Density-labelling

All of the geometry optimizations for acetyl radical produce similar structures. Here are the predicted spin densities (labeled Total atomic spin densities in the Gaussian output) ... 

Evidence that the increase in activity of at least four hydrolases induced by GA actually occurs as a result of de novo synthesis, rather than by activation of preformed enzyme, has been obtained in various ways. However, the most unequivocal proof comes from density labeling experiments, first performed by Filner and Varner in 1967 (13). Later Jacobsen and Varner (14) proved by the same procedures that protease also is synthesized de novo in response to GA. And Bennett and Chrispeels (15) proved, using D2O, GA-induced de novo synthesis of ribonuclease and 6-1,3-glucanase in barley aleurone cells. 

Preparative gradients have been widely used for preparing satellite DNAs such as the highly repetitous mouse satellite, the DNA sequences coding for ribosomal RNA and mitochondrial DNA (e.g. Flamm et al. 1969). They can be used for separating RNA-DNA hybrids from native and denatured DNA for preparative and analytical purposes (e.g. Birnstiel et al. 1968). DNA can be density-labelled with deuterium, N and bromo-deoxyuridine and normal and heavy DNA separated (e.g. Meselson, Stahl and Vinograd 1957 Braun and Will 1969). 

Density labeling procedures have been used along with synchronously growing populations of cells to study DNA synthesis in subfractions of the S phase in several eukaryote systems. In Physarum polycephalum, Braun et al. (1965) were able to radioactively label replicating DNA in the second half of one S i ase with tritiated thymidine and then density label all subsequent replicating DNA by the addition of bromodeoxyuridine to the cultures. Analysis in cesium chloride equilibrium gradients of DNA radioactively labeled, and then density labeled in this manner, indicated that DNA... 

These types of density labeling procedures provide a means of isolating replicating DNA sequences at any time during the S phase. There are other methods of analyzing the time of replication of specific classes of DNA sequences. For example, the time of replication of ribosomal RNA cistrons in synchronously growing hamster cells has been followed (Amaldi et al., 1969). Here DNA preparations were obtained from cultures at... 

The demonstration using autoradiographic and density labeling techniques that the replication of chromosomal DNA follows a heritable, sequential pattern through S implies that the initiation of DNA synthesis in the various replication units takes place many times in a controlled manner in the S phase. 

Surface density (labeled as ps f) is a calculated value that incorporates both surface oxide loading and specific surface area into a single metric. Surface density is, by nature, an averaged quantity and does not account for nonuniform surface oxide distribution. It does not contain structural information on surface oxide dispersion analogous to metal dispersion in supported metal catalysts (e.g., flat sheets vs. hemispherical islands of metal) [89-92],... 

The measurement itself would consist of illuminating the entire sample at a donor excitation wavelength and observing the resultant acceptor fluorescence intensity. In the reasonably attainable approximations that a) the bulk densities of the two lightly-labelled polymers are the same and b) the label density (labels/volume, ) on each side of the original Interface is the same, the observed fluorescence intensity, I(t), will increase with time like ... 

Definitive proof for the de novo synthesis of a-amylase by GA-stimulated barley aleurone tissue was obtained by Filner and Varner [36] in 1967. They used a density-labelling technique which involved incubation of aleurone layers in (plus GA). The in the water becomes introduced as a density-label in the carboxyl oxygen of amino acids formed during breakdown of protein in the aleurone cells. 

Most of the evidence suggests that the enzymes are newly synthesized, though in the majority of cases rigorous proof is unavailable. Enzyme development in a number of seeds is prevented by inhibitors of protein and/or RNA synthesis. For example, dipeptidase and isocitrate lyase development in Cucurbita maxima [92, 105] are suppressed by protein synthesis inhibitors, while actinomycin D, which inhibits some DNA-dependent RNA synthesis, prevents the increase in lipase and isocitrate lyase of castor beans [14, 77]. Studies with inhibitors can be criticized on several grounds especially since these chemicals may have previously unsuspected side effects. But more satisfactory evidence of the kind known for barley aleurone layers has been found in some seeds. De novo synthesis of isocitrate lyase in cotyledons of Citrullus vulgaris (watermelon) [52] and of endopeptidase in mung bean [21 a] have been shown to occur by means of density-labelling experiments with D2O. 

In repair replication experiments, cells are irradiated and then incubated in medium containing 3H-5-bromodeoxyuridine ( H-BrdUrd). After a suitable repair period the cells are harvested, and the DNA is extracted and centrifuged to equilibrium in cesium chloride. When the gradient fractions are collected, it is possible to follow the new DNA (semiconservatively synthesized during repair and at hybrid density) by counts. The new DNA is half substituted with bromouracil (BrUra) and sediments to a lower point on the gradient than the old or parental DNA, which is detected by absorbancy at 260 nm or by incorporation of a second radioactive label and is, of course, not density labeled. The appearance of counts in the old DNA region is indicative that repair has occurred and that the new incorporation is truly in the parental... 

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