Chromosomal PRINS DNA Labeling Combined with Indirect Immunocytochemistry

The technique of in situ hybridization developed initially by Pardue and Gall (1 ) and Jones (2 ) can be placed on a par with Southern (3 ) hybridization in the enormous contribution it has given to the fields of cellular and molecular biology in eukaryotes. Initially developed for repetitive DNAs in mammalian cells, it is now used to assign the chromosomal loci of all kinetic classes of DNA molecules throughout the animal and plant kingdoms. PRINS hybridization developed by Koch et al. (4 ) was a clever improvisation using oligonucleotide primers to anneal to chromosomal DNA sequences followed by extension using DNA polymerase. A reporter molecule (a digoxigenin or biotinylated deoxynucleotide triphosphate) was incorporated during the reaction. The reporter molecule was then detected using an immunocytochemical approach. Improvements to the basic PRINS technique involve direct incorporation of fluorochrome-tagged deoxynucleotide triphosphates, negating the need for secondary labeling and, the “cycling PRINS” (5 ) reactions. Here, multi-denaturation, annealing, and extension steps are carried out giving greater sensitivity and signal strength, especially when dealing with low-copy chromosomal repeat sequences. Chromosomes, however, do not contain only DNA molecules. More than half of the bulk of a chromosome consists of protein molecules, which themselves can be separated into two classes: the histone and the nonhistone proteins. The interactions of proteins from these two classes with chromosomal DNA lead to the formation of a visible chromosome when eukaryotic cells undergo mitotic and meiotic cell divisions. The precise mechanism underlying this process is not well understood. This is particularly the case for the nonhistone proteins. Some nonhistone proteins (like the histones, which interact with DNA sequences to form the basic nucleosomal structure) must also bind directly to specific nucleotide sequences along the backbone of the DNA molecule. One example consists of the scaffold proteins (6 ). These are nonhistones that are thought to be responsible for attaching specific DNA sequences to the chromosome scaffold. From the scaffold attachment sites, it is thought that loops of DNA, 100–200 kb in length, protrude into the surrounding matrix of the cell. The functional significance of these loops remains unclear, but it is thought that this is one of the intermediary higher-order structures necessary for the formation of a condensed chromosome. Nonhistone proteins are also thought to be integral parts of the kinetochore of mammalian chromosomes (7 ). This specialized structure is found at the primary constriction of mammalian chromosomes and is the site of attachment of spindle microtubules during cell division. An understanding of the proteins associated with this structure has come from the use of autoantibodies from some CREST patients who have a complex scleroderma syndrome (8 ).