Isolated Metaphase Chromosomes Research Articles

Entrapment of metaphase chromosomes into phospholipid vesicles (lipochromosomes): Carrier potential in gene transfer

Transfer of genes from one type of cultured mammalian cell to another by using isolated metaphase chromosomes has been reported with a frequency of one per 10(6)-10(8) cells. Very recently a rate of 16/10(6) has been reported with Chinese hamster ovary cells [Spandidos, D. A. & Siminovitch, L. (1977) Proc. Natl. Acad. Sci. USA 74, 3480-3484]. To increase the frequency of gene transfer, we isolated metaphase chromosomes from hypoxanthine guanine phosphoribosyltransferase (HGPRT) positive cells, entrapped them in liposomes, and fused the lipochromosomes with HGPRT-negative cells. Lipochromosomes were prepared with cholesterol and egg lecithin, using isolated metaphase chromosomes from a mouse-human somatic hybrid cell line (A9/HRBC2); the entire X chromosome, including the HGPRT, glucose-6-phosphate dehydrogenase, and phosphoglycerate kinase genes, is the only recognizable human genetic material retained by the hybrids. Enclosure of the chromosomes in the lipid envelope was confirmed by electron and fluorescence microscopy and differential centrifugation. These lipochromosomes were fused with HGPRT(-) mouse cells (A9) in the presence or absence of polyethylene glycol and transferents were selected in hypoxanthine/aminopterin/thymidine (HAT) medium. The frequency of transfer was at least once per 10(5) cells, a minimum 10-fold improvement over previous methods. The selected cells contained HGPRT activity similar to the amount found in the A9/HRBC2 cells. Starch gel electrophoresis verified that the observed HGPRT activity in the transferents is due to the human enzyme. Human glucose-6-phosphate dehydrogenase and phosphoglycerate kinase were also identified electrophoretically in the transferents. Karyotyping with C and Q banding did not reveal the presence of the whole human X chromosome or a visible extra fragment of a human chromosome associated with the mouse genome. The biochemical data strongly suggest, however, that transfer of a portion of the human X chromosome has occurred in these transferents. Thus, at least three X-linked genes have been transferred from one cell to another with high frequency, using metaphase chromosomes.

Transfer of the marker for morphologically transformed phenotype by isolated metaphase chromosomes in hamster cells.

IN spite of extensive investigations of the process of carcinogenesis in vitro, the basic mechanisms of the primary events are poorly understood1. In general, it is found that the first manifestation of the transformed phenotype involves morphological alteration of the cells. This is usually followed by an ill-defined process resulting in several further changes in phenotype including the ability of the cells to grow in agar (aga+), and to produce tumours in animals. In part because of the variety of phenotypes observed, but also because of the lack of suitable methods of analysis, it has been difficult to examine the process of transformation at the genetic level. We have recently initiated experiments designed to use chromosome transfer techniques to attempt such a genetic analysis2,3 and work in several laboratories has shown that single gene markers can be transferred from cell to cell at frequencies of 10−6–10−7 by means of the metaphase chromosome4–9. Most of the initial work was done with the genes for hypoxanthine phosphoribosyl transferase and thymidine kinase, but dominant markers such as resistance to methotrexate and ouabain can also be so transferred9, as can the wild-type alleles for three auxotrophic markers10. In seeking to delineate the genetic elements of carcinogenesis, we have examined the ability of purified metaphase chromosomes from Chinese hamster ovary (CHO) cells to transfer the first recognisable phenotype observed during cell carcinogenesis, that is, morphological transformation. Because of the low frequency of chromosome transfer, an appropriate method for selection of cells (transferents) which have incorporated the relevant markers from the background of recipient cells is necessary. Our experiments were based on the assumption that if transformation gene(s) could be transferred to senescent cells, then they should be able to produce colonies, whereas the majority of the cells which do not receive the appropriate marker, will not form colonies. The results presented here show that senescent hamster cells can be rescued and become transformed by transfer of purified metaphase chromosomes from CHO cells; the frequency of this event is similar to that of single gene markers.

