Reverse Transcriptase Domain Research Articles

Haploinsufficiency of t e lomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita

Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.

Soluble domains of telomerase reverse transcriptase identified by high‐throughput screening

Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

An extraordinary retrotransposon family encoding dual endonucleases

Retrotransposons commonly encode a reverse transcriptase (RT), but other functional domains are variable. The acquisition of new domains is the dominant evolutionary force that brings structural variety to retrotransposons. Non-long-terminal-repeat (non-LTR) retrotransposons are classified into two groups by their structure. Early branched non-LTR retrotransposons encode a restriction-like endonuclease (RLE), and recently branched non-LTR retrotransposons encode an apurinic/apyrimidinic endonuclease-like endonuclease (APE). In this study, we report a novel non-LTR retrotransposon family Dualen, identified from the Chlamydomonas reinhardtii genome. Dualen encodes two endonucleases, RLE and APE, with RT, ribonuclease H, and cysteine protease. Phylogenetic analyses of the RT domains revealed that Dualen is positioned at the midpoint between the early-branched and the recently branched groups. In the APE tree, Dualen was branched earlier than the I group and the Jockey group. The ribonuclease H domains among the Dualen family and other non-LTR retrotransposons are monophyletic. Phylogenies of three domains revealed the monophyly of the Dualen family members. The domain structure and the phylogeny of each domain imply that Dualen is a retrotransposon conserving the domain structure just after the acquisition of APE. From these observations, we discuss the evolution of domain structure of non-LTR retrotransposons.

Molecular characterization of a gender-linked DNA marker and a related gene in Mercurialis annua L.

The dioecious Mercurialis annua L. was used as a model plant to study some aspects of the molecular basis of sex determination in plants. We report in this paper the characterization of a previously identified male specific DNA marker, OPB01-1562, from diploid dioecious M. annua. The marker co-segregated with male sex in the progeny of hormonally feminized males. Sequence analysis showed the presence of approximately 0.6 kb retrotransposon-like sequence at its 3' end. Homologous sequences were isolated from diploid female, hexaploid male and monoecious plants. These sequences contained RNaseH and integrase domains of reverse transcriptase and were most similar to pineapple retrotransposon dea1, hence were named M. annua retrotransposon-like sequences (MARL-1 to MARL-5). A 771 bp fragment isolated from a diploid female, named fem771, was homologous to the 5' end of OPB01-1562. Results from DNA blot hybridization suggested OPB01-1562 and fem771 to be from the same locus and MARL-1 from a different one. RNA blot hybridization with OPB01-1562 and MARL-1 detected an approximately 2.8 kb transcript which was expressed strongly in stems and flowers of females but not males. This transcript was named M. annua female expressed (Mafex). Sex linkage of OPB01-1562 and expression of Mafex detected by OPB01-1562 strongly suggested Mafex to be a candidate gene involved in sex determination in M. annua.

Combinatorial Selection, Inhibition, and Antiviral Activity of DNA Thioaptamers Targeting the RNase H Domain of HIV-1 Reverse Transcriptase

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi <or= 0.005.

A new subtype (subgenotype) Ac (A3) of hepatitis B virus and recombination between genotypes A and E in Cameroon

Blood samples (n=544) from two different populations (Pygmies and Bantus) in Cameroon, West Africa, were analysed. Serological tests indicated that the anti-hepatitis C virus (HCV) prevalence in Bantus (20.3 %) was higher than that in Pygmies (2.3 %, P<0.0001), whereas the distribution of hepatitis B virus (HBV) serological markers was equally high in both populations: in total, 9.4, 17.3 and 86.8 % for HBsAg, anti-HBs and anti-HBc, respectively. HBV genotype A (HBV/A) and HBV/E were predominant (43.5 % each) in both populations, and HBV/D was found in a minority (13 %). The preS/S region was sequenced in nine cases (five HBV/A and four HBV/E) and the complete genome in six cases (four HBV/A and two HBV/E). Subsequent phylogenetic analysis revealed that the HBV/A strains were distinct from the subtypes (subgenotypes) described previously, Ae (A2) and Aa (A1), and in the preS/S region they clustered with previously reported sequences from Cameroon. Based on the nucleotide difference from Aa (A1) and Ae (A2), more than 4 % in the complete genome, the Cameroonian strains were suggested to represent a new subtype (subgenotype), designated HBV/Ac (A3). A high (3.9 %) nucleotide divergence in HBV/Ac (A3) strains suggested that the subtype (subgenotype) has a long natural history in the population of Cameroon. One of the HBV/Ac (A3) strains was found to be a recombinant with an HBV/E-specific sequence in the polymerase reverse transcriptase domain. Further cohort studies will be required to assess detailed epidemiological, virological and clinical characteristics of HBV/Ac (A3), as well as its recombinant form.

