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101.
102.
A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses. 相似文献
103.
Perry Barrett Peter-M. Kloetzel John Sommerville 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,740(4)
During early oogenesis in amphibia, most of the 5 S RNA and tRNA is stored in a ribonucleoprotein particle that sediments at 42 S. In Xenopus laevis the 42 S particle contains two major proteins: of Mr 48 000 (P48) and 43 000 (P43). It is shown that heterogeneity in composition of the 42 S particle reflects a changing situation whereby initially, both 5 S RNA and tRNA are complexed with P48 (1 molecule 5 S RNA: 1 molecule P48; 2 or 3 molecules tRNA: 1 molecule P48), but later, tRNA becomes increasingly associated with P43 (in a 1:1 ratio) although 5 S RNA remains complexed with a cleavage product of P48. These changes relate to the eventual utilization of the excess 5 S RNA and tRNA in ribosome assembly and protein synthesis. 相似文献
104.
Howard M. Laten Jane Harris Cramer Robert H. Rownd 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(1):1-6
By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm5s2Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification. 相似文献
105.
Piero Cammarano Filomena Mazzei Paola Londei Angela Teichner Mario de Rosa Agata Gambacorta 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,740(3):300-312
Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (Tm = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77°C) and E. coli counterparts (Tm = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions. 相似文献
106.
Mathias Sprinzl Karl-Heinz Scheit Hans Sternbach Friedrich von der Haar Friedrich Cramer 《Biochemical and biophysical research communications》1973,51(4):881-887
2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNAPhe by tRNA nucleotidyl transferase. tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNAPhe-C-C-3′dA proved to be chargeable. 相似文献
107.
我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91%可以被牛生长激素抗体沉淀。用SDS-11%PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。 相似文献
108.
David Dichek Thomas Quertermous 《In vitro cellular & developmental biology. Plant》1989,25(3):289-292
Summary Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time
in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to
early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plaminogen activator
inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's
Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC
cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species
was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial
cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression
in cultured endothelium. 相似文献
109.
110.
RICHARD A. ALBACH 《The Journal of eukaryotic microbiology》1989,36(2):197-205
This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating > 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of >48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes. 相似文献