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FISH physical mapping with barley BAC clones 总被引:7,自引:0,他引:7
Nora L.V. Lapitan Susan E. Brown Wayne Kennard Janice L. Stephens Dennis L. Knudson 《The Plant journal : for cell and molecular biology》1997,11(1):149-156
Fluorescence in situ hybridization (FISH) is a useful technique for physical mapping of genes, markers, and other single- or low-copy sequences. Since clones containing less than 10 kb of single-copy DNA do not reliably produce detectable signals with current FISH techniques in plants, a bacterial artificial chromosome (BAC) partial library of barley was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average barley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially characterized. FISH using these two BAC clones as probes showed specific hybridization signals near the end of the short arm of one pair of chromosomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the BAC clones corresponded to bands present in the genomic DNA, and the two BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with four BAC clones with inserts varying from 20 to 150 kb, showed distinct signals on paired chromatids. Physical mapping of single- or low-copy sequences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approach for saturating regions of interest with markers and for constructing contigs spanning those regions. 相似文献
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Quantitative measurement of guinea pig complement with liposomes 总被引:1,自引:0,他引:1
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Stimulation of glycosaminoglycan production in murine tumors 总被引:6,自引:0,他引:6
Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in part regulated by host-tumor interactions. 相似文献
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Rogers PA Dick GM Knudson JD Focardi M Bratz IN Swafford AN Saitoh S Tune JD Chilian WM 《American journal of physiology. Heart and circulatory physiology》2006,291(5):H2473-H2482
Hydrogen peroxide (H(2)O(2)) is a proposed endothelium-derived hyperpolarizing factor and metabolic vasodilator of the coronary circulation, but its mechanisms of action on vascular smooth muscle remain unclear. Voltage-dependent K(+) (K(V)) channels sensitive to 4-aminopyridine (4-AP) contain redox-sensitive thiol groups and may mediate coronary vasodilation to H(2)O(2). This hypothesis was tested by studying the effect of H(2)O(2) on coronary blood flow, isometric tension of arteries, and arteriolar diameter in the presence of K(+) channel antagonists. Infusing H(2)O(2) into the left anterior descending artery of anesthetized dogs increased coronary blood flow in a dose-dependent manner. H(2)O(2) relaxed left circumflex rings contracted with 1 muM U46619, a thromboxane A(2) mimetic, and dilated coronary arterioles pressurized to 60 cmH(2)O. Denuding the endothelium of coronary arteries and arterioles did not affect the ability of H(2)O(2) to cause vasodilation, suggesting a direct smooth muscle mechanism. Arterial and arteriolar relaxation by H(2)O(2) was reversed by 1 mM dithiothreitol, a thiol reductant. H(2)O(2)-induced relaxation was abolished in rings contracted with 60 mM K(+) and by 10 mM tetraethylammonium, a nonselective inhibitor of K(+) channels, and 3 mM 4-AP. Dilation of arterioles by H(2)O(2) was antagonized by 0.3 mM 4-AP but not 100 nM iberiotoxin, an inhibitor of Ca(2+)-activated K(+) channels. H(2)O(2)-induced increases in coronary blood flow were abolished by 3 mM 4-AP. Our data indicate H(2)O(2) increases coronary blood flow by acting directly on vascular smooth muscle. Furthermore, we suggest 4-AP-sensitive K(+) channels, or regulating proteins, serve as redox-sensitive elements controlling coronary blood flow. 相似文献
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The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo 总被引:3,自引:0,他引:3
Stallock J Molyneaux K Schaible K Knudson CM Wylie C 《Development (Cambridge, England)》2003,130(26):6589-6597
In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. We show that the pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, we show that Bax is downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages. 相似文献