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101.
Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA 总被引:7,自引:0,他引:7
Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition. 相似文献
102.
H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips 总被引:4,自引:3,他引:1
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The relationship between changes in H+ flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H+ efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H+-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H+ extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H+ secretion under the conditions of this investigation. 相似文献
103.
A Karara S Wei D Spady L Swift J H Capdevila J R Falck 《Biochemical and biophysical research communications》1992,182(3):1320-1325
Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase. 相似文献
104.
J. D. Wallach Ma Lan Wei Han Yu Bo-Qi Gu Feng Teng Yu Roy F. Goddard 《Biological trace element research》1990,24(2-3):189-205
The common denominator of a unique disseminated multi-focal milliary myocardial hyaline necrosis and fibrosis in Keshan disease (KSD) and cystic fibrosis (CF) and a commonality of the affected age groups of fetuses and preschool children led to the review of existing KSD autopsy material to search for pancreatic and hepatic lesions considered pathognomonic for CF. Pancreatic lesions considered pathognomonic for CF were found in 595, or 35% of 1700 documented cases of KSD. The pancreatic lesions were limited to tissues of fetuses and preschool children. Adults dying of KSD had diagnostic lesions limited to the cardiovascular system, liver, and skeletal muscle. Varying degrees of focal biliary cirrhosis were identified in 850, or 50% of the KSD autopsies, and 85, or 5% developed severe lobular cirrhosis. The common denominator in CF and KSD appears to be a primary or induced secondary selenium deficiency in age-susceptable humans, prenatally at or around 22 wk of fetal life, during early postnatal life, or during the rapid-growth preschool years. The basic difference between the natural history of CF and KSD is that the selenium deficiency is totally environmental in KSD and appears to be the result of a maternal malabsorptive syndrome or an abnormality of selenium transfer in CF. 相似文献
105.
本文用流式细胞光度术(FCM)等方法研究了MNNG,ENNG和DMS对HeLa细胞DNA含量分布的影响。经MNNG(6.8μmol/L)处理后,细胞分裂减少,DNA合成速率下降,S期细胞的比例随处理时间的延长而增加。DMS显示有类似的现象而ENNG的效应则较小。 相似文献
106.
质粒YRP7用氯霉素法扩增,碱变性裂解法提取,酸酚法及核糖核酸酶纯化后,得到了高产量(5.6mg/L培养液),高纯度(A260:A280=2.0)的质粒制品,经转化实验及酶切分析确定YRP7具有下列特征:大小为5.41±0.10kb,可赋予宿主细胞AmP~r、Tet~r的表型,对大肠杆菌C600的转化频率为10~(-6)、转化效率为1.5×10~6转化子/mgDNA。限制性内切酶BamH Ⅰ、ECoRⅠ、Hind Ⅲ及PstⅠ在其分子上的切点数分别为1、2、2、2,并确定了各酶切片段的分子大小,对BanHⅠ的单切点,经插入失活法证实其位于Tet~r的基因上。由上述特征可确定,质粒YRP7是一个比较理想的克隆载体。 相似文献
107.
Swelling and Ca2+-activated Anion Conductances in C127 Epithelial Cells Expressing WT and ΔF508-CFTR
CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations
in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism
of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion
channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, ΔF508-CFTR, or vector. Cell
lines were compared quantitatively using an 125I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, 125I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were
time- and voltage independent. All three lines responded to hypotonic challenge with large 125I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages.
Unexpectedly, basal 125I efflux was significantly smaller in the ΔF508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the
cell line expressing ΔF508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin
or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated
responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally
selected cell lines that are unrelated to CFTR expression.
Received: 15 November 1995/Revised: 16 February 1996 相似文献
108.
Ferritin, a protein widespread in nature, concentrates iron ∼1011–1012-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor
(based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin
gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants
but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals
and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization
in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning
of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals
but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the
absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In
plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin
gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary
protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin
genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding
sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional
constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin
gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid,
the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling.
Received: 25 July 1995 / Accepted: 3 October 1995 相似文献
109.
110.
Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication. 总被引:8,自引:6,他引:2
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Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule. 相似文献