Expression of AtMHX, an Arabidopsis vacuolar metal transporter, is repressed by the 5' untranslated region of its gene.

AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5' untranslated region (5' UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5' UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5' UTR of its gene.

Does hysteroscopy produce intraperitoneal spread of endometrial cancer cells?

Hysteroscopy is the tool of choice for the evaluation of the endometrial cavity, including the assessment of abnormal uterine bleeding (AUB). The combination of endometrial biopsy and diagnostic hysteroscopy could replace dilation and curettage in most patients. However, hysteroscopy might disseminate endometrial cells into the peritoneum, thereby potentially raising the stage and decreasing the survival of a patient with endometrial cancer. The purpose of this article is to explore the dilemma of whether hysteroscopy produces intraperitoneal spread of endometrial cancer cells, and, if the answer is yes, what is the prognostic significance of isolated malignant cells in the peritoneal cavity. We conducted a literature search using MEDLINE using the following key words: hysteroscopy, endometrial carcinoma, and dissemination for the years 1980 through 2001. Retrospective data shows a correlation between fluid-based hysteroscopy and the presence of cancer cells in the peritoneal cavity. It cannot, however, be determined whether positive peritoneal washings are the result of hysteroscopy or whether the endometrial cells are found in the peritoneum as a result of other mechanism. Because no prospective, randomized studies have been performed on the dissemination of cancer cells by diagnostic hysteroscopy, no definite conclusions can be made concerning the risk of diagnostic hysteroscopy. In addition, the prognostic significance of isolated malignant cells in the peritoneal cavity of women with endometrial cancer is unclear. Although there might be an increased risk of peritoneal contamination by cancer cells after hysteroscopy, there is currently no evidence that these patients face worse prognosis than patients who have undergone other diagnostic procedures.

Developmental expression of the Arabidopsis thaliana CycA2;1 gene.

The associations of cyclins with highly conserved cyclin-dependent kinases are key events in the regulation of cell cycle progression. The spatio-temporal expression of an Arabidopsis thaliana (L.) Heynh. mitotic cyclin, Arath;CycA2;1, was studied by histochemical beta-glucuronidase (GUS) analysis and in-situ hybridizations. The CycA2,1] promoter was active in the egg apparatus before fertilization. During embryogenesis, CycA2;1:gus expression was found in the embryo and the developing endosperm. Throughout plant development, CycA2;1 transcripts were found in both dividing and non-dividing cells, indicating that the expression of this cyclin is not a limiting factor for cell division. In the pericycle and stelar parenchyma, CycA2;1 transcripts were located at the xylem poles, a position that can be correlated with competence for lateral root formation. In addition, CycA2;1:gus expression was upregulated in roots by auxins and in the shoot apex by cytokinins. Transcription of CycA2;1 was shown by reverse transcription-polymerase chain reaction to be strongly induced by sucrose in A. thaliana cell suspensions.

Mycorrhiza-induced changes in disease severity and PR protein expression in tobacco leaves

The development of leaf disease symptoms and the accumulation of pathogenesis-related (PR) proteins were monitored in leaves of tobacco (Nicotiana tabacum cv. Xanthinc) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices. Leaves of mycorrhizal plants infected with the leaf pathogens Botrytis cinerea or tobacco mosaic virus showed a higher incidence and severity of necrotic lesions than those of nonmycorrhizal controls. Similar plant responses were obtained at both low (0.1 mM) and high (1.0 mM) nutritional P levels and with mutant plants (NahG) that are unable to accumulate salicylic acid. Application of PR-protein activators induced PR-1 and PR-3 expression in leaves of both nonmycorrhizal and mycorrhizal plants; however, accumulation and mRNA steady-site levels of these proteins were lower, and their appearance delayed, in leaves of the mycorrhizal plants. Application of 0.3 mM phosphate to the plants did not mimic the delay in PR expression observed in the mycorrhizal tobacco. Together, these data strongly support the existence of regulatory processes, initiated in the roots of mycorrhizal plants, that modify disease-symptom development and gene expression in their leaves.

Cloning and characterization of a novel Mg(2+)/H(+) exchanger.

