A soluble ubiquitin-depleted fraction from chicken skeletal muscle (fraction II), when incubated at neutral pH for several hours with 125I-ubiquitin and ATP, formed small amounts of a ubiquitin derivative (Mr 115,000) of the ubiquitin-activating enzyme E1 as well as certain similarly modified E2 species (Mr 37,000, 34,000 and 24,000). Treatment of such mixtures with NaOH during the incubations, even at early times, greatly enhanced the appearance of these entities; up to two-thirds of the thiolesters of ubiquitin bound to these proteins before alkali treatment were thus converted. The bonds involved had properties compatible with their being peptidic in nature, suggesting that auto-ubiquitination had occurred in each case. The protease inhibitor and alkylating agent tosyl-lysylchloromethane ('TLCK'), when preincubated at 50 microM with fraction II for 2 h at 37 degrees C before the addition of 125I-ubiquitin and ATP, promoted the subsequent auto-ubiquitination of E1 and inhibited its adenylate-forming and thiolester-transferring activities. The findings have a bearing on the physiological substrate- and site-specificity of ubiquitin-conjugating reactions. 相似文献
At dextran (Mw ≈ 500,000) concentrations from 2 to ≈10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations > 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations > 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.
We have characterized a murine monoclonal antibody (MAb 35), which was raised against human immunodeficiency virus type 1 (HIV-1) integration protein (IN), and the corresponding Fab 35. Although MAb 35 does not inhibit HIV-1 IN, Fab 35 does. MAb 35 (and Fab 35) binds to an epitope in the C-terminal region of HIV-1 IN. Fab 35 inhibits 3'-end processing, strand transfer, and disintegration; however, DNA binding is not affected. The available data suggest that Fab 35 inhibits enzymatic activities of IN by interfering with the ability of IN to form multimers that are enzymatically active. This implies that the C-terminal region of HIV-1 IN participates in interactions that are essential for the multimerization of IN. Titration of the various IN-mediated enzymatic activities suggests that different degrees of multimerization are required for different activities of HIV-1 IN. 相似文献
We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst. 相似文献
DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution. 相似文献
The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong
and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis
per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near
the telomeres. DSB-prone regions were associated with all of the known ”hot spots” for meiotic recombination on chromosomes
I, III and VI.
Received: 19 March 1996; in revised form: 26 July 1996 / Accepted: 18 August 1996 相似文献
In this paper we use cytonuclear disequilibria to test the neutrality of mtDNA markers. The data considered here involve sample frequencies of cytonuclear genotypes subject to both statistical sampling variation as well as genetic sampling variation. First, we obtain the dynamics of the sample cytonuclear disequilibria assuming random drift alone as the source of genetic sampling variation. Next, we develop a test statistic using cytonuclear disequilibria via the theory of generalized least squares to test the random drift model. The null distribution of the test statistic is shown to be approximately chi-squared using an asymptotic argument as well as computer simulation. Power of the test statistic is investigated under an alternative model with drift and selection. The method is illustrated using data from cage experiments utilizing different cytonuclear genotypes of Drosophila melanogaster. A program for implementing the neutrality test is available upon request. 相似文献
alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine
proteinase inhibitor superfamily, has a primary role in controlling
neutrophil elastase activity within the mammalian circulation. Several
studies have indicated that the reactive center region of alpha 1-PI, the
amino acid sequence of which is critical to recognition of and binding to
target proteinases, is highly divergent within and among species. This
appears to be a consequence of accelerated rates of evolution that may have
been driven by positive Darwinian selection. In order to examine this and
other features of alpha 1-PI evolution in more detail, we have isolated and
sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus
saxicola and Mus minutoides and have compared these with a number of other
mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent
of nonsynonymous substitution is generally high throughout the alpha 1-PI
mRNA molecule, indicating greater overall rates of amino acid substitution.
Within and among mouse species, the 5'-half of the mRNA, but not the
3'-half, has been homogenized by concerted evolution. Finally, the reactive
center is under diversifying or positive Darwinian selection in murid
rodents (rats, mice) and guinea pigs yet is under purifying selection in
primates and artiodactyls. The significance of these findings to alpha 1-PI
function and the possible selective forces driving evolution of serpins in
general are discussed.
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