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11.
S H Shakin-Eshleman A T Remaley J R Eshleman W H Wunner S L Spitalnik 《The Journal of biological chemistry》1992,267(15):10690-10698
Many eukaryotic proteins are modified by N-linked glycosylation, a process in which oligosaccharides are added to asparagine residues in the sequon Asn-X-Ser/Thr. However, not all such sequons are glycosylated. For example, rabies virus glycoprotein (RGP) contains three sequons, only two of which appear to be glycosylated in virions. To examine further the signals in proteins which regulate N-linked core glycosylation, the glycosylation efficiencies of each of the three sequons in the antigenic domain of RGP were compared. For these studies, mutants were generated in which one or more sequons were deleted by site-directed mutagenesis. Core glycosylation of these mutants was studied using two independent systems: 1) in vitro translation in rabbit reticulocyte lysate supplemented with dog pancreatic microsomes, and 2) transfection into glycosylation-deficient Chinese hamster ovary cells. Parallel results were obtained with both systems, demonstrating that the sequon at Asn37 is inefficiently glycosylated, the sequons at Asn247 and Asn319 are efficiently glycosylated, and the glycosylation efficiency of each sequon is not influenced by glycosylation at other sequons in this protein. High levels of cell surface expression of RGP in Chinese hamster ovary cells are seen with any mutant containing an intact sequon at Asn247 or Asn319, whereas low levels of cell surface expression are seen when the sequon at Asn37 is present alone; deletion of all three sequons completely blocks RGP cell surface expression. Thus, although core glycosylation at Asn37 is inefficient, it is still sufficient to support a biological function, cell surface expression. Future studies using mutagenesis of this model protein and its expression in these two well defined systems will aim to begin to unravel the rules governing core glycosylation of glycoproteins. 相似文献
12.
Statistical analyses of DNA sequences of globin genes (beta A, beta C, and
gamma) from goat and sheep (including new sequence information for the
second intron of sheep beta A and gamma, kindly provided by A. Davis and A.
W. Nienhuis) indicate that the rates of nonsynonymous substitution in these
genes have been greatly accelerated following the gene duplication
separating gamma and the ancestor of beta A and beta C and the gene
duplication separating beta A and beta C. In both cases the acceleration
was apparently due to relaxation of purifying selection (functional
constraints) rather than advantageous mutations because acceleration
occurred only in less important parts of the beta globin chain. The rates
of nonsynonymous substitution in these genes are estimated to be about 2.3
x 10(-9) per site per year, which is three times higher than that for the
divergence between human beta and mouse beta major globin genes. Our
analyses further suggest that the rate of synonymous substitution in
functional genes and the rate of substitution in pseudogenes are
approximately equal and are between 2.8 x 10(-9) and 5.0 x 10(-9) and that
the rate of substitution in introns is about 3.0 x 10(-9). The divergence
time between beta A and beta C and that between gamma and the beta A-beta C
pair are about 12 and 30 million years, respectively. The proportion of
transition mutations is estimated to be 64%, two times higher than expected
under random mutation but considerably lower than the 96% estimated for
animal mitochondrial DNA.
相似文献
13.
14.
Genetic and Physiological Properties of Temperature-Sensitive Mutants of Cocal Virus 总被引:5,自引:5,他引:0 下载免费PDF全文
Temperature-sensitive (ts) mutants of Cocal virus (VSV Cocal) were isolated after treatment with the base analogue mutagen, 5-fluorouracil. These mutants could be classified into four mutually complementing groups. Weak complementation was detected between certain pairs of VSV Cocal ts mutants and ts mutants of vesicular stomatitis virus (VSV) Indiana, but no complementation was observed with ts mutants of VSV New Jersey. Two complementing ts mutants of Chandipura virus, an unrelated rhabdovirus, did not complement any VSV mutant, Thus, ability to complement in the VSV group appears to be correlated with serological relationships.The RNA and protein-synthesizing capacities of these ts mutants have been determined, and it is possible to establish a correspondence between the VSV Cocal and the VSV Indiana complementation groups. 相似文献
15.
The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid 总被引:20,自引:17,他引:3
1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture. 相似文献
16.
Background
Many homeobox genes show remarkable conservation between divergent animal phyla. In contrast, the ARGFX (Arginine-fifty homeobox) homeobox locus was identified in the human genome but is not present in mouse or invertebrates. Here we ask when and how this locus originated and examine its pattern of molecular evolution. 相似文献17.
The genomes of homeothermic (warm-blooded) vertebrates are mosaic
interspersions of homogeneously GC-rich and GC-poor regions (isochores).
Evolution of genome compartmentalization and GC-rich isochores is
hypothesized to reflect either selective advantages of an elevated GC
content or chromosome location and mutational pressure associated with the
timing of DNA replication in germ cells. To address the present controversy
regarding the origins and maintenance of isochores in homeothermic
vertebrates, newly obtained as well as published nucleotide sequences of
the insulin and insulin-like growth factor (IGF) genes, members of a
well-characterized gene family believed to have evolved by repeated
duplication and divergence, were utilized to examine the evolution of base
composition in nonconstrained (flanking) and weakly constrained (introns
and fourfold degenerate sites) regions. A phylogeny derived from amino acid
sequences supports a common evolutionary history for the insulin/IGF family
genes. In cold- blooded vertebrates, insulin and the IGFs were similar in
base composition. In contrast, insulin and IGF-II demonstrate dramatic
increases in GC richness in mammals, but no such trend occurred in IGF- I.
Base composition of the coding portions of the insulin and IGF genes across
vertebrates correlated (r = 0.90) with that of the introns and flanking
regions. The GC content of homologous introns differed dramatically between
insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level
of noncoding regions in neighboring genes. Our findings suggest that the
base composition of introns and flanking regions is determined by
chromosomal location and the mutational pressure of the isochore in which
the sequences are embedded. An elevated GC content at codon third positions
in the insulin and the IGF genes may reflect selective constraints on the
usage of synonymous codons.
相似文献
18.
Homology between the glycoproteins of vesicular stomatitis virus and rabies virus 总被引:14,自引:9,他引:5 下载免费PDF全文
We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found. 相似文献
19.
20.
N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule 总被引:4,自引:0,他引:4
Wojczyk BS Takahashi N Levy MT Andrews DW Abrams WR Wunner WH Spitalnik SL 《Glycobiology》2005,15(6):655-666
Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined. 相似文献