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Anthony P. Fordham-Skelton F. Safadi M. Golovkin A. S. N. Reddy 《Plant Molecular Biology Reporter》1994,12(4):358-366
Calmodulin labeled with125I or34S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic
systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method
to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated
calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the
filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides.
The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated
calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA),
a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with35S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence
of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin.
All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate
cDNAs encoding CBPs from any eukaryotic organism. 相似文献
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Mitochondrial DNA sequence evolution in sharks: rates, patterns, and phylogenetic inferences 总被引:8,自引:0,他引:8
Abundant representation of sharks in the fossil record makes this group a
superb system in which to investigate rates and patterns of molecular
evolution and to explore the strengths and weaknesses of phylogenetic
inferences from molecular data. In this report, the molecular evolution of
the cytochrome b gene in sharks is described and the information related to
results from phylogenetic analysis of the data evaluated in the light of a
phylogeny derived independently of the molecular data. Across divergent
lineages of sharks there is evidence for significant substitution rate
variation, departure from compositional equilibrium, and substantial
homoplasy; nevertheless, the signal of evolutionary history is evident in
patterns of shared transversions and amino acid replacements.
相似文献
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There is marked heterogeneity of nucleotide composition in mitochondrial
DNA across divergent animals. Differences in nucleotide composition
presumably reflect differences in directional nucleotide substitution for
A+T or G+C nucleotides. In mitochondrial DNA, there is A+T directional
nucleotide substitution in most (if not all) animals surveyed, and the
magnitude of directional A+T nucleotide substitution differs greatly within
and among groups. Differences in directional nucleotide substitution among
lineages of mammals can be explained by changes in metabolic physiology.
This relationship is thought to be mediated by the effect of oxygen
radicals because these toxic compounds are by-products of aerobic
metabolism and are known mutagens. Association between metabolism and
nucleotide composition provides additional evidence in favor of the
hypothesis that rates and patterns of nucleotide substitution in
mitochondrial DNA can be influenced by factors that impinge on rates of
endogenous DNA damage.
相似文献
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Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections 总被引:15,自引:4,他引:11 下载免费PDF全文
A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules. 相似文献
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Kim J. Brolin Ribacke Alex J. van Duinen Helena Nordenstedt Jonas H?ijer Ragnhild Molnes Torunn Wigum Froseth AP Koroma Elisabeth Darj H?kon Angel Bolkan AnnaMia Ekstr?m 《PloS one》2016,11(2)