Telomere Gene Research Articles

Telomere Gene Complex Mutations Are Frequently Found with Shortened Telomeres in Bone Marrow Failure Syndromes and Idiopathic Pulmonary Fibrosis; Correlation with Haematological and Clinical Features

Shortened telomeres are seen in approximately a quarter of patients with idiopathic pulmonary fibrosis (IPF) and aplastic anemia (AA) and may have a role in disease pathogenesis. Mutations in the telomere gene complex (TGC) are less frequently seen in IPF (1%) and 5-10% in AA. Novel mutations affecting telomere function are increasingly being reported and involve the telomerase genes, the shelterin and helicase complexes, but their prognostic significance remains undefined. We report results on telomere length (TL) using multiplex quantitative real-time PCR (qPCR) and mutation analysis on a customised panel of 10 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1 and WRAP) using deeply parallel sequencing in a cohort of 69 patients with AA and IPF and their effects on clinical variables and response rates.69 patients (49 with AA, 14 with IPF and 6 with a bone marrow failure syndrome (BMFS) and pulmonary fibrosis; M/F =43/26) with a median age of 49.7 years (range 9-81) had TL (T/S ratio) tested using qPCR on peripheral blood mononuclear cells at diagnosis or pre-treatment. Massively parallel targeted sequencing of the TGC was performed in all cases and constitutional mutations identified using the exome variant server. Clinical variables studied included hemoglobin (Hb) level, neutrophil count (ANC), platelet count (PC), LDH and albumin levels, and response rates to immunosuppressive treatment (IST). In patients with AA and other BMFS, red cell and platelet transfusion dependency, bone marrow cellularity (<30), cytogenetics and p53 (Modified Quick score >1) on bone marrow (BM) staining were correlated with TL and presence of any TGC mutations. In patients with IPF, corrected transfer factor (Kco) was correlated with TL and mutation analysis.44/69 (64%) had TL below the 1st centile and 20 (29%) of them had TL very significantly below the 1st centile compared to a normal cohort (n=180) of age-matched individuals. Median Hb was (11g/dl), ANC was 1.69 and PC was 96x109/l. There was no statistical difference between patients whose TL was below or above the 1st centile for Hb levels, reticulocyte count or PC. 24 patients (34.7%) were red cell transfusion dependent and 15(21.7%) needed platelet transfusions. 42/55 (76.3%) had BM cellularity < 30% and was not statistically significant between patients with TL above or below the 1st centile. Similarly there was no statistical difference in LDH and albumin levels between the two groups. Equal number of patients were treated with IST between the two groups and response rates were similar. Median levels of Kco (0.95) were identical in IPF patients. 62 (89.8%) of patients were alive at a median follow up of 2.15 years (range 0.3-26.8).TGC mutations were seen 16 patients (23%) in the entire cohort with 13/44 (30%) were seen where TL was below the 1st centile as compared to 3/25 (12%) where TL was above the 1st centile (p<0.14). 11 patients had TERT (all missense), 4 TERC (non-coding transcript) and 1 RTEL mutations were identified. TERT A279T was the commonest mutation seen, in 4 patients.In our cohort, about two-thirds of patients had TL below the 1st centile. TL, however, does not affect blood counts, BM cellularity and p53 staining, red cell or platelet transfusion dependency or Kco in patients with IPF. A higher proportion of patients with TL below the 1st centile were found to have TGC mutations, although statistical significance was not achieved. Missense mutations in TERT were the most frequent followed by mutations in TERC. Patients with TL <1st centile were diagnosed at a younger age, and where TGC mutations were present, were also likely to show other features of telomereopathy.TableVariableTL < 1st centileTL > 1st centileMale/Female (Total)29/15 (44)14/11 (25)Age < 40 years at diagnosis23 (52.2%)7 (28%)TGC mutations %3012Hb (g/dl)1111.2Reticulocyte count ( x 10*9/L)5257ANC ( x 10*9/L)1.692.79PC ( x 10*9/L)90139Albumin g/dl4242LDH (IU)211210AA/BMFS3718Red cell transfusion dependency %3644.4Platelet transfusion dependency %18.138.8%Cytogenetic abnormality %15.60BM cellularity < 30% %77.772.2P53 (Modified quick score >1) %34.740Treatment with IST Response %23 (62.1%) 69.5%11 (64.7%) 72.7%IPF77Kco0.95 (0.6-1.9)0.95 (0.8-1.2) DisclosuresKulasekararaj:Alexion Pharmaceuticals: Speakers Bureau.