HTERT mRNA Partially Regulates Telomerase Activity in Gastric Adenocarcinoma and Adjacent Normal Gastric Mucosa

The extent to which human telomerase reverse transcriptase (hTERT) mRNA and its splice variants control telomerase activity in human cancers is controversial. Telomerase and hTERT mRNA were assessed quantitatively in paired samples of gastric adenocarcinoma and adjacent normal tissue. Splice variants within the hTERT reverse transcriptase domain (alpha, beta, alphabeta) were detected by RT-PCR. In gastric adenocarcinoma, compared to normal tissue, median telomerase activity increased significantly (from 0 total product generated [tpg; 95% confidence interval CI, 0-2.3] to 16.1 tpg [95% CI, 3.7-97]; P = 0.008) and median hTERT mRNA levels also increased (from 2.21 [95% CI, 1.40-4.62] to 7.08 [95% CI, 3.26-10.8]; P = 0.0054). hTERT mRNA and telomerase activity correlated in normal gastric mucosa (r = 0.819, P = 0.0002). Alpha, beta, and alphabeta deletions were similar in both groups. We conclude that hTERT mRNA partially regulates telomerase activity in normal gastric mucosa and gastric adenocarcinoma. In contrast, hTERT mRNA splicing is not involved in the regulation of enzyme activity.

Development of broadly targeted human endogenous gammaretroviral pol-based real time PCRs : Quantitation of RNA expression in human tissues

Endogenous retroviral sequences (ERVs) are dynamic genomic components with profound influences on gene expression and genomic structure. Their extent of expression is not well known. Several broadly targeted real-time reverse transcription PCR (QPCRs) systems for surveillance of RNA expression of the major groups of human gammaretroviral ERVs were constructed. The highly conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were used as targets for the PCRs, which were both probe-based (TaqMan ®) and probe-less (SYBR ® Green). Different levels of primer and probe degeneracy, with or without inosine, were tested. Several of the PCRs had sensitivities of a few HERV nucleic acid copies per PCR reaction. Specificities were approximately as expected from the fit of primers and probes. Gammaretroviral HERV RNA expression was studied in different human tissues. Each HERV group had a specific pattern of expression. HERV-E was highly expressed in testis, HERV-I/T in brain and testis, HERV-H in brain and testis, while HERV-W was highly expressed in placenta. Gammaretroviral RNA was not detected in plasma from 50 blood donors in saliva from 20 persons. In conclusion, a set of tools for investigation of gammaretroviral HERV RNA expression was created.

Identification of 18 new transcribed retrotransposons in Schistosoma mansoni

This work describes 18 new transcribed retrotransposons of the blood fluke Schistosoma mansoni. Among them, 9 were LTR, 8 non-LTR, and 1 Penelope-like element (PLE) retrotransposon. Sequences were generated by in silico reconstruction using S. mansoni ESTs and transcripts obtained by rapid amplification of cDNA ends, complemented in some cases by sequencing of genomic clones amplified by PCR. A novel element from the ancient R2/R4/CRE transposon group is described for the first time in S. mansoni. In addition, one non-LTR retrotransposon family displays long (40–450 bp) 3′-UTR with at least six different transcribed sequences among the copies, five LTR retrotransposons have abundantly transcribed incomplete copies lacking the sequence segment coding for the reverse transcriptase domain, and four non-LTR retrotransposons code for DNA-binding PHD domains that may give them a differential targeting. These results allow for a comprehensive description of the transcribed retrotransposon diversity of this complex human parasite.

Conservation of functional domains and limited heterogeneity of HIV-1 reverse transcriptase gene following vertical transmission.

