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31.
Left-handed Z-DNA helices in polymers, restriction fragments, and recombinant plasmids 总被引:4,自引:0,他引:4
T O'Connor M W Kilpatrick J Klysik J E Larson J C Martin C K Singleton S M Stirdivant W Zacharias R D Wells 《Journal of biomolecular structure & dynamics》1983,1(4):999-1009
Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition. 相似文献
32.
We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity. 相似文献
33.
G R Grotendorst A Igarashi R Larson Y Soma M Charette 《Journal of cellular physiology》1991,149(2):235-243
We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF-AB and PDGF-BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF-AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF-AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF-AA were absent. PDGF-AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti-Type alpha and beta PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF alpha and beta receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of beta receptors. These results indicate that cells possessing high levels of PDGF beta receptors only are capable of responding equally well to either PDGF AB or BB. 相似文献
34.
Molecular cloning of two linked loci that increase the transformability of transformation-deficient mutants of Haemophilus influenzae. 下载免费PDF全文
A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components. 相似文献
35.
D B Murphy S Rath E Pizzo A Y Rudensky A George J K Larson C A Janeway 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(11):3483-3491
MHC class I and class II molecules transport foreign and self peptides to the cell surface and present them to T lymphocytes. Detection of these peptide:MHC complexes has thus far been limited to analysis of the response of a T cell. Previously, we showed that a mAb, Y-Ae, reacts with 10 to 15% of class II molecules on peripheral B lymphocytes and on cells in the thymus medulla but not thymus cortex in mice that express both I-Ab and I-Eb molecules. Elsewhere, we show that Y-Ae detects a self E alpha peptide bound to I-Ab molecules. Data presented here suggest that the antibody binds over the peptide binding groove of class II molecules, and, like a TCR, appears to recognize both the self peptide and polymorphic class II residues. In addition to B lymphocytes, the Y-Ae determinant is expressed at comparable levels on other APC, including macrophages and dendritic cells. Finally, the antibody does not react with invariant chain-associated class II complexes, thus providing direct evidence that invariant chain:class II complexes and peptide:class II complexes are mutually exclusive. These data provide further evidence that immunologic self is of limited complexity, and have important implications for T cell selection, self tolerance, and autoreactivity. 相似文献
36.
Fate of the Benzene Ring of Linear Alkylbenzene Sulfonate in Natural Waters 总被引:5,自引:4,他引:1 下载免费PDF全文
The biodegradability of linear alkylbenzene sulfonate (LAS) was studied in water samples collected from a receiving stream at locations above and below the discharge of a municipal wastewater treatment plant. Rates of primary biodegradation were determined for a commercial LAS mixture by a modified methylene blue-active substance method. Rates of LAS ultimate degradation were determined by radiochemical methods, using a C12 LAS homolog uniformly labeled with 14C in the benzene ring. The C12 LAS was tested at low concentrations (50 and 500 μg/liter) comparable to those existing in the receiving stream. Loss of methylene blue-active substance response over time occurred rapidly in water samples containing sediment collected from below the treatment plant, with an estimated half-life for LAS of 0.23 days. Evolution of 14CO2 during mineralization of the benzene ring occurred rapidly in the same samples, with a half-life for the benzene ring of 0.73 day. Mineralization of the benezene ring was also observed in river water containing no sediments and in river water and sediment samples collected from above the treatment plant. However, the rate of degradation was reduced in these cases, with half-lives for ring carbon ranging from 1.4 to 14 days. Although LAS degradation was enhanced in the presence of sediments, adsorption of LAS to the clay-silt fraction of river sediments was low, and most of the radioactivity was bound to biomass. 相似文献
37.
Microautoradiography was used to follow the translocation pathways of 14C-labeled photosynthate from mature source leaves, through the stem, to immature sink leaves three nodes above. Translocation occurred in specific bundles of the midveins and petioles of both the source and sink leaves and in the interjacent internodes. When each of six major veins in the lamina of an exporting leaf was independently spot-fed 14CO2, label was exported through specific bundles in the petiole associated with that vein. When the whole lamina of a mature source leaf was fed 14CO2, export occurred through all bundles of the lamina, but acropetal export in the stem was confined to bundles serving certain immature sink leaves. Cross-transfer occurred within the stem via phloem bridges. Leaves approaching maturity translocated photosynthate bidirectionally in adjacent subsidiary bundles of the petiole. That is, petiolar bundles serving the lamina apex were exporting unlabeled photosynthate while those serving the lamina base were simultaneously importing labeled photosynthate. The petioles and midveins of maturing leaves were strong sinks for photosynthate, which was diverted from the export front to differentiating structural tissues. The data support the idea of bidirectional transport in adjacent bundles of the petiole and possibly in adjacent sieve tubes within an individual bundle.Abbreviations C
central leaf trace
- L
left leaf trace
- LPI
leaf plastochron index
- R
right leaf trace 相似文献
38.
39.
Thomas P. Caruso Dennis L. Larson Philip S. Portoghese A.E. Takemori 《Life sciences》1980,27(22):2063-2069
The nonequilibrium narcotic antagonist, chlornaltrexamine (CNA) was used to bind selectively and covalently pioid specific sites on brain membrane preparations. Selective binding of [3H]CNA occured with a saturation maximum of 185 fmol/mg protein. Bound [3H]CNA was extracted with Triton X-100, dialyzed against Brij 36T, precipitated with trichloroacetic acid and chromatographed on an ultrogel AcA 22 column. The elution profile suggests that this extract contains a minimum of four selective [3H]CNA complexes. At least two of these complexes migrate in a single large peak. Column calibration showed that this peak eluted at 590,000 daltons. One of these specific [3H]CNA complexes elutes at the elution volume of the column and is dialyzable. Finally, putative aggregate of these complexes elutes with the void volume. 相似文献
40.