Human leukocyte telomere length is associated with DNA methylation levels in multiple subtelomeric and imprinted loci.

In humans, leukocyte telomere length (LTL) is positively correlated with lifespan, and shorter LTL is associated with increased risk of age-related disease. In this study we tested for association between telomere length and methylated cytosine levels. Measurements of mean telomere length and DNA methylation at >450,000 CpG sites were obtained for both blood (N = 24) and EBV-transformed cell-line (N = 36) DNA samples from men aged 44–45 years. We identified 65 gene promoters enriched for CpG sites at which methylation levels are associated with leukocyte telomere length, and 36 gene promoters enriched for CpG sites at which methylation levels are associated with telomere length in DNA from EBV-transformed cell-lines. We observed significant enrichment of positively associated methylated CpG sites in subtelomeric loci (within 4 Mb of the telomere) (P < 0.01), and also at loci in imprinted regions (P < 0.001). Our results pave the way for further investigations to help elucidate the relationships between telomere length, DNA methylation and gene expression in health and disease.

No Impact of Lentiviral Transduction on Hematopoietic Stem/Progenitor Cell Telomere Length or Gene Expression in the Rhesus Macaque Model

The occurrence of clonal perturbations and leukemia in patients transplanted with gamma-retroviral (RV) vector-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions may perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We used two techniques to examine whether lentivirally transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral (LV) transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GF- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model.

Usefulness of Nucleic Acids (DNA/RNA) from Buccal Cells Isolated from Mouthwashes using a Modified Method

Human buccal cells are an easy source of nucleic acids (DNA/RNA) other than human blood. We have extracted buccal cells from mouthwashes of 10 individuals and tested its usefulness for genotyping, telomere length determination and gene expression. Genomic DNA was extracted using a salt precipitation method whereas total RNA was extracted using Hipura total RNA extraction kit. The quantity and quality of DNA and RNA was found to be satisfactory. PCR amplification was achieved using locus specific primers and genotyping was performed for minisatellites, microsatellites and Single nucleotide polymorphisms (SNPs). Telomere length was determined from the genomic DNA of buccal cells obtained from mouthwashes. Telomere length obtained from the DNA isolated from buccal cells and peripheral blood mononuclear cells was compared. Real time quantitative PCR was successfully performed for GADD45A, G6PD, B-Actin and B2M. In conclusion, our method of DNA /RNA extraction can be considered as an alternative method of sample collection for forensic medicine.

Abnormal Expression Of Shelterin Is Associated With Short Telomere and Immune Disorder In Severe Aplastic Anemia

ObjectiveTo identify changes in telomere length and gene expression of shelterin (which is composed of six core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia(SAA). Looking for the reason why telomere getting short in SAA patients and the pathway that abnormal telomeres cause AA. Methods15 controls and 27 cases of SAA were enrolled in this study. There were 9 untreated and 18 recovering patients. Peripheral white blood cells(PWBCs)' telomere length was tested by southern blot and gene expression of shelterin were detected by RT-PCR. Bone marrow mononuclear cells (BMMNC) from controls were cultured in different culture medium which were fetal calf serum only , SAA serum only, fetal calf serum containing TNF-α and fetal calf serum containing IFN-γ respectively. The apoptosis of these cells were detected by flowcytometry. The mRNA expression of POT1 were tested by RT-PCR , all the protein below including ATR, phosphorylated ATR, and phosphorylated ATM/ATR substrate were detected by western blot. ResultsTelomeres length of PWBCs were found significantly shorter in SAA untreated group (4.89±1.66)kb and recovering group (7.04± 1.47 )kb than control group (11.65±5.55)kb (P<0.05). Telomeres Length of PWBCs turned shortening in accordance with TH/S (CD4+Tcells/CD8+Tcells ratio) decreasing (r=0.593, p=0.007). The mRNA expression of POT1 decreased in SAA untreated group( 0.29(0.09-0.58)) than in recovering group( 0.78(0.22-2.69)) and controls (1.88(0.93-3.81)), (P<0.05). No differences about mRNA expression of TRF1、TRF2、TIN2、TPP1 and RAP1 were found among SAA untreated group, recovering group and controls in PWBCs. Undoubtedly, cells that cultured in SAA serum got more apoptosis(13.88±4.55) than that cultured in fetal calf serum(9.85±2.67)(P<0.05). The mRNA expression of POT1 was significantly declined in cells that from SAA patient uncultured group (0.45±0.29) , normal BMMNCs cultured by AA serum group (0.59±0.49) and IFN-γ group (0.40±0.35) than the cells cultured only by fetal calf serum group (1.19±0.62). ATR, phosphorylated ATR, and phosphorylated ATM/ATR substrate were found significantly increase in cells from AA serum group , TNF-α group and IFN-γ group than fetal calf serum group. ConclusionSAA patients have short PWBCs' telomeres length which was in accordance with TH/S.TNF-α and IFN-γ which were found at high concentration in SAA decades before may be the effective component that can trigger apoptosis through POT1 and the pathway of ATM/ATR and eventually lead to SAA. Disclosures:No relevant conflicts of interest to declare.

