114 search hits
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Phosphate nutrition in the Ricinus communis L. seedling
(2007)
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Dang Khoa Tran
- Phosphate (Pi) is one of the essential macronutrients required for growth and development of plants. Pi plays an important role in various metabolic processes, such as photosynthesis, respiration, energy conservation, carbohydrate metabolism and signal transduction. Although various Pi starvation induced genes have been isolated from different plant species grown under conditions of Pi starvation, information about their functions during germination and growth of seedlings is still lacking. During germination Pi stored in the endosperm is mobilized and transported to growing organs of seedlings, thus a phosphate transporters and acid phosphatases are expected to be involved in these processes. The aim of this study was to clarify the translocation of Pi within the seedlings and to identify the involvement of phosphate transporters and acid phosphatases in the growth of seedlings. Uptake into the phloem was analyzed by incubating the cotyledons in Pi. The movement of 32P-labeled applied as an inorganic phosphate (Pi) was detected from the cotyledons to the hypocotyl, in particular to its apical hook near the cotyledons, suggesting that Pi moves from the Ricinus communis L. cotyledons through the hypocotyl via phloem and partially re-circulates in the xylem or leaks out through the roots. Therefore reducing the efflux could be as important for the plant as increasing the efficiency of the uptake mechanism. Following the Pi uptake into the roots, the translocation of 32P-labeled Pi to the cotyledons through the hypocotyl via the xylem showed that a high amount of radiotracer accumulated in the cotyledons. The accumulated Pi in the cotyledons can be retranslocated to the roots via phloem. This work describes the cloning of the phosphate transporter RcPT1 and the acid phosphatase RcPS1 genes by RT-PCR from Ricinus seedlings grown under Pi starvation conditions. Phosphate transporter RcPT1 contains an open reading frame encoding a 530 amino acid polypeptide with a calculated molecular mass of 59 kD. The expression of RcPT1 in the yeast high-affinity phosphate transporter mutant strain complemented the mutant and enhanced the cell growth significantly. Southern blot analysis showed that the RcPT1 gene is present as a single or low-copy gene in the Ricinus genome. The transcripts of RcPT1 were expressed in the endosperm, cotyledons, hypocotyl and roots during germination. In detail in situ hybridization studies revealed RcPT1 expression in the adjacent area of endosperm to cotyledon, in the phloem and in the lower epidermis of cotyledons; Immunolocalization analysis showed RcPT1 accumulation at the same sites as its mRNA. In addition, RcPT1 transcripts were also found in the phloem of hypocotyl, and the epidermis and the steles of roots. These results implicated that RcPT1 is involved in the movement of Pi from endosperms to cotyledons and the redistribution of Pi within seedlings via phloem during germination. Acid phosphatase RcPS1 shows a 747 bp open reading frame encoding a 248 amino acids polypeptide with a calculated molecular mass of 27,5 kD. The amino acid sequence of RcPS1 shares significant similarity with the acid phosphatase LePS2 from tomato and highly conserved motifs, which are typical for a member of haloacid dehalogenase and DDDD superfamilies of enzymes catalyzing a diverse number of hydrolytic and phosphotransferase reactions. The functional analysis after expression of RcPS1 in E.coli showed significant acid phosphatase activity. The high transcript level of RcPS1 in endosperms, cotyledons and roots at the first few days of germination suggested that this acid phosphatase gene might be expressed during mobilization of storage products. RcPT1 and RcPS1 mRNA are detectable in the seedlings grown under Pi starvation and Pi sufficient conditions, indicating that both genes were expressed independently from exogenous Pi supply during germination. Moreover, RcPT1 and RcPS1 were expressed in leaves, stems and roots of plants grown under Pi starvation; furthermore, in situ hybridization studies localized RcPT1 and RcPS1 mRNA in the epidermis and the stele of roots of Pi-starved plants, suggesting that these genes also play a role in response to Pi starvation. Thus, it is concluded that there are different signals regulating RcPT1 and RcPS1 expression in response to Pi starvation and during germination.
