP-1. We have also extended our observations to the Arabidopsis app (AtPARP-2) gene, demonstrating for the first time that IR-induced DNA strand interruptions induce rapid and massive accumulation of AtPARP-1 and AtPARP-2 transcripts, whereas dehydration and cadmium preferentially induce the accumulation of AtPARP-2 transcripts. The IR-induced PARP gene expression seen in Arabidopsis is in striking contrast to the post-translational activation of the PARP-1 protein that is associated with genotoxic stress in animal cells. AtPARP-1 transcripts accumulate in all plant organs after exposure to ionising radiation, but this is followed by an increase in AtPARP-1 protein levels only in tissues that contain large amounts of actively dividing cells. This cell-type specific accumulation of AtPARP-1 protein in response to DNA damage is compatible with a role for the AtPARP-1 protein in the maintenance of DNA integrity during replication, similar to the role of "guardian of the genome" attributed to its animal counterpart.

Lipid phosphate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in response to stress.

An Arabidopsis thaliana gene (AtLPP1) was isolated on the basis that it was transiently induced by ionizing radiation. The putative AtLPP1 gene product showed homology to the yeast and mammalian lipid phosphate phosphatase enzymes and possessed a phosphatase signature sequence motif. Heterologous expression and biochemical characterization of the AtLPP1 gene in yeast showed that it encoded an enzyme (AtLpp1p) that exhibited both diacylglycerol pyrophosphate phosphatase and phosphatidate phosphatase activities. Kinetic analysis indicated that diacylglycerol pyrophosphate was the preferred substrate for AtLpp1p in vitro. A second Arabidopsis gene (AtLPP2) was identified based on sequence homology to AtLPP1 that was also heterologously expressed in yeast. The AtLpp2p enzyme also utilized diacylglycerol pyrophosphate and phosphatidate but with no preference for either substrate. The AtLpp1p and AtLpp2p enzymes showed differences in their apparent affinities for diacylglycerol pyrophosphate and phosphatidate as well as other enzymological properties. Northern blot analyses showed that the AtLPP1 gene was preferentially expressed in leaves and roots, whereas the AtLPP2 gene was expressed in all tissues examined. AtLPP1, but not AtLPP2, was regulated in response to various stress conditions. The AtLPP1 gene was transiently induced by genotoxic stress (gamma ray or UV-B) and elicitor treatments with mastoparan and harpin. The regulation of the AtLPP1 gene in response to stress was consistent with the hypothesis that its encoded lipid phosphate phosphatase enzyme may attenuate the signaling functions of phosphatidate and/or diacylglycerol pyrophosphate that form in response to stress in plants.

Molecular cloning and developmental expression of AtGR1, a new growth-related Arabidopsis gene strongly induced by ionizing radiation.

Screening for mRNAs that accumulate after DNA damage induced by ionizing radiation, we have isolated a 2.0-kb cDNA coding for a new Arabidopsis PEST-box protein named AtGR1 (A. thaliana gamma response 1) with an expression profile similar to that observed for several plant cell cycle-related proteins. Using an anti-AtGR1 antibody, we have shown that the AtGR1 protein is expressed at basal levels in mitotically dividing cells (meristematic tissues and organ primordia) and at a strongly enhanced level in endoreduplicating cells (stipules, trichomes). Using transgenic Arabidopsis plants that express the GUS reporter gene under the control of the AtGR1 promoter, we have demonstrated that the observed AtGR1 protein distribution is due to the promoter activity. Our results suggest that basal AtGR1 levels are associated with progression through mitosis, whereas elevated intracellular levels of AtGR1 seem to induce changes between the S and M phases of the cell cycle that trigger somatic cells to enter the endoreduplication cycle. Ionizing radiation-induced rapid and dose-dependent accumulation of AtGR1 mRNA in cell cultures and plant tissues leads to tissue-specific accumulation of AtGR1 protein, best observed in ovules, which never undergo an endoreduplication cycle. It therefore appears that the radiation-induced transient AtGR1 accumulation reflects DNA damage-dependent transient cell cycle arrest before mitosis, which is necessary to accomplish DNA repair prior to chromosome segregation and cytokinesis.

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells.

Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.

Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5.

Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1 gene. ATR1 encodes a protein of 692 amino acids, while ATR2 encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the choice of the initiation codon. Comparative analysis of ATR1 and ATR2-1 indicates 64% amino acid sequence identity and the absence of conservation in the third base of conserved amino acid codons. The two Arabidopsis reductases are encoded by distinct genes whose divergence is expected an early event in angiosperms evolution. A poly(Ser/Thr) stretch reminiscent of a plant chloroplastic targeting signal is present at the ATR2-1 N-terminal end but absent in ATR1. The cDNA open reading frames were expressed in yeast. The recombinant polypeptides were found present in the yeast endoplasmic reticulum membrane and exhibited a high specific NADPH-cytochrome c reductase activity. To gain more insight into the respective functions of the two reductases, the Arabidopsis cDNA encoding cinnamate 4-hydroxylase (CYP73A5) was cloned and co-expressed with ATR1 or ATR2 in yeast. Biochemical characterization of the Arabidopsis ATR1/CYP73A5 and ATR2-1/CYP73A5 systems demonstrates that the two distantly related Arabidopsis reductases similarly support the first oxidative step of the phenylpropanoid general pathway.

Catalytic properties of the plant cytochrome P450 CYP73 expressed in yeast. Substrate specificity of a cinnamate hydroxylase.

