Wayne Wray

Silver staining of proteins in polyacrylamide gels

The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified and further simplified so that it is stable, controllable, and even more rapid than previous silver-staining methods. The method retains its sensitivity to proteins at the nanogram level and may be used either before or after Coomassie blue staining. Reproducible staining patterns are obtained, and the method is inexpensive, completely under the control of the user, and effective with the common polyacrylamide gel electrophoresis methods.

Scanning-electron microscopy of chromosome aberrations.

This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. The technique reported allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques.

Use of direct current sputtering for improved visualization of chromosome topology by scanning electron microscopy.

Abstract The topological features of isolated Chinese hamster ovary metaphase chromosomes were studied with high resolution scanning electron microscopy (SEM) using the techniques of direct current sputtering for the deposition of metal on the specimens. Metaphase chromosome surfaces consist of numerous compact microconvules of an average diameter of 520 ± 78 A when corrected for the thickness of the gold-palladium coating (80 ± 2 A). These microconvules contain several orders of supercoiling. The superhelical structures were detected also in water-spread preparations. Most of the isolated chromosomes had membrane-like structures attached at the distal portions of the chromatids forming a terminal “plate”. Limited tryptic digests of such isolated chromosomes resulted in considerable stretching of the chromatids and revealed a series of interchromatidal fibers with diameters of 203 ± 38 A (corrected for gold coating). Treatment of these chromosomes with EDTA revealed a longitudinal array of fibers within the chromatids. The diameters of these fibers decreased as the concentration of EDTA was increased. The technique of direct current sputtering for the preparation of chromosomes for scanning microscopy is satisfactory for detailed topological ultrastructural studies in the 70 A range.

Chapter 3 Isolation of Nuclei Using Hexylene Glycol

Publisher Summary This chapter describes a general procedure for the isolation of nuclei in buffered 2-methyl-2,4-pentanediol (hexylene glycol) solutions that meets the requirements of nuclear purity and general applicability. The hexylene glycol-PIPES buffer is efficacious both in vivo and in vitro . The tissues from mouse brain, chicken liver, rat liver, rat uterus, chick oviduct, hen oviduct, rabbit oviduct, and Novikoff hepatoma ascites cells and from the tissue culture cell lines Chinese hamster ovary and HeLa are chosen to demonstrate the minor modifications necessary for general applicability of the isolation method. The purity and morphology of the hexylene glycol-isolated nuclei is established using several criteria. Phase microscopic observations indicate that the preparations are not contaminated with cytoplasmic structures, and electron microscopy shows that the nuclear fine structure is well preserved. Chemical composition data indicates that the RNA/DNA, protein/DNA, acid-soluble protein/DNA, and phospholipid/DNA ratios are similar to those reported for nuclei obtained by other isolation methods.

The Ultrastructural Organization of Prematurely Condensed Chromosomes

SUMMARY In an effort to understand the arrangement of the basic 30 nm chromatin fibre within metaphase chromosomes, changes in the organization of prematurely condensed chromosomes (PCC) were examined as a function of progression through the cell cycle. The structural features of PCC observed under the light microscope were compared with those obtained by scanning electron microscopy. PCC with varying levels of condensation were obtained by fusing mitotic HeLa cells with interphase cells synchronized at different times in the cell cycle. PCC from G1 cells are composed of rather tightly packed bundles of tortuous chromatin fibres. The density of fibre packing along the longitudinal axis of G1-phase PCC is lower and less uniform than that of metaphase chromosomes. Early G1 PCC exhibit gyres suggesting a despiralized chromonema. The condensed domains in G1 PCC appear to be organized as supercoiled loops; whereas fibre-sparse domains consist of longitudinal fibres running along the chromosome axis. As cells progressed towards S phase, a greater proportion of highly extended regions containing prominent longitudinal fibres became evident in the PCC. The pulverized appearance of S-phase PCC under the light microscope corresponded to the highly condensed, looping fibre domains separated by more extended segments containing longitudinal fibres that are visualized using the scanning electron microscope. Active sites of DNA synthesis are implicated to be localized within extended longitudinal fibres. Post-replicative chromosome maturation extends through the G2 period and appears to involve rearrangement of the extended longitudinal fibres into packed looping-fibre clusters, which then coalesce. These observations support the model for packing DNA into chromosomes proposed in 1980 by Mullinger & Johnson. Briefly, this model suggests that the chromonema of each metaphase chromatid contains regions composed of folded longitudinal chromatin fibres as well as looping fibres that emerge from the axis at distinct foci. The final level of chromatin packing in metaphase chromosomes is attained by spiralization of the chromonema.

