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51.
Rosemarie Weikard T. Goldammer Ch. Kühn W. Barendse M. Schwerin 《Mammalian genome》1997,8(11):836-840
A methodical strategy for the isolation of microsatellite markers specific for targeted regions of bovine chromosomes is
presented. The procedure involves directed microdissection of one defined subchromosomal area, its DOP-PCR-amplification and
cloning. With this approach, a library specific to the BTA 6q21-31 chromosomal region was constructed. Eleven unique microsatellite-containing
sequences were isolated, converted into sequence-tagged microsatellite sites, and characterized concerning their species-specific
origin. Seven primer pairs generated bovine-specific PCR products and provided a set of microsatellite markers that generally
revealed high informativity in the HF breed. Linkage analysis assigned six of them to their predefined subchromosomal origin
on BTA 6 corresponding to the specific rehybridization signal of the DOP-PCR product generated from the microdissected chromosome
area 6q21-31. The results underline the usefulness of the BTA 6q21-31 library for targeted isolation of unique sequences that
are specific for the dissected chromosomal region as demonstrated here by the isolation of microsatellite markers.
Received: 27 March 1997 / Accepted: 14 July 1997 相似文献
52.
Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum. 总被引:3,自引:0,他引:3 下载免费PDF全文
Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution. 相似文献
53.
Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain 总被引:2,自引:0,他引:2
Abstract: Elevated extracellular potassium concentration ([K+ ]e ) has been shown to induce reversal of glial Na+ -dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K+ ]e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K+ ]e (by equimolar substitution of K+ for Na+ ), and glutamate accumulation was measured by HPLC. With [K+ ]e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K+ ]e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K+ ]e attainable in brain. 相似文献
54.
Leslie A. Kuhn Craig A. Swanson Michael E. Pique John A. Tainer Elizabeth D. Getzoff 《Proteins》1995,23(4):536-547
Water-protein interactions drive protein folding, stabilize the folded structure, and influence molecular recognition and catalysis. We analyzed the closest protein contacts of 10,837 water molecules in crystallographic structures to define a specific hydrophilicity scale reflecting specific rather than bulk solvent interactions. The tendencies of different atom and residue types to be the nearest protein neighbors of bound water molecules correlated with other hydrophobicity scales, verified the relevance of crystallographically determined water positions, and provided a direct experimental measure of water affinity in the context of the folded protein. This specific hydrophilicity was highly correlated with hydrogen-bonding capacity, and correlated better with experimental than computationally derived measures of partitioning between aqueous and organic phases. Atoms with related chemistry clustered with respect to the number of bound water molecules. Neutral and negatively charged oxygen atoms were the most hydrophilic, followed by positively-charged then neutral nitrogen atoms, followed by carbon and sulfur atoms. Agreement between observed side-chain specific hydrophilicity values and values derived from the atomic hydrophilicity scale showed that hydrophilicity values can be synthesized for different functional groups, such as unusual side or main chains, discontinuous epitopes, and drug molecules. Two methods of atomic hydrophilicity analysis provided a measure of complementarity in the interfaces of trypsin:pancreatic trypsin inhibitor and HIV protease:U-75875 inhibitor complexes. © 1995 Wiley-Liss, Inc. 相似文献
55.
Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ 总被引:7,自引:3,他引:4
M. Germana Sanna Ronald V. Swanson Robert B. Bourret Melvin I. Simon 《Molecular microbiology》1995,15(6):1069-1079
CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos-phorylated form of the protein allow counter-ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephos-phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of inter-action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch. 相似文献
56.
Rosemarie Swanson 《Bulletin of mathematical biology》1984,46(2):187-203
The structure of the genetic code is related to a Gray code, which is a plausible theoretical model for an amino acid code.
The proposed model implies that the most important factor in shaping the code was the effects of mistakes in translation,
not effects of mutations. Another possible implication is that the preservation of stiffness and flexibility at appropriate
places in a protein chain is as important in protein structure as the appropriate placement of hydrophilic (external) and
hydrophobic (internal) residues. Other results are a simple conceptualization of the relationships among the 20 amino acids
and their relations to their codons. The detailed relationships are summarized in the following ‘similarity alphabet’: ala,
thr, gly, pro, ser; asp, asn, glu, gln, lys; his, arg, trp, tyr, phe; leu, met, ile, val, cys; (ATGPS DNEQK HRWYF LMIVC in
the one-letter code). This alphabet falls into four groups of amino acids: small, external, large, internal. The approximate
relation of the groups to their codons is expressed as: the first base of a codon controls size—a purine means a small amino
acid, a pyrimidine means large; the middle base controls cloisterednes—purine means external, pyrimidine means internal. These
relationships express the minimum change principle upon which the code appears to be founded. 相似文献
57.
H H Rotmensch M Vincent P H Vlasses B N Swanson J D Irvin M Hichens K E Harris R K Ferguson 《Federation proceedings》1984,43(5):1333-1335
MK-521 is a new orally active, nonsulfhydryl angiotensin-converting enzyme (ACE) inhibitor. Single doses of 2.5, 5.0, 10.0, and 20.0 mg were administered to nine hypertensive patients alternating with placebo. All doses of MK-521 caused profound suppression of ACE activity for more than 24 h and decreased standing diastolic blood pressure for more than 12 h without changes in pulse rate. Although there was no further reduction in blood pressure with doses above 5.0 mg, the duration of action was prolonged for more than 24 h with the higher doses. Serum MK-521 concentrations increased with dosage, and ACE was inhibited maximally at concentrations above 10 ng/ml. In this initial study, MK-521 was well tolerated and proved to be a potent and long-acting antihypertensive agent. 相似文献
58.
We have studied chemotaxis by individual Dictyostelium discoideum amoebae using strong, local gradients of the chemoattractant cyclic AMP. Gradients were provided by diffusion of cyclic AMP from a microneedle, which could be positioned at various points around the cell. Responses to changes in the gradient indicate how the cell is structurally organized for chemotactic movement. There is a polarity in the responsiveness of the surface to stimulation by cyclic AMP along the length of the amoeba. Furthermore, two aspects of chemotactic movement can be distinguished. The first response to cyclic AMP is a locally generated extension of a hyaline pseudopod from the region of the surface nearest the stimulus. The second response, the flow of cytoplasm in the direction of the stimulus, is coordinated and separate from the first response. The coordination appears to depend on the nucleus or on the microtubule-organizing center. 相似文献
59.
Summary The anterior end of the zoospore ofUlothrix belkae has been examined in detail and is compared toStigeoclonium and other filamentous green algae. The nature of the symmetry of green algal motile cells is discussed and the term, 180° rotational symmetry, is proposed to describe the type of arrangement of anterior end components seen inU. belkae, including the four basal bodies, rootlets and striated fibers. The four microtubular rootlets are cruciately arranged. A striated microtubule-associated component (SMAC) has a periodicity of 6.4 nm and extends with each 2-membered rootlet posteriorly into the cell. One 5-membered rootlet passes very near the eyespot. Phylogeny in green algal motile cells is discussed. 相似文献
60.