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P-B2-09 Regulatory sequences controlling the neutrophil-specific expression of the interleukin-8 receptor genes
B2 NA-protein interactions and gene regulation P-B2- 10 TRANSCRIPTIONAL INHIBITION BY HIGH PRESSURE in vitro L. ERIJMAN AND R. M. CLEGG MaxPlanckInstitutefor Biophysical Chemistry. Dept. of MolecularBiologyD-37077GBttingen, Germany.
P-B2-09 REGULATORY SEQURNCES CONTROLLING THE NEUTROPHIL-SPECIFIC EXPRRSSION OF THE 1NTERLEUKIN-g RECEPTOR GENES WLXINSON, N.; RAMAKRISHNAN, S.;COUSINS, B.; NAVARRO, N.Dept.of Physiology and Biophysics, Univ. of Texas Medical Branch at Galveston (USA) Purpose: Tocharacterize theregulatory elements dictating
Purpose: E. coli RNA Polymeraseundergoes cycles of sequence-dependentconformational changes in transcriptional complexes during RNA synthesis. Many mechanistic features remain unclear. We have used high pressure methods to investigate the stability and flexibility of elongating ternary complexes. Methods: Radioactivity and fluorescence assays were employed to examine the pressure stability and activity of RNAP participating in the elongation phase of transcription. Results: The rate of elongation by E. coli RNAP on DNA templates can be halted reversibly at high pressures. Analysis of solvent and temperature dependence of the pressure-induced inhibition shows evidence for major conformational changes in the core polymerase enzyme during elongation. Conclusions: Pressure perturbs nucleic acid-protein and protein-protein interactions, providing insight into the mechanism of transcription. We discuss our results in the framework of both the nucleic acid destabilization model and the “inchworm” elongation model.
thetissue-speciiic expression of the Interleukin-8 (lL-8) receptor A andB genes.
Methods: Northern blot analysis and IL-8 induced calcium mobilization were used to identify a cell line that proliferates continuously in culture and expresses the IL-8 receptor genes. 5’-sequences of the IL-8 receptor genes A and B were cloned upstream of a luciferase repotter gene, transiently transfocted by eleetroporation into IL-8 receptor-positive and -negative cell lines and expression levels of lwiferase were determined. Results:
A murine granulccytic blastcelllie, 3D,was
identified which expresses IL-8 receptor mRNA levels and induces calcium mobilization in response to human IL-8 similar to that in normal neutrophils. The -8OObp to +86bp of the human IL-8 receptor A gene demonstrated 140-fold higher activity compared with the promoter-less luciferase vector. Deletions of this sequence revealed strong negative (SOObp to -5Sbp) and positive (JSbp to +46bp) elements. Strong positive elements wcfc identified from the -192bp to +332bp sequence of the IL-8 receptor B gene. All sequences exhibital several fold less activity in IL-8 receptor-negative cd lines (NIH3T3,293, HL-60) compared to the IL-8 receptor-positive cell line (3D). Conclusions: The 3D blast cell line represents an excellent ceIhdar system for characterizing the reguhnory elements of the IG8 rweptor genes. 5’-sequences cloned upsbwn of the rcpatcr gene contain the regulatory elements directing the tissue-specific expression of the IL-8 receptor A and B genes.
P-B2-12
P-B2-11 THE TOPOLOGICAL COMPLEMENTARE TY OF THE CHARGED GROUPS CONTROLLING THE INTE RACTION OF DNA WITH CORE HISTONES IN THE NUCLEOSOME. ICHRAPUNOV S., DRAGAN A., SIVOLOB A. TShevchenkd
University
Inst.Genetics (japan) Purpose: In the Exoli RNA polymerase holoenzvme a&Yo, the re&te;e nslble fqr transcrifition aidtivation _by -eic Y nns or c~s factor iS Iocalized to the CXern&l i&&ofihe & subunit (aCTD). The functio al structure of a0 was ainbedto be solved by‘fu MR. Methods: aCTD of 98 mine m&Is was overex re ed and uniform “N and 13Cla&led. The 2Spbp%NA corns d mg lo a cis factor the UP element, was pie red with a T)NA synthesizer. The back$ one dynamics were measuredfor affD and whole a subunit. Results: The aCTD exceDt the 16 N-terminal residues has a compact st&cture of four l@ic$s two helical turns and-two lwg arms en&l
of Kiev (Ukraine)
Purpose. The goal of the present study is to investigatethe structural forms of histone octamer and energeticsof their interactions with DNA in a nucleosome. Methods. We have developed a method for recording the shifts in the Spectralmaximum of tyrosine fluorescencepermiting the recording of the formation/disruption of hydrogen bonds formed by phenolsof tyrosines. Results. The disruption of the contacts between the histone H2A-H2B dimers and (H3-H4)2 tetramer within the octamer as well as the two stage dissociation of histone complexes from DNA has been studied. It was established the energy of electrostatic interactions of histones with DNA within the nucleosome. Conclusions. We have derived four structural fonns of the histone octamer in the nucleosome.It is suggestedthat the spatial distribution of histone octamer cationic groups is precisely determined and they topologically correspond (are complementary)to the DNA phosphates. 82