Cellular functions require adequate homeostasis of several divalent metal cations, including Mg(2+) and Zn(2+). Mg(2+), the most abundant free divalent cytoplasmic cation, is essential for many enzymatic reactions, while Zn(2+) is a structural constituent of various enzymes. Multicellular organisms have to balance not only the intake of Mg(2+) and Zn(2+), but also the distribution of these ions to various organs. To date, genes encoding Mg(2+) transport proteins have not been cloned from any multicellular organism. We report here the cloning and characterization of an Arabidopsis thaliana transporter, designated AtMHX, which is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg(2+) and Zn(2+) ions. Functional homologs of AtMHX have not been cloned from any organism. Ectopic overexpression of AtMHX in transgenic tobacco plants render them sensitive to growth on media containing elevated levels of Mg(2+) or Zn(2+), but does not affect the total amounts of these minerals in shoots of the transgenic plants. AtMHX mRNA is mainly found at the vascular cylinder, and a large proportion of the mRNA is localized in close association with the xylem tracheary elements. This localization suggests that AtMHX may control the partitioning of Mg(2+) and Zn(2+) between the various plant organs.

Analysis of the Ac promoter: structure and regulation.

The Ac-encoded transposase, a factor that is essential for the mobility of the Ac element, is expressed under the control of a promoter that lacks a conventional TATA box. The regulation of this promoter is poorly understood. We have analyzed Ac promoter structure and activity, both in vitro and in vivo, using transgenic tobacco plants and cell suspensions. A deletion analysis of the Ac 5' region showed that the minimal promoter is located within 70 bp of the major transcription initiation site (at position 334). The minimal promoter includes the sequence TAAGAAATA at position 294 303, i.e., about 30 nucleotides upstream from the transcription start site. This sequence binds specifically to the TATA-binding protein (TBP), suggesting that it is functional as a TATA box. The regulation of the Ac promoter was studied throughout plant development. Levels of Ac mRNA were low in all tissues studied, with higher expression being observed in dividing cells. In order to test whether Ac promoter is regulated during the cell cycle, a tobacco cell suspension transformed with Ac, was grown synchronously. No differences were found in Ac mRNA levels between cells in S, G2, M, or G1 phases; however, expression was lower in the stationary phase. We conclude that Ac promoter is not cell-cycle regulated but is expressed at a higher level in dividing cells. The possible relationship between promoter features and the regulation of Ac element transposition is discussed.

The SV40 capsid protein VP3 cooperates with the cellular transcription factor Sp1 in DNA-binding and in regulating viral promoter activity.

Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells.

Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures. Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin- and dihydrozeatin-type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N- and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated form resulted from de novo synthesis. These findings suggest that zeatin- and dihydrozeatin-type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.

Two Arabidopsis cyclin promoters mediate distinctive transcriptional oscillation in synchronized tobacco BY-2 cells.

Cyclins are cell cycle regulators whose proteins oscillate dramatically during the cell cycle. Cyclin steady-state mRNA levels also fluctuate, and there are indications that both their rate of transcription and mRNA stability are under cell cycle control. Here, we demonstrate the transcriptional regulation of higher eukaryote cyclins throughout the whole cell cycle with a high temporal resolution. The promoters of two Arabidopsis cyclins, cyc3aAt and cyc1At, mediated transcriptional oscillation of the beta-glucuronidase (gus) reporter gene in stably transformed tobacco BY-2 cell lines. The rate of transcription driven by the cyc3aAt promoter was very low during G1, slowly increased during the S phase, peaked at the G2 phase and G2-to-M transition, and was down-regulated before early metaphase. In contrast, the rate of the cyc1At-related transcription increased upon exit of the S phase, peaked at the G2-to-M transition and during mitosis, and decreased upon exit from the M phase. This study indicates that transcription mechanisms that seem to be conserved among species play a significant role in regulating the mRNA abundance of the plant cyclins. Furthermore, the transcription patterns of cyc3aAt and cyc1At were coherent with their slightly higher sequence similarity to the A and B groups of animal cyclins, respectively, suggesting that they may fulfill comparable roles during the cell cycle.

