Analyze The Data

In culture, normal human cells undergo a finite number of cell divisions until they no longer proliferate and enter a state known as replicative senescence. The inability to maintain normal telomere length is thought to play an important role in this process. Telomerase is a ribonucleoprotein complex that regenerates the ends of telomeres lost during each round of DNA replication. Human telomerase consists of a template containing RNA subunit and a catalytic protein subunit known as human telomerase reverse transcriptase (hTERT). Most normal cells do not express telomerase; most cancer cells do express telomerase. Thus, telomerase is proposed to play a key role in the transformation of cells from a normal to a malignant state.

a. In the following experiments, the role of telomerase in the growth of human cancer cells was investigated (see W. C. Hahn et al., 1999, Nature Medicine 5:1164-1170). Immortal, telomerase-positive cells (cell A) and immortal, telomerase-

negative cells (cell B) were transfected with a plasmid expressing a wild-type or mutated hTERT. Telomerase activity in cell extracts was measured using the telomeric repeat amplification protocol (TRAP) assay, a PCR-based assay that measures the addition of telomere repeat units onto a DNA fragment. A six-base-pair ladder pattern is typically seen. Control indicates transfection of cells with just the plasmid. Wild type and mutant indicate transfection with a plasmid expressing a wild-type hTERT or the mutated hTERT, respectively. What do you conclude about the effect of the mutant hTERT on telomerase activity in the transfected cells? What type of mutation would this represent?

Cell A

Control Wild type Mutant

Cell B

Control Wild type Mutant

Cell A

Control Wild type Mutant

Cell B

Control Wild type Mutant

b. Telomere length in these transfected cells was examined by Southern blot analysis of total genomic DNA digested with a restriction enzyme and probed with a telomere-specific DNA sequence. What do you conclude about the lengths of the telomeres in cell A and cell B after transfec-tion with the wild-type or mutant hTERT? By what mechanism do cells A and B maintain their telomere lengths?

Cell A

Control Wild type Mutant

Cell B

Control Wild type Mutant

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