A method for analysis of chromosomes in hybrid cells employing sequential G-banding and mouse specific C-banding

A sequential banding technique is described for the identification of chromosomes of interspecific hybrid cells with a mouse parent. Metaphases were first G-banded using trypsin-Giemsa to identify individual chromosomes and then the centromeres of the same cells were differentially stained by a C-banding technique specific for mouse chromosomes. This mouse specific C-banding employs treatment with hot formamide-SSC before staining, and the effect of this treatment on the staining of chromosomes from a number of species was investigated. The specific staining of mouse centromeres confirms the parental origin of chromosomes identified by G-banding and allows the rapid recognition of mouse and non-mouse chromosomes in metaphases from many different hybrid combinations.     

G-banding of human sperm chromosomes

Summary G-banded human sperm chromosomes are routinely obtained in our laboratory using a modification of the method described by Martin et al. (1982). The study of banded sperm chromosomes is essential for the genetic counseling of male carriers of balanced chromosome rearrangements.     

The mechanism of C- and G-banding of chromosomes

A series of biochemical, staining and electron microscopy techniques were utilized to investigate the mechanisms of C- and G-banding. These led to the following conclusions.

1. 1. The treatment of fixed chromosomes with 0.07 N NaOH for 30 to 180 sec removes from 16 to 81% of the DNA from the chromosomes.

2. 2. On average, the complete C-band technique removes 60% of the DNA.

3. 3. This DNA is preferentially extracted from the non-C-band regions.

4. 4. In marked contrast to this, all G-band techniques (except 1) removed less than 9% of the chromosomal DNA.

5. 5. Most of the G-band techniques, including those using trypsin, remove very little protein from the chromosomes.

6. 6. Feulgen staining indicated that neither C- nor G-banding can be explained on the basis of different amounts of DNA along the length of the chromatid.

7. 7. Treatment of chromosomes with alkali or prolonged treatment with trypsin tends to destroy G-bands, while C-bands remain.

8. 8. The combined use of acridine orange and Giemsa staining indicate that, (a) repetitious DNA in situ renatures in seconds while non-repetitious DNA renatures in minutes; (b) Neither C- nor G-banding depends upon the differential renaturation of DNA for its effect.

9. 9. G-banding is more delicate and relatively mild conditions allow staining of both C- and G-bands. To obtain only C-bands the chromosome must be treated more harshly to disrupt or destroy the G-bands.

10. 10. DNA-non histone protein interactions probably play an important role in the production of both C- and G-banding.

     

Identification of flow-sorted chromosomes by G-banding and in situ hybridization

The identification of flow-sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G-banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest. In particular when sorting chromosomes from human-rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin-labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.     

Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine

Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: initially the BrdU is incorporated during the first part of the S-phase and then it blocks the cells at mid-S-phase. Within the cell cycle, mid-S-phase appears to be the most vulnerable time to various blocking agents. To differentiate the regions of BrdU incorporation from those that have not been substituted, the fluorescence-photolysis-Giemsa (FPG) technique was applied as modified for horse chromosomes. This dynamic technique, which produces many prometaphase and prophase chromosomes showing very sharp G-bands, is certain to enhance the accuracy of cytogenetic analysis and aid in the standardization of equine chromosomes.     

High resolution bands in maize chromosomes by G-banding methods

Summary It was demonstrated that G-bands are unequivocally present in plant chromosomes, in contrast to what had been formerly believed by plant cytologists. Maize chromosomes prepared by an enzymatic maceration method and treated with trypsin or SDS showed clear G-bands spreading along the chromosomes. The most critical point during the G-banding procedures was the post-fixation with glutaraldehyde solution. Banding patterns were processed by using the chromosome image analyzing system and a clearer image was obtained. Gbanding technique and the image manipulation method described here can be applied to many plant species, and would contribute new information in the field of plant cytology and genetics.     

Mapping of sister-chromatid exchanges in human chromosomes using G-banding and autoradiography.

Lumphocytes were pulse-labelled with [3H] thymidine. Following G-banding, the cells were autoradiographed and 46 in their third post-labelling division selected. The locations of 611 sister-chromatid exchanges (SCE's) which had occurred in the previous two cell cycles were recorded as label discontinuities along identified chromosomes. Between particular chromosomes, SCE frequency was proportional to chromosome length. SCE frequency distributions within particular chromosomes fitted Poisson expectations. There was no over-representation of exchanges in centromeric regions, or in the C-banded regions of chromosomes 1, 9 and 16. A trend of increased frequency of SCE in darkly G-banded regions and in relatively darkly banded chromosomes was evident. The apparent excess of SCE in dark G-bands could be considered to be a consequence of the more condensed state of the DNA in these regions in the interphase nucleus relative to the DNA in pale G-band regions. Such compaction could result in an enhanced probability of SCE and a reduced probability of gross inter- or intra-change involving these regions. In contrast, the more extended interphase state of the DNA in pale G-banded regions would allow non-homologous exchange and account for the preferred location of X-ray-induced exchange events to pale G-bands.     

