Telomere length analysis and in vitro telomerase assay

Barring exceptional instances, the DNA contained in eukaryotic chromosomes is linear. Linearity of the chromosomal DNA and the compartmentalized architecture of the eukaryotic cell are the two principle features that distinguish the prokaryotes from the eukary- otes and that have facilitated the evolution of totally different strat- egies for interaction with other species and the environment. Major changes in strategy that were made possible by linearity of chromo- somes were an enormous increase in the information content of the genome and the development of sexuality as a means for efficient exchange of genetic information. But linear chromosomes have ends, and the presence of ends produced two major biological prob- lems. One problem was identified early by Muller and by McClintock, as the requirement to protect natural ends of chro- mosomes from fusion and recombination with other chromosomes and from exonucleolytic erosion. It was thus recognized that the ends of chromosomes must have specialized structural and func- tional features required for chromosome stability. We know today that another potential cause of chromosomal instability that must be dealt with is that unless it is protected, a DNA end will be recog- nized as damaged DNA that the cell will attempt to heal with ensu- ing loss of chromosome integrity and cell viability. The second problem was identified more recently, when it became evident that all known DNA polymerases are unable to start synthesis de novo but can only add a nucleotide onto an already existing 3'-OH group, normally provided by an RNA primer. Such a primer positioned at the very end of the chromosomal DNA would be degraded, shorten- ing the lagging DNA strand. Both these sets of problems have found a solution through the development of a unique chromosomal organelle, the telomere, comprised of particular telomeric DNA sequences complexed with particular non-histone proteins, that are organized at least in part in a non-nucleosomal heterochromatic form of chromatin, defined as the telosome.