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排序方式: 共有107条查询结果,搜索用时 15 毫秒
1.
Branch migration mediated DNA labeling and cloning 总被引:2,自引:0,他引:2
The sequence-dependent attachment (capture) of an oligodeoxynucleotide duplex containing a single-stranded tail can be mediated by branch migration into the end of a DNA molecule. Substitution of bromodeoxycytidine (BrdC) for deoxycytidine (dC) increased DNA-DNA hybrid stability. BrdC-containing oligodeoxynucleotides displaced dC-containing strands from duplexes with blunt ends or 3'-overhangs. In the later case the rate of displacement was of the same order of magnitude as DNA reassociation. A BrdC-containing displacer oligodeoxynucleotide was used for transient sequence-specific invasion at a particular PstI site. The product was captured by use of T4 DNA ligase and a linker oligodeoxynucleotide. The capture rate was more than 300 times the rate observed for an unrelated PstI site. This high degree of specificity required BrdC substitution. In addition, deliberate incorporation of an incorrect nucleotide into a displacer strand demonstrated that branch migration was terminated at a mismatch. A branched, BrdC-containing ligated product of a capture reaction was cloned and sequenced. The specific capture reaction may be used to label a particular DNA fragment prior to electrophoresis, to mark the specific fragment for affinity chromatography, or to facilitate cloning by introducing a new overhanging sequence compatible with a restriction endonuclease site in a cloning vector. 相似文献
2.
Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking. 相似文献
3.
We have broadly defined the DNA regions regulating esterase6 activity in
several life stages and tissue types of D. melanogaster using P-
element-mediated transformation of constructs that contain the esterase6
coding region and deletions or substitutions in 5' or 3' flanking DNA.
Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and
the primary sequences regulating its activity lie between -171 and -25 bp
relative to the translation initiation site: deletion of these sequences
decrease activity approximately 20-fold. Hemolymph activity is also
modulated by four other DNA regions, three of which lie 5' and one of which
lies 3' of the coding region. Of these, two have positive and two have
negative effects, each of approximately twofold. Esterase6 activity is
present also in two male reproductive tract tissues; the ejaculatory bulb,
which is another ancestral activity site, and the ejaculatory duct, which
is a recently acquired site within the melanogaster species subgroup.
Activities in these tissues are at least in part independently regulated:
activity in the ejaculatory bulb is conferred by sequences between -273 and
-172 bp (threefold decrease when deleted), while activity in the
ejaculatory duct is conferred by more distal sequences between -844 and
-614 bp (fourfold decrease when deleted). The reproductive tract activity
is further modulated by two additional DNA regions, one in 5' DNA (-613 to
-284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to
+2731 bp; threefold decrease when deleted) that probably overlaps the
adjacent esteraseP gene. Collating these data with previous studies
suggests that expression of EST6 in the ancestral sites is mainly regulated
by conserved proximal sequences while more variable distal sequences
regulate expression in the acquired ejaculatory duct site.
相似文献
4.
Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans 总被引:2,自引:0,他引:2
Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and
D. simulans for two common allozyme forms, as well as for several other
less common variants. Parallel latitudinal clines in the frequencies of the
common EST6-F and EST6-S allozymes in these species have previously been
interpreted in terms of a shared amino acid polymorphism that distinguishes
the two variants and is subject to selection. Here we compare the sequences
of four D. simulans Est-6 isolates and show that overall estimates of
nucleotide heterozygosity in both coding and 5' flanking regions are more
than threefold higher than those obtained previously for this gene in D.
melanogaster. Nevertheless, the ratio of replacement to exon silent-site
polymorphism in D. simulans is less than the ratio of replacement to silent
divergence between D. simulans and D. melanogaster, which could be the
result of increased efficiency of selection against replacement
polymorphisms in D. simulans or to divergent selection between the two
species. We also find that the amino acid polymorphisms separating EST6- F
and EST6-S in D. simulans are not the same as those that separate these
allozymes in D. melanogaster, implying that the shared clines do not
reflect shared molecular targets for selection. All comparisons within and
between the two species reveal a remarkable paucity of variation in a
stretch of nearly 400 bp immediately 5' of the gene, indicative of strong
selective constraint to retain essential aspects of Est-6 promoter
function.
相似文献
5.
delta-Aminolevulinate dehydratase deficient porphyria: identification of the molecular lesions in a severely affected homozygote. 总被引:3,自引:0,他引:3 下载免费PDF全文
M Plewinska S Thunell L Holmberg J G Wetmur R J Desnick 《American journal of human genetics》1991,49(1):167-174
delta-Aminolevulinate dehydratase deficient porphyria, a recently recognized inborn error of heme biosynthesis, results from the markedly deficient activity of the heme biosynthetic enzyme, delta-aminolevulinate dehydratase (ALA-D). The four homozygotes described to date with this disorder have remarkably distinct phenotypes, ranging from a severely affected infant with failure to thrive to an essentially asymptomatic 68-year-old male. To investigate the molecular nature of the lesions causing the severe infantile-onset form, total RNA was isolated from cultured lymphoblasts of the affected homozygote, RNA was reverse-transcribed to cDNA, and the 990-bp ALA-D-coding region was amplified by the PCR. Heterozygosity for an RsaI RFLP within the ALA-dehydratase-coding region permitted identification of the paternal and maternal mutant alleles prior to sequencing. The maternal mutation (designated G133R), a G-to-A transition of nucleotide 397, predicted a glycine-to-arginine substitution at residue 133 at the carboxyl end of the highly conserved zinc-binding site in the enzyme subunit. The G133R mutation created a PstI site and permitted the confirmation and rapid detection of this lesion in amplified genomic DNA from maternal relatives. The paternal mutation, a G-to-A transition of nucleotide 823, predicted a valine-to-methionine substitution of residue 275 (designated V275M). This mutation was confirmed in genomic DNA from family members by the competitive PCR technique. Both missense mutations, which occurred at CpG dinucleotides, resulted in the synthesis of enzyme subunits such that the activity of the homooctameric enzyme was markedly reduced, thereby causing the severe infantile-onset phenotype in the affected homozygote. 相似文献
6.
7.
Rod and cone photoreceptors project from the outer retinal surface into a
carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM
glycoconjugates are distributed around rods and cones. Wheat germ
agglutinin (WGA) strongly decorates the rod matrix domains and weakly
decorates the cone matrix domains. This study characterizes the major
WGA-binding glycoprotein in the human IPM, which we refer to as SPACR
(sialoprotein associated with cones and rods). SPACR, which has a molecular
weight of 147 kDa, was isolated and purified from the IPM by lectin
affinity chromatography. A polyclonal antibody to SPACR was prepared that
colocalizes in tissue preparations with WGA-binding domains in the IPM.
Sequential digestion of SPACR with N- and O- glycosidases results in a
systematic increase in electrophorectic mobility, indicating the presence
of both N- and O-linked glycoconjugates. Complete deglycosylation results
in a reduction in the relative molecular mass of SPACR by about 30%.
Analysis of lectin binding allowed us to identify some of the structural
characteristics of SPACR glycoconjugates. Treatment with neuraminidase
exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut
agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia
amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in
alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA),
specific for alpha2-6 linked sialic acid, does not, indicating that the
dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate,
NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR
suggests that this glycoprotein may contribute substantially to the
polyanionic nature of the IPM. The carbohydrate chains present on SPACR
could also provide sites for extensive crosslinking and participate in the
formation of the ordered IPM lattice that surrounds the elongate
photoreceptors projecting from the outer retinal surface.
相似文献
8.
The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity. 相似文献
9.