Isolation Of Metaphase Chromosomes Research Articles

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry ofchromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Interphase FISH for prenatal diagnosis of common aneuploidies.

Cytogenetic analysis is currently a standard prenatal diagnostic test. It is routinely offered to pregnant patients who have an increased risk of carrying chromosomally abnormal fetuses. The traditional “gold standard” for prenatal diagnosis of chromosome abnormalities is metaphase analysis by G-banding. The primary advantages of standard cytogenetic analysis are the ability to detect aneuploidies as well as structural chromosomal aberrations with great accuracy (<99.5%). Traditional karyotyping, however, requires isolation of metaphase chromosomes from cultured fetal cells and therefore is time consuming. Although reporting time has decreased dramatically during the last 3 decades, conventional karyotyping still requires 7–12 d of which culture time is the most time consuming ().

A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L.

A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G1/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 μM amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 10(6) chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.

A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.

A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.

Mass isolation of plant chromosomes and nuclei

A method is presented for mass isolation of metaphase chromosomes and nuclei from plant protoplasts. The isolation procedure was developed for both monocotyledonous and dicotyledonous species using wheat (Triticum monococcum) and poppy (Papaver somniferum) cell cultures. Metaphase chromosomes were isolated from partially synchronized mitotic protoplasts, while for the isolation of nuclei unsynchronized protoplasts were used. Light and electron-microscopic studies revealed that isolated chromosomes and nuclei preserved their intact morphology. A preliminary biochemical study of chromosomal proteins was made by polyacrylamide-gel electrophoresis. Because of the purity and high quantity of isolated chromosomes and nuclei, the given isolation procedure can supply useful material for structural and biochemical studies, and for genetic manipulation.

Chinese hamster metaphase chromosomes isolated under physiological conditions: A partial characterization of associated non-histone proteins and protein cores

In a previous report [2] we have described a non-histone protein core which could be isolated from Chinese hamster metaphase chromosomes. This core structure maintained the overall morphology of the metaphase chromosome even after removal of all of the histones, together with many of the non-histone proteins and the bulk of the DNA. As part of our work on the characterization of these core structures, we have developed a novel procedure for the isolation of metaphase chromosomes which avoids the use of high pH buffers and hexylene glycol, as well as eliminating the numerous centrifugation and resuspension steps previously employed. Chromosome cores prepared by 2 M NaCl extraction and DNase I digestion from metaphase chromosomes isolated under these more gentle, quasi-physiological conditions, are shown to contain a relatively simple subset of non-histone proteins. One-dimensional SDS-polyacrylamide gel electrophoresis shows two major groups of polypeptides having molecular weights 48 000–52 000 and 65 000–72 000 D respectively, with similarities in mobilities to the nuclear pore complex-lamina polypeptides and tubulins. However, more detailed analysis by two-dimensional gel electrophoresis and peptide mapping has failed to detect these proteins. A 52 000 D polypeptide component of the core is tentatively identified as the intermediate filament protein vimentin. The in vivo significance of chromosome cores is discussed.

Isolation of chromosome clusters from metaphase-arrested HeLa cells.

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosome from a cell remain together in a bundle which we call a "metaphase chromosome cluster". Cells are arrested to 90-95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a PercollTM density gradient. -- This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.

Insect chromosome isolation: a method for metaphase chromosome harvest from cultured cells of the mosquito Aedes albopictus.

A modified technique for bulk isolation of metaphase chromosomes from an in vitro cell line of the mosquito Aedes albopictus (Skuse) is described.

Isolation and structural organization of human mitotic chromosomes.

New methods are presented for the bulk isolation of metaphase chromosomes from HeLa cells, and an electron microscopic study of thin sections of these chromosomes is presented. The techniques for chromosome isolation were developed to utilize solution conditions that are as mild as possible, so that further biochemical and structural studies can be directly related to the in situ state of chromosomes. - Electron micrographs of thin sections of isolated HeLa metaphase chromosomes reveal the general organization of the nucleosome-containing fibers. Chromosomes in isolation buffer show a dense, relatively uniform distribution of material across the chromatids. Swollen chromosomes reveal the primary mode of organization of the fibers to be a radial distribution from the central axes of the chromatids. A significant proportion of the fibers could also be oriented longitudinally.

Rapid isolation of metaphase chromosomes containing high molecular weight DNA.

Metaphase chromosomes with high molecular weight DNA were isolated from Chinese hamster ovary (CHO) cells in a neutral buffer containing polyamines and chelators. The individual, unfixed chromosomes retained their centromeric and secondary constrictions, distinct sister chromatids, and complex banding patterns. The DNA from these chromosomes was 100-fold larger (2 x 10(8) daltons) than DNA from chromosomes isolated by other procedures. These characteristics indicate preservation during isolation of considerable native structure. In contrast to chromosomes produced by other methods, these chromosomes were stable in storage and did not aggregate, thus providing useful material for studies of the structure and biochemistry of individual chromosomes.

Dephosphorylation of histones H1 and H3 during the isolation of metaphase chromosomes.

