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141.
Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth 总被引:13,自引:0,他引:13
Parmender P. Mehta Agnes Hotz-Wagenblatt Birgit Rose David Shalloway Warner R. Loewenstein 《The Journal of membrane biology》1991,124(3):207-225
Summary Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals. 相似文献
142.
An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis 总被引:4,自引:0,他引:4
Martin Schnorf Gabriele Neuhaus-Url Alessandro Galli Shigeru Iida Ingo Potrykus Gunther Neuhaus 《Transgenic research》1991,1(1):23-30
A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded
in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts
regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l−1 kanamycin or 5 mg l−1 paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of
theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin
resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown
by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization
to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range
from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian
fashion. 相似文献
143.
J. Donald Weaver G. Stetten J. W. Littlefield 《In vitro cellular & developmental biology. Animal》1991,27(8):670-675
Summary During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies
arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc
or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm
of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates
the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly
long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere.
Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than
in fibroblasts and that a triple dose of one or more genes may be the initial event in this process.
This work was supported by grant CA16754 from the National Cancer Institute, Bethesda, MD. 相似文献
144.
145.
Fiona R. Murray Garrick C. M. Latch D. Barry Scott 《Molecular & general genetics : MGG》1992,233(1-2):1-9
Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum. 相似文献
146.
Sonication: A new method for gene transfer to plants 总被引:9,自引:0,他引:9
Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects are reviewed. The state of the art of acoustic transformation is presented and possible mechanisms are discussed. 相似文献
147.
Krishan K. Arora David M. Parry Peter L. Pedersen 《Journal of bioenergetics and biomembranes》1992,24(1):47-53
Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed. 相似文献
148.
149.
150.
Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts 总被引:9,自引:0,他引:9
Swapan K. Datta Karabi Datta Nouchine Soltanifar Gunter Donn Ingo Potrykus 《Plant molecular biology》1992,20(4):619-629
The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hgr and 170 PPTr IR72 plants from protoplasts through selection. 31 Hgr and 70 PPTr plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT. 相似文献