G banding

Abstract: A GIEMSA staining procedure that preferentially stains centromeric heterochromatin in mouse chromosomes has been described1. This specificity was observed when fixed preparations were treated with sodium hydroxide to denature the DNA, and then incubated in warm saline to allow annealing, in the presence of 3H-labelled single stranded satellite DNA or its complementary RNA. In this way mouse satellite DNA was located in the centromeric heterochromatin1,2. It is known to consist of highly repetitive sequences3 and to anneal much more rapidly than non-repetitive DNA4. It seems probable, therefore, that the darker staining with Giemsa of these regions, after denaturation and annealing, indicates the presence of highly repetitive DNA.

Abstract: Combining high resolution in situ hybridization with quantitative solid state imaging, we show that human metaphase chromosome Giemsa/Quinacrine and Reverse bands are each characterized by distinct families of interspersed repeated sequences: the SINES, Alu family dominates in Reverse bands, and the LINES, L1 family dominates in Giemsa/Quinacrine positive bands. Alu is 56% guanine plus cytosine, and L1 is 58% adenine plus thymine, and each may comprise 13%-18% of the total DNA in a chromosome band. Therefore, the distribution of these sequences alone may account for a large part of human chromosome banding seen with fluorescent dyes. With the exception of some telomeric regions, and the chromosomal regions of simple sequence DNA, Alu and L1 are precisely inversely distributed, suggesting an inverse functional relationship. This finding links genome organization with chromosome structure and function.

Abstract: 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.

Abstract: A significant improvement in fluorescence in situ hybridization, enabling the detection of single-copy genes as small as 500 bp directly on banded chromosomes, is presented. The induction of chromosome banding, which does not require additional handling or any system of amplification, is obtained simply by using an alkaline (pH 11) p-phenylenediamine anti-fade solution. As the banding produced is related to the timing of 5-bromodeoxyuridine incorporation, either R- or G-banding, constitutive heterochromatin staining, or chromosome asymmetry can be observed simultaneously with the fluorescent hybridized spots. Results of hybridization of small cDNA probes for the human genes for motilin, thymidylate synthetase, and lymphocyte activation-3 are provided as examples of the high-resolution mapping obtainable with this technique.