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排序方式: 共有177条查询结果,搜索用时 15 毫秒
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A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions 总被引:55,自引:0,他引:55
Philip Youngman John B. Perkins Richard Losick 《Molecular & general genetics : MGG》1984,195(3):424-433
Summary A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cmr) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cmr
B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl2-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes. 相似文献
4.
The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates. 相似文献
5.
In isolated protoplasts of Vicia faba L. (cv. Weißkernige Hangdown) which were immobilised in a cross-linked alginate matrix, ethane production and ribulose bisphosphate carboxylase (RuBP carboxylase) aetivity were determined after treatment of the alginate strips with various chemical substances.
The stimulation of ethane formation and the inhibition of RuBP carboxylase activity by the toxic materials might provide the basis for a simple, rapid and sensitive system for the determination of chemicals in the environment. 相似文献
The stimulation of ethane formation and the inhibition of RuBP carboxylase activity by the toxic materials might provide the basis for a simple, rapid and sensitive system for the determination of chemicals in the environment. 相似文献
6.
Sandra Youngman Mansoor Sarfarazi Maja Bucan Marcy MacDonald Barbara Smith Michael Zimmer Conrad Gilliam Anna-Maria Frischauf John J. Wasmuth James F. Gusella Hans Lehrach Peter S. Harper Duncan J. Shaw 《Genomics》1989,5(4)
Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene. 相似文献
7.
Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements. 总被引:6,自引:2,他引:4
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J A Gutierrez P J Crowley D P Brown J D Hillman P Youngman A S Bleiweis 《Journal of bacteriology》1996,178(14):4166-4175
New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus. 相似文献
8.
Variation in heat shock proteins within tropical and desert species of poeciliid fishes 总被引:8,自引:0,他引:8
Norris CE; diIorio PJ; Schultz RJ; Hightower LE 《Molecular biology and evolution》1995,12(6):1048-1062
The 70-kilodalton heat shock protein (hsp70) family of molecular
chaperones, which contains both stress-inducible and normally abundant
constitutive members, is highly conserved across distantly related taxa.
Analysis of this protein family in individuals from an outbred population
of tropical topminnows, Poeciliopsis gracilis, showed that while
constitutive hsp70 family members showed no variation in protein isoforms,
inducibly synthesized hsp70 was polymorphic. Several species of
Poeciliopsis adapted to desert environments exhibited lower levels of
inducible hsp70 polymorphism than the tropical species, but constitutive
forms were identical to those in P. gracilis, as they were in the
confamilial species Gambusia affinis. These differences suggest that
inducible and constitutive members of this family are under different
evolutionary constraints and may indicate differences in their function
within the cell. Also, northern desert species of Poeciliopsis synthesize a
subset of the inducible hsp70 isoforms seen in tropical species. This
distribution supports the theory that ancestral tropical fish migrated
northward and colonized desert streams; the subsequent decrease in
variation of inducible hsp70 may have been due to genetic drift or a
consequence of adaptation to the desert environment. Higher levels of
variability were found when the 30- kilodalton heat shock protein (hsp30)
family was analyzed within different strains of two desert species of
Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In
both cases the distribution of hsp30 isoform diversity was similar to that
seen previously with allozyme polymorphisms.
相似文献
9.
10.
Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP 总被引:8,自引:5,他引:3
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We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase. 相似文献