Strand Extension Research Articles

Förster resonance energy transfer as a probe of membrane protein folding

The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.

56 ESTABLISHMENT OF TRANSGENIC RED FLUORESCENCE PROTEIN (RFP) CLONE DOGS THROUGH A STABLE TRANSMISSION OF RFP GENE TO NEXT GENERATION

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.

Protocorm-like Bodies Initiation from Root Tips of Cyrtopodium paranaense (Orchidaceae)

Cyrtopodium paranaense is a tropical terrestrial orchid, which propagates mainly through sexual seed germination. In this study, we document the asexual morphogenesis of the root tip to protocorm-like body (PLB) conversion in Cyrtopodium paranaense. Protocorm-like bodies sporadically developed from root tips of flask-grown seedlings in the absence of any exogenous plant growth regulators (PGRs). The compact PLBs ultimately gave rise to normal plantlets. Histological observations revealed that the root cap became dissociated from the root apex at an early stage followed by dispersed extension of root vascular strands into nascent PLBs. Protocorm-like bodies also developed from the root central stele tissue. In root tip segment cultures, PLBs were not formed without providing PGRs but were efficiently formed from root tips in Murashige and Skoog (MS) medium supplemented with 10.2 μM indole-3-acetic acid (IAA) and 9.0 μM thidiazuron (TDZ). Both IAA and TDZ promoted the formation of PLBs; however, TDZ did not induce PLB formation in the absence of IAA, indicating a synergistic effect of the two PGRs. Protocorm-like bodies were proliferated and subsequently plants regenerated in PGR-free MS medium. Root tip culture may be used as an alternative method for the propagation of Cyrtopodium paranaense.

Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Combining electrochemistry and direct force measurements: from the control of surface properties towards applications

Direct force measurements contributed in the last years much to our understanding of the diffuse double layer of charged interfaces in electrolyte solutions. Such measurements have been performed with the atomic force microscope or the surface force apparatus. This review gives an overview over the recent studies based on force measurements with electrode surfaces. Not only bare metal electrodes but also electrodes modified by different organic layers, including electroactive films, have been studied by these techniques. Direct force measurements indicate that further effects besides classical Gouy–Chapman–Stern theory have to be taken into consideration in order to describe the force profiles. In addition to the long-range forces also the adhesion between surfaces can be tuned by potentiostatic control. New single-molecule techniques based on the atomic force microscope allow to probe the extension of polymer strands or their desorption from solid interfaces. In combination with electrochemistry, it became now possible to tune the desorption behavior of polymer strands or to measure the electromechanical coupling of motors from single strands of electroactive polymers.

Functions of Alternative Replication Protein A in Initiation and Elongation

Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair, and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative replication protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to those of the canonical RPA complex but does not support DNA replication. We describe studies that aimed to identify what properties of aRPA prevent it from functioning in DNA replication. We show aRPA has weakened interaction with DNA polymerase alpha (pol alpha) and that aRPA is not able to efficiently stimulate DNA synthesis by pol alpha on aRPA-coated DNA. Additionally, we show that aRPA is unable to support de novo priming by pol alpha. Because pol alpha activity is essential for both initiation and Okazaki strand synthesis, we conclude that the inability of aRPA to support pol alpha loading causes aRPA to be defective in DNA replication. We also show that aRPA stimulates synthesis by DNA polymerase alpha in the presence of PCNA and RFC. This indicates that aRPA can support extension of DNA strands by DNA polymerase partial differential. This finding along with the previous observation that aRPA supports early steps of nucleotide excision repair and recombination indicates that aRPA can support DNA repair synthesis that requires polymerase delta, PCNA, and RFC and support a role for aRPA in DNA repair.

Efficiency and specificity of microRNA-primed nucleotide analog incorporation by various DNA polymerases

MicroRNAs (miRNAs) as endogenous regulators of gene expression have spurred a surge of interest for their quantification and expression analysis. High-sensitivity and high-specificity miRNA detection techniques, such as real-time polymerase chain reaction and recently introduced bioluminescent miRNA detection, require systematic study of DNA polymerases for use with miRNAs. In this study, a variety of DNA polymerases were studied to assess their capabilities of using miRNA as a primer and incorporating 2′-deoxyadenosine-5′- O-(1-thiotriphosphate) as a dATP alternative during DNA strand extension. Five DNA polymerases were investigated: mesophilic DNA polymerase I large (Klenow) fragment, 3′ → 5′ exo − Klenow DNA polymerase, thermophilic Bst DNA polymerase large fragment, Therminator DNA polymerase, and Taq DNA polymerase. The experimental results show that, except for Taq DNA polymerase, the polymerases can use miRNA as a primer and have both common and divergent properties of the nucleotide analog incorporation and miRNA discrimination. DNA polymerase I large (Klenow) fragment showed no detectable polymerization product with the thio-modified dATP as a substrate. Thermophilic Bst DNA polymerase had the highest specificity for miRNA recognition on a DNA template. The study provides a novel method for miRNA detection without reverse transcription to complementary DNA that is faster, simpler, and less prone to biases and errors.

