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191.
A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.  相似文献   
192.
During sequence analysis of the first intron of the human c-fms oncogene, we identified an open reading frame encoding the ribosomal protein L7 (RPL7). The presence of this sequence within intron 1 of the c-fms gene was confirmed by Southern blot hybridization and by sequence analysis of two independent cosmid clones (cos2-e and cos1-22) that span the human genomic c-fms locus. The RPL7 sequence was detected in a region of sequence overlapped by the cos2-e and cos1-22 cosmid clones but oriented opposite to the c-fms gene. We demonstrated that the sequence is identical to the full-length RPL7 cDNA sequence, but lacks any recognizable introns, has a 30-bp poly(A) tail, and is bracketed by two perfect direct repeats of 14 bp. We also showed that despite the fact that the 5′ flanking region of the RPL7 sequence contains a potential TATA box upstream of an intact open reading frame, this pseudogene (RPL7P) is not actively transcribed.  相似文献   
193.
The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strepr) progeny, but in many of these families a small proportion of strepr seedlings were also resistant to hygromycin (hygr). Nevertheless, 70% of families tested did give rise to at least one strepr, hygr seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer streprhygr progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strepr seedlings were also hygr than after activation by 35S::TPase. We also examined the genotype of strepr, hygr seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strepr progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.  相似文献   
194.
Protoplasts ofBifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO4. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.  相似文献   
195.
Photosystem II (PS II) particles isolated from spinach in the presence of 10 M CuSO4 contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO4 was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1–1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.Abbreviations CP 26 and CP 29 chlorophyll a/b protein complex 26 and 29 - LHC II light-harvesting chlorophyll a/b protein complex of Photosystem II - SB14 sulfobetaine 14 A preliminary report of these results was presented at the IX Int. Congress on Photosynthesis, Nagoya, Japan, 1992.  相似文献   
196.
Sediments were sampled from 62 sites in the Kattegat and Skagerrak, which are located between the Baltic and the North Sea in the Western Atlantic, during autumn 1989 and spring 1990. From each site 5 to 6 samples were taken wit ha box-corer. After mixing to composite samples on board, transport and storage (at 4 °C for 2 to 4 weeks), the samples were tested for toxicity to Daphnia magna and Nitocra spinipes. Immobility in Daphnia after exposure to 16 percent sediment (wet wt) in reconstituted standardized water (ISO, 1982) ranged from 0 to 88 percent after 24 h and from 3 to 95 percent after 48 h. For Nitocra the toxicity, determined as the 96-h LC50 (% wet wt) at 7 salinity, ranged from > > 32 percent (nontoxic) to 1.8 percent (most toxic). All exposures were made in duplicates and the effects obtained in the duplicates with the same sediment were correlated to each other. However, sediment toxicity to Daphnia and Nitocra was not. The test with Nitocra, which was made at several concentrations of sediment, was considered to give the most reliable picture of sediment toxicity in the Kattegat and Skagerrak. This ambient toxicity assessment identified three areas with toxic sediment, (1) the Göta älv estuary (outside the city of Göteborg) and its surroundings, (2) the Bay of Laholm in southern Kattegat, which is an area with periodic oxygen depletion and where repeated mussel kills have occurred during the last decade, and (3) an area in the open Skagerrak northwest of Skagen (the tip of the Jutland peninsula). Sediments, which had been stored at 4 °C, were tested again after 6 to 13 mos with the Nitocra test. Stored sediment toxicity was poorly correlated with fresh sediment toxicity. The average detoxification during storage was 5 times, but the range was 3 orders of magnitude, from 17 times more toxic to 73 times less toxic. The reasons for the observed areal and storage differences in sediment toxicity are so far not understood.  相似文献   
197.
What is Microthrix parvicella ?   总被引:1,自引:0,他引:1  
Microthrix parvicella is one of the filamentous bacteria that is known to create problems in the operation of activated sludge plants. Its physiology has already been investigated, although primarily in the context of its being a bulking species. It is now recognized that it is one of the major foam-forming organisms and, as such, needs further study. The initial isolation of M. parvicella did not prove to be as easy as would be expected from the earlier work and, eventually, micromanipulation was required. Growth studies showed that it exhibited several morphological forms, only one of which was that described previously. This has led to doubts about the classification of this species which does not yet have a clearly defined bacteriological designation.  相似文献   
198.
Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.  相似文献   
199.
The epiphytic vegetation on host trees along an altitudinal transect on the west slope of the Central Cordillera in Colombia was sampled. Bark-type restricted sampling comprised four full-grown forest trees, not necessarily four species, at altitudinal intervals of c. 200 meters from 1000 to 4130 m a.s.l. and included the canopy. Both vascular and non-vascular epiphytes, the latter often more abundant in the montane rain forest, were included in the analyses. Using a method of direct gradient analysis, canonical correspondence analysis, the variation explained by various environmental variables could be discriminated from a great amount of variation that seemed not related to any ecological factor. To a large extent, the randomness in propagule supply appears to determine the floristic composition on branch/trunk segments. The grouping of relevés in a phytosociological analysis concurred with a clustering of samples in an ordination diagram of the first two extracted constrained axes. The sample scores on these two axes were strongly correlated with the complex variables altitude and height within the host tree. Specialists and generalists with respect to the two variables were defined based on tolerances provided by the canonical correspondence analysis as was the position of species on the gradients involved. Independently from any of the other variables entered, a relation between the epiphytic vegetation and host species was detected, particularly in the case of Brunellia occidentalis, a fast growing tree species of higher altitude. No relation between chemical characteristics of suspended soil in the Upper Montane Rain forest and its supporting species could be demonstrated.  相似文献   
200.
In the primordial thoracic ganglia of locust embryos, the bromodeoxiuridine (BrdU) technique for labelling proliferating cells and their progeny was combined with intracellular dye injection to investigate the origin and the clonal relationship of common inhibitory motoneurons. Common inhibitors 1 (CI1) and 3 (CI3) were found to be siblings, that is, they are produced by the division of one ganglion mother cell. This ganglion mother cell results from the first division of neuroblast 5–5, at about 30% of embryonic development. A large portion, at least, of the ganglion mother cells produced by subsequent divisions of neuroblast 5–5 give rise to interneurons with contralaterally ascending or descending axons and GABA-like immunoreactivity. Thus, CI1 and CI3 are more closely related to putative inhibitory interneurons than they are to other, that is, excitatory, motoneurons. Consistent with this, the CI somata are associated with cell bodies of putative inhibitory interneurons rather than with clusters of excitatory motoneuron somata. These results elicit speculations regarding the evolutionary origin of inhibitory motoneurons. 1994 John Wiley & Sons, Inc.  相似文献   
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