SummaryGlutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. A family of seven related proteins named AtGPX1± AtGPX7 in Arabidopsis was identi®ed, and the genomic organization of this family was reported. The putative subcellular localizations of the encoded proteins are the cytosol, chloroplast, mitochondria, and endoplasmic reticulum. Expressed sequence tags (ESTs) for all the genes except AtGPX7 were identi®ed. Expression analysis of AtGPX genes in Arabidopsis tissues was performed, and different patterns were detected. Interestingly, several genes were up-regulated coordinately in response to abiotic stresses. AtGPX6, like human phospholipid hydroperoxide GPX (PHGPX), possibly encodes mitochondrial and cytosolic isoforms by alternative initiation. In addition, this gene showed the strongest responses under most abiotic stresses tested. AtGPX6::GUS analysis in transgenic Arabidopsis showed that AtGPX6 is highly expressed throughout development in most tissues, thus supporting an important role for this gene in protection against oxidative damage. The different effects of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and auxin on the expression of the genes indicate that the AtGPX family is regulated by multiple signaling pathways. Analysis of the upstream region of the AtGPX genes revealed the presence of multiple conserved motifs, and some of them resembled antioxidant-responsive elements found in plant and human promoters. The potential regulatory role of speci®c sequences is discussed.

Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36⌬50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36⌬50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36⌬50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36⌬50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36⌬50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.

Amplification of the human cytomegalovirus (HCMV) lytic origin (oriLyt) in human fibroblasts is dependent upon six core replication proteins and UL84, IE2, and UL36-38. Using a telomerase-immortalized human fibroblast cell line (T-HFs), oriLyt-dependent DNA replication no longer required the gene products of UL36-38. To determine the role of IE2 in DNA replication in human fibroblasts, we examined potential IE2-binding sites within HCMV oriLyt. We now show that a strong bidirectional promoter (oriLyt PM ) (nucleotides 91754 to 92030) is located in the previously identified core region of the origin and is required for efficient amplification of oriLyt. It was determined that a 14-bp novel DNA motif (oriLyt promoter activation element), which was initially identified as a binding element for the immediate-early protein IE2, was essential for oriLyt PM activity. In Vero cells the oriLyt PM was constitutively active and strongly repressed by IE2, but it was reactivated by UL84. In contrast, transfection of the oriLyt PM into human fibroblasts resulted in a very low basal level of promoter activity that was dramatically up-regulated upon infection with HCMV. Cotransfection assays demonstrated that the transfection of UL84 along with IE2 transactivated the oriLyt PM in human fibroblasts. Further activation was observed upon cotransfection of the set of plasmids expressing the entire replication complex. Efficient oriLyt amplification in the absence of IE2 in human fibroblasts was observed by replacing the oriLyt PM with the simian virus 40 early promoter. Under these conditions, however, UL84 was still required for amplification of oriLyt. These results suggest that the mechanism of initiation of HCMV lytic replication in part involves transcriptional activation.Human cytomegalovirus (HCMV) contains a single lytic origin for DNA replication, oriLyt, located near the center of the U L region and upstream of the open reading frame (ORF) encoding the single-stranded DNA-binding protein ppUL57 (1,19,40). The HCMV oriLyt is remarkable among viral replication origins for its apparent size and complexity. The entire oriLyt region of HCMV is located from nucleotides (nt) 90500 to 93930 and is composed of a core (nt 91751 to 93299) which contains two essential regions (1,40,66). These essential regions (I and II) contain a pyrimidine-rich sequence (Y-block), various reiterated sequences, several transcription factor-binding sites, direct and inverted repeat sequences, base composition biases and strand asymmetries, and RNA-DNA hybrid structures and is a site of active transcription (the small replication transcript [SRT]) (1,22,40,49,66). Despite the apparent exhaustive investigation of elements within oriLyt that contribute to DNA synthesis, few data exist with respect to the actual function of these elements in initiation of DNA replication.Some information concerning the initiation of DNA synthesis can be inferred from the required virus-encoded transacting factors necessary to replicate oriLyt. In human fibroblasts, oriLy...

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.Al is one of the most important limiting factors for crop production on acid soils. The most important effect of Al toxicity is a dramatic reduction in root growth, which leads to poor productivity. Severe Al stress threatens the survival of many sensitive crop genotypes. Al has been shown to affect a large number of cellular processes, especially the uptake of K (Jones and Kochian, 1995). Al also induces the secretion of organic acids (e.g. citrate, malate, and oxalate) from roots (Delhaize and Ryan, 1995). These organic acids form a stable complex with Al, preventing the toxic effects of Al and providing the most valuable source of tolerance in the majority of plant species studied. Despite the considerable progress made over the last decade, the rather modest progress in isolating Al-regulated genes has limited our understanding of the molecular mechanisms underlying Al toxicity and tolerance.Changes in gene expression control normal physiological processes and are also the main effectors of cellular responses to biotic or abiotic stresses (Jiang et al., 2000). Since the cloning of the Wali genes (Snowden and Gardner, 1993;, other genes have been shown to respond to Al stress, such as those identified in rice (Oryza sativa; Yu et al., 1998), tobacco (Nicotiana tabacum; Ezaki et al., 1995;Ezaki et al., 1996), wheat (Triticum aestivum; Cruz-Ortega et al., 1997;Hamel et al., 1998;Delhaize et al., 1999;Hamilton et al., 2001), Arabidopsis (Richards...

Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent