Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3-phosphoadenosine-5-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector YES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35 S-labeled substrate to acid-volatile 35 S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel ''APS reductase'' enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of Ϸ1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.Sulfur in its reduced form plays an important role in plant metabolism being involved in the biosynthesis of a wide range of primary and secondary S-containing metabolites. However the pathway of reductive assimilation of inorganic sulfate to sulfide in higher plants is the subject of some controversy (reviewed in ref. 1). After sulfate uptake, and its activation to adenosine 5Ј-phosphosulfate (APS) (5Ј-adenylylsulfate) by the enzyme ATP sulfurylase, it has been unclear whether APS is further metabolized by the enzyme APS sulfotransferase to ''bound-sulfite'' and then reduced to ''bound sulfide'' by a thiosulfonate reductase or whether the ''free-sulfite'' pathway, found in most enterobacteria and certain fungi and yeast, operates. In this latter pathway APS is converted to 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) (3Ј-phosphoadenylylsulfate) by APS kinase and PAPS is reduced to free sulfite by PAPS reductase. Sulfite is then reduced to sulfide by sulfite reductase. Discovery of a thioredoxin-dependent PAPS reductase activity in spinach (2) and the more recent suggestion that APS sulfotransferase activity is a side-reaction of APS kinase (3) provide strong support for the notion that the ''free-intermediate'' pathway operates not only in...

Previous space research conducted during short-term flight experiments and long-term environmental monitoring on board orbiting space stations suggests that the relationship between humans and microbes is altered in the crewed habitat in space. Both human physiology and microbial communities adapt to spaceflight. Microbial monitoring is critical to crew safety in long-duration space habitation and the sustained operation of life support systems on space transit vehicles, space stations, and surface habitats. To address this critical need, space agencies including NASA (National Aeronautics and Space Administration), ESA (European Space Agency), and JAXA (Japan Aerospace Exploration Agency) are working together to develop and implement specific measures to monitor, control, and counteract biological contamination in closed-environment systems. In this review, the current status of microbial monitoring conducted in the International Space Station (ISS) as well as the results of recent microbial spaceflight experiments have been summarized and future perspectives are discussed.

The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.

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The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy. We present here one such strategy focusing on the homeologous pairing suppressor (Ph1) locus of wheat. This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control. The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content. As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A). These loci are located within 400 kb of each other in the rice genome. By sequencing this region of the rice genome, it should now be possible to define the nature of this factor.

Comparative genome analysis enables the sites of centromeres, telomeres and nucleolar organiser regions to be aligned with borders that define the sets of linked genes conserved across the cereal genomes. This provides a basis for studying cereal genome evolution.

We have cloned an Arabidopsis thaliana cDNA encoding serine acetyltransferase (EC 2.3.1.30) by functional complementation of the Escherichia coli cysE mutant JM15. The cDNA clone Sat-1 conferred serine acetyltransferase activity (with apparent Km for the two substrates acetyl CoA and L-serine of 0.043 and 3.47 mmol/dm3 respectively) on the cysE mutant. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids which includes a putative chloroplastic targeting presequence. Northern analysis revealed a single message of 1.5 kb, while Southern hybridisation suggests a small multigene family of related sequences.

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