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21.
Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana 总被引:1,自引:0,他引:1
Joaquín Ariño Encarna Pérez-Callejón Nuria Cunillera Manel Camps Francesc Posas Albert Ferrer 《Plant molecular biology》1993,21(3):475-485
Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants. 相似文献
22.
Yannick Azou Monique Laval 《Biology of the cell / under the auspices of the European Cell Biology Organization》1993,77(2):155-164
Summary— We have previously shown the presence, in the amplified DNA of a Drosophila cell line resistant to N-phosphonacetyl-L-aspartate (PALA), of two units of 150 kb and 120 kb respectively duplicated and amplified. The two joints (J1 and J2) linking these units as well as their respective wild-type counterparts have been sequenced. Sequence analysis indicates that a region of the Drosophila genome which corresponds to the proximal boundary of the 150 kb unit is common to both joints. In addition to this common region, the J1 junction possesses a 26-nucleotide sequence belonging to the J2 junction. This indicates that the J2 junction was the first formed, and that J1, therefore, results from recombination between J2 and a region of the wild-type genome 120 kb distal to J2. Sequence analysis also reveals that the joints result from illegitimate recombination between unrelated regions. AT-rich sequences, strand bias composition and putative topoisomerase I and II sites were found in at least one of the two parental sequences involved in the formation of the joints. On the basis of these results we can hypothesize that after two illegitimate recombinations between sister chromatids, leading first to J2 and then to J1, the amplification may have arisen by a series of homologous (unequal crossing-over) or illegitimate recombinations, or by an intrachromosomal rolling circle. 相似文献
23.
Esterina Pascale Christine Liu Eulalia Valle Karen Usdin Anthony V. Furano 《Journal of molecular evolution》1993,36(1):9-20
Summary All modern mammals contain a distinctive, highly repeated (⩾50,000 members) family of long interspersed repeated DNA called
the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation
∼80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently
we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family
that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family
to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx
amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that
Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary
intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved
under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replicationcompetent
L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the
evolution of L1 DNA elements and the mammalian genome. 相似文献
24.
An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture. 相似文献
25.
The autoxidation of tetralin is treated as a model reaction system to define the applicability of stereospecific autocatalysis. This concept, predicting a spontaneous amplification of enantiomeric excess generated by an autocatalytic chemical reaction, is used in several theoretical models as an explanation for the origin of natural optical activity. The reaction system investigated obeys the basic criteria of these models: a chiral intermediate (tetralin hydroperoxide) is produced from an achiral substrate (tetralin) via an autocatalytic pathway where the feedback mechanism is expected to generate a state of broken chiral symmetry. In order to test the amplification capacity of this reaction a computer analysis of the kinetic scheme is performed. This simulation is derived from the known kinetic scheme of autoxidation and is validated by fitting the experimentally observed data of hydroperoxide evolution. Calculations show that this model allows powerful amplification of enantiomeric excess and a transient amplification of the optical rotation. It is also demonstrated that the model system exhibits pronounced sensitivity toward any loss of absolute configuration of the involved chiral species. Since an amplification effect results exclusively at a high degree of stereoselectivity, it is concluded that stereospecific autocatalysis is possible in systems which show template reactions, crystallization, or colloidal effects. © 1993 Wiley-Liss, Inc. 相似文献
26.
【背景】肺炎支原体是导致儿童和青少年呼吸道感染的重要病原体,长期以来由于其临床表现不特异而容易错过最佳治疗时期。【目的】结合多酶恒温扩增(multienzyme isothermal rapid amplification,MIRA)技术和核酸试纸条建立一种快速检测肺炎支原体的方法。【方法】以肺炎支原体社区获得性肺炎呼吸窘迫综合征(community acquired respiratory distress syndrome, CARDS)毒素编码基因为靶基因设计引物和探针,对反应体系的温度、时间等进行优化,评估其敏感性,通过检测肺炎支原体和其余7种病原体分析其特异性,并对35份临床样本进行验证。【结果】MIRA核酸试纸条法在37℃条件下,15 min内便可完成对肺炎支原体的检测,最低检出限为10 copies/μL;除肺炎支原体外,其余7种病原体均不能扩增,特异性较好。以实时荧光PCR检测为标准,MIRA核酸试纸条法对35份临床样本检测后的诊断特异度为100.00%、灵敏度为96.15%、阴性预测值为90.00%、阳性预测值为100.00%。【结论】本研究建立了MIRA核酸试纸条法... 相似文献
27.
F J Livesey 《Briefings in Functional Genomics and Prot》2003,2(1):31-36
One of the critical limitations of current microarray technologies for use in expression analyses is the relatively large amount of input RNA required to generate labelled cDNA populations for array analysis. In situations where RNA is limiting, the options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell. 相似文献
28.
马铃薯卷叶病毒基因间隔区的克隆及序列分析 总被引:6,自引:0,他引:6
根据已报道的马铃薯卷叶病毒基因组序列.设计合成一对特异性引物,以马铃薯卷叶病毒中国分离株(PLRV-Ch)的RNA为模板,反转录合成cDNA第一条链,经PCR扩增后克隆于pUC19质粒中,进一步用PCR鉴定、限制酶切分析和序列分析,结果表明:PLRV-Ch基因间隔区由197个核苷酸组成,与国外报道的荷兰PLRV-N加拿大PLRV-C,澳大利亚PLRV-A,苏格兰PLRV-S各株系核苷酸序列具有很高的同源性,同源率依次为99%、98%、93%、98%。 相似文献
29.
A. Depeiges C. Goubely A. Lenoir S. Cocherel G. Picard M. Raynal F. Grellet M. Delseny 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,91(1):160-168
The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles. 相似文献
30.
Hyung-Joo Jin Jeong–Ha Kim Chul Hyun Sohn R.E. DeWreede Tae–Joo Choi G.H.N. Towers J.B. Hudson Yong–Ki Hong 《Journal of applied phycology》1997,9(4):383-388
Fifty-nine species of marine macrophytes from the coasts of British Columbia, Canada and Korea have been screened for the
presence of PCR inhibitors, namely inhibitors of Taq DNA polymerase. Eleven of the species displayed some inhibitor activity.
At the concentration of 5 μg of methanol extract in 25μL reaction mixture of PCR containing 1.5 unit of Taq DNA polymerase,
one (Ulva sp.) of 8 Chlorophyta, eight (Colpomenia bullosa, Ecklonia cava, Endarachne binghamiae, Fucus distichus, Hizikia
fusiformis, Sargassum confusum, Sargassum sagamianum, and Sargassum thunbergii) of 28 Phaeophyta, and one (Symphyocladia latiuscula)
of 34 Rhodophyta showed inhibition in PCR amplification. In the case of the water extract, two (Cladophora columbiana, Ulva
sp.) Chlorophyta, seven (Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum,
Sargassum horneri, Scytosiphon dotyi) Phaeophyta, no Rhodophyta and one (Phyllospadix scouleri) seagrass showed inhibition
in PCR amplification. the methanol fraction of Sargassum confusum and the water fraction of Fucus gardneri (mid–intertidal)
have been found to inhibit PCR at level as low as 0.5 μg in 25μL of PCR reaction mixture.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献