The simian virus 40 packaging signal ses is composed of redundant DNA elements which are partly interchangeable.

Using the experimental system of simian virus 40 (SV40) pseudovirions we have previously shown that SV40 requires a specific DNA element for packaging, ses, which was mapped to the SV40 regulatory region. ses was previously found to play a role in facilitating the nucleosomal rearrangement required for chromatin condensation and viral packaging. Here, the fine structure of ses was investigated by genetic studies. Analyses of ses+ revertants indicated that in order to function, ses must be present in close proximity to the origin of replication (ori), supporting a role in the regulation of the viral life cycle. Fine dissection of ses was performed using a series of plasmids carrying mutations and deletions in this region. The results suggest that multiple DNA elements participate in the SV40 packaging process, including the GC-boxes and elements derived from the enhancer. The elements are redundant, and they can function in various combinations. Packaging efficiency correlated with the number of GC-boxes, known to bind Sp1. In addition, AP-2 binding elements appeared to more important than others. These findings were supported by experiments which showed that packaging was significantly enhanced by adding AP-2 binding sites to plasmids with large deletions and lacking those sites. The results imply that binding of Sp1 and/or AP-2 may participate in the packaging process.

Lysine synthesis and catabolism are coordinately regulated during tobacco seed development.

The regulation of synthesis and accumulation of the essential amino acid lysine was studied in seeds of transgenic tobacco plants expressing, in a seed-specific manner, two feedback-insensitive bacterial enzymes: dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4). High-level expression of the two bacterial enzymes resulted in only a slight increase in free lysine accumulation at intermediate stages of seed development, while free lysine declined to the low level of control plants toward maturity. To test whether enhanced catabolism may have contributed to the failure of free lysine to accumulate in seeds of transgenic plants, we analyzed the activity of lysine-ketoglutarate reductase (EC 1.5.1.7), an enzyme that catabolizes lysine into saccharopine. In both the control and the transgenic plants, the timing of appearance of lysine-ketoglutarate reductase activity correlated very closely with that of dihydrodipicolinate synthase activity, suggesting that lysine synthesis and catabolism were coordinately regulated during seed development. Notably, the activity of lysine-ketoglutarate reductase was significantly higher in seeds of the transgenic plants than in the controls. Coexpression of both bacterial enzymes in the same plant resulted in a significant increase in the proportions of lysine and threonine in seed albumins. Apparently, the normal low steady-state levels of free lysine and threonine in tobacco seeds may be rate limiting for the synthesis of seed proteins, which are relatively rich in these amino acids.

Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase.

The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.

Threonine Overproduction in Transgenic Tobacco Plants Expressing a Mutant Desensitized Aspartate Kinase of Escherichia coli.

In higher plants, the synthesis of the essential amino acid threonine is regulated primarily by the sensitivity of the first enzyme in its biosynthetic pathway, aspartate kinase, to feedback inhibition by threonine and lysine. We aimed to study the potential of increasing threonine accumulation in plants by means of genetic engineering. This was addressed by the expression of a mutant, desensitized aspartate kinase derived from Escherichia coli either in the cytoplasm or in the chloroplasts of transgenic tobacco (Nicotiana Tabacum cv Samsun NN) plants. Both types of transgenic plants exhibited a significant overproduction of free threonine. However, threonine accumulation was higher in plants expressing the bacterial enzyme in the chloroplast, indicating that compartmentalization of aspartate kinase within this organelle was important, although not essential. Threonine overproduction in leaves was positively correlated with the level of the desensitized enzyme. Transgenic plants expressing the highest leaf aspartate kinase activity also exhibited a slight increase in the levels of free lysine and isoleucine, both of which share a common biosynthetic pathway with threonine, but showed no significant change in the level of other free amino acids. The present study proposes a new molecular biological approach to increase the limiting content of threonine in higher plants.

A cis-acting DNA signal for encapsidation of simian virus 40.

Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the sesdeleted plasmid 2 kbp away from the ori, suggesting that for encapsidation the two DNA elements have to be close to each other. These findings afford novel strategies for the investigation of viral encapsidation.

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts.

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.