^^ Chromatid

Metaphase chromosome

▲ FIGURE 10-27 Microscopic appearance of typical metaphase chromosome. Each chromosome has replicated and comprises two chromatids, each containing one of two identical DNA molecules. The centromere, where the chromatids are attached, is required for their separation late in mitosis. Special telomere sequences at the ends function in preventing chromosome shortening.

DNA is organized into 22 pairs of homologous autosomes and two physically separate sex chromosomes. In contrast, the other species contains only three pairs of autosomes; one sex chromosome is physically separate, but the other is joined to the end of one autosome.

During Metaphase, Chromosomes Can Be Distinguished by Banding Patterns and Chromosome Painting

Certain dyes selectively stain some regions of metaphase chromosomes more intensely than other regions, producing characteristic banding patterns that are specific for individual chromosomes. Although the molecular basis for the regularity of chromosomal bands remains unknown, they serve as useful visible landmarks along the length of each chromosome and can help to distinguish chromosomes of similar size and shape.

G bands are produced when metaphase chromosomes are subjected briefly to mild heat or proteolysis and then stained with Giemsa reagent, a permanent DNA dye (Figure 10-28). G bands correspond to large regions of the human genome that have an unusually low G+C content. Treatment of chromosomes with a hot alkaline solution before staining with Giemsa reagent produces R bands in a pattern that is approximately the reverse of the G-band pattern. The dis-

tinctiveness of these banding patterns permits cytologists to identify specific parts of a chromosome and to locate the sites of chromosomal breaks and translocations (Figure 10-29a). In addition, cloned DNA probes that have hybridized to specific sequences in the chromosomes can be located in particular bands.

A recently developed method for visualizing each of the human chromosomes in distinct, bright colors, called chromosome painting, greatly simplifies differentiating chromosomes of similar size and shape. This technique, a variation of fluorescence in situ hybridization, makes use of probes specific for sites scattered along the length of each chromosome. The probes are labeled with one of two dyes that fluoresce at different wavelengths. Probes specific for each chromosome are labeled with a predetermined fraction of each of the two dyes. After the probes are hybridized to chromosomes and the excess removed, the sample is observed with a fluorescent microscope in which a detector determines the fraction of each dye present at each fluorescing position in the microscopic field. This information is conveyed to a computer, and a special program assigns a false color image to each type of chromosome. A related technique called multicolor FISH can detect chromosomal translocations (Figure 10-29b). The much more detailed analysis possible with this technique permits detection of chromosomal translocations that banding analysis does not reveal.

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