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1.
Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background. 相似文献
2.
Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle. 相似文献
3.
A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations. 相似文献
4.
Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of Mr 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe. 相似文献
5.
6.
Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation. 相似文献
7.
B. C. Loughman 《Plant and Soil》1987,99(1):63-74
Summary Little is known about the biochemical basis of the genotypic differences in the capacity for ion absorption and transport
shown by many crop species. If these differences reflect the abundance of a specific membrane component or the activity of
an enzyme we need to have some indication of thein vivo operation of these systems in whole plants. Thein vivo assessment of glycolytic enzymes is illustrated by the effects of mannose on the transport of phosphate in maize varieties.
The application of high resolution31P-NMR to the study of intermediary metabolismin vivo is also helpful in following transport capacity.
The five-fold rise in respiratory rate that occurs when freshly cut potato slices are maintained in aerated water for 24 hours
is accompained by the turning on of a wide range of biochemical systems. Major increases in the capacity for absorption of
phosphate from low concentrations (0.1 μM–10 μM) and in the phosphorylative ability of the tissue are seen, indicating the synthesis of a carrier involved in phosphate transport.
These capacities differ markedly between individual tissues of the tuber,i. e. pith, parenchyma, cortex and buds and large differences have been observed between comparable tissue from different varieties.
Varieties grown under similar conditions have been compared and shown to exhibit different kinetics with respect to the development
of the low concentration absorption site and in their sensitivity to the effects of uncouplers such as 2,4-dinitrophenol. 相似文献
8.
Norma J. Greenfield Mussadeq Hussain John Lenard 《Biochimica et Biophysica Acta (BBA)/General Subjects》1987,926(3)
The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH higher Pi content, and polyphosphates of longer average chain lenght, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an icnrease in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments. 相似文献
9.
Summary Time-course experiments of phosphate uptake by size-fractionated phytoplankton were conducted in oligotrophic Kennedy and Sproat Lakes. The objective was to determine if large phytoplankton obtained more phosphate than smaller cells, when the nutrient was present at higher concentrations. Studies at Kennedy Lake revealed that uptake rates in the 0.2–3.0 m fraction were very sensitive to the time they were exposed to elevated concentrations; rates determined over the 60–120 min interval were less than 30% of those recorded over the 0–60 min interval. In contrast, there was little difference in uptake rates over these intervals for cells>3.0 m. At Sproat Lake phosphate incorporation into the two size fractions was followed after the aerial fertilization of the lake with inorganic nutrients. Following nutrient addition the proportion of phosphate entering the>3.0 m size fraction increased from ca. 35% to ca. 85%. Despite these observations, it is doubtful that larger cells are able to sequester enough phosphate from pulses to realize the same specific growth rates as their smaller counterparts. 相似文献
10.
The aim of this work was to examine the effect of abrupt changes in temperature in the range 5 to 30°C upon the rate of photosynthetic carbon assimilation in leaves of barley (Hordeum vulgare L.). Measurement of the CO2-assimilation rate in relation to the intercellular partial pressure of CO2 at different temperatures and O2 concentrations and at saturating irradiance showed that as the temperature was decreased photosynthesis was saturated at progressively lower CO2 partial pressures and that the transition between the CO2-limited and ribulose-1,5-bisphosphate-regeneration-limited rate became more abrupt. Feeding of orthophosphate to leaves resulted in an increased rate of CO2 assimilation at lower temperatures at around ambient or higher CO2 partial pressures both in 20% O2 and in 2% O2 and it removed the abruptness in the transition between the CO2-limited and ribulose-1,5-bisphosphate-regeneration-limited rates. Phosphate feeding tended to inhibit carbon assimilation at higher temperatures. The response of carbon assimilation to temperature was altered by feeding orthophosphate, by changing the concentrations of CO2 or of O2 or by leaving plants in the dark at 4°C for several hours. Similarly, the response of carbon assimilation to phosphate feeding or to changes in 2% O2 was altered by leaving the plants in the dark at 4°C. The mechanism of limitation of photosynthesis by an abrupt lowering of temperature is discussed in the light of the results.Abbreviations A
rate of CO2 assimilation
-
P
i
intercellular partial pressure of CO2
- RuBP
ribulose-1,5-bisphosphate 相似文献