Embryonic regulation of histone ubiquitination the sea urchin

We have used quantitative 2-D protein electrophoresis and immunoprecipitation to study the patterns of histone ubiquitination at 10 h and 36 h of embryonic development in Strongylocentrotus purpuratus. Variants csH2A, ΑH2A, ΒH2A, ΓH2A, ΔHA, H2AF./Z, ΑH2B, ΒH2B, and ΓH2B showed up to sevenfold differences in level of monoubiquitination between variants, and individual variants showed up to sixfold changes during development. At 36 h of embryogenesis, the late variants were less ubiquitinated than the early variants, althoug h the overall level of ubiquitination was appreciably greater than at 10 h. Antiubiquitin antibodies were used to precipitate formaldehyde-fixed chromatin fragments in order to estimate the degree of ubiquitination of the early histone genes. The 5′ regulatory region of the active H3 gene appeared to be at least twice as ubiquitinated as the adjacent upstream spacer. However, the absolute level of ubiquitination of the early histone gene repeat seemed to be independent of transcriptional activity. These results show that variant-specific ubiquitination of histones is a part of the developmental program in sea urchin embryos, but is not clearly correlated with transcriptional activity of the early histone genes, except perhaps in the regulatory regions. © 1995 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50179/1/1020160308_ftp.pd

Quantitative energy-filtered electron microscopy of biological molecules in ice

The theoretical and experimental bases for quantitative electron microscopy of frozen-hydrated specimens are described, with special considerations of energy filtration to improve the images. The elastic and inelastic scattering from molecules in vacuum and in ice are calculated, and simple methods to approximate scattering are introduced. Multiple scattering calculations are used to describe the scattering from vitreous ice and to predict the characteristics of images of frozen-hydrated molecules as a function of ice thickness and accelerating voltage. Energy filtration is predicted to improve image contrast and signal-to-noise ratio. Experimental values for the inelastic scattering of ice, the energy spectrum of thick ice, and the contrast of biological specimens are determined. The principles of compensation for the contrast transfer function are presented. Tobacco mosaic virus is used to quantify the accuracy of interpreting image intensities to derive the absolute mass, mass per unit length, and internal mass densities of biological molecules. It is shown that compensation for the contrast transfer function is necessary and sufficient to convert the images into accurate representations of molecular density. At a resolution of 2 nm, the radial density reconstructions of tobacco mosaic virus are in quantitative agreement with the atomic model derived from X-ray results.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29814/1/0000160.pd

Highly Purified Micro- and Macronuclei from Tetrahymena thermophila Isolated by Percoll Gradients 1

A new procedure is described that utilizes Percoll gradients for purifying micronuclei (MIC) and macronuclei (MAC) from Tetrahymena thermophila . Separation of MIC from MAC during certrifugation in Percoll gradients occurs as a result of their difference in size rather than density. Three kinds of tests were used to evaluate the purity of the nuclei: visualization of the nuclei by light microscopy; examination of the nuclei by electron microscopy; and Southern blots of MIC and MAC DNA probed with the 5s rRNA genes or a fragment from the MAC extrachromosomal rDNA molecule. When examined under the light microscope, the isolated MIC and MAC have much lower nuclear cross contamination levels than previous methods have reported. MIC's contaminated with less than 1 MAC in 1000 MIC and MAC's contaminated with less than 1 MIC in 500 MAC can be routinely prepared. Quantitative analyses of electron micrographs gave higher estimates of cross contamination in our purified nuclei, which may, in part, be explained by the difficulty in identifying small MIC or MAC fragments. Southern blots of MIC and MAC DNA probed with 5s rDNA confirmed the level of MAC contamination in the MIC estimated by light microscopy during purification of the nuclei. The level of nucleolar contamination in the MIC was estimated at 10% by Southern blots of MIC and MAC DNA, derived from a heterokaryon with distinctive MIC and MAC Bam HI sites, using an rDNA probe.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75629/1/j.1550-7408.1983.tb01027.x.pd

The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of [alpha]2-macroglobulin and thrombin

A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of [alpha]2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was [alpha]2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When [alpha]2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular [alpha]2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh [alpha]2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of [alpha]2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29323/1/0000388.pd

Variability in Avian Eggshell Colour: A Comparative Study of Museum Eggshells

Background: The exceptional diversity of coloration found in avian eggshells has long fascinated biologists and inspired a broad range of adaptive hypotheses to explain its evolution. Three main impediments to understanding the variability of eggshell appearance are: (1) the reliable quantification of the variation in eggshell colours; (2) its perception by birds themselves, and (3) its relation to avian phylogeny. Here we use an extensive museum collection to address these problems directly, and to test how diversity in eggshell coloration is distributed among different phylogenetic levels of the class Aves. Methodology and Results: Spectrophotometric data on eggshell coloration were collected from a taxonomically representative sample of 251 bird species to determine the change in reflectance across different wavelengths and the taxonomic level where the variation resides. As many hypotheses for the evolution of eggshell coloration assume that egg colours provide a communication signal for an avian receiver, we also modelled reflectance spectra of shell coloration for the avian visual system. We found that a majority of species have eggs with similar background colour (long wavelengths) but that striking differences are just as likely to occur between congeners as between members of different families. The region of greatest variability in eggshell colour among closely related species coincided with the medium-wavelength sensitive region around 500 nm. Conclusions: The majority of bird species share similar background eggshell colours, while the greatest variability among species aligns with differences along a red-brown to blue axis that most likely corresponds with variation in the presence and concentration of two tetrapyrrole pigments responsible for eggshell coloration. Additionally, our results confirm previous findings of temporal changes in museum collections, and this will be of particular concern for studies testing intraspecific hypotheses relating temporal patterns to adaptation of eggshell colour. We suggest that future studies investigating the phylogenetic association between the composition and concentration of eggshell pigments, and between the evolutionary drivers and functional impacts of eggshell colour variability will be most rewarding.Phillip Cassey, Steven J. Portugal, Golo Maurer, John G. Ewen, Rebecca L. Boulton, Mark E. Hauber and Tim M. Blackbur

Chromatin : By K. E. van Holde. New York: Springer-Verlag. (1989). 497 pp. $98.00

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27714/1/0000102.pd

Low-angle X-ray-scattering Of Chromatin - Reply

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62800/1/291359d0.pd

Long G Tails at Both Ends of Human Chromosomes Suggest a C Strand Degradation Mechanism for Telomere Shortening

AbstractThe chromosomes of lower eukaryotes have short telomeric 3′ extensions. Using a primer-extension/nick-translation technique and nondenaturing hybridization, we find long 3′ G-rich tails at human chromosome ends in mortal primary fibroblasts, umbilical vein endothelial cells, and leukocytes, as well as in immortalized fibroblasts. For all cells tested, >80% of the telomeres have long G-rich overhangs, averaging 130–210 bases in length, in disagreement with the conventional model for incomplete lagging-strand replication, which predicts overhangs on 50% of the chromosome ends. The observed G tails must exist during most of the cell cycle and probably result from degradation of both chromosome ends. The average lengths of the G tails are quantitatively consistent with the observed rates of human chromosome shortening