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1.
Nicholas Cianciotto Terry Serwold-Davis Neal Groman Giulio Ratti Rino Rappuoli 《FEMS microbiology letters》1990,66(1-3):299-301
Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes. 相似文献
2.
L Morbiato F Tombola S Campello G Del Giudice R Rappuoli M Zoratti E Papini 《FEBS letters》2001,508(3):479-483
The protein vacuolating toxin A (VacA) of Helicobacter pylori converts late endosomes into large vacuoles in the presence of permeant bases. Here it is shown that this phenomenon corresponds to an accumulation of permeant bases and Cl(-) in HeLa cells and requires the presence of extracellular Cl(-). The net influx of Cl(-) is due to electroneutral, Na(+), K(+), 2Cl(-) cotransporter-mediated transport. Cell vacuolation leads to cell volume increase, consistent with water flux into the cell, while hyper-osmotic media decreased vacuole formation. These data represent the first evidence that VacA-treated cells undergo an osmotic unbalance, reinforcing the hypothesis that the VacA chloride channel is responsible for cell vacuolation. 相似文献
3.
On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells. 总被引:10,自引:3,他引:7 下载免费PDF全文
Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed. 相似文献
4.
Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide
Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH4)2SO4 fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same Mr(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.Both activities could be eluted from the same position of the native gel.Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa. 相似文献
5.
To date, the most significant use of recombinant-DNA technologies has been to hyperproduce natural molecules that are difficult to obtain in large quantities by conventional methods. However, genetic manipulation can also be an efficient way to modify the properties of natural molecules in order to make them more suitable for human use. In the development of third-generation whooping cough vaccines, recombinant-DNA methods were used to remove the enzymatic activity of pertussis toxin in order to obtain a new molecule which is devoid of toxicity, and can be used for safer vaccination against this disease. 相似文献
6.
Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. 总被引:7,自引:1,他引:6 下载免费PDF全文
Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level. 相似文献
7.
Detection and physical map of a omega tox+-related defective prophage in Corynebacterium diphtheriae Belfanti 1030(-)tox-. 总被引:1,自引:0,他引:1 下载免费PDF全文
A library of chromosomal DNA from Corynebacterium diphtheriae Belfanti 1030(-)tox- was cloned in the lambda phage vector EMBL4 and screened for sequences homologous to corynephage omega tox+ and the attB1-attB2 region of the C7(-)tox- chromosome. Two portions of the 1030(-)tox- chromosome, 35 and 30.5 kilobases long which contain, respectively, the entire region homologous to corynephage omega tox+ and the attB1-attB2 sites, were mapped with the restriction endonucleases BamHI and EcoRI. Chromosomal DNA from 1030(-)tox- was shown to contain a 15.5-kilobase region that was homologous to ca. 42% of the corynephage omega tox+ genome. These sequences were found to hybridize to three regions of the phage genome and do not contain either the diphtheria tox operon or the attP site. These sequences are distant from the chromosomal region that contains the attB1-attB2 sites. Moreover, unlike other known defective prophages, the physical map of this prophage starts at the cos site and is colinear with the vegetative phage map. The 30.5-kilobase region of the 1030(-)tox- chromosome, which contains the attB1-attB2 sites, has a central core region that is almost identical to the corresponding region of the C7(-)tox- chromosome; however, the flanking sequences in these two strains of C. diphtheriae are different. 相似文献
8.
Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2. 总被引:2,自引:1,他引:1 下载免费PDF全文
The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth. 相似文献
9.
Isolation and characterization of Corynebacterium diphtheriae nontandem double lysogens hyperproducing CRM197. 总被引:7,自引:0,他引:7 下载免费PDF全文
R Rappuoli 《Applied microbiology》1983,46(3):560-564
Phage beta 197tox-, which codes for CRM197, a nontoxic protein immunochemically indistinguishable from diphtheria toxin, was UV induced from a culture of the C7(beta 197)tox- strain. A total of 191 C7(beta 197)tox- lysogens were isolated and selected according to the halo produced on TYE agar containing antidiphtheria toxin serum and were further characterized by Southern blots of their chromosomal DNA. Most of the isolates turned out to be monolysogens, but some tandem and nontandem double lysogens were also found. The nontandem double lysogens were stable and capable of giving high yields of CRM197, up to threefold higher than monolysogens. They are, therefore, suitable for large-scale industrial production. 相似文献
10.
The complete nucleotide sequence of the gene coding for diphtheria toxin in the corynephage omega (tox+) genome. 总被引:24,自引:4,他引:20 下载免费PDF全文
A segment of corynephage omega (tox+) DNA, containing the gene for diphtheria toxin (tox) was fragmented with restriction enzymes and the fragments cloned into M13 vectors for nucleotide sequence determination. A long open reading frame was shown to encode the tox gene by comparing the predicted amino acid sequence with that of peptides derived from the mature toxin molecule. Analysis of the nucleotide sequence shows RNA polymerase and ribosome binding signals preceding a GTG codon in the open reading frame: if this is the correct starting signal for translation, then a 25 amino acid signal peptide can be predicted for the toxin molecule. 相似文献