Histones have been extracted from isolated metaphase chromosomes prepared by the method of Wray and Sutbblefield [Exp. Cell Res 59, 469-478 (1970)] and by a Nonidet P-40 detergent procedure based on the method of Wigler and Axel [Nucleic Acids Res. 3, 1463-1471 (1976)]. Analysis of the densitometer profiles of long polyacrylamide gels shows that the mitotic phosphorylations of histone H1 (H1M) and histone H3 are extensively depleted during chromosome isolation. These data indicate that CHO metaphase chromosomes prepared by standard methodologies do not represent in vivo chromosomes with respect to their histone phosphorylations; therefore, current chemical and structural studies of isolated metaphase chromosomes may require further clarification.

Bulk isolation of metaphase chromosomes from an in vitro cell line of Drosophila melanogaster.

Bulk isolation of metaphase chromosomes from an in vitro cell line of Drosophila melanogaster.

Chapter 9: Isolation of Metaphase Chromosomes with High Molecular Weight DNA at pH 10.5

Publisher Summary This chapter discusses the isolation of metaphase chromosomes with high molecular weight DNA at pH 10.5. Since DNA is the most interesting molecule in the chromosome from a genetic standpoint, it was felt that the size distributions present in metaphase chromosomes might lead to an insight as to their arrangement and/or packaging. Molecular weight may be estimated by the use of alkaline sucrose velocity gradients similar to the way radiation damage and repair processes have been analyzed. With the excellent methods of obtaining large populations of metaphase chromosomes and the ease of fractionation of these into size groups, the experimental design was to analyze and compare the molecular weight of the DNA in large and small chromosomes. Hopefully if the length of DNA is different, the emerging distributions will fall into a predictable or at least interpretable pattern.

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

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.

F1-histone modification at metaphase in Chinese hamster cells

When the histones of metaphase and interphase Chiness hamster, HeLa S 3 and rat nephroma cells are compared by polyacrylamide gel elecrophoresis, a shift to slower mobility of metaphase F1-histone is observed. This change in mobility of the F1-histone results from enhanced phosphorylation since it is not observed after treatment of the extracted proteins with alkaline phosphatase. The phosphorylation is found in mitotic cells collected without use of a metaphase arrest agent. Further, the extent of phosphorylation of F1 is independent of the elapsed time of metaphase arrest with vinblastine. The phosphoryl-rich F1 subcomponents are dephosphorylated upon allowing the metaphase cell to enter the G 1 phase. The observed protein phosphorylation may be related to the unique physical state of metaphase chromatin. It has a pronounced influence on the chemical extraction of lysine-rich histone during isolation of metaphase chromosomes.

Isolation of metaphase chromosomes from HeLa cells.

The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of RNA for each milligram of DNA. The RNA bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA.

STUDIES ON THE ISOLATION OF METAPHASE CHROMOSOMES.

A method for the isolation of metaphase chromosomes from mouse L1210 leukemia cells has been developed. Cells, arrested at metaphase with colchicine, were exposed to hypotonic solution and the pH was then adjusted to 5.6 to stabilize the chromosomes. The metaphase figures were subsequently disrupted and the chromosomes isolated by a series of differential centrifugations in sucrose. The isolated chromosomes were well preserved, as judged by morphological criteria. The effect of various enzymes and chemical agents on the isolated chromosomes was studied. Chymotrypsin, trypsin, and deoxyribonuclease caused a marked disintegration of the chromosomes, whereas treatment with pepsin and ribonuclease induced no significant morphological alterations.

Isolation of chromosomes

TECHNIQUES for the isolation of interphase chromosomes from nuclei of vertebrate tissues were described in 1947 by Mirsky and Ris [19]. The determination of the chemical composition of interphase chromosomes from these preparations [20, 21, 221 aided in the initial definition of deoxyribonucleic acid (DNA) as the genetic material. Although information in more recent years regarding nucleoprotein relationships has been obtained from other cellular fractions, such as isolated nuclei, the earlier information provided by Mirsky and Ris is often used in the interpretation of studies on chromosome structure and function. Mirsky and Ris [ 191 used a Waring blendor for cellular and nuclear disruption to isolate interphase chromosomes. This technique has been claimed by some to render nuclear and chromosomal fragmentation during the isolation procedure [ 15, 171. Others, however, regard the final preparation to consist of intact interphase chromosomes [6, 231. The degree of chromosomal fragmentation, if any, cannot be strictly ascertained. Unfortunately, techniques are not available at present to allow morphological identification of the individual chromosomes at interphase. Chromosomes in division stages have always been the cytologist’s point of reference for defining morphology. Therefore, a method has been sought for the isolation of metaphase chromosomes from cells grown in vitro. Preservation of chromosome morphology could be used as a significant criterion in the evaluation of the techniques used. Recently, a method has been described for the mass isolation of metaphase chromosomes employing a formamide treatment to provide cellular disruption [4]. Individual metaphase complements can also be isolated, free from cytoplasm, if cells are disrupted in 3 : 1, alcohol: acetic acid, fixative [24]. The present report describes several attempts to isolate metaphase chromosomes from mammalian cells grown in vitro. ’