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101.
Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments. 总被引:23,自引:12,他引:11 下载免费PDF全文
Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro. Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices. The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively. The two strands also vary with respect to A-methylation in GATC sites. In cases of asymmetrical methylation, transfection of E. coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand. This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector. The results suggest how these procedures can be optimized. Precise construction of a 93 bp insertion of 9.5% marker yield is described. 相似文献
102.
Fritz Herz Alexander Schermer Murray Halwer Lee H. Bogart 《Archives of biochemistry and biophysics》1981,212(2):581-588
HT-29, a cell line derived from a human colon carcinoma, exhibits very low alkaline phosphatase activity. The enzyme is thermolabile and is of the intestinal type. Hyperosmolality and/or sodium butyrate induce increased levels of activity. The increase is most pronounced with HT-29 cells growing in hyperosmolar medium containing sodium butyrate. Under these conditions specific activity rises over 1000-fold. The effect of hyperosmolality is blocked by cycloheximide and that of sodium butyrate by thymidine, cordycepin, and cycloheximide. By contrast to other human cancer cell lines, the enzyme of HT-29 is not influenced by cell density and glucocorticoid hormones. 5-Bromo-2′-deoxyuridine and inhibitors of DNA synthesis cause a slight increase in specific activity. 相似文献
103.
The Crustacea of some chalk streams in southern England 总被引:1,自引:1,他引:0
Stephanie F. Ham 《Hydrobiologia》1982,97(3):193-201
Records of Crustacea from chalk streams in southern England are described. Classification of sites suggested that flow regime was an important influence on the fauna and the distribution of individual species are discussed in this respect. 相似文献
104.
A modified latex agglutination (LA) test was compared with Gram-staining and counterimmunoelectrophoresis (CIE) for the rapid detection in the cerebrospinal fluid (CSF) of antigen to Haemophilus influenzae type b, Neisseria meningitidis groups A, B and C, Escherichia coli K1, Streptococcus pneumoniae and group B streptococci, seven frequent causes of bacterial meningitis in children. Of 50 CSF samples from patients with culture-proven bacterial meningitis 90% were correctly shown by the LA test to contain antigen of the responsible organism. Gram-staining revealed organisms in 80% of 45 of these samples. In 75% of the 40 samples that were of sufficient volume for CIE, positive results for the appropriate antigen were obtained. The concentration of antigen detected in the CSF by the LA test varied from undetectable to 800 000 ng/ml. Patients with a high concentration (more than 2000 ng/ml or a positive result at dilutions of CSF over 1/8) were significantly more likely to have a poor response to therapy (two died and two had persistent pleocytosis or bacteria in the CSF) than patients with a lower concentration (4/16 v. 0/18, P < 0.05). After appropriate therapy was begun the concentration of antigen fell dramatically, but measurable amounts of antigen persisted in the CSF for up to 6 days. The LA test detected bacterial antigen at concentrations 2 to 70 times below the lower limit detected by CIE. In seven additional patients who had received antibiotics before lumbar puncture was performed the LA test detected antigen from meningitis-causing bacteria even though cultures of the CSF were sterile. In another 145 patients who did not have meningitis the results of the LA test were negative. The LA test, done as described in this article, is easier to perform than CIE and should be a useful addition to the diagnostic tests carried out on the CSF of any patient suspected of having meningitis. 相似文献
105.
Stephanie Gordon Phillips Shiu-Lan Lui David M. Phillips 《In vitro cellular & developmental biology. Plant》1982,18(8):727-738
Summary Epithelial cells may relate to their basement membrane substrates via lectin-like interactions. In a model system for study
of this type of interaction, lectin-coated bacteriological plastic petri dishes were presented as substrates for epithelial
cell adhesion. Of 21 lectins tested by mixed agglutination against two epithelial cell types, Madin-Darby canine kidney (MDCK),
and human embryonic kidney cells (HEK), nine gave less than 5% rosettes and 12 gave 5 to 50% rosettes. Wheat germ agglutinin
(WGA) andGeodia cydonium lectin gave the highest percentage of rosettes. Wheat germ agglutinin was readily adsorbed to plastic surfaces and maintained
specificity in binding interactions. Both MDCK and HEK cells attached as well to WGA coated petri dishes as to conventional
tissue culture dishes. Furthermore, both spread over the lectin-coated surfaces. The MDCK cells grew to confluence and could
be subcultured and maintained indefinitely on such surfaces, although WGA in solution was toxic to the cells in concentrations
as low as 0.1 to 1.0 μg/ml. Cell attachment to WGA coated dishes was blocked by cycloheximide only if the cells had been preincubated
with the inhibitor for several hours. Cell attachment was not inhibited by pretreatment of cells with neuraminidase. Precoating
cells with WGA blocked binding to both WGA-coated surfaces and untreated tissue culture dishes. Cells attached to WGA-coated
dishes could not be readily dislodged by trypsin-EDTA for the first 2 h after subculture. By 4 h, attachment was again trypsin
sensitive, suggesting that the cells synthesized a trypsin-sensitive material that was laid down between the cell surface
and the WGA-coated dish. Regeneration of trypsin sensitivity was not blocked by cycloheximide.
This work was supported by Research Grant AG01986 from the National Institutes of Health, Bethesda, Maryland. 相似文献
106.
