Induced Telomerase Loss And Behavior Of The Resulting Culture

Two ways to lose telomerase Telomerase loss is typically induced by deleting a gene for an essential telomerase component (usually the catalytic

TERT subunit or the TER template RNA) and then maintaining that strain by providing this gene in trans. One common method is to construct a diploid yeast strain and then delete one copy of the gene using standard techniques. The diploid is phenotypically wild type because one active copy of the gene remains in the diploid cell. The diploid yeast can then be sporulated to produce two haploid wild-type spores and two haploid spores bearing the gene deletion (e.g., Lendvay et al., 1996). Because the gene deletion is accomplished by replacing the gene with a selectable marker, the deletion spores can be identified on medium requiring the selectable marker for growth. These haploid spores are born with wild-type length telomeres that gradually shorten over repeated mitotic divisions.

A second method is to delete the gene for the telo-merase component in a haploid cell, identify the correct transformant, and introduce a normal copy of the gene on a plasmid that replicates as an episome in the cell. Episomal yeast plasmids are lost at rates that vary from 1 to 50% per cell division even when grown under selection (Murray and Szostak, 1983). Since most yeast selectable markers complement auxotrophic markers, cells that have lost the plasmid continue to divide until the gene product is diluted away or degraded and then arrested due to starvation for the auxotrophy. The fraction of plasmidfree cells in a selectively grown culture can vary from 10 to 70%, depending on the type of plasmid used. These cells can grow again if plated on medium that supplements the auxotrophic requirement (Newlon, 1988). Thus, plating cells from a selectively grown culture onto complete medium and testing portions of the resulting colonies for the presence of the plasmid's selectable marker allows one to identify the colony that lacks the gene for the telomerase component. The original colony can then be grown and tested in the same way as the haploid spore generated in the method described earlier. The disadvantage of this method is that the initial transformant lacks telomerase activity, and some mutant combinations with lack of telomerase cause rapid death (e.g., haploid spores bearing deletions of a telomerase component gene and the YKU70 gene die very rapidly (Nugent et al., 1998)). While this disadvantage can be overcome using more complex yeast methods to disrupt the chromosomal copy of the gene in a cell that already bears a plasmid-borne copy of the same gene, it is probably easier for the beginner to use the diploid method described earlier.

Observing senescence in yeast Two common methods are used for different purposes in yeast. Serial streaking of individual colonies is a method used to demonstrate that senescence occurs, while serial dilution and regrowth of small cultures are used to show that the culture senesces and to isolate survivor/ALT cells whose rapid growth allows these cells to quickly overtake the slow growing cells with very short telomere tracts.

Serial streaking is streaking a colony of yeast on a plate to isolate single cells and to allow them to grow into colonies. A freshly grown colony is then restreaked on a fresh plate. A yeast colony ~2 mm in diameter contains 3 x 105 to 106 cells. If one approximates yeast cell growth as cell number = 2N where N is the number of cell doublings, then each yeast colony is 18-20 population doublings. Cells that have just lost a gene for a telomerase component lose 3-5 bp per cell doubling, and so give colonies for the first 3 streaks, and then show slower growth on the fourth streak (Lundblad and Szostak, 1989; Singer and Gottschling 1994). Not all cells senesce at the same time, which explains why some cells continue to grow on the fourth streak. This difference may reflect the fact that telomere tract length is not clonal and telomeres do not shorten at the same rate (e.g., Li and Lustig, 1996), and some small amount of lengthening may occur in these cells by nonreciprocal gene conversion mechanisms (Pluta and Zakian, 1989). Some cells in the colony may therefore have different telomere lengths than other cells, and so senesce after different numbers of cell divisions. Because each colony is a single cell that has grown into ~106 cells, there has only been ~106 cell divisions in which mutation(s) can occur to give rise to a survivor/ALT cell that can elongate its telomeres in the absence of active telomerase. Survivors can be readily identified on the fourth restreak because they grow much faster than the cells that are senescing, and form larger colonies (Teng and Zakian, 1999).