Bioluminescent assay for quantitation microRNAs.

A novel bioluminescent enzymatic assay for high‐throughput detection and quantification of microRNA has been developed. The assay is based on the time‐resolved luminescence detection of inorganic pyrophosphate (PPi) released during microRNA initiated polymerase reaction, which utilizes a specially designed long oligonucleotide template with complementary sequence to the microRNA target of interest. The subsequent primer strand extension gives rise to the nucleotide incorporation and the generation of PPi molecules with the amount proportional to the number of miRNA target molecules in sample. The assay does not need the thermal cycling of PCR reaction. Compared to the current RT‐PCR, microarray, and hybridization‐based microRNA detection methods, this assay provides a simpler, faster, and less expensive platform to be used in life science research, drug discovery, and clinical diagnosis.

Time‐lapse imaging of Arabidopsis leaf development shows dynamic patterns of procambium formation

The principles underlying the formation of leaf veins have long intrigued developmental biologists. In leaves, networks of vascular precursor procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Understanding dynamics of procambium formation has been hampered by the difficulty of observing the process in vivo. Here we present a live-imaging technique that allows visual access to complex events occurring in developing leaves. We combined this method with stage-specific fluorescent markers in Arabidopsis (Arabidopsis thaliana) to visualize preprocambial strand formation and procambium differentiation during the undisturbed course of development and upon defined perturbations of vein ontogeny. Under all experimental conditions, we observed extension, termination and fusion of preprocambial strands and simultaneous initiation of procambium differentiation along entire individual veins. Our findings strongly suggest that progressiveness of preprocambial strand formation and simultaneity of procambium differentiation represent inherent properties of the mechanism underlying vein formation.

Characterization of nucleoprotein complexes in plants with human-type telomere motifs

A conserved feature of telomeres is the 3′-overhang of their G-rich strand. These G-overhangs function as substrates for telomerase-mediated strand extension, and are critical for end-protection of telomeres. These functions and their regulations are mediated by specific G-overhang binding proteins. In species of the plant order Asparagales, telomere motifs have diverged from a type typical of the plant Arabidopsis thaliana (TTTAGGG) n to a type typical of human (TTAGGG) n . Presumably, this change in motif had an impact on the structure of the telomere and/or the binding of telomeric proteins, including the G-overhang binding proteins. Therefore, we analyse here nucleoprotein complexes formed by protein extracts from plants possessing human-type telomeres ( Muscari armeniacum and Scilla peruviana). Proteins were characterized that bind to the G-rich strand of both telomere motifs, or to the ancestral Arabidopsis-type motif alone, but none bound to double-stranded or C-rich complementary strand telomere motifs. We demonstrate the size, sequence-specificity and thermostability of these DNA-binding proteins. We also analysed the formation of complexes from renatured protein fractions after SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide-gel-electrophoresis). We discuss the evolutionary consequences of protein binding flexibility, to act on both ancestral and present telomeric sequences. Of particular interest is that the ancestral repeat, which is thought not to form the telomere, binds the proteins most strongly. These data are discussed in line with other known plant telomere-binding proteins and with the complex nature of the telomere in Asparagales carrying a human-type motif.

Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction

A method, termed hierarchical oligonucleotide primer extension (HOPE), is developed for quantitative, multiplexing detection of DNA targets present in PCR-amplified community 16S rRNA genes. It involves strand extension reaction and multiple oligonucleotide primers modified with different lengths of polyA at the 5′ end and targeting 16S rRNA genes at different phylogenetic specificities. On annealing to the targets, these primers are extended with a single fluorescently labeled dideoxynucleoside triphosphate or a dye-terminator. Using a DNA autosequencer, these extended primers are separated and identified by size and dye color, and quantified and normalized based on the fluorescence intensities and internal size standards. Using a primer-to-target ratio >1000, constant primer extension efficiencies can be obtained with individual primers to establish a ‘calibration factor’ between individual primers and a universal or domain-specific primer, providing the relative abundance of targeted rRNA genes with respect to total rRNA genes. HOPE up to 10-plexing is demonstrated to correctly identify 20 different bacterial strains, and quantify different Bacteroides spp. in 16S rRNA gene amplicons from different model bacteria mixtures and the influent and effluent of a wastewater treatment plant. Single mismatch discrimination with detection sensitivity of a target down to 0.01–0.05% of total DNA template is achieved.