BackgroundThe reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the life cycle of the virus by converting the single stranded RNA genome into double stranded DNA that integrates into the host chromosome. In addition, RT is also responsible for the generation of mutations throughout the viral genome, including in its own sequences and is thus responsible for the generation of quasi-species in HIV-1-infected individuals. We therefore characterized the molecular properties of RT, including the conservation of functional motifs, degree of genetic diversity, and evolutionary dynamics from five mother-infant pairs following vertical transmission.ResultsThe RT open reading frame was maintained with a frequency of 87.2% in five mother-infant pairs' sequences following vertical transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' sequences. Both mothers and infants RT sequences were under positive selection pressure, as determined by the ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 132 mother-infant RT sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked mother-infant pairs. The functional domains of RT which are responsible for reverse transcription, DNA polymerization and RNase H activity were mostly conserved in the RT sequences analyzed in this study. Specifically, the active sites and domains required for primer binding, template binding, primer and template positioning and nucleotide recruitment were conserved in all mother-infant pairs' sequences.ConclusionThe maintenance of an intact RT open reading frame, conservation of functional domains for RT activity, preservation of several amino acid motifs in epidemiologically linked mother-infant pairs, and a low degree of genetic variability following vertical transmission is consistent with an indispensable role of RT in HIV-1 replication in infected mother-infant pairs.

Identification and Characterization of Reverse Transcriptase Domain of Transcriptionally Active Retrotransposons in Wheat Genomes

: To clarify activation characterization of wheat (Triticum aestivum L.) retrotransposons, transcriptionally active Ty1-copia retrotransposons were found in wheat by using RT-PCR to amplify the RT domain. Sequence analysis of random RT-PCR clones reveals that Ty1-copia retrotransposons are highly heterogeneous and can be divided into at least four groups, which are tentatively named TaRT-1 to TaRT-4. Dot blot hybridization indicates that TaRT-1 exists in the wheat genome as multiple copies (at 30 000 copies/ a hexaploid genome (ABD)). Northern blot hybridization showed that TaRT-1 is only expressed at a low level under normal conditions in seedlings, but at a high level when induced by powdery mildew fungus, jasmonic acid (JA) and salicylic acid (SA). These results suggest that the TaRT-1 expression is highly sensitive to biotic and abiotic stresses. (Managing editor: Li-Hui ZHAO)

Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT

Several in vitro strategies have been developed to selectively screen for nucleic acid sequences that bind to specific proteins. We previously used the SELEX procedure to search for aptamers against HIV-1 RNase H activity associated with reverse transcriptase (RT) and human RNase H1. Aptamers containing G-rich sequences were selected in both cases. To investigate whether the interaction with G-rich oligonucleotides (ODNs) was a characteristic of these enzymes, a second in vitro selection was performed with an isolated RNase H domain of HIV-1 RT (p15) as a target and a new DNA library. In this work we found that the second SELEX led again to the isolation of G-rich aptamers. But in contrast to the first selection, these latter ODNs were not able to inhibit the RNase H activity of either the p15 domain or the RNase H embedded in the complete RT. On the other hand, the aptamers from the first SELEX that were inhibitors of the RT-associated RNase H did not inhibit the activity of the isolated p15 domain. This suggests that the active conformation of both RNase H domains is different according to the presence or absence of the DNA polymerase domain. HIV-1 RNase H and integrase both belong to the phosphotransferase family and share structural similarities. An interesting result was obtained when the DNA aptamers initially raised against p15 RNase H were assayed against HIV-1 integrase. In contrast to RNase H, the HIV-1 integrase was inhibited by these aptamers. Our results point out that prototype structures can be exploited to develop inhibitors of two related enzymes.

Two Purified Domains of Telomerase Reverse Transcriptase Reconstitute Sequence-specific Interactions with RNA

Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) function together to create a uniquely specialized polymerase. Here we have described for the first time domains of bacterially expressed Tetrahymena TERT that interacted directly with TER in the absence of assembly chaperones. We used quantitative binding assays to define TER sequence requirements for recognition by the high affinity RNA binding domain and an independent N-terminal RNA interaction domain. The TERT RNA binding domain and N-terminal RNA interaction domain had distinct, nonoverlapping requirements for TER sequence and structure that together accounted for all of the sites of TER contact inferred for full-length TERT. The TER residues important for TERT binding are only a subset of the residues required for catalytic activity. Our findings demonstrate telomerase functional specialization by an elaborate ribonucleoprotein architecture physically separable from the active site.

