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21.
22.
An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP
+ transformants). Plasmids isolated from the transformants (URA
+
TRP
+) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs. 相似文献
23.
S. S. Kanj J. E. Corkill Z. A. Kanafani G. F. Araj C. A. Hart R. Jaafar G. M. Matar 《Clinical microbiology and infection》2008,14(5):501-504
The prevalence of bla CTX-M , bla TEM and bla SHV genes among extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Escherichia coli ( n = 50) and Klebsiella spp. ( n = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla CTX-M-15 , bla TEM-1 , bla OXA-1 and six bla SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants. 相似文献
24.
L. Poirel T. Vu Nguyen A. Weintraub C. Leviandier P. Nordmann 《Clinical microbiology and infection》2006,12(10):1021-1023
PCR was used to investigate the occurrence of the plasmid-encoded quinolone resistance determinants qnrA and qnrS among diarrhoeagenic enterobacterial isolates recovered from Hanoi, Vietnam, during the period March 2001 to April 2002. In total, 162 Escherichia coli isolates, 28 Shigella isolates and three Enterobacter cloacae isolates were negative for qnrA, while a single Ent. cloacae isolate harboured a 50-kb qnrS-positive conjugative plasmid. Cloning and sequencing identified a qnrS gene bracketed by open reading frames identical to those surrounding the qnrS gene of a Shigella flexneri isolate from Japan, thereby suggesting a common mechanism of acquisition. 相似文献
25.
We have constructed a series of promoter or upstream activating sequence (UAS)-probe plasmids carrying the Tn5-derived neomycin resistance gene whose seven additional ATG codons in the 5-untranslated region were completely or partially removed. When the deleted version of the neo sequence retaining only one additional ATG (NeoD) was expressed under the control of a TDH3 promoter whose UAS was deleted, the transformed cells were unable to grow at a low concentration of the antibiotic G418. In contrast with this, yeast cells expressing the NeoC sequence and having no additional ATG exhibited a high level of G418-resistance. Moreover, the UAS-probe system using NeoD has been successfully applied for the identification of several E. coli DNA sequences that clearly function as UASs in yeast cells. Two of these prokaryotic sequences with UAS activity were identified as a part of the coding region of the tgt and the hydG gene, respectively. 相似文献
26.
27.
Philippe Normand Pascal Simonet Luc Giasson Patrick Ravel-Chapius J. -André Fortin Maurice Lalonde 《Current genetics》1987,11(4):335-338
Summary A plasmid-like molecule was detected in a strain of the ascomycete Ceratocystis fimbriata Ell. & Halst., a pathogenic fungus of Populus spp. The DNA replicon, designated pFQ501, was found to have a linear structure with a length of 6.0 kb (3.9 × 106 daltons) and a density of 1.685 g/cc. This molecule was found to be associated with the mitochondria and was isolated from the gel; its restriction map was deduced from single and double digestions. 相似文献
28.
The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5′ ends of pMLP1 and pMLP2
DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp
with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3′-CCCCC-5′, similar to those
of Escherichia coli phage PRD1. The pMLP1 plasmid harbors two long open reading frames (ORF1 and ORF2) and at least one minor ORF (mORF1). The
deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of
ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most
likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products
from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of
pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products.
Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis
indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase.
Received: 9 February 2000 / Accepted: 18 July 2000 相似文献
29.
de Carvalho MC Barca FN Agnez-Lima LF de Medeiros SR 《Environmental and molecular mutagenesis》2003,42(3):185-191
An extract (decoction) from pepper tree stem bark (Schinus terebinthifolius Raddi) is widely used in Brazil as a topical antiinflammatory agent and to cicatrize wounds. The extract contains catechin, tannins, terpenes, flavonoids, and saponins; of these components, both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study, we evaluated an extract of S. terebinthifolius in a series of cell-free and bacterial assays in order to determine its genotoxic potential. The extract was negative in a cell-free plasmid DNA test, indicating that it did not directly break DNA. Positive results, however, were obtained in the SOS chromotest, in a forward mutagenesis assay employing CC104 and CC104mutMmutY strains of Escherichia coli, and in the Salmonella reversion assay, using strains TA97, TA98, TA100, and TA102. All the bacterial tests were performed without exogenous metabolic activation due to the topical use of this preparation. The results indicate that pepper tree stem bark extract produces DNA damage and mutation in bacteria, and that oxidative damage may be responsible for the genotoxicity. 相似文献
30.
生长抑制因子1基因核定位序列-绿荧光蛋白融合表达载体的构建及表达 总被引:1,自引:0,他引:1
目的构建p33^ING1b核定位序列(nuclear locating sequence,NLS)-绿荧光蛋白融合表达载体,将其转染到人胚肺纤维母细胞系MRC-5,建立稳定表达该融合蛋白的细胞模型。方法应用逆转录PCR获得p33^ING1b的NLS序列,然后将NLS序列插入绿荧光蛋白融合表达载体pEGFP-C1的多克隆位点,构建pEGFP-C1-NLS-绿荧光蛋白融合表达载体,再用此载体转染MRC-5细胞系,观察活细胞绿荧光蛋白的亚细胞定位。结果成功构建了pEGFP-C1-NLS-绿荧光蛋白融合表达载体,由该载体表达的绿荧光蛋白-NLS肽段融合蛋白产生的绿色荧光信号全部定位于胞核部位,而空载体转染的细胞表达的绿色荧光蛋白,绿色荧光信号定位于细胞浆中。结论在活细胞内,生理情况下p33^ING1b完全定位于细胞核,并且在其亚细胞定位的转运过程中,NLS肽段起着决定性作用。 相似文献