Isolation of specific human metaphase chromosomes

Abstract The human karyotype can be subdivided into seven fractions containing specific chromosomes to provide material for recombinant DNA research. The isolated metaphase chromosomes are sorted according to size by velocity zonal centrifugation, and specific chromosome groups are further purified by electrostatic deflection in a flow microfluorometer. Rapid improvements in technology should soon provide preparations of single chromosomes.

Functional homologies of acidic chromatin proteins in higher and lower eukaryotes

Within a specific fraction of acidic chromatin-associated proteins from HeLa and the lower eukaryote Physarum polycephalum numerous similarities exist. Several of the similar polypeptides in both cell types are synthesized and appear in the residual chromatin material while still others disappear in response to starvation, a common and universal stimulus. Proteins which incorporate no radioactive amino acids during starvation and ultimately disappear from the residual chromatin material are resynthesized upon refeeding. This resynthesis must be complete before mitosis will again occur. These observations suggest that within the complement of acidic chromatin proteins functional homologies exist in diverse eukaryotes.

Chapter 8: Isolation of Metaphase Chromosomes, Mitotic Apparatus, and Nuclei

Publisher Summary This chapter discusses the isolation of metaphase chromosomes, mitotic apparatus, and nuclei. The methods employed for the mass isolation of any cell organelle are limited by the physical and chemical nature of the structure, the adherence of contaminating materials from the cell, and the final desired state of the isolated component. Isolation of subcellular components for experimental study has generally been aimed at a specific cell organelle with little or no consideration of the slight modifications which might allow nearly identical isolation conditions for a number of related structures. The advantage of subjecting biochemical isolations of organelles to similar conditions is that the observations may be compared without concern that differences in experimental parameters may prevent parallel or related observations.

Isopycnic centrifugation of mammalian metaphase chromosomes in metrizamide

Isopycnic centrifugation is a powerful separation technique, but when applied to metaphase chromosome research it has been technically frustrating. High concentrations of salt rapidly dissociate metaphase chromosomes, and MacGillivray, et al. [l] show that high concentrations of salt dissociate nucleoproteins. Chromatin will band isopycnically in CsCl after fixation with formaldehyde [2], but the reaction is irreversible and the material of little use thereafter. Obviously, formaldehyde treated chromosomes are equally useless. We have used sucrose, Ficoll, and fructose as the support media for isopycnic banding of metaphase chromosomes, but at the density required to band the chromosomes (p=l.3 1 g/cm3) the viscosity of the sugar solutions is so high as to make their use prohibitive. Chromatin has been shown to band in chloral hydrate gradients [3], but these gradients are very difficult to handle and are also quite viscous which again make them unsuitable for banding metaphase chromosomes. In order to circumvent these problems an elaborate system of nonaqueous gradients was developed by Stubblefield and Wray [4] for buoying chromosomes. These organic gradients accomplish their objective of isopycnically banding metaphase chromosomes co= 1.36 g/cm3) in a low viscosity media. However, the technical problems in preparing these gradients, the solvent properties of the organic chemicals and especially their ability to extract certain proteins from the chromosomes make this system amenable only to certain specified applications. The introduction of iodinated density gradient media for biological separations represents a signifi-

Mitotic cell populations obtained from a micro-carrier culturing system☆

Abstract Colcemid treatment of cell culture beads confluent with cells released a mitotic population of cells which was as efficient as that obtained by conventional shake-off procedures. The bead system provides an easy and efficient means for harvesting large quantities of mitotic cells, costs about one-tenth as much as conventional culturing techniques, and is at least 100 times more rapid.