Biomedical subjects

I Szatmari

Publications and source records attributed to I Szatmari.

3 recordsLinked to original sources

PubMed2001

Telomeric repeat amplification, without shortening or lengthening of the telomerase products: a method to analyze the processivity of telomerase enzyme.

The Telomeric Repeat Amplification Protocol (TRAP) and its modified versions (including ours, TP-TRAP) change the size and/or the ratio of the telomerase products in the amplification stage of the assay. Based on our recently published method we developed a new TRAP. This method ensures that the number of telomeric repeats present in the original telomerase products does not change on PCR amplification. The usefulness of the method was proved with amplification of chemically synthesized telomerase products and a newly designed telomerase substrate oligonucleotide. This is the first report in which the PCR products directly reflect the size distribution of telomerase products generated by the enzyme.

Cell Line↗

PubMed2000

Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.

A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)].

Cell Extracts↗

PubMedDate not supplied

Activation of telomerase rna gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3.

Expression of the human telomerase RNA component gene, hTERC is essential for telomerase activity. The hTERC gene is expressed during embryogenesis and then downregulated during normal development, leaving most adult somatic cells devoid of hTERC expression. During oncogenesis, however, hTERC is re-expressed consequently contributing to the unrestricted proliferative capacity of many human cancers. Thus the identification of the molecular basis for the regulation of the telomerase RNA component gene in normal cells and its deregulation in cancer cells is of immediate interest. We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner. Whereas Sp1 and Sp3 act on the human, bovine, and mouse TERC promoters, pRb activates only the human and bovine promoter, and NF-Y is only essential for the human TERC gene.

Animals↗