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71.
Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process. 相似文献
72.
Molecular cloning of the chicken avidin cDNA. 总被引:3,自引:1,他引:2
M L Gope R A Kein?nen P A Kristo O M Conneely W G Beattie T Zarucki-Schulz B W O'Malley M S Kulomaa 《Nucleic acids research》1987,15(8):3595-3606
A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe. 相似文献
73.
Characterization and localization of myosin in the brush border of intestinal epithelial cells 总被引:28,自引:26,他引:2
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The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web. 相似文献
74.
Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations
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Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester. 相似文献
75.
Hytönen VP Nyholm TK Pentikäinen OT Vaarno J Porkka EJ Nordlund HR Johnson MS Slotte JP Laitinen OH Kulomaa MS 《The Journal of biological chemistry》2004,279(10):9337-9343
The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T(m) = 107.4 +/- 0.3 degrees C) than avidin (T(m) = 83.5 +/- 0.1 degrees C) or bacterial streptavidin (T(m) = 75.5 degrees C). AVR4/5 also exhibits a high affinity for biotin (K(d) approximately 3.6 x 10(-14) m) comparable to that of avidin and streptavidin (K(d) approximately 10(-15) m). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed. 相似文献
76.
Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci 总被引:8,自引:0,他引:8
Roy MS; Geffen E; Smith D; Ostrander EA; Wayne RK 《Molecular biology and evolution》1994,11(4):553-570
Genetic divergence and gene flow among closely related populations are
difficult to measure because mutation rates of most nuclear loci are so low
that new mutations have not had sufficient time to appear and become fixed.
Microsatellite loci are repeat arrays of simple sequences that have high
mutation rates and are abundant in the eukaryotic genome. Large population
samples can be screened for variation by using the polymerase chain
reaction and polyacrylamide gel electrophoresis to separate alleles. We
analyzed 10 microsatellite loci to quantify genetic differentiation and
hybridization in three species of North American wolflike canids. We
expected to find a pattern of genetic differentiation by distance to exist
among wolflike canid populations, because of the finite dispersal distances
of individuals. Moreover, we predicted that, because wolflike canids are
highly mobile, hybrid zones may be more extensive and show substantial
changes in allele frequency, relative to nonhybridizing populations. We
demonstrate that wolves and coyotes do not show a pattern of genetic
differentiation by distance. Genetic subdivision in coyotes, as measured by
theta and Gst, is not significantly different from zero, reflecting
persistent gene flow among newly established populations. However, gray
wolves show significant subdivision that may be either due to drift in past
Ice Age refugia populations or a result of other causes. Finally, in areas
where gray wolves and coyotes hybridize, allele frequencies of gray wolves
are affected, but those of coyotes are not. Past hybridization between the
two species in the south-central United States may account for the origin
of the red wolf.
相似文献
77.
Changgui Shi MD Vaskar Das PhD Xin Li MD PhD Ranjan Kc PhD Sujun Qiu MD InSug O‐Sullivan PhD Richard L. Ripper CVT Jeffrey S. Kroin PhD Fackson Mwale PhD Atiyayein A. Wallace MS Bingqian Zhu MSN Lan Zhao PhD Andre J. van Wijnen PhD Mingliang Ji MD PhD Jun Lu MD PhD Gina Votta‐Velis MD PhD Wen Yuan MD Hee‐Jeong Im PhD 《Journal of cellular physiology》2018,233(10):6589-6602
78.
Raffael AC Oliveira Ricardo VM Almeida Márcia DA Dantas Felipe N Castro Jo?o Paulo MS Lima Daniel CF Lanza 《BMC bioinformatics》2014,15(1)
Background
The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.Results
A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.Conclusion
In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users. 相似文献79.
Background
The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in α2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%. 相似文献80.
Farrowing survival is usually analysed as a trait of the sow, but this precludes estimation of any direct genetic effects associated with individual piglets. In order to estimate these effects, which are particularly important for sire lines, it is necessary to fit an animal model. However this can be computationally very demanding. We show how direct and maternal genetic effects can be estimated with a simpler analysis based on the reduced animal model and we illustrate the method using farrowing survival information on 118 193 piglets in 10 314 litters. We achieve a 30% reduction in computing time and a 70% reduction in memory use, with no important loss of accuracy. This use of the reduced animal model is not only of interest for pig breeding but also for poultry and fish breeding where large full-sib families are performance tested. 相似文献