The distribution of pol containing human endogenous retroviruses in non-human primates

Few human endogenous retroviruses (HERVs) have been extensively studied in non-human primates. Such investigations have demonstrated that several element classes are primate unique, contain members with important biological function, are conserved in specific primate lineages, and have in some cases expanded in copy number. We have examined multiple sub-families of all major groups of HERVs using a DNA microarray based on the reverse transcriptase (RT) domain of the viral polymerase gene ( pol). The microarray was used to investigate the distribution of HERVs in non-human primates with particular focus on the differences between New World monkeys (NWMs) and other anthropoids. This is the first study examining most HERV families in multiple non-human primate DNAs using a uniform and sensitive method and suggests that major differences exist between primate groups. The results indicate that a major invasion and expansion of pol containing HERVs occurred after the platyrrhine (NWM) lineage separated from the catarrhines (Old World Monkeys and apes).

Genome constitution and classification using retrotransposon-based markers in the orphan crop banana

We have exploited the repetitive and dispersed nature of many long terminal repeat (LTR)-retrotransposon families for characterizing genome constitutions and classifying cultivars of the genusMusa. Insertional polymorphisms of the elements were studied using seven published and two newly designed primers facing outwards from the LTRs and reverse transcriptase (RT) domain of the retrotransposon. The primers generated specific amplification patterns showing the universal applicability of this marker type. The Inter-Retrotransposon Amplified Polymorphism (IRAP) markers distinguished the A and B genomes of the banana species (Musa acuminata Col la andMusa balbisiana Colla) and between banana cultivars. The IRAP markers enabled phylogenetic analysis of 16 Malaysian banana cultivars and determination of the genome constitution of hybrid banana (AAB, ABB, AABB, and AAAB), and gave information about ancestral genotypes of the hybrids. In addition, the IRAP detected new retrotransposon insertions into the genome of tissue culture regenerants. This PCR-based IRAP assay is amenable to large-scale throughput demands in screening breeding populations and is applicable for any crop.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylogenetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylo-genetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster

ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg 2+ or Mn 2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 °C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.

Telomerase, hTERT and splice variants in Barrett??s oesophagus and oesophageal adenocarcinoma

The enzyme telomerase is re-activated in most cancers but its mechanism of regulation in oesophageal carcinogenesis is unclear. The aim of this study was to determine the roles of human telomerase reverse transcriptase (hTERT) mRNA expression and hTERT mRNA splicing in the regulation of telomerase enzyme activity in Barrett's oesophagus and oesophageal adenocarcinoma. Paired samples from oesophageal adenocarcinoma (n=21) and adjacent macroscopically normal mucosa, and paired samples from Barrett's oesophagus (n=16) and adjacent cardia mucosa were obtained. Telomerase activity was measured by the telomeric repeat amplification protocol assay. hTERT mRNA was quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Splice variants within the hTERT reverse transcriptase domain (alpha, beta, alpha beta) were detected by RT-PCR. In oesophageal adenocarcinoma, compared to adjacent mucosa, median telomerase activity increased significantly (from 5 to 229 total product generated (tpg), P=0.0002), but median hTERT mRNA levels were not significantly different. Similarly, median telomerase activity was significantly higher in oesophageal adenocarcinoma compared to Barrett's oesophagus (229 vs 20 tpg, P=0.001), but hTERT mRNA levels were not significantly different. There was no significant difference in telomerase activity and hTERT mRNA levels between Barrett's oesophagus and adjacent cardia. The frequency of detection of all variants increased from cardia to Barrett's oesophagus to oesophageal adenocarcinoma (P<0.05). A major increase in telomerase activity occurs after the Barrett's oesophagus stage in oesophageal carcinogenesis. Levels of hTERT mRNA and hTERT mRNA splicing patterns did not correlate with telomerase activity and do not appear to regulate enzyme activity. In this cancer, an important clinical diagnostic role for the transcripts of the telomerase gene is improbable.