共查询到20条相似文献,搜索用时 31 毫秒
1.
A new, high-efficiency, DNA-cloning vector pJ1-8 was derived in two steps from the chimeric plasmid pD7 consisting of RSF
0885(amp
r) andHaemophilus influenzae chromosomal DNA. pJl-8 has only oneEcoRI site and a molecular weight of only 2.5 × 106. No detectableamp
r transformation was obtained with pJl-8 DNA. However,amp
r transformation increases markedly ifHaemophilus influenzae chromosomal DNA segments are spliced into it, providing a very facile assay for detecting inserts. 相似文献
2.
A temperature sensitive kanamycin (Km) resistant R plasmid, Rtsl, was found to confer cupric ion (Cu2+) resistance on its hosts in . At conjugal transfer, two kinds of segregants were obtained from Rtsl, i.e. Cu2+ resistant, Km sensitive and Km resistant, Cu2+ sensitive plasmids. Protein T existed in cells harboring Rtsl or the CurKms-plasmid. The inhibitory effect on the host cell growth at 43°C was observed with Rtsl+ or the KmrCus-plasmid+ cells. A relationship between these Rtsl derivatives and Rtsl in which has been studied was discussed. 相似文献
3.
H Kung M Tainsky H Weissbach 《Biochemical and biophysical research communications》1978,81(3):1000-1010
The regulation of the synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacps or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of repressor in the S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)1 for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacps DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen. 相似文献
4.
A Sugino 《Biochemical and biophysical research communications》1979,91(4):1321-1329
Mitochondrial DNA from contains high “A+T”-rich region. Its DNA replication starts in the “A+T”-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the “A+T”-rich region of mitochondrial DNA from has been cloned in . The chimeric plasmid DNA containing the “A+T”-rich region stimulates DNA replication system from mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA. 相似文献
5.
A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant 总被引:3,自引:0,他引:3
This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (ampr) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the ampr gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the ampr gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid. 相似文献
6.
Molecular characterization of a stable Flac plasmid 总被引:2,自引:0,他引:2
F L Macrina E Balbinder A Bassel 《Biochemical and biophysical research communications》1973,54(2):737-743
FS is a thermostable extrachromosomal element isolated in which is altered in its replication as compared to its precursor Fts114. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of Fts114 to be 81 × 106 daltons while that of FS is 109 × 106 daltons. FS may carry a segment of chromosomal or cryptic plasmid DNA. 相似文献
7.
The ionic influence and ouabain sensitivity of lymphocyte Mg2+-ATPase and ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of was located inside the membrane.Concanavalin A induced an early stimulation of Mg2+-ATPase and both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). activity was undetectable in thymocytes. However, in spleen lymphocytes activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation. 相似文献
8.
The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in the range of 30–100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4–26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adynyah, J.T. Penniston and E. Carafoli, 1981, J.Biol.Chem.256,. 395 – 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that of the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter. 相似文献
9.
Hiromichi T. Narahara Vincent G. Vogrin John D. Green Ronald A. Kent Michael K. Gould 《生物化学与生物物理学报:生物膜》1979,552(2):247-261
A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. , Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed () pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction. 相似文献
10.
Sam W. Woo Anthony W. Linnane Phillip Nagley 《Biochemical and biophysical research communications》1982,109(2):455-462
strain DC5-E2 that lacks mtDNA () can be cotransformed with a mixture of mtDNA and the plasmid YEp13 (LEU2/2μ/pBR322) to produce Leu+ transormants which, on being mated to tester strains, generate respiratory competent diploids (such events are denoted marker rescue). In this work strain DC5-E2 was transformed with recombinant molecules consisting of a mtDNA segment including the gene inserted into YEp13. The Leu+ transformants made with the recombinant plasmids were unable to rescue testers carrying mutations in the region, in contrast to Leu+ cotransformants made with mixtures of YEp13 and mtDNA. We conclude that marker rescue events occur as a result of interactions between the mtDNA of the tester and the mtDNA sequences introduced by transformation. Such interactions cannot occur when the latter mtDNA is forced to replicate in covalent association with YEp13, probably in the nucleus. 相似文献
11.
A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ()-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [3H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated ), there are several minor surface glycopeptides () which are apparent extrinsic membrane components, and two surface glycopeptides () which are intrinsic membrane components. 相似文献
12.
Peter J. Abbott 《Molecular & general genetics : MGG》1985,201(1):129-132
Summary Previous studies have shown that transformation of Escherichia coli by plasmid DNA modified in vitro by carcinogens leads to RecA-dependant recombination between homologous plasmid and chromosomal DNA sequences. The mechanism of this recombination has now been studied using recombination-deficient mutants, and the influence of induction of the SOS response on the level of recombination investigated. Plasmid pNO1523, containing the str
+ operon (Sms), has been modified in vitro by either irradiation with UV light, or by reaction with (±) trans-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and used to transform streptomycin-resistant hosts. The formation of Ampr transformants which also carry streptomycin resistance was used as a measure of the level of recombination between plasmid and chromosomal DNA. Transformation of recB and recC mutants produced no change in the level of recombination while in the recF mutant a significant decrease was observed compared to the wild type host. Thermal induction of the SOS response in tif-1 and tif-1 umuC mutants followed by transformation led to a four-fold increase in recombination in both cases. The results suggest that the streptomycin-resistant transformants arise exclusively via a recombinational pathway which is largely dependant on the recF gene product, and that this pathway is influenced by induction of the SOS response. These results are discussed in terms of the mechanism of this recombination. 相似文献
13.