Telomere length and gene expression of shelterin in CD3(+) T cell of severe aplastic anemia patients

To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA). Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR). Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P < 0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P < 0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P < 0.05). Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

65. No Impact of Lentiviral Transduction on Hematopoietic Stem and Progenitor Cell Telomere Length or Gene Expression Profile in the Rhesus Macaque

65. No Impact of Lentiviral Transduction on Hematopoietic Stem and Progenitor Cell Telomere Length or Gene Expression Profile in the Rhesus Macaque

Impact of D-bifunctional Protein Deficiency on Telomere Length and Gene Expression in a Child

Impact of D-bifunctional Protein Deficiency on Telomere Length and Gene Expression in a Child

Impact of D-bifunctional Protein Deficiency on Telomere Length and Gene Expression in a Child

Aim: To explore, in one patient, the possibility that D-bifunctional protein (D-BP) deficiency affects telomere length, and to determine the profile of genetic expression. Presentation of Case: Due to the symptoms of a newborn and his family background, a peroxisomal panel was performed. There were high levels of very long chain fatty acids and abnormal peroxisomes. At 8 months the patient exhibited other complications, including progressive multi systemic deterioration, and at 15 months died of pneumonia. Discussion: Analysis of the patient’s fibroblasts provided evidence of a defect in the peroxisomes and in the oxidation of fatty acids, leading to a diagnosis of D-BP deficiency. Significant alterations were found in the genetic expression profile, with the

Identification of the Functional Domains of the Telomere Protein Rap1 in Schizosaccharomyces pombe

The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1 + have revealed that the long N-terminal region (1–456 a.a. [amino acids]) of Rap1 (full length: 693 a.a.) is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457–693 a.a.) containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus) and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.

Shorter peripheral blood telomeres are a potential biomarker for patients with advanced colorectal adenomas.

Colorectal cancer (CRC) can be prevented by the early detection and removal of advanced adenomas (AAs) by colonoscopy. Our aim was to evaluate peripheral blood leukocyte (PBL) telomere length as a potential biomarker for the presence of AAs. PBL telomere length was measured in patients with AAs (n=35), in a control group of similarly aged patients who had a normal colonoscopy (n=145) and in a separate population group with no history of cancer, again similarly aged (n=495). Telomere measurements were performed using a quantitative PCR assay and reported as ratios of telomere and single copy gene measurements. Telomere lengths tended to be lower in patients with AAs than in patients in the normal colonoscopy group (p<0.001) as well as those in the population group (p=0.011). A telomere/single copy gene ratio of 0.5 was found to have an estimated 94% sensitivity and a 56% specificity for AAs; a combination of sensitivity and specificity for which a value of >0.5 would reduce the odds of a patient having AAs by a factor of 0.11 (the negative likelihood ratio). Thirty three percent of individuals in the population group tested above this cutoff and could be considered at low risk for AAs. PBL telomeres are shortened in patients with colorectal neoplasia, suggesting that PBL telomere length could be a promising non-invasive blood biomarker to pre-screen for risk of AAs prior to colonoscopy.