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.

Transfer of codominant markers by isolated metaphase chromosomes in Chinese hamster ovary cells

Transfer of codominant markers by isolated metaphase chromosomes in Chinese hamster ovary cells

Transfer of anchorage independence by isolated metaphase chromosomes in hamster cells

The cellular property of being able to grow on agar (aga +) or to show anchorage independence has been transferred by means of metaphase chromosomes from CHO cells to BHK and other permanent transformed hamster lines unable to grow on agar. As with other genetic markers, the transferents are unstable when grown under non-selective conditions. The aga + transferents are tumorigenic, providing further evidence for the association between the ability to grow in agar or anchorage independence and tumorigenicity. Evidence has been obtained in these experiments for the existence of at least two discrete events in the transformation of normal into tumorigenic cells. The ability to transfer and select for the aga + marker in recipient cells indicates that tumorigenicity behaves dominantly phenotypically.

Transfer of codominant markers by isolated metaphase chromosomes in Chinese hamster ovary cells.

Codominant mutations to methotrexate and ouabain resistance in Chinese hamster ovary cells can be transferred to recipient Chinese hamster ovary cells by isolated metaphase chromosomes. For methotrexate, both the structural change and the increased activity of dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase EC 1.5.1.3), characteristic of the donor cells, are observed in the transferents (cells that carry and express functions derived by chromosome transfer). The transferents are unstable in the absence of selection although stable clones can be isolated. From results obtained by fractionation of chromosomes and transfer to recipients, the methotrexate and ouabain markers can be assigned to the middle and large size-classes of chromosomes, respectively. By fractionation and transfer of chromosomes, from transferents to new recipients, evidence has been obtained that chromosome integration is not restricted to a particular chromosomal site in the recipient.

Bandign isolated metaphase chromosomes by a sequential flurescent G/Q technique.

G/Q-banding is a new, rapid, fluorescent technique for banding isolated chromosomes that incorporates characteristics of both G- and Q- banding. G-bands, while easily characterized, are often inconsistent when using isolated chromosomes, and Q-bands, while reliable, fade rapidly under UV exposure, making prolonged observation and photography difficult. G/Q-banding combines these techniques to sequentially utilize quinacrine staining over Giemsa banding to produce slow-fading fluorescent G/Q-bands. The background fluorescence in G/Q preparations fades quickly under continued UV exposure, while the chromosomes remain brightly banded and can be observed and photographed for at least five minutes. G/Q-banding was extended to whole cell chromosome spreads and produced results identical to those obtained with isolated chromosomes. Whole cell karyotypes indicate that G/Q-bands generally correspond to Q-bands. Advantages of G/Q-banding as a unique and universal technique over current double-staining procedures are discussed.

Transfer of the human gene for hypoxanthine-guanine phosphoribosyltransferase via isolated human metaphase chromosomes into mouse L-cells.

We have transferred the human gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferease) via isolated metaphase chromosomes from human HeLa S3 cells into murine A9 cells which lack functional murine HPRT activity, using the technique of McBride and Ozer (Proc, Nat. Acad. Sci. USA 70, 1258-1262, 1973). Three transformed clones were isolated which contained human HPRT activity as determined by electrophoretic and immunochemical assays. Twenty human isozymes other than HPRT whose genes have been assigned to 14 human chromosomes were found to be absent in our transformed clones. Moreover, the human isozymes of hlucose-6-phosphate dehydrogenase (EC 1.1.1.49; D-glucose 6-phosphate:NADP 1-oxidoreductase) and phosphoglycerate kinase (EC 2.7.2.3;ATP:3-phospho-D-glycerate 1-phosphotransferase), whose genes have been linked with the HPRT gene to the long are of the human X chromosome, were also absent. On the basis of the known linkage relationships of the three markers, we thereby suggest that the transferred piece of human genetic material is smaller than 20% of the human X chromosome or less than 1% of the human genome. This estimate assumes a normal syntenic relationship for the long arm of the X chromosome in HeLa S3 cells. In agreement with this conclusion, no human chromosomes could be detected in our transformed clones. When grown under nonselective conditions about 3% of the gene transfer cells lost the human HPRT marker per cell generation. Transformants that had lost human HPRT activity were subjected to hypoxanthine-aminopterin-thymidine selection. The frequency of revertants to the HPRT(+) phenotype was less than 1 x 10(-6), and two revertants that were obtained possessed the mouse electrophoretic phenotype. These results argue against a stable integration of the human donor genetic material into the mouse recipient genome.

