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71.
72.
An equilibrium between distorted and undistorted DNA in the adult chicken beta A-globin gene 总被引:2,自引:0,他引:2
We have used single strand specific nucleases to map DNA distortion in the adult chicken beta A-globin gene. We have detected two structures of that kind and have mapped nuclease-cutting sites at one base resolution. One prominent site is centered at -190 relative to the RNA capping site and is positioned at the center of a stretch of contiguous C residues. The second site is near the first intron/exon junction (+620) and appears as a series of discrete 1-base-long enzyme-cutting sites. Based upon the pattern of nuclease cutting and the kinetics of nuclease cutting we conclude that the "poly(C)" stretch may assume a looped geometry in supertwisted DNA molecules which is similar to that proposed by Felsenfeld (Nickol, J. M., and Felsenfeld, G. (1983) Cell 35, 467-477). We show that S1 nuclease cuts within the intron occur mainly at the end points of polypurine segments and suggest that such end points may assume a distorted transitional geometry. We find that Neurospora crassa endonuclease cuts both the promotor and intron sites in linear DNA molecules but that in linear DNA the cutting process is limited by a first order conformation change of the DNA substrate. Based upon those kinetics we propose that in unstressed DNA, each of the two sites can convert between a distorted and undistorted geometry. In the enzyme assay buffer at 37 degrees C, the time constant for the equilibrium is nearly 10 h for the promotor site and 7 h for the intron. 相似文献
73.
Localization of tyrosine kinase-coding region in v-abl oncogene by the expression of v-abl-encoded proteins in bacteria 总被引:26,自引:0,他引:26
A series of plasmids containing different segments of the v-abl oncogene have been constructed to express different portions of the v-abl protein in bacteria. The tyrosine kinase activity of these proteins was determined by an in vitro assay employing histones or angiotensin II as substrates for the v-abl-encoded tyrosine kinase. These experiments show that the 5'-1.2 kilobases of v-abl is necessary and sufficient to produce an active tyrosine kinase which is functional as a monomeric soluble protein. The kinase-coding region corresponds to the minimal region of v-abl required for the transformation of fibroblasts. The kinase-coding region also coincides with the conserved protein sequences which are found in other tyrosine kinases. A compact domain of the v-abl protein including this kinase-coding region can accumulate to high levels in bacteria. The C-terminal region of the v-abl protein is not needed for the kinase activity and is rapidly degraded in bacteria. 相似文献
74.
Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin 总被引:5,自引:0,他引:5
B Martin C J Pallen J H Wang D J Graves 《The Journal of biological chemistry》1985,260(28):14932-14937
Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low molecular weight phosphate esters. The low molecular weight phosphatase activity of calcineurin has been studied by utilizing tyrosine phosphate derivatives. Kinetic studies suggest that the substrate specificity is dependent upon the electronic nature of the substrate in contrast to results obtained with alkaline phosphatase from Escherichia coli. Comparison of calcineurin and acid-catalyzed hydrolyses indicates a 1:1 correlation between the rate constants for the two processes. This correlation and other model studies have been utilized to provide insight into the chemical mechanism of calcineurin. Possible chemical mechanisms for calcineurin are discussed. 相似文献
75.
M X Zuber S M Wang K V Thammavaram D K Reed B C Reed 《The Journal of biological chemistry》1985,260(26):14045-14052
Sulfonylurea compounds are hypoglycemic agents which by unknown mechanisms alter the amount of insulin receptor and the rate of glucose utilization in tissues exposed to the drugs. In this study the effects on insulin binding and uptake of 2-deoxyglucose by 3T3-L1 adipocytes were assessed after maintaining cell monolayers for 1-3 days in medium containing different concentrations of the sulfonylurea, tolbutamide. The amount of 125I-insulin bound by treated monolayers gradually increased to values 150-250% of those of control monolayers after 2-3 days of exposure to 1.5 mM tolbutamide. Such increases in insulin binding capacity arose primarily from an increase in receptor number and not from an alteration in the affinity of the receptor for insulin. Concomitant with the changes observed for the insulin receptor, tolbutamide-treated monolayers expressed 1.5-2-fold higher rates of uptake of 2-deoxyglucose relative to control monolayers at concentrations of insulin between 0 and 10(-10) M. This study thus demonstrates the responsiveness of adipocytes to tolbutamide and also establishes the usefulness of 3T3-L1 cells as a model system in which to study the mechanism of tolbutamide action, both as it relates to the use of sulfonylurea compounds in clinical applications and as possible probes for perturbing and studying relatively uncharacterized regulatory pathways controlling receptor level and biological responses to insulin. 相似文献
76.
