Cis-acting motifs in artificially synthesized expression cassette leads to enhanced transgene expression in tomato (Solanum lycopersicum L.).

Efficacy of artificial synthetic expression modules was compared with native CaMV35S and DECaMV35S promoter in transgenic tomato developed by Agrobacterium-mediated transformation. The promoters under trial were CaMV35S-mec (PcamI), CaMV35S (PcamII), DECaMV35S (PcamIII), synthetic minimal expression cassette (Pmec), complete expression cassette (Pcec), double enhancer expression cassette (Pdec) and triple enhancer expression cassette (Ptec) for driving the uidA gene for ß-glucuronidase (GUS) activity. The promoter efficiency based on average of GUS expression in T(0) and T(1) transgenic tomato was in the order Pcec > Pdec > PcamIII > PcamII > PcamI > Ptec > Pmec. The two promoters Pcec and PcamIII were deployed for development of insect-resistant transgenic tomato with optimal expression of modified cry1Ac insecticidal toxin gene from Bacillus thuringiensis (Bt). The transgenic status and copy number of the cry1Ac in T(0) transgenic tomato was confirmed through PCR, Southern hybridization, RT-PCR and Western immunoassay, while toxin expression was monitored by DAS-ELISA. The expression level of Cry1Ac toxin driven by Pcec in T(0) population ranged from 0.08 to 0.8% of total soluble protein (TSP) that was significantly higher to PcamIII which ranged from 0.02 to 0.13% of TSP. The outcome of insect mortality bioassay with Helicoverpa armigera correlated well with the results of DAS-ELISA. The higher expression of cry1Ac gene driven by Pcec promoter in transgenic tomato did not show any yield penalty and reflected complete protection, while low recovery of promising transgenics expressing Cry1Ac toxin driven by PcamIII was a major limitation for complete protection against the fruit borer insect.

QSPR of the bioconcentration factors of non-ionic organic compounds in fish using extended topochemical atom (ETA) indices.

Bioconcentration refers to the absorption or uptake of a chemical from the media to an organism's tissues leading to greater concentration in tissues than that in the surrounding environment. Considering the importance of bioconcentration from the viewpoint of ecological safety assessment, a QSPR study was conducted based upon log BCF of 122 non-ionic organic compounds in fish using the recently introduced extended topochemical atom (ETA) indices. In deriving the models, principal component factor analysis (FA) followed by multiple linear regression (MLR), stepwise regression, partial least squares (PLS) and principal component regression analysis (PCRA) were applied as statistical tools. This was repeated with non-ETA (topological and physicochemical) descriptors and a combination set including both the ETA and non-ETA descriptors. The ETA indices suggested negative contributions of functionalities of nitro, amino and hydroxy substructures and positive contributions of branching, volume and functionality of chloro substituents. Again, the predictive ability of the developed models was compared with the previously reported models. Finally the validation of all the QSAR models was discussed based on random division, sorted log BCF data and K-means clusters for the factor scores of the original variable (ETA) matrix without the response property values. The results suggest that ETA parameters are sufficiently rich in chemical information to encode the structural features contributing to the bioconcentration of the non-ionic organic compounds in fish and thus these merit further assessment to explore their potential in QSAR/QSPR/QSTR modelling.

Analysis of eosinophils and myeloid progenitor responses to modified forms of MPIF-2.

Myeloid progenitor inhibitory factor (MPIF)-2 is a beta-chemokine with select and potent activities on eosinophils and myeloid progenitors. In the beta-chemokine family, biological activity is modulated by differential processing of the amino-terminus. Here, for MPIF-2, we describe the biological activities of NH(2)-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities. Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors. For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity. One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays. Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation. In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities.

[2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase.

The type and properties of the Fe-S cluster in recombinant Escherichia coli biotin synthase have been investigated in as-prepared and dithionite-reduced samples using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman spectroscopies. The results confirm the presence of one S = 0 [2Fe-2S]2+ cluster in each subunit of the homodimer in aerobically purified samples, and the Fe-S stretching frequencies suggest incomplete cysteinyl-S coordination. However, absorption and resonance Raman studies show that anaerobic reduction with dithionite in the presence of 60% (v/v) ethylene glycol or glycerol results in near-stoichiometric conversion of two [2Fe-2S]2+ clusters to form one S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination. The stoichiometry and ability to effect reductive cluster conversion without the addition of iron or sulfide suggest that the [4Fe-4S]2+ cluster is formed at the subunit interface via reductive dimerization of [2Fe-2S]2+ clusters. EPR and VTMCD studies indicate that more than 50% of the Fe is present as [4Fe-4S]+ clusters in samples treated with 60% (v/v) glycerol after prolonged dithionite reduction. The [4Fe-4S]+ cluster exists as a mixed spin system with S = 1/2 (g = 2. 044, 1.944, 1.914) and S = 3/2 (g = 5.6 resonance) ground states. Subunit-bridging [4Fe-4S]2+,+ clusters, that can undergo oxidative degradation to [2Fe-2S]2+ clusters during purification, are proposed to be a common feature of Fe-S enzymes that require S-adenosylmethionine and function by radical mechanisms involving the homolytic cleavage of C-H or C-C bonds, i.e., biotin synthase, anaerobic ribonucleotide reductase, pyruvate formate lyase, lysine 2, 3-aminomutase, and lipoic acid synthase. The most likely role for the [4Fe-4S]2+,+ cluster lies in initiating the radical mechanism by directly or indirectly facilitating reductive one-electron cleavage of S-adenosylmethionine to form methionine and the 5'-deoxyadenosyl radical. It is further suggested that oxidative cluster conversion to [2Fe-2S]2+ clusters may play a physiological role in these radical enzymes, by providing a method of regulating enzyme activity in response to oxidative stress, without irreversible cluster degradation.