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Esterase 2-oligodeoxynucleotide conjugates as enzyme reporter for electrochemical detection of DNA and identification of bacterial species
(2007)
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Yiran Wang
- Electrical Chip (E-Chip) system offers a fast, sensitive and cost-effective way to detect analyte. To improve its application of nucleic acids detection, a suitable enzyme reporter is expected. Esterase 2 (EST2) from Alicyclobacillus acidocaldarius was introduced and mutated to have an accessible cysteine residue at 118th codon. This esterase was purified by a single-step affinity chromatography with trifluoromethyl ketone as a ligand and covalently conjugated to a 5’-amino modified oligodeoxynucleotide. The purified conjugate served as a reporter enzyme for electrochemical detection of nucleic acids. Being an optimal substrate, p-aminophenylbutyrate exerts maximal signal response to EST2 in E-Chip, as determined by comparison of p-aminophenyl esters with acyl chain length from two to eight carbons. An assay of 15 pM of soluble esterase 2 in 1 ml was obtained exploiting p-aminophenylbutyrate. E-Chip detection of nucleic acids requires three essential steps: immobilization of thiol-modified capture oligodeoxynucleotides onto electrode, recruiting EST2 to electrode vicinity by means of nucleic acids hybridization, and amperometric determination of p-aminophenol produced by EST2 catalytic hydrolysis of p-aminophenylbutyrate. Generally, EST2 reporter allows a detection of approximately one million molecules/0.6 mm2 electrode. EST2 covalently attached by an oligodeoxynucleotide significantly increased the ability of mismatch discrimination as compared to the streptavidin conjugated EST2. Moreover, single nucleotide mismatch in analyte could be reliably discriminated in the set-up, as demonstrated by single nucleotide mismatch in a 49-mer oligodeoxynucleotide as well as in a 510-nucleotide ssDNA. Application of E-Chip to bacterial species identification through 16S rRNA was demonstrated. Escherichia coli and Listeria innocua were easily identified as judged by signals given by rRNA hybridization with species-specific capture ODNs. This system allows a detection of 1,000 Escherichia coli cells. As a further optimization, a stem-loop structured molecular beacon with 5’-thiol and 3’-biotin modifications was synthesized and tested on the chip using EST2-streptavidin as reporter. The presence of target oligodeoxynucleotides complementary to the whole stem-loop sequence enhanced signal for a moderate 2-fold. The future work should focus on combination of continuous flow PCR with EST2-oligodeoxynucleotide conjugate reporter to do faster and more automatic disease related DNA analysis, as well as construction of EST2 based biosensor for toxic agents detection.
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Convex hulls of polyominoes
(2007)
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Sascha Kurz
- In this article we prove a conjecture of Bezdek, Brass, and Harborth concerning the maximum volume of the convex hull of any facet-to-facet connected system of $n$ unit hypercubes in $mathbb{R}^d$. For $d=2$ we enumerate the extremal polyominoes and determine the set of possible areas of the convex hull for each $n$.
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Regulation of Progression through Unperturbed Mitosis and in the Presence of Environmental Stress
(2007)
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Rahul Pandey
- During mitosis, the replicated genome is distributed onto two daughter cells. Perfect regulation of progression through mitosis is essential for an error-free segregation and propagation of the genome. Surveillance pathways like the mitotic spindle checkpoint have evolved to prevent mitotic defects effectively. The molecular basis of these regulatory mechanisms is particularly well understood in budding yeast. It is not entirely clear to what extent these findings are valid for other eukaryotes as well. Therefore, in a first part, I have addressed the function of Drosophila Separase, a protease which has been implicated in several regulatory processes in budding yeast. Although an essential role of this protease for sister chromatid separation has been demonstrated in a wide range of eukaryotes, its involvement in additional processes still remains controversial. In budding yeast, separase promotes rapid exit from mitosis in a dedicated regulatory network known as FEAR. In Drosophila, a FEAR-like role could not be confirmed. Similarly, an essential involvement in the duplication of centrosomes, which form the poles of mitotic spindles in metazoan cells and are highly divergent from the functionally equivalent fungal spindle pole bodies, could not be demonstrated. In a second part I have analyzed the importance of surveillance mechanisms (in particular the mitotic spindle checkpoint) for progression through mitosis in the presence of environmental stress like anoxia and hypothermia. So far, the interaction of environmental stress with mitotic regulation has been largely neglected. However, oxygen deprivation leads to a rapid and reversible mitotic arrest. Here, anoxia is shown to have rapid effects on spindle and kinetochore function which are proposed to cause the observed efficient activation of the mitotic spindle checkpoint. Moreover, the consequences of anoxia were found to be very similar to those caused by inhibitors of oxidative phosphorylation. This suggests that the reduction in ATP levels is responsible for metaphase arrest in anoxia. Interestingly, the mitotic spindle checkpoint was also found to be important for survival in hypothermia, and the early syncytial stages which are characterized by an extremely rapid progression through mitotic cycles, were found to be the most cold-sensitive stages of Drosophila embryogenesis.
