Stefania Petrucco

Coordinate modulation of maize sulfate permease and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by L-cysteine.

To gain insight into the regulatory mechanisms and the signals responsible for the adaptation of higher plants to conditions of varying sulfate availability, we have isolated from a sulfate- deprived root library maize cDNAs encoding sulfate permease (ZmST1) and ATP sulfurylase (ZmAS1), the two earliest components of the sulfur assimilation pathway. The levels of ZmST1 and ZmAS1 transcripts concomitantly increased in both roots and shoots of seedlings grown under sulfate-deprived conditions, and rapidly decreased when the external sulfate supply was restored. This coordinate response, which was not observed under conditions of limiting nitrate or phosphate, correlated with the depletion of glutathione, rather than sulfate stores. However, drastically reducing glutathione levels through treatment with buthionine sulfoximine, a specific inhibitor of γ-glutamyl cysteine synthetase, did not provide an adequate stimulus for the up- regulation of either sulfate permease or ATP sulfurylase messengers. Indeed, L-cysteine, but not D-cysteine, effectively down-regulated both transcripts when supplied to sulfur-deficient seedlings under conditions of blocked glutathione synthesis. Altogether, these data provide evidence for the coordinate regulation of sulfur assimilation mRNAs in higher plants and for the glutathione-independent involvement of cysteine as a stereospecific pretranslational modulator of the expression of sulfur status-responsive genes.

Oxidative Stress and Age-Related Cataract

The authors review the available evidence supporting the possible role of oxidative stress in cataract formation from an epidemiological and a clinical point of view. They discuss in more detail what is presently known about the molecular mechanisms of response of the mammalian lens to an oxidative insult and report unpublished data on gene modulation upon oxidative stress in a bovine lens model. Main research endeavors that seem to be a most promising source of new insights into the problem of age-related cataract formation are briefly discussed.

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

Conserved Alternative Splicing of Arabidopsis Transthyretin-Like Determines Protein Localization and S-Allantoin Synthesis in Peroxisomes

Conserved alternative splicing of an unusual internal targeting sequence turns the enzyme completing the peroxisomal pathway of ureide biosynthesis into a cytosolic protein with a different function. S-allantoin, a major ureide compound, is produced in plant peroxisomes from oxidized purines. Sequence evidence suggested that the Transthyretin-like (TTL) protein, which interacts with brassinosteroid receptors, may act as a bifunctional enzyme in the synthesis of S-allantoin. Here, we show that recombinant TTL from Arabidopsis thaliana catalyzes two enzymatic reactions leading to the stereoselective formation of S-allantoin, hydrolysis of hydroxyisourate through a C-terminal Urah domain, and decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline through an N-terminal Urad domain. We found that two different mRNAs are produced from the TTL gene through alternative use of two splice acceptor sites. The corresponding proteins differ in the presence (TTL1−) and the absence (TTL2−) of a rare internal peroxisomal targeting signal (PTS2). The two proteins have similar catalytic activity in vitro but different in vivo localization: TTL1− localizes in peroxisomes, whereas TTL2− localizes in the cytosol. Similar splice variants are present in monocots and dicots. TTL originated in green algae through a Urad-Urah fusion, which entrapped an N-terminal PTS2 between the two domains. The presence of this gene in all Viridiplantae indicates that S-allantoin biosynthesis has general significance in plant nitrogen metabolism, while conservation of alternative splicing suggests that this mechanism has general implications in the regulation of the ureide pathway in flowering plants.

A general one-step method for the cloning of PCR products

A very fast, highly efficient, versatile and low‐cost cloning of PCR products is described. PCR amplicons, obtained with any set of primers, is directly integrated into circular plasmid vectors by means of a one‐step restriction–ligation procedure. When using proof‐reading DNA polymerases, 100% cloning efficiency is easily achieved, implying that direct cloning into ‘final‐use’ vectors (i.e. avoiding any intermediate cloning step) is a feasible task. Albeit with a lower efficiency, the same procedure is also suitable for the cloning of PCR products generated by ‘non‐proof‐reading’ DNA polymerases. Furthermore, with a simple modification of the vector polylinker site, the present method can be easily adapted to the directional cloning of open‐reading‐frame‐encoding amplicons. This one‐step procedure thus couples high efficiency with high reliability and versatility, and lends itself as the method of choice for routine cloning of PCR products.

A nick-sensing DNA 3'-repair enzyme from Arabidopsis

DNA single-strand breaks, a major cause of genome instability, often produce unconventional end groups that must be processed to restore terminal moieties suitable for reparative DNA gap filling or ligation. Here, we describe a bifunctional repair enzyme from Arabidopsis (named AtZDP) that recognizes DNA strand breaks and catalyzes the removal of 3′-end-blocking lesions. The isolated C-terminal domain of AtZDP is by itself competent for 3′-end processing, but not for strand break recognition. The N-terminal domain instead contains three Cys3-His zinc fingers and recognizes various kinds of damaged double-stranded DNA. Gapped DNA molecules are preferential targets of AtZDP, which bends them by ∼73° upon binding, as measured by atomic force microscopy. Potential partners of AtZDP were identified in theArabidopsis genome using the human single-strand break repairosome as a reference. These data identify a novel pathway for single-strand break repair in which a DNA-binding 3′-phosphoesterase acts as a “nick sensor” for damage recognition, as the catalyst of one repair step, and possibly as a nucleation center for the assembly of a fully competent repair complex.

Structural recognition of DNA by poly(ADP‐ribose)polymerase‐like zinc finger families

PARP‐like zinc fingers (zf‐PARPs) are protein domains apt to the recognition of multiple DNA secondary structures. They were initially described as the DNA‐binding, nick‐sensor domains of poly(ADP‐ribose)polymerases (PARPs). It now appears that zf‐PARPs are evolutionary conserved in the eukaryotic lineage and associated with various enzymes implicated in nucleic acid transactions. In the present study, we discuss the functional and structural data of zf‐PARPSs in the light of a comparative analysis of the protein family. Sequence and structural analyses allow the definition of the conserved features of the zf‐PARP domain and the identification of five distinct phylogenetic groups. Differences among the groups accumulate on the putative DNA binding surface of the PARP zinc‐finger fold. These observations suggest that different zf‐PARP types have distinctive recognition properties for DNA secondary structures. A comparison of various functional studies confirms that the different finger types can accomplish a selective recognition of DNA structures.

A Plant 3′-Phosphoesterase Involved in the Repair of DNA Strand Breaks Generated by Oxidative Damage

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia colidiphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg2+-dependent and Li+-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3′-phosphoadenosine 5′-phosphate (3′-PAP) and 2′-PAP without any discrimination between the 3′- and 2′-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li+ sensitivity, a strong preference for 3′-PAP, and the unique ability to utilize double-stranded DNA molecules with 3′-phosphate- or 3′-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3′-phosphate- or 3′-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3′-phosphatase that is thought to be involved in DNA repair, but is devoid of 5′-kinase activity. ZmDP2 is the first DNA 3′-phosphoesterase thus far identified in plants capable of converting 3′-blocked termini into priming sites for reparative DNA polymerization.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism.

PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed.