Metaphase Chromosome Preparations Research Articles

Chromosome-mediated gene transfer between closely realted strains of cultured mouse cells.

Gene transfer between two closely related mouse cell lines has been carried out, using as the vector a cell-free preparation of metaphase chromosomes and nuclei. Distinction between gene transferents and revertants of the recipient mutant phenotype was achieved by the use of a donor strain carrying a mutationally altered (8-azaguanine-resistant) hypoxanthine-guanine phosphoribosyltransferase (HPRTase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8). The transferred HPRTase gene is initially unstable; in nonselective medium, it is lost at a rate of about 0.1 per cell per generation. Stabilization occurs as a rare event, with a frequency on the order of 1 X 10(-5) per cell per generation. The unstable state can be maintained for at least 200 generations through serial passages of the transferent in selective medium. Under the conditions of cultivation used in these experiments, the unstable HPRTase-positive cells are eventually replaced by the stable HPRTase-positive cells in the population.

Chromosome preparations of human blood lymphocytes—Evaluation of techniques

Several parameters of human lymphocyte culturing techniques and metaphase chromosome preparation procedures were studied and quantitatively evaluated in regard to their influence on the results of Q and G banding procedures. The culturing conditions were studied using3H thymidine incorporation as a parameter. A whole blood culturing technique using Ham's F12 medium was found to give optimal and consistent results. Colcemid concentration proved to be of no influence on chromosome contraction or on the number of metaphases obtained over the concentration range investigated. Prolonged exposure to colcemid was found to cause a decrease in the mean chromosome length but the absolute number of metaphases with a low degree of chromosomal contraction hardly decreased. Different spreading techniques were quantitatively analysed and factors important for the spreading of chromosomes were evaluated. Based on the results obtained, an optimal procedure is described which over a period of one year has given consistent results.

Preparation and spread of unfixed metaphase chromosomes for immunofluorescence staining of nuclear antigens

A new chromosome preparation technique, which allows immunofluorescence staining of acidlabile antigens of metaphase cells, is described. Hypotonically treated, unfixed cells are centrifuged on a microscope slide. The hypotonic medium is composed of organic buffers, glycerol, and low concentrations of Mg 2+, Ca 2+, Na +, K + and Cl −. This technique allows immunofluorescence staining and microfluorimetry of both chromosomal and nucleoplasmic antigens of flattened but unbroken metaphase cells. The method can be used to demonstrate the Epstein-Barr virus associated nuclear antigen, SV 40-induced T-antigen and autoantigens, which previously could not be detected in conventional acid-fixed chromosome preparations. Only a small sample of cells is needed for the chromosome preparation.

Polymorphism of C-heterochromatin regions in chromosomes of different lines of mice

The C-heterochromatin regions were studied in the chromosomes of mice of lines C3HA and CBA and of mice homozygous for chromosomal translocations T6T6 and T1IEM. Preparations of metaphase chromosomes were obtained from bone marrow cells by Ford's method. The pericentromeric heterochromatin was revealed by the method of Arrighi and Hsu. Different chromosomes in the karyotypes of mice of different lines were found to contain different quantities of pericentromeric heterochromatin. Polymorphism of the C-heterochromatin regions is observed not only in mice of different lines, but also in different individuals belonging to the same line.

The gorilla karyotype: chromosome lengths and polymorphisms.

Metaphase chromosome preparations of three male and one female <i>Gorilla gorilla </i>were stained to demonstrate quinacrine, Giemsa, centromeric heterochromatin, and, in one case, reverse-Giemsa bands. A standard karyotype is proposed based on chromosome banding pattern, centromeric index, and length. Three types of variation between homologous chromosomes are described: presence or absence of very bright fluorescence of the short arm or satellite region of acrocentric chromosomes, presence or absence of bright quinacrine bands at the distal ends of chromosome arms, and large differences in the size of the heterochromatic region on each of two biarmed chromosomes. At least half the chromosome pairs show polymorphisms of these types. Satellite associations were scored for each animal. In one case the two smallest pairs of chromosomes were preferentially involved in associations.

Staining of human metaphase chromosomes with fluorescent conjugates of polylysine

The interaction of polylysine and partially substituted dansyl, fluorescein, and quinacrine conjugates of polylysine with cytological preparations of human metaphase chromosomes has been studied by fluorescence microscopy. The fluorescence intensity along chromosomes stained with the dansyl and fluorescein conjugates exhibits little variation, suggesting that regions capable of binding these polycations are nearly evenly distributed. In contrast, the quinacrine derivatives of polylysine stain the chromosomes in a banded fluorescence pattern resembling that observed following quinacrine or quinacrine mustard treatment.