High resolution R- and G-banding on the same preparation

Summary A simple method using bromodeoxyuridine (BrdU), for both cell synchronization and incorporation into replicating DNA is described. Many prophasic and prometaphasic mitoses were observed, and due to the probable blocking at different times of the cell cycle, very good R-banding and G-banding were obtained simultaneously on the same preparation.     

Polytene chromosomes in mouse trophoblast giant cells

Mouse trophoblast giant cells undergo successive rounds of DNA replication resulting in amplification of the genome. It has been difficult to determine whether giant cell chromosomes are polyploid as in liver cells or polytene as in Dipteran salivary glands because the chromosomes do not condense. We have examined the pattern of hybridization of mouse giant cells with a variety of in situ chromosome markers to address this question. Hemizygous markers displayed one hybridization signal per nucleus in both diploid and giant cells, while homozygous markers displayed two signals per nucleus in both cell types. These patterns are consistent with cytological evidence indicating that giant cell chromosomes are polytene rather than polyploid. However, in contrast to the situation in Dipteran salivary glands, the two homologues do not appear to be closely associated. We conclude that the mechanism of giant cell DNA amplification involves multiple rounds of DNA replication in the absence of both karyokinesis and cytokinesis, and that sister chromatids, but not homologous chromosomes, remain closely associated during this process.     

Influence of Q- and G-banding on the Feflgen-stainability of human metaphase chromosomes

Synopsis In this paper, model experiments on chicken red blood cell nuclei are described concerning the influence of methanol-acetic acid fixation and irradiation at different wavelengths, with and without prior Atebrin staining on subsequent Feulgen-stainability. In addition, data are reported on the influence on Feulgenstainability of Giemsa-banding procedures, illumination of unstained chromosomes at 220 and 515 nm and exposure of unstained and Atebrin-stained chromosomes to illumination at 440 nm.The ASG and especially the trypsin-Giemsa technique appeared to reduce markedly Feulgen-stainability. The same holds true for Atebrin fluorescence of chromosomes. The data are discussed in relation to their implications for the assumed cause of the Q- and G-banding phenomena. Techniques are described that allow reliable Feulgen DNA measurements of individual chromosomes after application of either G- or Q-banding.     

Differential decondensation of mitotic chromosomes during the hypotonic treatment of cells as a possible cause of G-banding

Chinese hamster metaphase chromosomes were studied after treating the cells with a hypotonic 0.075 M KCl solution, routinely used for differential staining of chromosomes. After the incubation of cells in KCl for 5 seconds-40 minutes and fixation with glutaraldehyde, the chromosomes displayed DNP fibrils about 20 nm in diameter. These fibrils were unevenly packed along the chromosomal arms and formed sites of differential density. In sister chromatids, the even density sites were located symmetrically. The use of serial ultrathin sections of marker chromosomes (e.g., chromosomes with a telomeric disposition of nucleolar organizing regions) made it possible to establish a good correlation between the sites with the light packing of DNP-fibrils and G-bands, identified in the same chromosomes by the standard Giemsa-staining technique. Fixation of the KCl-incubated cells with the methanol: glacial acetic acid (3:1) solution did not change the DNP packing heterogeneity. The chromosomal banding state is reversible, for with the transfer of cells from KCl to the normal cultural medium the chromosomes become homogeneous in length. Thus, the data obtained allow to propose that the capacity of chromosomes for differential staining may be based on an uneven sensitivity of G- and R-bands to the decondensing effect of hypotonic treatment.     

Chinese hamster X mouse hybrid cells segregating mouse chromosomes and isozymes.

Hybrid cells are readily formed by fusing clonal Chinese hamster cells to fresh, noncultured, adult mouse spleen cells followed by isolation in selective medium. The vast majority of such hybrids retain Chinese hamster chromosomes and isozymes while segregating mouse chromosomes and isozymes. The growth, plating efficiency, ease of karyology, and rapid segregation of mouse markers allows linkage tests in primary clones. Analysis of 13 isozymes showed 12 to be asyntenic and on epair (PGD-PGM2) to be syntenic This system will allow extensive somatic cell hybrid gene mapping in the mouse and permit a comparison of human and mouse linkage relationships.