The catalytic properties of CYP73, a cinnamate 4-hydroxylase isolated from Helianthus tuberosus tuber [Teutsch, H. G., Hasenfratz, M. P., Lesot, A., Stoltz, C., Garnier, J. M., Jeltsch, J. M., Durst, F. & Werck-Reichhart, D. (1993) Proc. Natl Acad. Sci. USA 90, 4102-4106] and expressed in an optimised yeast system [Urban, P., Werck-Reichart, D., Teutsch, G. H., Durst, F., Regnier, S., Kazmaier, M. & Pompon, D. (1994) Eur. J. Biochem. 222, 843-850] have been investigated. Microsomes from transformed yeast catalysed trans-cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p-coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism-based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.

Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast. Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway.

Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisiae strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced iron/carbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data.

DNA binding properties of the LexA repressor.

The LexA repressor from Escherichia coli negatively regulates the transcription of about 20 different genes upon binding with variable affinity to single-, double- or even triple-operators as in the case of the recN gene. Binding of LexA to multiple operators is cooperative if the spacing between these operators is favorable. LexA recognizes DNA via its amino-terminal domain. The three-dimensional structure of this domain has been determined by NMR measurements. It contains three alpha-helices spanning residues 8-20, 28-35 and 41-54. In view of this structure, but also according to homology considerations and the unusual contact pattern with the DNA backbone, the LexA repressor is not a normal helix-turn-helix DNA binding protein like for example phage lambda repressor. LexA is at best a distant relative of this class of transcription factors and should probably be considered as a protein that contains a new DNA binding motif. A cluster of LexA mutant repressors deficient in DNA binding falling into the third helix (residues 41-54 bp) suggests that this helix is involved in DNA recognition.

In vitro assembly of U1 snRNPs.

An efficient system for the in vitro assembly of U1 snRNPs is described. RNA-protein interactions in a series of U1 snRNA mutants assembled both in vivo and in vitro were studied in order to verify the accuracy of the system. Two discrete protein binding sites are defined by immunoprecipitation with antibodies against different protein components of the U1 snRNP and a newly developed protein sequestering assay. The U1 snRNP-specific proteins 70K and A require only the 5'-most stem-loop structure of U1 snRNA for binding, the common U snRNP proteins require the conserved Sm binding site (AUnG). Interactions between these two groups of proteins are detected. These results are combined to derive a model of the U1 snRNP structure. The potential use of the in vitro system in the functional analysis of U1 snRNP proteins is discussed.

Functional characterization of X. laevis U5 snRNA genes.

Xenopus laevis U5 snRNA genes are found in several genomic arrangements, represented by a predominant tandem repeat of 583 bp and other minor repeats. Several copies of the major tandem repeat have been cloned and expressed in Xenopus oocytes. The transcripts assemble into U5 snRNPs which are recognized by anti-Sm antibodies. We have identified functional elements in the U5 gene promoter. Although similar in organization to other U snRNA gene promoters, U5 contains significant differences and is more efficiently expressed than the Xenopus U2 gene in oocytes. The proximal sequence element (PSE), although homologous to a mammalian consensus for this region (Skuzeski et al., 1984), does not resemble the previously characterized Xenopus U1 and U2 PSEs closely in sequence. The ATGCAAAT (octamer) part of the distal sequence element (DSE 1) is found in U5 in the orientation opposite to that in U1 and U2 gene promoters. DNase I protection experiments led to the identification of a third element (DSE 2), situated close to the octamer motif. Analysis of deletion mutants showed that both DSE 1 and 2 are essential parts of the U5 gene enhancer, and provides evidence that U snRNA enhancers are complex structures consisting of more than one site of DNA-factor interaction.

Post-transcriptional regulation of albumin gene expression in Xenopus liver.

To clarify on what level of gene expression estrogen represses albumin synthesis in Xenopus hepatocytes, we have analyzed nuclear RNAs and the transcriptional rates in isolated nuclei. Since in nuclear RNA the quantity of albumin mRNA and its precursors does not change and the transcription remains constant during estrogen treatment, we conclude that a posttranscriptional control, possibly involving destabilization of cytoplasmic mRNA, is responsible for the repression of albumin synthesis by estrogen. This post-transcriptional control is in contrast to the well-known transcriptional induction of vitellogenin gene activity. The results can be reproduced in liver cube cultures thereby establishing that estrogen interferes directly with hepatic albumin synthesis. In these liver cube cultures albumin mRNA levels are reduced compared with the liver used to set up the culture whereas the transcription of the albumin genes is not influenced. This reveals another post-transcriptional control of hepatic albumin synthesis.

Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the mature C3 beta chain was predicted to be Ile-Pro-Met-Tyr-Ser-Ile-Ile-Thr-Pro-Asn-Val-Leu-Arg-Leu-Glu. This sequence is in good agreement with the reported amino acid sequences of human and guinea pig C3 beta chains. These data position the C3 beta subunit to the NH2-terminal portion of the precursor C3 molecule (pro-C3) and establish the order of subunits in pro-C3 to be NH2-beta-alpha-COOH. In addition, the cDNA sequence indicates that an NH2-terminal extension peptide precedes the beta chain in pro-C3. The amino acid sequence of the mouse C3a fragment and its flanking regions was determined. The data indicate the presence of four arginine residues located between the COOH terminus of the C3 beta and the NH2 terminus of the C3 alpha subunits in pro-C3. The coding sequences of the amino acids that constitute the internal thioester domain in C3 were determined. Unexpectedly, the glutamyl residue that has been shown to participate in the thioester bond in native C3 was found to be encoded as a glutamine.