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81.
Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure 总被引:3,自引:0,他引:3
J E Mueller C J Newton F Jensch B Kemper R P Cunningham N R Kallenbach N C Seeman 《The Journal of biological chemistry》1990,265(23):13918-13924
Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction. 相似文献
82.
Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase 总被引:5,自引:0,他引:5
The importance of the interdomain bridging interactions observed only in the R-state structure of Escherichia coli aspartate transcarbamylase between Glu-50 of the carbamoyl phosphate domain with both Arg-167 and Arg-234 of the aspartate domain has been investigated by using site-specific mutagenesis. Two mutant versions of aspartate transcarbamylase were constructed, one with alanine at position 50 (Glu-50----Ala) and the other with aspartic acid at position 50 (Glu-50----Asp). The alanine substitution totally prevents the interdomain bridging interactions, while the aspartic acid substitution was expected to weaken these interactions. The Glu-50----Ala holoenzyme exhibits a 15-fold loss of activity, no substrate cooperativity, and a more than 6-fold increase in the aspartate concentration at half the maximal observed specific activity. The Glu-50----Asp holoenzyme exhibits a less than 3-fold loss of activity, reduced cooperativity for substrates, and a 2-fold increase in the aspartate concentration at half the maximal observed specific activity. Although the Glu-50----Ala enzyme exhibits no homotropic cooperativity, it is activated by N-(phosphonoacetyl)-L-aspartate (PALA). As opposed to the wild-type enzyme, the Glu-50----Ala enzyme is activated by PALA at saturating concentrations of aspartate. At subsaturating concentrations of aspartate, both mutant enzymes are activated by ATP, but are inhibited less by CTP than is the wild-type enzyme. At saturating concentrations of aspartate, the Glu-50----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
83.
B J Feldman S F Gheller G F Bailey W E Newton F A Schultz 《Analytical biochemistry》1990,185(1):170-175
Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4. 相似文献
84.
Pattern of growth in weight of alate and apterous nymphs of the English grain aphid, Sitobion avenae
Daily increase in fresh weight was recorded for apterous and alate nymphs of S. avenae at 20°C. Comparison with a control group indicated that daily disturbance and weighing of nymphs did not affect significantly their growth, developmental time or survival. The increase in fresh weight of apterous and alate virginoparae at 20°C was best described by logistic equations. Alate virginoparae were significantly heavier than apterous virginoparae at birth and throughout most of their nymphal life, but they experienced a weight loss at the final ecdysis. The relative growth rate did not remain constant, but declined during development. The decline is associated with a decline in honeydew production per unit body weight. The implications of an inconstant relative growth rate and the marked loss in weight at the adult moult in alates are discussed.
Résumé L'enregistrement de l'augmentation quotidienne du poids frais à 20°C des larves ailées et aptères de S. avenae a montré que des perturbations quotidiennes n'affectent pas significativement la croissance, la durée du développement et la survie. Les équations logistiques décrivent plus exactement l'augmentation de poids frais des aptères et des ailés virginipares à 20°C. Les virginipares ailés étaient significativement plus lourds que les virginipares aptères à la naissance et pendant la plus grande partie de la vie larvaire, mais présentaient une perte de poids à la mue finale. Le taux de croissance relative ne restait pas constant, mais diminuait au cours du développement. La diminution était associée à une diminution de la production de miellat par unité de poids du corps. La discussion porte sur les conséquences de la variation de l'augmentation du poids relatif et de la perte marquée de poids à la mue imaginale.相似文献
85.
A. P. W. Newton 《Polar Biology》1982,1(3):167-172
Summary Studies of colonization of snow slopes by the snow algae Chlamydomonas nivalis (Chlorophyta volvocales) show that various environmental factors can be correlated with regions of algal colonization. All colonized sites discovered faced west, and when transects were made across the slopes it was found that areas of maximum colonization showed minima in temperature and pH and maxima of conductivity and chloride ion concentration.Svalbard Expedition (1980) Perse School, Cambridge 相似文献
86.
Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition 总被引:7,自引:0,他引:7
S S Yang W H Pan G D Friesen B K Burgess J L Corbin E I Stiefel W E Newton 《The Journal of biological chemistry》1982,257(14):8042-8048
Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation. 相似文献
87.
Ribosomal DNA fragments of 1.0, 3.4, 3.7 and 6.1 kb2 produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RNA probe. These genomic sequences were further characterized by the isolation of 13 hybrid λ Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Cc1880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to 32P-labeled rRNA. Thus, the two clones contain different DNA inserts which together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S-5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 × 109 daltons. The relatively simple organization of rDNA sequences in the C. crescentus chromosome compared to Escherichia coli is discussed. 相似文献
88.
C A Frolik A B Roberts T E Tavela P P Roller D L Newton M B Sporn 《Biochemistry》1979,18(10):2092-2097
Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay. 相似文献
89.
23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration. 相似文献
90.
Purification and characterization of a polyhook protein from Caulobacter crescentus. 总被引:2,自引:0,他引:2 下载免费PDF全文
A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins. 相似文献