Human gene expression in rodent cells after uptake of isolated metaphase chromosomes.

Permanent transfer of genetic information from chromosomes isolated from human diploid cells to recipient cells has been demonstrated. Human metaphase chromosomes were incubated with mouse A9 fibroblasts deficient in hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7). Colonies of cells containing hypoxanthine phosphoribosyltransferase appeared during growth in a selective medium. The hypoxanthine phosphoribosyltransferase gene product in four independent colonies was identified as human donor species by both gel electrophoresis and isoelectric focusing; hence these colonies did not result from reversion of ta9 parental cells. Other X-linked human genes, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD(+) 1-oxidoreductase, EC 1.1.1.49) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), were not expressed in these same colonies. Dissociation of expression of these X-linked genes probably results from chromosomal fragmentation during uptake, but other mechanisms have not been excluded.

Contractile proteins: Major components of nuclear and chromosome non-histone proteins

Two of the major non-histone proteins from Physarum polycephalum have been isolated under nondenaturing conditions and identified as actin and myosin. A third protein has been purified from crude nuclear actomyosin and from residual nonhistone fractions and found to bind actomyosin in the presence of Mg 2+. In Physarum these proteins are not components of the nuclear membrane. Based on sodium dodecylsulfate-polyacrylamide gel electrophoresis, similar proteins are also present in nuclei of HeLa cells and mouse embryo fibroblasts. Isolated metaphase chromosomes from Physarum show a several-fold enrichment in myosin and an altered ratio of actin to the Mg 2+-dependent actomyosin binding protein as compared to interphase nuclei. When non-proliferative states are induced in any of these cells, the Mg 2+-dependent actomyosin binding protein decreases while actin increases several fold in intranuclear concentration; concomitantly, there is a generalized condensation and inactivation of chromatin. Experiments with added purified radioactive nuclear actomyosin; comparative studies on nuclear protein during stepwise nuclear purification; and studies on isolated metaphase chromosomes indicate that these proteins exist in nuclei in vivo. These observations suggest that contractile proteins may function in the structural interconversions of chromatin and in the regulation of cell proliferation.

THE MECHANISM OF GIEMSA-BANDING OF MAMMALIAN CHROMOSOMES, WITH SPECIAL ATTENTION TO THE ROLE OF NON-HISTONE PROTEINS

Some cytological, biochemical and biophysical studies were made on the G-banding mechanism of chromosomes, using isolated metaphase chromosomes of the Chinese hamster. The results obtained are:1. All G-banding techniques tested cause the loss of certain macromolecules including DNA and proteins, leading to an uneven distribution of chromatin substances. The amounts of such macromolecules resided in the G-banded chromosomes approximate the morphologically estimated genome size of the Giemsa-positive bands.2. The acrylamide gel electrophoresis reveals that the banding procedures remove non-histone proteins of relatively larger molecular sizes.3.The major non-histone proteins reside in the G-banded chromosomes represent relatively small molecules containing a large number of SS bonds.4. The residual G-band chromatin shows higher Tm and hyperchromicities as compared to the extracted one, suggesting that DNA in the former is more stabilized.5. A slight difference in the DNA base composition between the residual and extracted chromatins does not seem to account for the banding mechanism.6. Removal of RNA and histone does not affect the band formation.7. In conclusion, the Giemsa-positive bands represent relatively thermostable chromatins constituting of smaller non-histone protein molecules.