Philip L. Smith Stephanie A. Orellana Michael Field M.D. 《The Journal of membrane biology》1981,63(3):199-206
Summary Unidirectional fluxes of35SO4 across and into rabbit ileal epithelium were measured under short-circuit conditions, mostly at a medium SO4 concentration of 2.4mm. Unidirectional mucosa (m)-to-serosa (s) ands-to-m fluxes (J
ms,J
sm) were 0.456 and 0.067 moles hr–1 cm–2, respectively.J
ms was 2.7 times higher in distal ileum than in mid-jejunum. Ouabain abolished net SO4 transport (J
net) by reducingJ
ms. Epinephrine, a stimulus of Cl absorption, had no effect on SO4 fluxes. Theophylline, a stimulus of Cl secretion, reducedJ
ms without affectingJ
sm, causing a 33% reduction inJ
net. Other secretory stimuli (8-Br-cAMP, heat-stable enterotoxin, Ca-ionophore A23187) had similar effects. Replacement of all Cl with gluconate markedly reducedJ
net through both a decrease inJ
ms and an increase inJ
sm. The anion-exchange inhibitor, 4-acetoamido-4-isothiocyano-2,2-sulfonic acid stilbene (SITS), when added to the serosal side, reducedJ
ms by 94%, nearly abolishingJ
net. SITS also decreasedJ
sm by 75%. Mucosal SITS (50 m) was ineffective. 4,4-diisothiocyano-2,2-sulfonic acid stilbene (DIDS) had effects similar to SITS but was less potent. Measurements of initial rates of epithelial uptake from the luminal side (J
me) revealed the following: (1)J
me is a saturable function of medium concentration with aV
max of 0.94 moles hr–1 cm–2 and aK
1/2 of 1.3mm; (2) replacing all Na with choline abolishedJ
me; (3) replacing all Cl with gluconate increasedJ
me by 40%; (4) serosal SITS had no effect onJ
me; and (5) stimuli of Cl secretion had no effect onJ
me or increased it slightly. Determination of cell SO4 with35SO4 indicated that, at steady-state, the average mucosal concentration is 1.1 mmoles per liter cell water, less than half the medium concentration. Cell SO4 was increased to 3.0mm by adding SITS to the serosal side. Despite net transport rates greater than 1.4 Eq hr–1 cm–2, neither addition of SO4 to the SO4-free medium nor addition of SITS to SO4-containing medium altered short-circuit current. The results suggest that (1) ileal SO4 absorption consists of Na-coupled influx (symport) across the brush border and Cl-coupled efflux (antiport) across the basolateral membrane; (2) the overall process is electrically neutral; (3) the medium-to-cell Cl concentration difference may provide part of the driving force for net SO4 absorption; and (4) since agents affecting Cl fluxes (both absorptive and secretory) have little effect on SO4 fluxes, the mechanisms for their transcellular transports are under separate regulation. 相似文献
107.
Fritz Schöffl Walter Arnold Alfred Pühler Josef Altenbuchner Rüdiger Schmitt 《Molecular & general genetics : MGG》1981,181(1):87-94
Summary The 10.7 kilobase (kb) tetracycline resistance transposons Tn1721 and Tn1771, isolated from disparate sources, are completely homologous on the basis of heteroduplex analyses. Both transposable elements are capable of forming multiple duplications of a 5.3 kb portion encompassing the resistance genes (tet region). A model accounting for both, recA-independent translocation and recA-dependent amplification, postulates two direct and one inverted repeat as essential constituents of the transposons. DNA sequence analyses of Tn1721 and Tn1771 have substantiated this model. They demonstrated three identical 38 base pair repeats identically in both transposons dividing them into a minor transposon and a tet region. Identical sequences of at least 87 base pairs providing recombination hot spots for gene duplication have been found at the ends of the repetitious tet region. Translocation of Tn1721 and Tn1771 generates five base pair direct repeats at the respective sites of insertion. On the basis of the heteroduplex molecules and sequences analyzed the two transposons are identical.To Professor Wolfram Heumann on the occasion of his 65th birthday 相似文献
108.
Summary Milkweed (Asclepias syriaca L.) umbels and stems attended by ants (Lasius neoniger Emery and Tapinoma sessile (Say)) initiated significantly fewer pods and showed a trend to produce fewer mature pods than did umbels and stems not attended by ants. Since similar numbers of pollinia were inserted in ant-attended flowers, we hypothesize that ant-excluded flowers obtain more pollinia from other clones than do ant-attended flowers of this normally non-selfing species. If pollinators from other clones first visited flowers not depleted of nectar by ants, they could provide this source of foreign pollinia. Our experiments suggest that nocturnal noctuid and geometrid moths can provide these pollinia. 相似文献
109.
Fritz A. Henn Rick Venema David Anderson Ake Sellstrom 《Journal of neurochemistry》1980,34(6):1671-1677
Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [3 H]GABA receptor binding and [3 H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high-affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β-alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation. 相似文献
110.
Gerhard Toggenburger Max H?sermann Beat Mütsch Giulio Genoni Markus Kessler Fritz Weber Dietrich Hornig Brigitte ONeill Giorgio Semenza 《生物化学与生物物理学报:生物膜》1981,646(3):433-443
l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na+-dependent and potential-sensitive transport process. This uptake shows saturation () and is strongly stimulated by low concentrations of N3?. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter. 相似文献