Cloning of human telomeres by complementation in yeast

Telomeres confer stability on chromosomes by protecting them from degradation and recombination and by allowing complete replication of the end. They are genetically important as they define the ends of the linkage map. Telomeres of lower eukaryotes contain short repeats consisting of a G-rich and a C-rich strand, the G-rich strand running 5'-3' towards the telomere and extending at the end. Telomeres of human chromosomes share characteristics with those of lower eukaryotes including sequence similarity as detected by cross-hybridization. Telomeric repeats from many organisms can provide telomere function in yeast. Here we describe a modified yeast artificial chromosome (YAC) vector with only one telomere which we used to clone human telomeres by complementation in yeast. YACs containing human telomeres were identified by hydridization to an oligonucleotide of the trypanosome telomeric repeat. A subcloned human fragment from one such YAC is immediately subtelomeric on at least one human chromosome.     

Telomere reduction in human colorectal carcinoma and with ageing

We have hypothesized that end-to-end chromosome fusions observed in some tumours could play a part in genetic instability associated with tumorigenesis and that fusion may result from the loss of the long stretches of G-rich repeats found at the ends of all linear chromosomes. We therefore asked whether there is telomere loss or reduction in common tumours. Here we show that in most of the colorectal carcinomas that we analysed, there is a reduction in the length of telomere repeat arrays relative to the normal colonic mucosa from the same patient. We speculate on the consequences of this loss for tumorigenesis. We also show that the telomere arrays are much smaller in colonic mucosa and blood than in fetal tissue and sperm, and that there is a reduction in average telomere length with age in blood and colon mucosa. We propose that the telomerase is inactive in somatic tissues, and that telomere length is an indicator of the number of cell divisions that it has taken to form a particular tissue and possibly to generate tumours.     

Telomeric repeat from T. thermophila cross hybridizes with human telomeres

The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.