Escherichia coli biotin synthase: an investigation into the factors required for its activity and its sulfur donor.

Biotin synthase catalyzes the chemically difficult final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. In the present work, we extend our characterization of this enzymatic reaction and the extensive set of factors required by it. A defined mixture of components that supports the biotin synthase reaction has been found. The mixture contains biotin synthase, flavodoxin, flavodoxin reductase, NADPH, Ado-Met, Fe, fructose-1,6-bisphosphate, cysteine, and dithiothreitol. Even though this defined mixture supports the biotin synthase reaction, and in that regard is an important step forward in the study of this enzyme, it is unlikely that it contains all the physiologically significant factors involved in the biotin synthase reaction since it supports as an upper limit the synthesis of only 2 mol of biotin per mole of biotin synthase monomer. Progress in our efforts to identify additional physiologically significant factors is also reported. First, we describe evidence that the fructose 1,6-bisphosphate in the defined reaction mixture is substituting for an unknown factor of considerably higher potency present in crude extracts. Second, we have found that a labile low-molecular-weight product of the 7,8-diaminopelargonic acid aminotransferase reaction stimulates the rate of biotin formation in the defined biotin synthase reaction mixture and can increase the final amount of biotin formed by threefold. This product seems to be derived from Ado-Met, which 7,8-diaminopelargonic acid aminotransferase uses as its amino donor. However, 5'-deoxy-5'-methylthioadenosine, the postulated breakdown product from the action of 7,8-diaminopelargonic acid aminotransferase on Ado-Met, cannot be the active material since it has no stimulatory effect when added to the biotin synthase reaction mixture. Third, with a defined reaction mixture in hand, [35S]cysteine and [35S]Ado-Met, two potential sulfur donors present in the defined reaction mixture, were tested separately as sulfur donors. No 35S was incorporated into newly formed biotin when either [35S]cysteine or [35S]Ado-Met was added to the defined biotin synthase reaction mixture.

Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product.

We report here the first purification of the protein encoded by the Escherichia coli bioB gene. One species of this protein runs on native gels with an electrophoretic mobility typical of a protein with m = 82 kDa, suggesting the protein is a dimer (gene sequence predicts m = 38.7 kDa). There are two iron- and two acid-labile sulfur atoms per protein monomer. Solutions containing the protein are red and have an absorbance spectrum characteristic of proteins with [2Fe-2S] clusters. In its oxidized native state, the protein is EPR-silent. Upon addition of dithionite, the protein's UV-visible absorbance spectrum is very slowly bleached, and an EPR active species is produced that displays a signal at gavg = 1.95. All these results are consistent with this protein containing one [2Fe-2S] cluster per monomer. The EPR spin quantitation is only 5-10% of expected. Since this protein loses iron upon reduction with dithionite, the low-spin quantitation is probably due to cluster instability in the reduced state. Another species of the bioB gene products has also been purified which runs on native gels with an electrophoretic mobility typical of a protein with m = 104 kDa. This species appears to be a dimer with one [2Fe-2S] cluster per dimer. The 104-kDa protein can be converted to the 82-kDa protein upon incubation with Fe3+ and S2-. The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro in the presence of NADPH, AdoMet, Fe3+ or Fe2+, and additional unidentified factors from the crude extracts of E. coli. The Km for dethiobiotin in this reaction has been found to be 2 microM.

The gene for biotin synthase from Saccharomyces cerevisiae: cloning, sequencing, and complementation of Escherichia coli strains lacking biotin synthase.

Biotin synthase catalyzes the insertion of a sulfur atom between two carbon atoms of dethiobiotin to form biotin in the last step of the biotin biosynthesis pathway. In Escherichia coli, biotin synthase is coded for by bioB gene. We report here cloning, sequencing, and initial functional characterization of the yeast gene for biotin synthase in Saccharomyces cerevisiae. We have named this gene BIO2. It consists of a 355-codon open reading frame near the ZUO1 gene. Analysis of the yeast protein encoded by the BIO2 gene reveals that it shares extensive homology with biotin synthases of E. coli and Bacillus sphaericus. The yeast and the two bacterial biotin synthase proteins have similar molecular weights, amino acid compositions, and hydropathies. The plasmid pUCBIO2 containing the yeast BIO2 gene completely complements E. coli bioB- and delta bio mutants and enables these mutants to grow on dethiobiotin. Although BIO2 is physically linked to ZUO1, which encodes the putative left-handed Z-DNA binding protein zuotin, it appears to be regulated independently from it. The yeast BIO2 and ZUO1 genes reside near ADE3 gene on chromosome VII. BIO2 is the first eukaryotic gene reported from the biotin biosynthetic pathway.