Distribution of constitutive heterochromatin in HeLa and HEp-2 cell lines.

Despite the fact that HeLa and HEp-2 cell lines have been established respectively from a female and a male patient, the frequent loss of the Y chromosome makes it difficult to discriminate the two cell types. The technique of centromeric localization of heterochromatin, however, facilitates more definite identification of the two cell types. Giemsa positive centromeric constitutive heterochromatin blocks were induced within the intact metaphase chromosome preparations of Hela and HEp-2 cells. The Hela cell population had a single heterochromatin marker chromosome, whereas the HEp-2 cell line was found to possess two types of heterochromatin markers. The distinctive patterns of distribution of constitutive heterochromatin of these three chromosomes served as the guides in tracing the presumptive cytological pathways for their orgin.

Das W-Chromosom von Ephestia kuehniella (Lepidoptera) und die Ableitung des Geschlechtschromatins

The W-chromosome, though not conspicuous in normal metaphase chromosome preparations, can be demonstrated in early oocyte and nurse cell pachytene stages using a fluorescent Feulgen stain (BAO-S02). In such preparations, it appears as a continuously fluorescing thread, whereas Z-chromosome and autosomes give a distinct pattern of chromomeres and interchromomeres. Four γ-ray induced chromosome-fusions which had been shown genetically to include the W, combine this continuously fluorescing thread with a thread having an autosomal pattern of chromomeres, differing in the four fusion-chromosomes. Concomitantly the sex-chromatin is atypically separated into several smaller bodies in highly polyploid cells of individuals possessing these W-fusions, thus showing that sex-chromatin properties depend on the W. This abnormal behaviour of the sex-chromatin is interpreted as the result of the opposing tendencies of the autosomes to form separate chromocenters and the W-chromosome to produce a single big chromocenter during polyploidisation.

Turner's syndrome karyotypes.

To the Editor.— The letter of Miller et al ( 214 :2337, 1970) describes a young woman with some of the features of Turner's syndrome. The karyotype reproduced in the article is likely to mislead readers who are not familiar with chromosome morphology. The third pair of chromosomes in group A is mislabeled as is the sex chromosome pair (XX). The latter pair is actually number 3—arms and legs are about equal. The pair placed by the authors in the A3 position are C group chromosomes and the larger of the two might be an X. However, actual identification of the two Xs would require examination of other more favorable metaphase chromosome preparations. Barr body (or sex chromatin) studies should also include description of the size of the sex chromatin body, a smaller than normal one might be a lead toward recognizing a partial deletion of the short arm of an

Association of nuclear membrane fragments with metaphase and anaphase chromosomes as observed by whole mount electron microscopy

Whole mount preparations of metaphase and anaphase chromosomes from a wide variety of mammalian tissues, prepared with and without hypotonic, with and without colchicine, and with and without critical point drying, all showed that fragments of the nuclear membrane may remain attached at multiple points along the chromosomes. No consistent patterns were observed, indicating that the attachment of these fragments was random and fortuitous. Preparations of purified nuclear membrane showed morphological features identical to that of the fragments attached to the chromosomes. The major significance of these observations is that they indicate that the metaphase chromosomes were attached at multiple sites to the nuclear membrane during interphase.

Heterochromatization in opossum, Didelphys virginiana.

Somatic heterochromatization of the opossum,D. virginiana, was studied from prophase and tritiated thymidine-labeled metaphase chromosome preparations. In both sexes of this mammal, a number of prophase autosomes and the sex chromosomes were observed displaying deeply stained condensed areas. These chromosomal areas were interpreted as evidence for heterochromatization. The extent of heterochromatization in the opossum was found much greater in certain autosomes than either theX orY chromosome alone. This assertion that some opossum autosomes possess more heterochromatin than the sex chromosomes was supported by data collected on the terminal labeling patterns of the chromosomes. Metaphase autoradiographs prepared from cultured leucocytes of the animals unequivocally suggested that certain opossum autosomes completed replication later than the sex chromosomes. If one assumes that there is heterochromatin in the autosomes, as the evidence suggests, then it merely becomes a question as to what block of heterochromatin replicates last — a phenomenon that is probably size dependent.

Template functions in the enzymic formation of polyribonucleotides, II. Metaphase chromosomes as templates in the enzymic synthesis of ribonucleic acid.

