Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins. 相似文献
Summary Bifunctional reagents, namely bis-(2-chloroethyl)-amine (nitrogen mustard) and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid (bromo-ketone reagent) are used to cross-link protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing 32P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11, and L2 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful for topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies. 相似文献
Primary hamster embryo cells infected with bovine papilloma virus (BPV) or treated with BPV DNA-calcium phosphate precipitates
showed striking morphological alterations characteristic of transformed cells. Long, spindle-shaped cells grew into dense
foci, eventually overgrowing monolayers of normally shaped cells. Samples of these cells were tested for anchorage-independent
growth in dilute agarose medium. Cells were able to grow in agarose to form colonies which, when removed from agarose and
transferred to liquid medium, established clones. Mockinfected cultures inoculated with plain medium displayed normal cell
morphology and growth properties. This is the first report of BPV-transformed cells demonstrating anchorage-independent growth
in agarose and the establishment of BPV transformed clones. 相似文献
Mature seeds of Arabidopsis thaliana strain “Columbia” were soaked for 7.5 hr in an aqueous solution of the chemical mutagen ethyl methanesulfonate (0.05, 0.10, or 0.50%, v/v). Embryo-lethal mutants were identified in the resulting M-1 chimeral plants by screening the first five siliques of each plant and noting the frequency of aborted seeds. Three hundred sixty seeds were treated at each mutagen dose; the frequency of embryo-lethal mutants ranged from 1–3% of the M-1 plants grown from seeds exposed to 0.05% EMS, to 20–30% of the M-1 plants at the highest mutagen dose. Six embryo-lethal mutants identified through screening of M-1 plants were chosen for detailed studies in subsequent generations. All six mutants segregate as nonallelic, Mendelian recessive lethals, and are maintained as heterozygotes since homozygotes die as embryos. Fruits of heterozygous plants contain 25% aborted seeds and 75% phenotypically normal seeds ( heterozygotes and wild type). Segregation ratios are not temperature sensitive; the same frequency of aborted seeds is found in plants grown at 18, 25, and 32°C. Embryo arrest and eventual lethality in each mutant occur at a characteristic stage of early embryo development: globular-heart, globular, early globular, or preglobular. Arrested embryos from five of the six mutants resemble normal embryos at early stages of development. Developmental arrest of the embryo proper in the remaining mutant is followed by abnormal growth of the suspensor, an embryonic structure that attaches the embryo proper to the maternal tissue. 相似文献
25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness.
These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.
Calicioid lichens and fungi are a polyphyletic grouping of tiny ascomycetes that accumulate a persistent spore mass (mazaedium) on top of their usually well-stalked ascomata (‘mazaediate fungi’). In addition to extant forms, six fossils of the group were previously known from European Paleogene amber. Here we report nine new fossils and analyze the preserved features of all fossils to assess their applicability for dating molecular phylogenies. Many fossils are extremely well preserved, allowing detailed comparisons with modern taxa. SEM investigation reveals that even fine details of ascospore wall ultrastructure correspond to those seen in extant specimens. All fossils can confidently be assigned to modern genera: three to Calicium (Caliciaceae, Lecanoromycetes), five to Chaenotheca (Coniocybaceae, Coniocybomycetes), six to Chaenothecopsis (Mycocaliciaceae, Eurotiales), and one to Phaeocalicium (Mycocaliciaceae, Eurotiales). Several Calicium and Chaenotheca fossils are assignable to specific lineages within their genera, while the Chaenothecopsis fossils demonstrate the extent of intraspecific variation within one such lineage. Some features in the morphology of Chaenotheca succina nov. sp. seem to be ancestral as they have not been reported from modern species of the genus. 相似文献
We employed a genetic approach to study protein glycosylation in the
procyclic form of the parasite Trypanosoma brucei. Two different mutant
parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures
by selecting cells which resisted killing or agglutination by concanavalin
A. Both mutant cells show reduced concanavalin A binding. However, the
mutants have different phenotypes, as indicated by the fact that ConA 1-1
binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot
probed with concanavalin A revealed that many proteins in both mutants lost
the ability to bind this lectin, and the blots resembled one of wild type
membrane proteins treated with PNGase F. This finding suggested that the
mutants had altered asparagine- linked glycosylation. This conclusion was
confirmed by studies on a flagellar protein (Fla1) and procyclic acidic
repetitive protein (PARP). Structural analysis indicated that the N- glycan
of wild type PARP is exclusively Man5GlcNAc2 whereas that in both mutants
is predominantly a hybrid type with a terminal N- acetyllactosamine. The
occupancy of the PARP glycosylation site in ConA 4-1 was much lower than
that in ConA 1-1. These mutants will be useful for studying trypanosome
glycosylation mechanisms and function.
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