Biology Reference
In-Depth Information
constitutive expression of the catalytic component of human telomerase
(hTERT ) gene enabled cells to maintain telomere length and continue dividing
inde®nitely (Bodnar et al., 1998; Vaziri and Benchimol, 1998). The seemingly
immortal cells appear to be normal in every way, allaying fears that bypassing
replicative senescence might automatically lead to malignant transformation
(Jiang et al., 1999). Whether similar manipulations in T cells will lead to par-
allel outcomes is currently being investigated in several laboratories.
TELOMERE LENGTHS AND HIV DISEASE
Identi®cation of the speci®c telomere sequence led to the rapid development of
a variety of techniques to evaluate telomere length. Telomeres can be measured
by isolating total genomic DNA from a population of cells, incubating this
DNA with restriction enzymes that digest all but the telomeric and sub-
telomeric sequences, and perfoming Southern analysis (either gel or slot blot)
with radiolabeled telomeric probe (Bryant et al., 1997; Valenzuela and E¨ros,
2000a). The results of such analyses provide the mean terminal restriction
fragment ( TRF ) length of a cell population, which, in turn, re¯ects the com-
posite of the telomere lengths of all the chromosomes in each of the cells in the
starting population. Cell sorting techniques to enrich for particular cell sub-
populations ( E¨ros et al., 1996; Monteiro et al., 1996), analysis of TRF lengths
from sorted chromosomes (Martens et al., 1998), or probing speci®c chromo-
somes containing telomeres that have been seeded with plasmids (Murnane et
al., 1994) may, in some cases, increase the precision of the telomere assay. A
second strategy, developed by Landsorp and colleagues, uses ¯uorescence in
situ hybridization (FISH) technology on metaphase spreads or on interphase
nuclei, with ¯ow cytometric visualization and quantitation of the amount of
telomere sequences on individual chromosomes (Lansdorp et al., 1996). Flow
cytometric analysis of telomere size in concert with cell cycle analysis adds an
additional dimension to telomere studies ( Hultdin et al., 1998). Finally, tech-
niques currently under development that combine FISH telomere analysis with
¯ow cytometric cell surface phenotype or tetramer-binding will provide critical
information on speci®c subpopulations of cells. Telomere length assays have
contributed remarkable insights into a variety of facets of cell biology in both
normal and transformed cells. The speci®c telomere sequence also forms the
basis for assays to detect telomerase, using techniques involving telomere repeat
ampli®cation protocol (TRAP) ( Kim et al., 1994).
In the context of HIV disease, telomere length analysis has been used as
a tool to analyze the dynamics of lymphocyte homeostasis and its relation to
ultimate immune collapse that is fundamental to HIV disease pathogenesis.
Research in this area has provided extraordinary insights into both T and B
function in HIV-infected persons, as will be described below.
In a longitudinal analysis of peripheral blood mononuclear cell ( PBMC)
samples collected over a period of 9 years, Miedema and colleagues docu-