C W Chi D X Zhu N Q Lin L X Xu F L Tan L X Wang 《Biological chemistry Hoppe-Seyler》1985,366(9):879-885
After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family. 相似文献
77.
Simplified purification and testing of colloidal gold probes 总被引:2,自引:0,他引:2
A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates. 相似文献
78.
Segmental pulmonary vascular resistance in progressive hydrostatic and permeability edema 总被引:1,自引:0,他引:1
Wang C. G.; Hakim T. S.; Michel R. P.; Chang H. K. 《Journal of applied physiology》1985,59(1):242-247
We used the in situ blood-perfused left lower lobe preparation of the dog to examine the effect of hydrostatic and permeability edema on the slope and intercept of the vascular pressure-flow (P/Q) relationship and on the longitudinal distribution of vascular resistance with the arterial and venous occlusion technique. Hydrostatic edema (HE) was induced by raising the venous pressure, and permeability edema (PE) was induced with alpha-naphthylthiourea. When the hematocrit (Hct) of the perfusate was kept normal (approximately 40%), HE had no significant effect on either the slope or the intercept of the P/Q relationship or on the distribution of vascular resistance. PE caused a small increase in the intercept of the P/Q relationship and a small rise in the resistance of the vessels in the middle segment. In another series of HE experiments in which Hct was allowed to increase during edema formation, there was a marked increase in vascular resistance. We conclude that edema per se does not increase vascular resistance significantly and that the increases in vascular resistance which were observed previously by other investigators in the isolated lungs may be due to increases in blood hematocrit. 相似文献
79.
白头翁的受精及组织化学研究 总被引:2,自引:1,他引:1
成熟花粉为二细胞,生殖细胞的蛋白质染色较营养细胞的深,淀粉粒充满营养细胞。花粉管常见穿人正在退化中的助细胞,并释放出两个精子,大量稠浓的蛋白质和淀粉,个别的释放在助细胞与胚囊壁之间。在一些卵细胞核,次生核(或极核)中,受精前后有1—3个小核仁(约1微米);助细胞具丝状器;反足细胞宿存,具多核和多核仁。胚囊中各个细胞的原生质稠浓程度和蛋白质染色深浅不同,其中以反足细胞和助细胞的原生质最稠浓,蛋白质染色最深。淀粉十分贫乏,绝大多数卵细胞,中央细胞和几乎全部助细胞和反足细胞均不含淀粉。双受精属于有丝分裂前类型,两性核融合步骤为:精子核接近和贴附在卵核和次生核上(或极核上),两性核的核膜溶解,其染色质沉入融合核,并随之松解,同时出现雄性核仁,最终,雄性核仁和雌性核仁合并,形成具单核和单核仁的合子和初生胚乳细胞。另外,雄性核仁同卵核仁合并较雄性核仁和次生核的晚,所以卵细胞完成受精较次生核晚。 相似文献
80.
Summary The structural gene encoding human -aminolevulinate dehydratase has been assigned to the long arm of chromosome 9 by somatic cell hybridization techniques using murine erythroleukemia-human fibroblast somatic cell hybrids. Dimethyl sulfoxide induction of erythroid differentiation in these hybrid cells resulted in a 3 to 12-fold increase in the levels of total -aminolevulinate dehydratase. Human -aminolevulinate dehydratase was detected by an immunodiscrimination assay using polyclonal mouse anti-human aminolevulinate dehydratase antibodies. Of four primary hybrid clones, each from an independent fusion, one hybrid line, XX-8, was positive for human -aminolevulinate dehydratase. Examination of 23 secondary, tertiary, and quaternary XX-8 subclones revealed that the expression of the human isozyme segregated with human chromosome 9q, confirming the provisional regional assignment made by classical linkage studies. One positive quaternary clone, XX-8-H21-H7-2, expressed human -aminolevulinate dehydratase activity and contained only human 9q13»qter. In addition, studies of tetiary and quaternary subclones from two series, XX-8-A31 and XX-8-H21-H7, indicated that murine regulatory factors increased the human as well as the murine enzymatic activity following induction of erythroid differentiation. 相似文献