No evidence of telomere length attrition in newborns from high level natural background radiation areas in Kerala coast, south west India

Purpose: The tandemly repeated hexamers (TTAGGG)n present at the telomeric ends protect the human genome from a variety of environmental exposures including ionizing radiation. In order to find out the effect of chronic low dose radiation exposure, we have determined telomere length among newborns from high level natural radiation areas (HLNRA) of the Kerala coast in South west India.Methodology: Umbilical cord blood samples were collected from 128 newborns from HLNRA and 43 newborns from normal level natural radiation areas (NLNRA) and genomic DNA was isolated using a salt precipitation method. The mean telomere length was determined using SYBR green-based real time quantitative Polymerase chain reaction (qPCR), where telomere gene specific (T) and single copy gene specific (S) primers were used. The average of telomere versus single copy gene (T/S) ratio was calculated which was proportional to telomere length of each individual.Results: The mean relative telomere length was found to be 1.03 ± 0.01 (95% CI, 0.99–1.05) and 1.10 ± 0.03 (95% CI, 1.04–1.17) in HLNRA and NLNRA newborns, respectively (P > 0.05). Based on the level of background radiation dose, samples were divided into four groups, i.e., NLNRA: < 1.50 mGy/year and three HNLRA groups: 1.51–3.00 mGy/year, 3.01–5.00 mGy/year, and > 5.00 mGy/year. The mean relative telomere length in these groups were found to be 1.10 ± 0.03 (95% CI, 1.03–1.17), 0.98 ± 0.01 (95% CI, 0.95–1.01), 1.05 ± 0.02 (95% CI, 1.01–1.10) and 1.07 ± 0.03 (95% CI, 1.04–1.10), respectively. No significant difference was observed between the mean telomere length of male and female newborns.Conclusions: The elevated level of natural chronic background radiation prevailing in Kerala coast did not show any significant effect on telomere length of newborns. To our knowledge, this is the first report on newborn telomere length from a high level natural radiation area.

Association Between Telomere Shortening and Ageing During Occupational Exposure

Association Between Telomere Shortening and Ageing During Occupational ExposureTelomere length is considered as a biomarker of ageing, resulting in shortening during the process. The present investigation was an attempt to determine the relative telomere length in mechanical workshop workers. Telomere length shortening in cells during occupational exposure causes accelerated ageing. Genomic DNA was isolated from buccal epithelial cells collected from 240 individuals, comprising two groups of 120 exposed workers and 120 unexposed controls. Telomere length was measured by using real time PCR. Both telomere (T) and single copy gene (S) specific primers were used to compute the relative T/S ratio and expressed as the relative telomere length. Telomere length differed significantly between the workers and controls (p&lt;0.05). The results showed an indirect and significant association (r=-0.356, p=0.001) between age and telomere length in the workers. This study showed that the difference in telomere length shortening was statistically significant (p&lt;0.05) between the workers and controls. It was concluded that occupational exposure acts as a risk factor to enhance telomere length shortening and accelerate ageing.

Newly Discovered Quick, Non-Invasive Screening Method of Bone Marrow Malignancies Including Various Leukemias, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, &amp; Multiple Myeloma by Abnormality of Small Rectangular Area within Bone Marrow Organ Representation Areas of the Face

