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.     

Non random distribution of lesions induced by deoxyribonuclease I in human chromosomes

We studied the action of deoxyribonuclease I on human lymphocytes in order to determine the localization of the DNAase induced aberrations. Our results indicate a non-random distribution of the lesions on chromosome regions which may reflect a differential pattern of sensitivity to the enzyme. Furthermore we observed a correspondence between the preferential DNAase induced breaks and fragile sites that are expressed in lymphocytes maintained in medium without folic acid. A possible interpretation of our findings is that the accessibility to DNAase and/or the efficiency of the repair systems depend on the chromatin structure that influences also the expression of some common fragile sites.Abbreviations DNAase I deoxyribonuclease I E.C.3.1.4.5.     

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.     

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.

     

A rapid, simple and reliable combined method for G-banding mammalian and human chromosomes

A simple and reliable method for G-banding chromosomes from human and mammalian cells is described. This rapid method combines hot saline and trypsin treatments and yields high quality G-bands in both bone marrow and cultured cells.     

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.     

Cryptic variants of acrocentric human chromosomes as analysed by restriction endonucleases

Ten phenotypically normal human individuals have been analysed by in situ treatments with restriction endonucleases in order to obtain a better characterization of some cryptic variants of acrocentric chromosomes. Treatments with AluI, NdeII and Sau 3AI confirm the existence of two cryptic amplified regions on the short arms of both one chromosome 15 and one chromosome 22, in one female. These amplifications seem to be of different origin involving the nucleolar organizer region of chromosome 15 and the satellite of chromosome 22.     

Direct preparation and G-banding of chromosomes of mouse liver cells

A method has been developed for the direct preparation and G-banding of chromosomes of mouse liver cells by combining the techniques of liver perfusion and preparation of G-banded chromosomes with partial hepatectomy and colcemid treatment of the animal. The results indicate that cytogenetic investigations of isolated preneoplastic liver cells are possible. The method offers an increased possibility for the use of the liver as an in vivo test system for mutagens and carcinogens.     

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.     

Longitudinal patterns similar to G-banding in untreated human chromosomes: evidence from atomic force microscopy

The structure of human metaphase chromosomes, fixed according to standard procedures for optical microscopy but not treated for banding, was exammed by atomic force microscopy (AFM). The images show that chromosomes display a banding pattern very similar to G-banding, detected by the AFM as a variation in the thickness of chromatin. This similarity allows the identification of individual chromosomes.     

Distribution of spontaneous chromosome breaks in human chromosomes

Summary Localization of chromosome breaks in human chromosomes was analyzed in 264 peripheral lymphocyte cultures. Three hundred and sixty-nine chromosome breaks could be exactly localized to a chromosome band or region of the Paris Conference nomenclature. The distribution of breaks in the chromosome regions was found to be nonrandom. Chromosome 3 alone had 23% of the breaks and region 3p2 had 13% of the total breaks. Some other chromosome regions, such as 5p1, 9q1, 14q2, and 16q2 also displayed clustering of breaks. Sex chromosomes had less breaks than expected. Spontaneous chromosome breaks were almost exclusively located in the lightly stained G bands.Supported by grants from the Foundation for Pediatric Research and Research Foundation of Orion Corporation Ltd.     

Relationship between patterns of late S DNA synthesis and C- and G-banding in muntjac chromosomes

In pulse-labelled muntjac chromosomes, segmental labelling corresponding to the large blocks of G-bands was observed during late S; the termination sequence of DNA synthesis was light bands → C-bands → G-bands.     

Localization of chemically induced breaks in chromosomes of human leucocytes

The distribution of 16942 chromosome breaks induced by 3 chemical agents— N,N,N-triethylene thiophosphamide (thiosphosphamide, Thio-TEPA), dihydrochloride-1,6-di(choloroethyl)-amino-1,6-desoxymannitol (Degranol), and mitomycin-C (MC)—and their dependence on the moment at which the mutagen was introduced to the cultures, the dose of the mutagen, the time of fixation of the cultures and the sex and age of the donor, were statistically investigated.As control served the distribution of spontaneous chromosome breaks found in a group of 1649 newborns.The spontaneous breaks were randomly localized on the chromosomal groups, whereas the induced breaks showed a non-random distribution. It was demonstrated that the experimental conditions, which have been investigated, had no influence on the distributions of chromosome breaks within the groups. “Hot-spots” and “cold-spots” could be established along the length of the chromosomes. The localization of these spots did not depend on the experimental parameters investigated, but on the chemical agent by which they were induced.The possible cause of discrepancies between the present results and those reported by other authors are discussed.     

Changes in elemental composition of human chromosomes during a G-banding (ASG) and a C-banding (BSG) procedure.

Human chromosomes fixed in methanol-acetic acid have been examined by X-ray microanalysis, before, during and after a G-banding and a C-banding procedure. Phosphorus (representing mainly DNA), sulphur and calcium are the most prominent elements in untreated chromosomes. In the G-banding procedure, the calcium is lost during 2 x SSC treatment. In the C-banding procedure, calcium is lost in the preliminary HCl treatment. During the following barium hydroxide treatment a large amount of barium becomes attached to the chromosomes, but is lost again during the subsequent 2 x SSC treatment. In both banding techniques Giemsa staining produces large peaks for sulphur (thiazine dyes) and bromine (eosin), showing that both types of dyes are involved in the staining. Reduction in the phosphorus peak during these procedures may be partly due to extraction of DNA and other chromosomal components, but could also be due to absorption of phosphorus X-rays by heavy elements (barium and bromine).     

G-banding of germ line limited chromosomes in Acricotopus lucidus (Diptera,Chironomidae)

The germ line limited (K) chromosomes of Acricotopus lucidus (Diptera, Chironomidae) were stained for G-banding on gonial mitoses, along with the somatic (S) chromosomes. Nine different types of K chromosomes could be distinguished by the G-banding pattern and other cytological criteria. Various combinations of K chromosomes were found in the complements of different individuals and cells: some Ks were missing and others were present up to as many as five times. No two animals were completely alike in the composition of their gonial chromosome complement. Thus none of the different K types can be essential. These results are discussed in view of the complex chromosome cycle of the Orthocladiinae.