To understand what chromosomal bands represent, it is helpful to understand the structure of chromosomes. Eukaryotic chromosomes are composed of chromatin, a combination of nuclear DNA and proteins. At metaphase, which is a phase in the cell cycle after the DNA in the nucleus has been replicated, each chromosome contains two identical strands of DNA. (Each strand contains two complementary strands of nucleotides.) The two strands of DNA, or chromatids, are arranged in a double-helix and are held together at a single point, the centromere, or primary constriction point.
During mitosis, each chromatid becomes condensed approximately ten-thousand fold reaching maximal condensation at metaphase. DNA that was roughly 5 centimeters (2 inches) long is compacted to 5 micrometers. The DNA wraps around proteins called histones, forming complexes called nucleosomes. The nucleosomes twist around each other and assume a loop formation projecting out from the chromosome's protein backbone, or scaffold. The loops weave and condense further to package the DNA into a chromosome. Some of the looped segments of DNA remain close together and condense more than others, forming regions known as domains. These domains are the darkly-stained chromosomal bands that appear when specific stains are applied (such as Giemsa staining; see below).
Looped domains are also seen in polytene chromosomes, which are found mainly in insects of the order Diptera, including Drosophila, which are fruit flies. Polytene chromosomes are large chromosomes that are formed after DNA undergoes repeated rounds of replication without cell division. A polytene chromosome in a Drosophila salivary gland cell can contain as many as five thousand alternating dark and light bands. The dark bands correspond to the folded and looped DNA, and the lighter bands are composed of less condensed DNA. The DNA in polytene chromosomes becomes less condensed when genes become active, permitting DNA to be transcribed into messenger RNA. This unraveling is observed as "puffing" of the polytene chromosome. The puffing resolves (the DNA condenses again) as the genes become inactive.