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71.

Background

Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.

Results

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.

Conclusion

We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.  相似文献   
72.
The reduction of linoleic acid hydroperoxide catalysed by rat liver cytosol was previously shown to be catalysed by a selenium-dependent glutathione peroxidase. In contrast, the enzyme responsible in guinea-pig liver cytosol is not selenium-dependent and appears to be a glutathione transferase.  相似文献   
73.
Chemokine receptor CCR2 and stromal-derived factor (SDF-1) are involved in HIV infection and AIDS symptom onset. Recent cohort studies showed that point mutations in these two genes, CCR2-64I and SDF1-3'A, can delay AIDS onset > or = 16 years after seroconversions. The protective effect of CCR2-64I is dominant, whereas that of SDF1-3'A is recessive. SDF1-3'A homozygotes also showed possible protection against HIV-1 infection. In this study, we surveyed the frequency distributions of the two alleles at both loci in world populations, with emphasis on those in east Asia. The CCR2-64I frequencies do not vary significantly in the different continents, having a range of 0.1-0.2 in most populations. A decreasing cline of the CCR2-64I frequency from north to south was observed in east Asia. In contrast, the distribution of SDF1-3'A in world populations varies substantially, and the highest frequency was observed in Oceanian populations. Moreover, an increasing cline of the SDF1-3'A frequency from north to south was observed in east Asia. The relative hazard values were computed to evaluate the risk of AIDS onset on the basis of two-locus genotypes in the east Asian and world populations.  相似文献   
74.
We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.  相似文献   
75.
76.
Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any unfolding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin’s visco-elastic properties appear to depend on the Ig domain un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.  相似文献   
77.
Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.  相似文献   
78.
Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field‐grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB‐exposed plants accumulated higher levels of ultraviolet (UV)‐absorbing compounds (rutin, chlorogenic acid, crypto‐chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl‐L‐isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N. attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) showed strongly altered accumulation patterns. While constitutive HGL‐DTGs levels were higher under UVB, N. attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL‐DTGs under UVB, and mirids preferred to feed on HGL‐DTGs‐silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL‐DTGs accumulation, and modulates ecological interactions of N. attenuata with its herbivores in nature.  相似文献   
79.
1. Glycopeptides and glycolipids were isolated from normal cat liver and liver from a cat affected with GM1 gangliosidosis. 2. Bio-Gel P-6 chromatography of the crude glycopeptide fractions demonstrated three major peaks of hexose-containing compounds that were greatly increased in the mutant liver sample; these peaks contained oligosaccharides that comprised over 2% of the liver wet weight. 3. Two of the major pathological oligosaccharides, GP5 and GP6, were purified by chromatography on charcoal/Celite and Sephadex G-25. Oligosaccharides GP5 and GP6 had apparent mol.wts. of 1800 +/- 200 and 1350+/-200 respectively, and contained galactose, mannose and N-acetylglucosamine in molar proportions of 2.0:3.1:4.1 (GP5) and 1.0:2.2:2.7 (GP6). Periodate oxidation studies demonstrated the presence of galactose in a non-reducing terminal position. 4. The neutral glycolipid fraction from the mutant cat liver has a 1.3-fold increase in hexose content accompanied by an increased concentration of asialo-(ganglioside GM1). 5. There was a 2-fold increase of gangliosides in the mutant cat liver compared with normal cats. Ganglioside GM1 and a compound tentatively identified as N-glycolloyl-(ganglioside GM1) were the major glycolipids accumulated.  相似文献   
80.
Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction.  相似文献   
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