Diagnoses of bone marrow associated malignancies such as Acute & Chronic Lymphocytic Leukemia, Acute & Chronic Myelogenous (Myeloid) Leukemia, Hodgkin's Lymphoma & Non-Hodgkin's Lymphoma, and Multiple Myeloma are often missed without a blood test. However, in 2008, Omura Y reported several newly discovered organ representation areas that exist between the lower end of the eyebrows and upper end of the upper eyelid. This space was divided into 5 organ representation areas. The first space (more than 1/4 of entire space) near the side of the face (temple) is the bone marrow representation area (BMRA). Therefore, we examined the bone marrow representation areas non-invasively using the Bi-Digital O-Ring Test (BDORT). When the small rectangular shaped part of the BMRA is strong negative (-) with more than -2, often there is a malignancy associated with bone marrow. In this area, we found 1) Integrin alpha5beta1 & Oncogen C-fos Ab2 increased very significantly between 125-300 ng BDORT units; 2) very high Chrysotile Asbestos (0.11-0.14 mg); 3) markedly reduced Acetylcholine of less than 1 ng; 4) significantly reduced telomere of less than 1 yg (= 10(-24) g); and 5) Increased 8-OH-dG (often more than 5 ng). Once the abnormal small rectangular area is localized by BDORT, by detecting the specific microscope slide which produces EMF (electromagnetic field) resonance, one can diagnose these malignancies non-invasively in about 10 minutes. When a subject has any one of the above 7 types of bone marrow associated malignancies, the 5 aforementioned abnormal parameters can be detected. When Acetylcholine is markedly reduced to 0.25 ng or less, 8-OH-dG is 10 ng or higher, and Sirtuin 1 (one of the 7 mammalian longevity genes products) in both the Hippocampus and the body is 0.025 pg or less, most of the patients have a very poor prognosis. However, we found that increasing normal cell telomere & longevity gene product Sirtuin 1 can often improve both pathology & prognosis. All measurements are in BDORT units (the weight required to produce maximum EMF resonance).

Acetylation of the SUN protein Mps3 by Eco1 regulates its function in nuclear organization

The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of certain types of persistent DNA double-stranded breaks. How these diverse SUN protein functions are regulated is unknown. Here we show that the Mps3 N-terminus is a substrate for the acetyltransferase Eco1/Ctf7 in vitro and in vivo and map the sites of acetylation to three lysine residues adjacent to the Mps3 transmembrane domain. Mutation of these residues shows that acetylation is not essential for growth, SPB duplication, or distribution in the nuclear membrane. However, analysis of nonacetylatable mps3 mutants shows that this modification is required for accurate sister chromatid cohesion and for chromosome recruitment to the nuclear membrane. Acetylation of Mps3 by Eco1 is one of the few regulatory mechanisms known to control nuclear organization.

HTLV‐1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells

Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression.

G‐quadruplexes in RNA biology

G-quadruplexes are noncanonical structures formed by G-rich DNA and RNA sequences that fold into a four-stranded conformation. Experimental studies and computational predictions show that RNA G-quadruplexes are present in transcripts associated with telomeres, in noncoding sequences of primary transcripts and within mature transcripts. RNA G-quadruplexes at these specific locations play important roles in key cellular functions, including telomere homeostasis and gene expression. Indeed, RNA G-quadruplexes appear as important regulators of pre-mRNA processing (splicing and polyadenylation), RNA turnover, mRNA targeting and translation. The regulatory mechanisms controlled by RNA G-quadruplexes involve the binding of protein factors that modulate G-quadruplex conformation and/or serve as a bridge to recruit additional protein regulators. In this review, we summarize the current knowledge on the role of G-quadruplexes in RNA biology with particular emphasis on the molecular mechanisms underlying their specific function in RNA metabolism occurring in physiological or pathological conditions.

Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA

In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C (t)) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4-4.7%) and inter-assay (RSD range: 3.1-5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples.

Locked Nucleic Acid Flow Cytometry-fluorescence &lt;em&gt;in situ&lt;/em&gt; Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

A pyridyl carboxamide molecule selectively stabilizes DNA G-quadruplex and regulates duplex–quadruplex competition

G-Quadruplexes formed by G-rich DNA are of broad interest due to their involvement in telomere function, gene transcription and recombination. Small ligands that interact strongly with G-quadruplexes have been considered to further influence telomeric function and gene transcription. Because most G-rich sequences are trapped in duplex structures in gene promoters, ligands that can stabilize G-quadruplexes in the presence of their complimentary strands would likely have strong effects on gene transcription. Here, we report a novel simple small molecule (pyridyl carboxamide), consisting of three pyridine rings and four amide bonds. Comparing with some reported G-quadruplex ligands, this molecule not only selectively stabilizes G-quadruplexes rather than duplexes, but also maintains a G-quadruplex structure even if the G-rich region was trapped in long double-stranded DNA (dsDNA). It is widely believed that the dissociation of duplexes is involved in gene transcription and that the formation of the G-quadruplex influences some oncogene expression. Py-Am exhibited strong G-quadruplex-forming ability within a long dsDNA sequence, suggesting it would have potent effects on the G-quadruplex-forming sequences involved in gene transcription.