全文获取类型
收费全文 | 110562篇 |
免费 | 13263篇 |
国内免费 | 412篇 |
出版年
2018年 | 991篇 |
2017年 | 967篇 |
2016年 | 1258篇 |
2015年 | 1555篇 |
2014年 | 1944篇 |
2013年 | 2470篇 |
2012年 | 2864篇 |
2011年 | 2818篇 |
2010年 | 1886篇 |
2009年 | 1847篇 |
2008年 | 2362篇 |
2007年 | 2344篇 |
2006年 | 2335篇 |
2005年 | 2135篇 |
2004年 | 2074篇 |
2003年 | 2105篇 |
2002年 | 2079篇 |
2001年 | 9643篇 |
2000年 | 9509篇 |
1999年 | 7200篇 |
1998年 | 1616篇 |
1997年 | 1792篇 |
1996年 | 1580篇 |
1995年 | 1408篇 |
1994年 | 1308篇 |
1993年 | 1249篇 |
1992年 | 4794篇 |
1991年 | 4525篇 |
1990年 | 3994篇 |
1989年 | 4014篇 |
1988年 | 3610篇 |
1987年 | 3084篇 |
1986年 | 2776篇 |
1985年 | 2678篇 |
1984年 | 1974篇 |
1983年 | 1729篇 |
1982年 | 1231篇 |
1981年 | 983篇 |
1980年 | 913篇 |
1979年 | 1762篇 |
1978年 | 1361篇 |
1977年 | 1199篇 |
1976年 | 1029篇 |
1975年 | 1149篇 |
1974年 | 1166篇 |
1973年 | 1162篇 |
1972年 | 1033篇 |
1971年 | 955篇 |
1970年 | 822篇 |
1969年 | 743篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
941.
Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. 总被引:67,自引:9,他引:58
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
P Stanssens C Opsomer Y M McKeown W Kramer M Zabeau H J Fritz 《Nucleic acids research》1989,17(12):4441-4454
An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model. 相似文献
942.
943.
944.
Sequence divergence and open regions of RNA secondary structures in the envelope regions of the 17 human immunodeficiency virus isolates. 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Genetic variation during the course of infection of an individual is a remarkable feature of the acquired immune deficiency syndrome (AIDS) disease. This variation has been studied for the envelope protein encoding regions of seventeen different sequences from various isolates of human immunodeficiency virus (HIV) using multiple sequence comparison and calculation of variability. The open regions with little intramolecular base pairing in these envelope sequences are predicted by a recently developed statistical method. The minimum length L for a run of hypervariable sites, conserved sites, or open regions that gives significance at the 1% (or 0.1%) level is then determined by a scan statistical method. The results show that significant clusters of open regions predicted at the RNA levels correlate with significant clusters of hypervariable sites in the HIV envelope gene. Those significant genomic variations in HIVs seem to be manifested mainly in the extracellular portion of the envelope protein. Twelve potential antigenic determinants are predicted using an antigenic index method. Interestingly, the majority of the significant hypervariable regions in the exterior envelope protein (gp120) were predicted potential epitopes. 相似文献
945.
946.
Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation 总被引:1,自引:0,他引:1
S Yoshimura Y Mizuno K Kimura K Yatsunami J Fujisawa K Tomita A Ichikawa 《Biochimica et biophysica acta》1989,981(1):69-76
The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself. 相似文献
947.
Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro 总被引:1,自引:0,他引:1
Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner. 相似文献
948.
Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut
area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times
as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was
significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates
from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the
clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth
medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors,
phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly
or. indirectly - through inhibition of seed germination or of mycorrhizal fungi. 相似文献
949.
Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid 总被引:2,自引:0,他引:2
We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis. From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell. The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs. The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative. Moreover, the complexes are extremely stable and do not dissociate after 90 min. These properties are consistent with a role of the HPB12 protein in the structure of the B. subtilis chromosome. 相似文献
950.
Prepubertal (28-30 days old) female rats were infused s.c. over a 60-h period with a purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH specific activity 8.4 times that of NIH-FSH-S1 and luteinizing hormone (LH) specific activity less than 0.005 times that of NIH-LH-S1, based on radioreceptor assays. When the FSH infusion rate of this preparation was increased over the range of 0.5-2 units/day (mg NIH-FSH-S1 equivalent), an all-or-none response was observed, with the threshold dose for superovulation being between 1 and 2 units/day. Eleven of twelve rats receiving the 2 units/day dose ovulated a mean +/- SEM of 67 +/- 8 oocytes on the morning of the third day after the beginning of FSH infusion. Addition of human chorionic gonadotrophin (hCG), as a source of LH activity, to a subthreshold (1 U/day) FSH infusion rate resulted in 20% of rats ovulating at an hCG dosage of 50 mIU/day; increasing the hCG infusion to 200 mIU/day concomitant with the subthreshold FSH infusion rate increased ovulation rate to a mean of 69 +/- 8/rat, with 100% of rats ovulating. To determine the effect of varying both FSH infusion rates and LH:FSH ratios, FSH was infused at several rates, with hCG added to give varying hCG:FSH ratios for each FSH infusion rate. Administration of hCG alone was ineffective in causing ovulation except at the highest infusion rates. Adding hCG to FSH to reach a ratio of 0.2 IU hCG/U FSH significantly increased the superovulatory response to an intermediate, 1 U/day FSH dose, but not to the low, 0.5 U/day dose.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献