Conservation of the Homeobox
In the early 1980s Drosophila geneticists started sequencing the DNA from the homeotic genes. Much to their surprise they found that all the homeotic genes contained a 180-basepair region. This region, named the homeobox after the genes in which it was first found, encodes for a sixty-amino-acid sequence that is very well conserved among the homeotic genes. Homeobox refers to the sequence of DNA; the amino acid sequence it encodes is called a homeodomain. Sequences at the homeobox usually differ by ten percent or less between pairs of homeotic genes in Drosophila. Homeoboxes are not restricted to homeotic genes and have been found in several other classes of developmentally important genes. The amino acids encoded by the homeobox region contain a motif called a helix-turn-loop, which is associated with binding to DNA sequences. Thus, a gene with a homeobox would be a prime candidate for a gene that encodes a transcription factor.
More surprising than the discovery of homeoboxes themselves was their ubiquity. Soon after homeoboxes were found in Drosophila, William McGinnis and his colleagues went on a "fishing" expedition looking for homeotic genes. They looked in a variety of organisms using a method called "zoo blotting," a modified type of Southern blot. (See the Genetically Modified Organisms unit.) The process consisted of using gel electrophoresis to separate the DNA by size from each species they were interested in. The DNA was then heated to separate it into single-stranded DNA (ssDNA). Next the ssDNA was blotted and trapped on nitrocellulose filter paper. The researchers then added single-stranded homeobox DNA, which had been labeled with a radioactive isotope, to the filter paper. If the ssDNA on the blot was sufficiently similar to the labeled homeobox ssDNA, the two ssDNAs would hybridize on the filter paper. The filter paper would be radioactive wherever there was hybridization. To their surprise, McGinnis's group found homeobox sequences everywhere - in insects, crustaceans, vertebrates (including humans and mice), echinoderms, and mollusks. Almost all multicellular animals had genes with homeoboxes. Moreover, they all expressed these genes during development, often in very similar ways.
Most invertebrates have a single cluster of homeotic genes. In Drosophila that cluster is broken in two. Vertebrates have four copies of the cluster, strongly suggesting that the cluster had been duplicated twice in vertebrates.
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