X-ray diffraction from isolated metaphase chromosomes

Metaphase chromosomes were isolated from Chinese hamster fibroblasts. They were found to give the same characteristic diffraction pattern in the wet state as isolated nucleohistone but when dry did not give the reflections normally associated with dry nucleohistone. No higher structural order than that associated with the nucleohistone supercoil was detected.

STRUCTURE OF CHROMOSOMES VISUALIZED BY SCANNING ELECTRON MICROSCOPY

Isolated metaphase chromosomes of Chinese hamster observed under the scanning electron microscope appear to have a distinct rough surface composed of chromonemata. The chromonema is a fine filament coated with nucleoprotein, which can be easily removed by hypotonic sodium citrate solution. The centromere, a narrowed, tight portion of the chromosome, is also multistranded. No membrane has been seen in metaphase chromosomes.

Replication of chromosomal fibres from isolated metaphase chromosomes

The 3H-thymidine labelling patterns of isolated, enzyme-treated L cell metaphase chromosomes indicate that DNA synthesis occurs at numerous sites within each chromosomal fibre, and that each fibre contains many tandemly-joined replicating units. It is suggested that there may be clusters of adjacent replication units in the chromosome which undergo DNA synthesis relatively synchronously within the period of the 3H-thymidine pulse. The length of the chromosomal fibres was quite variable, but some fibres attained a length of over 1 mm.

Fractionation of isolated mammalian metaphase chromosomes

Isolated metaphase chromosomes from HeLa cells, mouse L cells, and Syrian hamster embryonic fibroblast cells have been fractionated on the basis of size by linear sucrose density gradient centrifugation. At present, this technique only permits a partial separation of the chromosomes into several size groups and has not yet proved sensitive enough to permit the fractionation of one specific type or group of chromosomes from all other chromosomes.

Uptake of isolated metaphase chromosomes by mammalian cells in vitro

In an autoradiographic investigation, it was found that isolated 3H-thymidine-labelled metaphase chromosomes can penetrate into mammalian cells in vitro. The progressive changes in the cellular labelling patterns with time indicate that, in most cases, the ingested chromosome is degraded in the cytoplasm of the recipient cell and the chromosomal DNA is integrated into the host nuclear DNA. This integration of extracellular chromosomal DNA into the recipient cell DNA may involve DNA macromolecules rather than free nucleotides, and in most cases, the chromosomal DNA becomes randomly integrated among the chromosomes of the recipient cell. Rarely, whole extracellular chromosomes may become incorporated into a cell without being degraded, although this has not been established with certainty.

Isolated mammalian metaphase chromosomes: II. Fractionated chromosomes of mouse and Chinese hamster cells

Isolated metaphase chromosomes of mouse L-cells, mouse L-5178Y lymphoblast tumor cells and Chinese hamster cells have been separated into several distinct size classes by velocity sedimentation in steep sucrose gradients. Fractions showing relatively homogeneous chromosome size distributions were readily obtained, but none of the fractions was composed entirely of chromosomes of one specific type. Fractions of the smallest chromosomes were the most homogeneous in size, and the heterogeneity of the fractions increased progressively with increasing average chromosome length. Mouse satellite DNA was not restricted to chromosomes of a limited size class or type but was distributed throughout all fractions of the isolated chromosomes in nearly the same proportion of the total DNA. These results are consistent with the interpretation that the amount of satellite DNA in each chromosome is proportional to the total DNA of the chromosome and that it is not limited to a fixed amount per chromosome. Approximately 70% of the total chromosomal DNA remained in the supernatant or sodium chloride-soluble fraction after extracting the isolated L-cell chromosomes twice in 2 m-NaCl and centrifuging after each extraction. The sodium chloride-soluble DNA was main-band DNA, whereas satellite DNA remained associated with the insoluble chromosome pellet fraction. Isolated chromosomes of the Chinese hamster were also separated into several size classes by centrifugation in sucrose gradients. The DNA's from the various fractions showed no significant differences either in densities or band profiles in cesium chloride density-gradient centrifugation. The DNA's from two fractions showing highly restricted chromosome size-distributions also failed to renature after heating and annealing and their behavior in this respect resembled that of the total DNA of mammalian cells.

