No abstract

Individual pairs of human chromosomes can be reliably identified by a new method that does not require special optical equipment and that results in permanent preparations. This method, which is based on treatment of the chromosomes in situ with NaOH, followed by incubation in sodium chloride-trisodium citrate and Giemsa staining, results in highly specific banding patterns in characteristic regions of the chromosome arms. It should prove useful for the detection of small structural changes in chromosomes.During the past 15 years remarkable progress has been made in the descriptive morphology of both normal and abnormal human chromosomes, but with conventional cytological and autoradiographic techniques we have thus far failed to identify every chromosome in the human complement. Furthermore, these techniques have revealed very little differentiation within a chromosome arm, so that structural rearrangements that do not alter the length or centromere position, or produce only slight changes in these, will escape detection.Since the original description of specific fluorescent staining of human chromosomes (1), investigations have led to renewed hope for subdividing the classical chromosome groups into individual pairs. This paper describes a new method of differentiating human chromosomes that does not require fluorescence microscopy and that results in permanent preparations. The chromosomes exhibit banding patterns in specific regions. In addition to the sex chromosomes, 20 of the 22 autosomal pairs have been identified by this technique, and the remaining two are tentatively classified. MATERIALS AND METHODSLymphocyte and fibroblast cultures from normal persons were prepared and harvested in the usual manner. Colchicine, 0.04 /Ag/ml of medium, was added to the cultures 2 and 6 hr before harvest. After hypotonic treatment with 1% sodium citrate and fixation in methanol-acetic acid 3:1, flame-dried slides were prepared. The method to be described consists of treatment of the chromosomes in situ with NaOH, followed by incubation in several concentrations of a saline-citrate solution (SSC). Specific regions of the chromosome arms then stain differentially with buffered Giemsa. Because a number of parameters have varied in our pilot experiments and we do not yet know the optimal conditions for treatment, we will present first a basic procedure that we have found to produce bands fairly consistently, and then a range of times and concentrations that have been tested.The slides are treated for 30 sec in a solution of 0.07 N NaOH in 0.112 M NaCl (pH 12.0) at room temperature and then rinsed three times in 12 X SSC (pH 7.0) for 5-10 min each time. They are then incubated in 12X SSC at 650C for 60-72 hr. After incubation, the slides are passed through three changes of 70% ethanol and three changes of 95% ethanol (3 min each). After air-drying, slides are stained for 5 min in buffered Giemsa solution (pH 6.6), rinsed briefly in distilled water, air-dried again, and mounted in Permount.The alkaline solution is pr...

No abstract

Lymphocytes from 20 notmal subjects (11 male and 9 female) were examined for the frequency and location of sister chromatid exchanges (SCE) by the BrdU--Giemsa method. The mean frequency of SCE was 6.37 with little significant variation. One subject had a high number of exchanges in chromosome 1 while the remainder showed a random distribution of exchanges between chromosomes. The frequency of exchanges generally increased with chromosome length. However, chromosome 1, 2 and the B group had more exchanges than expected while the E, F and G grous had less than expected. The distribution of exchanges in chromosomes 1, 2 and the B group was non-random with a concentration of exchanges below the centromere and to a lesser extent on the distal portion of the long arm. The majority of exchanges appeared to occur at the junction between the dark and light G bands. It is suggested that the concentration of exchanges may reflect differences in BrdU incorporation along the length of the chromosome.

No abstract

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.

No abstract

No abstract