ABSTRACT It was shown for the first time the presence of the soluble cytokinin�binding protein wi... more ABSTRACT It was shown for the first time the presence of the soluble cytokinin�binding protein with a mol wt of 67 kD (CBP67) in the cyanobacterium Synechocysti sp. PCC 6803. CBP67 was isolated by two indepen� dent methods: by affinity chromatography on zeatin�Sepharose and by immunoaffinity chromatography on zeatin�Sepharose using monoclonal antibodies (mAb) raised against maize CBP70 (70 kD). The cytokinin� binding properties of CBP67 were assessed by its interaction with anti�idiotype Ab (Aba�i) raised against Ab to zeatin. Aba�i are, in fact, Ab to zeatin�binding site of the protein. The high specificity of interaction between trans�zeatin and CBP67 was demonstrated. Zeatin riboside and cis�zeatin mahifested a reduced interaction with CBP67; other phytohormones (auxin, gibberellic acid, and ABA) and adenine did not interact with the protein at all. Cyanobacterial CBP67 activated in vitro transcription in the Synechocystis lysate. The highest effect on transcription was observed at a combined action of CBP67 and trans�zeatin. Cyanobacterial CBP67 activated transcription in the barley chloroplast lysate as well. The totality of the results indicates a possibility of the existence of systems of cytokinin signal perception in the evolutionary precursor of chloroplasts, cyanobacteria, which could bring this system into a plant cell.
Temperature-dependent changes in genomic DNA supercoiling might play an important role in tempera... more Temperature-dependent changes in genomic DNA supercoiling might play an important role in temperature perception and responsive gene regulation. In the cyanobacterium Synechocystis, low temperatures induce the expression of the genes for fatty acid desaturases that introduce double bonds into acyl chains of lipid-bound fatty acids, thus regulating the membrane fluidity. I studied the effects of low temperature on supercoiling of the genomic DNA region that contains the regulatory elements of the desB gene for the omega3 desaturase, which is strongly induced by cold. Upon decrease in temperature, the degree of DNA supercoiling increased in this region. Novobiocin, an inhibitor of the DNA gyrase, prevented low-temperature-induced changes in DNA supercoiling and affected the expression of several desaturase genes with the most effect on desB. Decreasing in temperature induces three genes of FA desaturases encoding delta12, delta6 and omega3 desaturases in Synechocystis cells. Novobiocin inhibited completely low-temperature-induced transcription of desB, accumulation of the corresponding protein, and the formation of the omega3 unsaturated fatty acids. In the presence of novobiocin, the novobiocin-resistant mutant cells of Synechocystis responded to the low-temperature treatment in the same way as the wild-type cells in the absence of the antibiotics. Thus, temperature-induced changes in DNA supercoiling might form an essential part of a global regulation pathway leading to low-temperature acclimation in this mesophilic cyanobacterium.
ABSTRACT It was shown for the first time the presence of the soluble cytokinin�binding protein wi... more ABSTRACT It was shown for the first time the presence of the soluble cytokinin�binding protein with a mol wt of 67 kD (CBP67) in the cyanobacterium Synechocysti sp. PCC 6803. CBP67 was isolated by two indepen� dent methods: by affinity chromatography on zeatin�Sepharose and by immunoaffinity chromatography on zeatin�Sepharose using monoclonal antibodies (mAb) raised against maize CBP70 (70 kD). The cytokinin� binding properties of CBP67 were assessed by its interaction with anti�idiotype Ab (Aba�i) raised against Ab to zeatin. Aba�i are, in fact, Ab to zeatin�binding site of the protein. The high specificity of interaction between trans�zeatin and CBP67 was demonstrated. Zeatin riboside and cis�zeatin mahifested a reduced interaction with CBP67; other phytohormones (auxin, gibberellic acid, and ABA) and adenine did not interact with the protein at all. Cyanobacterial CBP67 activated in vitro transcription in the Synechocystis lysate. The highest effect on transcription was observed at a combined action of CBP67 and trans�zeatin. Cyanobacterial CBP67 activated transcription in the barley chloroplast lysate as well. The totality of the results indicates a possibility of the existence of systems of cytokinin signal perception in the evolutionary precursor of chloroplasts, cyanobacteria, which could bring this system into a plant cell.
Temperature-dependent changes in genomic DNA supercoiling might play an important role in tempera... more Temperature-dependent changes in genomic DNA supercoiling might play an important role in temperature perception and responsive gene regulation. In the cyanobacterium Synechocystis, low temperatures induce the expression of the genes for fatty acid desaturases that introduce double bonds into acyl chains of lipid-bound fatty acids, thus regulating the membrane fluidity. I studied the effects of low temperature on supercoiling of the genomic DNA region that contains the regulatory elements of the desB gene for the omega3 desaturase, which is strongly induced by cold. Upon decrease in temperature, the degree of DNA supercoiling increased in this region. Novobiocin, an inhibitor of the DNA gyrase, prevented low-temperature-induced changes in DNA supercoiling and affected the expression of several desaturase genes with the most effect on desB. Decreasing in temperature induces three genes of FA desaturases encoding delta12, delta6 and omega3 desaturases in Synechocystis cells. Novobiocin inhibited completely low-temperature-induced transcription of desB, accumulation of the corresponding protein, and the formation of the omega3 unsaturated fatty acids. In the presence of novobiocin, the novobiocin-resistant mutant cells of Synechocystis responded to the low-temperature treatment in the same way as the wild-type cells in the absence of the antibiotics. Thus, temperature-induced changes in DNA supercoiling might form an essential part of a global regulation pathway leading to low-temperature acclimation in this mesophilic cyanobacterium.
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Papers by Dmitry Los