Behavior of enteric neural crest‐derived cells varies with respect to the migratory wavefront

Neural crest-derived cells colonize the entire gastrointestinal tract. The migration of these enteric neural crest-derived cells (ENCCs) occurs by their formation of cellular strands that extend into the intestinal mesenchyme. We have studied the behavior of crest cells that underlies the formation and extension of these strands by time-lapse microscopy. ENCCs expressing fluorescent marker molecules were visualized in situ in the embryonic mouse and chick gut. The major contributor to strand extension is from cells located within a region approximately 300 microm behind (rostral to) the most caudal cells in the migratory wavefront. Cells in the region immediately behind the leading cell of the strand either move intermittently in parallel with the leading cell, or advance caudally toward the wavefront over other ENCCs. Another addition to the strands arises from isolated cells located caudal to the wavefront. These cells showed a range of behavior including attachment and separation from the strands. The extending strands converged to form nodes, and then diverged along independent paths to form new strands, a behavior suggestive of attraction and repulsion. This behavior is probably responsible for the unique reticulated arrangement of ganglia in the enteric nervous system. As cells become positioned farther behind the wavefront, they exhibit more restricted movement and varied trajectories. We conclude that ENCCs exhibit different behaviors, depending on their position with respect to the wavefront. These different behaviors suggest a critical role for cell-cell interaction in the migratory process.

P53 Monitors Replication Fork Regression by Binding to “Chickenfoot” Intermediates

The tumor suppressor protein, p53, utilizes multiple mechanisms to ensure faithful transmission of the genome including regulation of DNA replication, repair, and recombination. Monitoring these pathways may involve direct binding of p53 to the DNA intermediates of these processes. In this study, we generated templates resembling stalled replication forks and utilized electron microscopy to examine p53 interactions with these substrates. Our results show that p53 bound with high affinity to the junction of stalled forks, whereas two cancer-derived p53 mutants showed weak binding. Additionally, some of the templates were rearranged to form "chickenfoot" structures in the presence of p53. These were mostly formed due to p53 trapping intermediates of spontaneous fork regression; however, in a small population, the protein appeared to be promoting their formation. Collectively, these results demonstrate the importance of sequence-independent binding in p53-mediated maintenance of genomic integrity.

Efficient Primer Strand Extension beyond Oxadiazole Carboxamide Nucleobases

Two oxadiazole carboxamide deoxyribonucleoside analogues are described that can be incorporated and efficiently extended by Taq DNA polymerase. The primer strand extension beyond oxadiazole nucleoside analogues occurs at rates similar to the values observed for the canonical Watson-Crick base pairs irrespective of the template nucleobase. These distinctive chemical effects in DNA polymerase extensions are attributed to the smaller size and unique electronic properties of the oxadiazole nucleobase.

Crystal Structure of the Mosquito-larvicidal Toxin Cry4Ba and Its Biological Implications

Cry4Ba, isolated from Bacillus thuringiensis subsp. israelensis, is specifically toxic to the larvae of Aedes and Anopheles mosquitoes. The structure of activated Cry4Ba toxin has been determined by multiple isomorphous replacement with anomalous scattering and refined to R(cryst) = 20.5% and R(free)= 21.8% at 1.75 Angstroms resolution. It resembles previously reported Cry toxin structures but shows the following distinctions. In domain I the helix bundle contains only the long and amphipathic helices alpha3-alpha7. The N-terminal helices alpha1-alpha2b, absent due to proteolysis during crystallisation, appear inessential to toxicity. In domain II the beta-sheet prism presents short apical loops without the beta-ribbon extension of inner strands, thus placing the receptor combining sites close to the sheets. In domain III the beta-sandwich contains a helical extension from the C-terminal strand beta23, which interacts with a beta-hairpin excursion from the edge of the outer sheet. The structure provides a rational explanation of recent mutagenesis and biophysical data on this toxin. Furthermore, added to earlier structures from the Cry toxin family, Cry4Ba completes a minimal structural database covering the Coleoptera, Lepidoptera, Diptera and Lepidoptera/Diptera specificity classes. A multiple structure alignment found that the Diptera-specific Cry4Ba is structurally more closely similar to the Lepidoptera-specific Cry1Aa than the Coleoptera-specific Cry3Aa, but most distantly related to Lepidoptera/Diptera-specific Cry2Aa. The structures are most divergent in domain II, supporting the suggestion that this domain has a major role in specificity determination. They are most similar in the alpha3-alpha7 major fragment of domain I, which contains the alpha4-alpha5 hairpin crucial to pore formation. The collective knowledge of Cry toxin structure and mutagenesis data will lead to a more critical understanding of the structural basis for receptor binding and pore formation, as well as allowing the scope of diversity to be better appreciated.