Radiation-Induced Double-Strand Scission of the DNA of Mammalian Metaphase Chromosomes

The molecular weight distribution of double-stranded mammalian (hamster) DNA was determined by ultracentrifugation of isolated metaphase chromosomes previously layered onto sucrose gradients containing high salt concentrations to dissociate the protein and nucleic acid components. In untreated controls the distribution (as determined by counting the incorporated radioactivity in the resultant fractions) exhibited a peak at 225 X 10(6) daltons. Inclusion of mercaptoethanol and hydroxylamine into the gradients produced no significant change of these sedimentation patterns. Gamma-radiation-induced reduction in the number and weight average molecular weights was used to calculate a value of 1.05 X 10(11) double-strand breaks/gram rad, equivalent to about 600 eV/break. No significant difference was observed for chromosomes irradiated either before or after isolation from intact mitotic cells. Irradiation in the presence of cystamine resulted in at least a sevenfold reduction in the apparent double-strand scission. The observed sedimentation patterns were compared with those generated by a theoretical computer simulation of radiation-induced degradation which assumed random selection and breakage of molecules. These results suggested that at least 80 to 90 % of the isolated DNA was distributed approximately normally with a mean molecular weight of about 200 X 10(6) daltons and a standard deviation of about 50 X 10(6) daltons.

Isolated mammalian metaphase chromosomes: I. General characteristics of nucleic acids and proteins

The metaphase chromosomes of tissue culture cells derived from four different mammalian species have been isolated in relatively large quantities at neutral pH and essentially free of nuclei and microscopically visible cytoplasmic debris. Some characteristics of the nucleic acids and proteins associated with these chromosome preparations are given below. The average chemical composition of the HeLa chromosome preparations is 16% DNA, 12% RNA and 72%, protein. Similar chemical compositions are reported for the chromosomes from Syrian and Chinese hamster cells and mouse L-cells. The DNA and RNA associated with the HeLa chromosomes have G + C contents of 41 and 63%, respectively. The DNA from the isolated metaphase chromosomes of the four cell types studied here exhibits the same density and very similar band profiles in CsCl density-gradient centrifugation as DNA extracted directly from the interphase nuclei of the respective cells. The satellite band of mouse L-cell DNA is present in chromosomal DNA in the same proportion as in DNA from interphase nuclei. More than 80% of the RNA associated with the HeLa chromosome preparations is 28 s and 16 s in the proportion of 2:1. At least 50% of the chromosomal protein is acid-soluble and shows an acrylamide gel electropherogram similar to that of acid-soluble proteins extracted from the nuclei of the respective cells.

The fine structure of isolated metaphase chromosomes

Chromosomes have been isolated directly from cells in metaphase for study under the electron microscope by spreading on a Langmuir trough, followed by critical point drying. The morphology of metaphase chromosomes obtained by this method, when compared with the chromosome-like threads isolated from cells by the Waring blender method, indicates that the latter are artifacts resulting from the distortion of interphase nuclei by the blender. The presence in the blender isolate of various stages in the conversion of interphase nuclei to chromosome-like elongated masses supports this interpretation. Because the blender threads show a fibrous fine structure similar to the basic 250–280 Å fibers making up interphase nuclei and metaphase chromosomes isolated by the Langmuir trough method, they are considered to be of nuclear origin, which would account for their staining characteristics.