Cyclic blockade of initiation sites by streptomycin-damaged ribosomes in Escherichia coli: an explanation for dominance of sensitivity 总被引:13,自引:0,他引:13
In bacterial extracts streptomycin is known not only to inhibit ribosomal activity but also to cause gradual release of ribosomes from polysomes. Nevertheless, we now find that after streptomycin has virtually halted protein synthesis in cells of Escherichia coli K12 a substantial (though reduced) level of polysomes persists. These polysomes are evidently maintained by turnover rather than by static blockade, for in streptomycin-treated cells [3H]uracil pulses are rapidly incorporated in the polysomal messenger RNA; moreover, if the synthesis of RNA or the formylation of methionyl-transfer RNA is blocked the polysome level decreases rapidly. Streptomycin thus appears to cause a cycle of ribosomal initiation, blockage of chain extension, gradual release, and reinitiation.The resulting cyclic blockade of initiation sites can account for the dominance of streptomycin sensitivity over resistance in 2 heterozygotes. In confirmation of this model, the inactive resistant ribosomes in treated heterozygotes were found to resume activity if the cells were lysed and excess messenger was provided. These findings further suggest that in sensitive cells damage to only a fraction of the ribosomal population by streptomycin may be sufficient to block protein synthesis. 相似文献
14.
Localization of the structural gene for the ' subunit of RNA polymerase in Escherichia coli 总被引:3,自引:0,他引:3
A minicell-producing strain of carrying an F′ factor, KLF10-1, forms minicells that contain plasmid but not chromosomal DNA. These minicells were found to synthesize two polypeptides corresponding precisely to the β and β′ subunits of RNA polymerase in SDS-polyacrylamide gel electrophoresis. In contrast, minicells obtained from an isogenic strain carrying F13-1 do not synthesize these proteins under similar conditions. These results indicate that the structural genes for the β′ as well as β subunits of the polymerase are located on the chromosomal segment (78 to 81 min on the standard genetic map of ) carried by KLF10-1. 相似文献
15.
S F Jackson B R Wentzell D R McCalla K B Freeman 《Biochemical and biophysical research communications》1977,78(1):151-157
L(+)--chloramphenicol induces reversion of His? strains TA100 and TA1535 in the conventional Ames' assay without microsomal activation. Any mutagenicity of D(?)--chloramphenicol was masked by toxicity. Similarly, a sensitive fluctuation test showed mutagenesis with L(+)--chloramphenicol at concentrations of 0.5 μM and above but the D(?) isomer proved to be toxic even at these low levels. The L(+) isomer caused single strand breaks in the DNA of and strains TA1535, TA100 and TA1976. The D(?) isomer caused breaks in and TA1976 although it was less effective and it did not produce DNA breaks in TA1535 or TA100. 相似文献
16.
Y Iwasa K Yonemitsu K Matsui K Fukunaga E Miyamoto 《Biochemical and biophysical research communications》1981,98(3):656-660
A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca2+-dependent cyclic AMP phosphodiesterase of . The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca2+,Mg2+)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca2+-dependent fashion with an apparent Ka of 5 × 10?5. The factor and brain calmodulin had no effect on the phosphodiesterase of . It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes. 相似文献
17.
When strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [3H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome. 相似文献
18.
Insertion of a genetic marker into the ribosomal DNA of yeast 总被引:38,自引:0,他引:38
Plasmid pBR322 carrying the yeast LEU2+ gene transforms leu− yeast into LEU+ at a low frequency by integration at homologous chromosomal DNA. When one-half of the yeast rDNA repeat unit (BglII-A) is inserted into the plasmid, the frequency of yeast transformation increases 100- to 200-fold, in proportion to the increased amount of homologous repetitive rDNA available for integration. When the other half of the repeat unit (BglII-B) is inserted into the plasmid, the transformation frequency increases by a factor of 104, and the transformants are very unstable. It is likely that this fragment of rDNA contains a yeast origin of replication. This plasmid is a useful vector for cloning fragments of yeast DNA in yeast. We have used the LEU2+ gene, inserted into the rDNA locus, as a genetic marker for mapping the rDNA, in a procedure analogous to the use of antibiotic resistance transposons in the mapping of bacterial genes. Yeast ribosomal DNA is on chromosome XII between asp5 and ura4 as determined by mitotic linkage. Genetic analysis of markers inserted at the rDNA locus should be a useful tool for studying the conservation of sequence homology and the conservation of copy number of repeated genes. 相似文献
19.
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 μM) in a similar way as the from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte . In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal . In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes. 相似文献
20.
A Rosner K M Jakob J Gressel D Sagher 《Biochemical and biophysical research communications》1975,67(1):383-391
A 0.5 × 106r RNA found in plastids of the aquatic angiosperm , is synthesized at a much higher rate than any other rapidly labeling RNA species about h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 106r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in , poly(A) sequences are not detected in the 0.5 × 106r RNA; yet a sucrose gradient fraction which includes RNA of this r stimulates amino acid incorporation by an cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 106r RNA as a chloroplast messenger is discussed. 相似文献