Serial dilutions of liquid cultures involve growing cells in a liquid culture, often 10 ml or so, for 24 hours. Usually, yeast grow to densities of ~108 cells/ml in the first growth phase without telomerase, in the same way wild-type cells do. Cells are then diluted to a constant number (from 104 cells/ml to 5 x 105 cells/ml, depending on the lab) in fresh medium and grown for another 24 hours for the second cycle (Le et al., 1999; Teng and Zakian, 1999). Each subsequent 24-hr cycle involves diluting cells down to 5 x 105 cells/ml and allowing regrowth; however, cells do not grow to saturation by the 4th or 5th day. Instead, they begin to show slower rates of growth as the cells in the population begin to senesce, and the number of cells/ml that grow up in 24 hours steadily decreases on days 4 to 6. By the 7th day, cells begin to grow rapidly again as survivor/ALT cells take over the culture. It should be noted that this serial dilution method selects for faster growing mutants compared to the serial restreaking method because while serial restreaking restarts from a single cell each time, serial dilution restarts from 5 106 cells in a culture. Thus, any mutants that arose in an earlier dilution culture can be transferred to the new culture and undergo further selection for rapid growth. If the goal of the experiment is to isolate fast-growing survivors, the serial dilution method is superior. If the goal is to determine if the cells senesce, it is probably faster to use the serial restreaking method because more individual strains can be easily and rapidly assayed in parallel. The serial restreaking method may also allow one to isolate suppressors that grow at different rates (Teng and Zakian, 1999).

The serial dilution method has been frequently used to monitor the requirements for cell senescence and the two types of survivor/ALT cells that form in yeast cells lacking active telomerase. By starting with cells that lack active telomerase and one DNA damage checkpoint gene, it has been shown that a subset of checkpoint genes are required for the yeast G2/M cell cycle arrest caused by short telomeres, namely, MEC1, DDC2, MEC3 and RAD24 (Enomoto et al., 2002; IJpma and Greider, 2003). Similar experiments have used cells lacking active telomerase and one or more recombination proteins to determine the requirements for forming Type I survivors and Type II survivors (Chen et al., 2001).

Monitoring telomere structure during cell senescence The slow loss of telomere repeats in yeast cells lacking active telomerase, about 3-5 bp per generation (Lundblad and Szostak, 1989), allows the experimenter to isolate large numbers of cells at different population doublings for analysis. Examining the length of the terminal TG1-3 repeat tracts is necessary to ensure that the cells are behaving as predicted. Two methods are routinely used: Southern blotting and Telomere PCR. Once telomere shortening has been verified, the chromatin components of telomeres can by monitored by chromatin immuno-precipitation at different time points during the telomere shortening.

Standard Southern blotting of Xho I cut yeast DNA using TG1-3 or TG sequences as probes has long been used to monitor telomere length. Most lab yeast strains have two types of subtelomeric middle repetitive elements, X and Y' (reviewed by Louis 1995) (Figure 17.3).

Y' is a highly conserved family of elements that contain an Xho I site 0.83-0.86 kb from the beginning of the TG1-3 repeats. As more than half of the telomeres in the common lab yeast strains have Y' elements adjacent to the TG1-3 repeats, the 1.1-1.3 Y' telomere restriction fragment provides an indicator of the behavior of many telomeres at once. In contrast, the X family of elements is less well conserved, and the Xho I restriction site is present at varying distances from the beginning of the TG1-3 repeat tract. Thus, the Xho I digest provides information on individual telomeres, which run as bands in the 2-4 kb range in many strains. When telomerase activity is absent, all of these bands shorten. When Type I survivors containing many tandem Y' elements take over the culture, all of the X telomere bands disappear. When Type II survivors with heterogeneous lengths of TG1-3 arise, the telomere band pattern becomes highly variable (see Lundblad and Blackburn (1993); Teng and Zakian (1999)).

Telomere PCR is a more recent technique that allows one to monitor the length and sequence of the TG1-3 tract at the end of an individual telomere (Forstemann

telomeres

Xho I site

Xho I site

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