A recent study from this laboratory1 gave a preliminary account of experiments aiming at the isolation of chromosome preparations from synchronized HeLa cells in metaphase arrest. It appeared of interest to examine the template activity of these preparations in directing the incorporation of ribonucleotides by RNA polymerase, with respect to the amount and, more importantly, to the composition of the polymeric product formed; and to do this in comparison with the template behavior of the total free DNA isolated from the same HeLa cell cultures. The RNA normally present in the preparations of metaphase chromosomes is, similarly to cytoplasmic RNA, of a high GC-type, quite unlike the DNA of HeLa cells. It was one of the purposes of this study to ascertain whether the enzymic product formed with chromosomal preparations as templates resembled in any way the RNA component of the chromosomes. This proved not to be the case. Materials and Procedures.-HeLa cells in suspension culture were grown in phosphate medium containing 0.3% (w/v) Methocel HG (Standard Grade, 4000 cP, Dow Chemical Co.) and 5% (v/v) whole horse serum.2 The cells were synchronized and arrested at metaphase as described previously for stationary cultures.1 Three cultures, each containing 1.2-1.5 X 10' cells of which about 40 c were in metaphase, were fractionated so as to yield the preparations rich in chromosomes and free of nuclei and soluble DNA described previously as fraction C.' Two cycles of resuspenision in 0.16 A/IT NaCl solution and centrifugation (1100 X g, 15 mim, 40) resulted in the recovery of 95%0 of the chromosomes, as judged from DNA assay. In the incorporation experimenits the washed chromosomes were tested as a suspension in 0.16 M NaCl solution. Four preparations of DNA were used. Pireparations 1-3 were isolated from HeLa cells with the aid of Duponol' and purified in the usual manner (compare p. 327 of ref. 4). The fibers obtained by precipitation with ethanol were dissolved in, and dialyzed against, physiological saline, and the solution was stored in the frozen state at -35?. For the isolation of DNA preparation 4, another procedure described recently1 was adapted. The preparation from HeLa cells comprising intact nuclei and aggregated chromosomes, designated as fraction P in a recent paper,' was washed twice by centrifugation (1100 X g, 15 min, 40) with 0.15 M NaCI-0.015 M sodium citrate (pH 7) and then treated with trypsin (0.3 mg/ml) in the same saline-citrate solution for 4 hr at 37?. After cenitrifugation the entire DNA was found in the supernatanlt layer which was then subjected to ilncubation with pronase and ribonuclease anid to extractioni with phenol, as described in the papers mentioned before.' The final preparation was essentially free of protein and RNA. Two preparations of RNA were isolated with the use of a procedure applied previously to the preparatioln of RNA from HeLa cells.' The starting material for the specimen designated as cytoplasmic RNA was the cell lysate freed by centrifugation of n-uclei and chromosomal particles.' The chromosomal RNA preparation was made from fraction C.' The base composition of DNA was determined in the usual manner.7 RNA was hydrolyzed to the mononucleotides and analyzed by the two-dimensional chromatography of the latter (isopropanol-NH3, buffered isobutyrate) in the arrangement described before.8 RNA polymerase was prepared from frozen cells of Escherichia coli, strain W (Grain Processing Co., Muscatine, Iowa) by a procedure patterned after one described previously,9 with the modifications menitioned in a recent paper. Other enzymes used were commercial preparations.

Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl.

Leukocytes were cultured from 0.2 ml of whole blood inoculated into 5 ml portions of a medium consisting of Eagle's basal amino acids and vitamins at double strength in Earle's balanced salt solution brought to pH 7.0 with 7.5% NaHCO3, and containing additives: glutamine, 2 mM; penicillin, 100 units/ml; streptomycin, 100 μg/ml; phenol red, 7 μg/ml; fetal or newborn agammaglobulin bovine serum, 15%; phytohemagglutinin M, 2%; and U.S.P. heparin sodium, 20,000 units/liter. Cultures were incubated in closed 60 × 28 mm screw-cap vials, in a gas phase initially of room air, for 3 days at 37 C, with colchicine to make 0.2 μg/ml added for the final 3-5 hr. After incubation, the cells were separated from the medium by centrifugation, the medium replaced by 0.075 M KCI plus 16 U.S.P. units/ml of heparin sodium at 37 C, cells resuspended and allowed to incubate 10 min. Removal of the hypotonic KCI was followed by fixation in methanol-acetic acid, 3:1 (changed twice), spreading cells on slides by the air-drying metho...