P-B2-09 REGULATORY SEQURNCES CONTROLLING THE NEUTROPHIL-SPECIFIC EXPRRSSION OF THE 1NTERLEUKIN-g RECEPTOR GENES WLXINSON, N.; RAMAKRISHNAN, S.;COUSINS, B.; NAVARRO, N.Dept.of Physiology and Biophysics, Univ. of Texas Medical Branch at Galveston (USA) Purpose: Tocharacterize theregulatory elements dictating
Purpose: E. coli RNA Polymeraseundergoes cycles of sequence-dependentconformational changes in transcriptional complexes during RNA synthesis. Many mechanistic features remain unclear. We have used high pressure methods to investigate the stability and flexibility of elongating ternary complexes. Methods: Radioactivity and fluorescence assays were employed to examine the pressure stability and activity of RNAP participating in the elongation phase of transcription. Results: The rate of elongation by E. coli RNAP on DNA templates can be halted reversibly at high pressures. Analysis of solvent and temperature dependence of the pressure-induced inhibition shows evidence for major conformational changes in the core polymerase enzyme during elongation. Conclusions: Pressure perturbs nucleic acid-protein and protein-protein interactions, providing insight into the mechanism of transcription. We discuss our results in the framework of both the nucleic acid destabilization model and the “inchworm” elongation model.
thetissue-speciiic expression of the Interleukin-8 (lL-8) receptor A andB genes.
Methods: Northern blot analysis and IL-8 induced calcium mobilization were used to identify a cell line that proliferates continuously in culture and expresses the IL-8 receptor genes. 5’-sequences of the IL-8 receptor genes A and B were cloned upstream of a luciferase repotter gene, transiently transfocted by eleetroporation into IL-8 receptor-positive and -negative cell lines and expression levels of lwiferase were determined. Results:
A murine granulccytic blastcelllie, 3D,was
identified which expresses IL-8 receptor mRNA levels and induces calcium mobilization in response to human IL-8 similar to that in normal neutrophils. The -8OObp to +86bp of the human IL-8 receptor A gene demonstrated 140-fold higher activity compared with the promoter-less luciferase vector. Deletions of this sequence revealed strong negative (SOObp to -5Sbp) and positive (JSbp to +46bp) elements. Strong positive elements wcfc identified from the -192bp to +332bp sequence of the IL-8 receptor B gene. All sequences exhibital several fold less activity in IL-8 receptor-negative cd lines (NIH3T3,293, HL-60) compared to the IL-8 receptor-positive cell line (3D). Conclusions: The 3D blast cell line represents an excellent ceIhdar system for characterizing the reguhnory elements of the IG8 rweptor genes. 5’-sequences cloned upsbwn of the rcpatcr gene contain the regulatory elements directing the tissue-specific expression of the IL-8 receptor A and B genes.
P-B2-12
P-B2-11 THE TOPOLOGICAL COMPLEMENTARE TY OF THE CHARGED GROUPS CONTROLLING THE INTE RACTION OF DNA WITH CORE HISTONES IN THE NUCLEOSOME. ICHRAPUNOV S., DRAGAN A., SIVOLOB A. TShevchenkd
University
Inst.Genetics (japan) Purpose: In the Exoli RNA polymerase holoenzvme a&Yo, the re&te;e nslble fqr transcrifition aidtivation _by -eic Y nns or c~s factor iS Iocalized to the CXern&l i&&ofihe & subunit (aCTD). The functio al structure of a0 was ainbedto be solved by‘fu MR. Methods: aCTD of 98 mine m&Is was overex re ed and uniform “N and 13Cla&led. The 2Spbp%NA corns d mg lo a cis factor the UP element, was pie red with a T)NA synthesizer. The back$ one dynamics were measuredfor affD and whole a subunit. Results: The aCTD exceDt the 16 N-terminal residues has a compact st&cture of four l@ic$s two helical turns and-two lwg arms en&l
of Kiev (Ukraine)
Purpose. The goal of the present study is to investigatethe structural forms of histone octamer and energeticsof their interactions with DNA in a nucleosome. Methods. We have developed a method for recording the shifts in the Spectralmaximum of tyrosine fluorescencepermiting the recording of the formation/disruption of hydrogen bonds formed by phenolsof tyrosines. Results. The disruption of the contacts between the histone H2A-H2B dimers and (H3-H4)2 tetramer within the octamer as well as the two stage dissociation of histone complexes from DNA has been studied. It was established the energy of electrostatic interactions of histones with DNA within the nucleosome. Conclusions. We have derived four structural fonns of the histone octamer in the nucleosome.It is suggestedthat the spatial distribution of histone octamer cationic groups is precisely determined and they topologically correspond (are complementary)to the DNA phosphates. 82