DNA Adduct of the Mitomycin C Metabolite 2,7-Diaminomitosene Is a Nontoxic and Nonmutagenic DNA Lesion in Vitro and in Vivo

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.

Capturing in vivo dynamics of the actin cytoskeleton stimulated by auxin or light.

We present here a transient expression system that allows the response of actin microfilaments to physiological stimuli (changes in auxin content, light) to be observed in single cells in vivo. Etiolated, intact rice seedlings are attached to glass slides, transfected biolistically with talin fused to yellow-fluorescent protein to visualize actin microfilaments, and either treated with auxin or irradiated. The talin marker labels distinct populations of actin that are differentially expressed depending on the physiological state of the coleoptile (active elongation versus ceased elongation). Whereas longitudinal transvacuolar bundles prevail in cells that have ceased to elongate, fine cortical strands are characteristic for elongating cells. The visualized actin structures remain dynamic and responsive to signals. Exogenous auxin triggers a loosening of the bundles and an extension of the cortical strands, whereas irradiation reorientates cortical strands into longitudinal arrays. These responses correspond in quality and timing to the signal responses inferred previously from fixed specimens and biochemical studies. In big advantage over those methods it is now possible to observe them directly at the single cell level. Thus, the rice coleoptile system can be used as a convenient model to study actin dynamics in vivo, in response to physiologically relevant stimuli.

Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation.

During leaf development, ground meristem cells along continuous lines undergo coordinated oriented cell divisions and differentiate to form procambial cells, the precursors of all vascular cells. The molecular genetic dissection of early procambial development suffers from the lack of easily identifiable markers, especially of cell states preceding procambium formation. In this study, we have identified and characterized three reporter gene expression markers that reflect three distinct preprocambial stages, as well as one marker whose expression seems to be perfectly congruent with the appearance of procambial cells. All four markers are invariably expressed in continuous domains connected to pre-existing vasculature and their expression profiles reveal a common spatiotemporal pattern of early vein formation. We observed progressive extension of vascular strands at the preprocambial stage, suggesting that veins are initiated as freely ending preprocambial domains and that network formation occurs through subsequent fusion of these domains. Consistent with this interpretation, we demonstrate that veins are generally not programmed to become freely ending or interconnected network elements. Instead, we found that the progressive extension of preprocambial domains can be interrupted experimentally and that this leads to less complex vein patterns consisting of fewer vein orders, in which even lower-order veins become freely ending. Mesophyll differentiation turned out to be strictly correlated with the termination of preprocambial domain extension. These findings suggest that Arabidopsis vein pattern is not inherently determinate, but arises through reiterative initiation of new preprocambial branches until this process becomes terminated by the differentiation of mesophyll.

Nonlinear stress relaxation of an entangled linear polystyrene in single step‐strain flow: A quantitative theoretical investigation

AbstractThe nonlinear stress relaxation of a nearly monodisperse, moderately entangled polystyrene solution (i.e., roughly seven entanglements per chain at equilibrium) in single step‐strain flow is investigated quantitatively by a detailed comparison of an existing set of experimental data with a simulation based on the tube model. The proposed simulation enables the effects of primary nonlinear relaxation mechanisms other than chain retraction to be identified more clearly and investigated individually. Two peculiar nonlinear relaxation behaviors are observed in this experiment. One is concerned with an apparent enhancement in the stress relaxation at short times, and the other is responsible for a seeming slowdown of the stress relaxation at long times. These findings are discussed within the tube model, in view of the effects of convective constraint release, partial strand extension, and nonaffine deformation. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1281–1293, 2003

Detection of activity of telomerase in tumor cells using fiber optical biosensors

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80–90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 °C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.