Cellular senescence - The limited capacity of cells to divide beyond a finite number of population doublings (finite growth potential). Irreversible growth-arrest state that depends on the age or cell doublings of a cell.

Cellular immortalization - Cells are capable of indefinite proliferation (or unlimited lifespan). In long lived multicellular organisms, immortality may be thought of as an abnormal escape from cellular senescence.

End replication problem - The mechanisms of DNA replication in linear chromosomes is different for each of the two strands (called leading and lagging strands). Semiconservative DNA replication requires a labile short RNA primer to begin DNA polymerization in the 5' to 3' direction. After DNA polymerization, the RNA primers are degraded and replaced by DNA synthesized from an upstream primer. Because there is no DNA beyond the end of the chromosome to serve as a template for an RNA priming event, the gap between the final lagging strand segment (Okazaki fragment) and the end of the chromosome cannot be filled in. Thus the 5' end of the lagging strand will lose some nucleotides each time a cell replicates its DNA.

ITAS (Internal Telomerase Assay Standard) - Telomerase products visualized by gel electrophoresis are quantitated by Phosphorimager analysis by evaluating each lane of the gels separately. The lanes are separated into two peak areas for evaluation: one peak for the internal telomerase amplification standard or ITAS (40) or the 36bp short internal standard (for the TRAP-ezeTM kit) and the other peak for all telomerase products. A ratio of telomerase products to ITAS or the short internal standard is determined and subsequently plotted graphically using a logarithmic scale.

Telomeres - Repeated DNA sequences [(TTAGGG)n in humans] found at the ends of linear chromosomes that protect the ends of the chromosome from degradation. Telomere shortening has been suggested to be a "clock" that regulates how many times an individual cell can divide. At birth, as determined by terminal restriction fragment (TRF) analysis, telomeres consist of about 15,000 base pairs of repeated TTAGGG DNA sequences, which become shorter with each cell division owing to the end replication problem. Every time a cell divides it loses 25-200 DNA base pairs off the telomere ends. Once this pruning has occured about 100 times a cell senesces (or ages) and does not continue dividing.

TRFs (Terminal Restriction Fragments) - an estimate of telomere length is measured by digesting cellular DNA with restriction enzymes having 4-base recognition sites, so that most of the DNA is reduced to short fragments. Because telomeric repeats are non palindromic and lack restriction sites, they remain as relatively long Terminal Restriction Fragments (TRFs), which can be identified by probing with labeled telomeric oligonucleotides.

Telomerase - Telomere terminal transferase or telomerase is a ribonucleoprotein enzyme (composed of both RNA and proteins) that uses its internal RNA component (complementary to the telomeric single stranded overhang) as a template in order to synthesize telomeric DNA (TTAGGG)n, directly onto the ends of chromosomes. Telomerase is present in most fetal tissues, normal adult male germ cells, inflammatory cells, in proliferative cells of renewal tissues, and in most tumor cells. After adding six bases, the enzyme is thought to pause while it repositions (translocates) the template RNA for the synthesis of the next six base pair repeat. This extension of the 3' DNA template end in turn permits additional replication of the 5' end of the lagging strand, thus compensating for the end replication problem. Telomerase is often referred to as an immortalizing enzyme.

TRAP (Telomeric Repeat Amplification Protocol) - a highly sensitive, polymerase chain reaction-based assay for measuring telomerase activity. The TRAP assay includes an improved detergent lysis method to allow more uniform extraction of telomerase from a small number of cells and uses a single tube reaction in which telomerase first synthesizes extension products which then serve as templates for amplification. A commericially available research kit (TRAP-ezeTM) for the detection of telomerase is available (41).

 

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