TG Repeats Research Articles

Phenotypes of California CF Newborn Screen-Positive Children with CFTR 5T Allele by TG Repeat Length.

At the cystic fibrosis transmembrane conductance regulator (CFTR) gene (IVS8)-(TG)m(T)n locus, a lower number of thymidines (legacy names 9T vs. 7T vs. 5T) and a higher number of (TG) repeats (TG-11 vs. 12 vs. 13) are associated with decreasing translation of functional CFTR protein in vitro. Retrospective cohort study comparing phenotypes of California CF newborn screen-positive children (followed 2-8 years) who had two CF-causing mutations (diagnosed as CF) with those who had one mutation from a panel of 40 CF-causing mutations (CF40mut) and one (IVS8)-(TG)11, 12, or 13-5T mutation detected by sequencing (diagnosed as CFTR-related metabolic syndrome [cRMS]). The study included 428 children, of which 234 had two CF-causing mutations, and were used to compare with the other 194 children with one CF-causing mutation and one isolated 5T allele [CF40mut/(TG)13-5T = 21, CF40mut/(TG)12-5T = 85, and CF40mut/(TG)11-5T = 88]. Among children with CF40mut/(TG)13-5T, 38% were diagnosed with CF by 8 years, based on sweat chloride results and clinical presentation. Six percent of those with CF40mut/(TG)12-5T, and none with CF40mut/(TG)11-5T, reached diagnostic criteria. CFTR (IVS8)-(TG)m-5T allele (TG) tract length determination provides valuable information in predicting the risk of developing a CF phenotype. Of the three types of 5T alleles evaluated, screen-positive children with genotype CF40mut/(TG)13-5T progressed from CRMS to CF at a high rate, while there was little evidence of clinical disease in those with CF40mut/(TG)11-5T. Additional data from longer follow-up intervals are needed to fully understand the natural history of individuals with a CF40mut/(TG)m-5T genotype.

Molecular screening of CFTR gene in Egyptian patients with congenital bilateral absence of the vas deferens: a preliminary study.

In the current study, we enrolled 14 Egyptian infertile males with isolated congenital bilateral absence of the vas deferens (CBAVD). Screening for the most commonly reported 36 CFTR mutations, and the intron 8 (T)n splice variant was performed by multiplex PCR followed by reversed hybridisation. Samples with the 5T variant were picked for DNA sequencing of intron 8/exon 9 region to identify the number of adjacent TG repeats. The p.Phe508del and the p.Ser1251Asn mutations were detected in heterozygous state in three patients (10.7% of alleles) and in one patient (3.6% of alleles), respectively, while the 5T variant was detected in five patients (28.6% of alleles). Among those five patients, four had TG12 repeats and one had TG13 repeats confirming the pathogenic penetrance of all 5T alleles in Egyptian CBAVD patients. The allelic frequencies of the mutations p.Phe508del, p.Ser1251Asn and the 5T variant in 60 Egyptian cystic fibrosis patients were 24.2%, 3.3% and 2.5% respectively. The mutation p.Ser1251Asn was detected for the first time in isolated CBAVD patient in our study. Due to the high prevalence of p.Phe508del mutation and 5T variant in Egyptian CBAVD patients, we recommend their screening initially, ideally followed by full CFTR gene sequencing in unidentified patients.

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

Identification of the first Fusarium sibiricum isolate in Iran and Fusarium langsethiae isolate in Siberia by morphology and species-specific primers.

Fusarium langsethiae is an European species, while Fusarium sibiricum is mainly distributed in northern Asia. Morphological characters alone do not allow a clear separation of F. langsethiae from F. sibiricum. The long TG repeat in the ribosomal IGS region is the only DNA sequence that has been used to design the species-specific primer pair CNL 12/PulvIGSr for the identification of F . sibiricum isolates. Another way to identify F . sibiricum is to use a combination of F. sporotrichioides- and F. langsethiae-specific primer pairs. The aim of this study was to find new ways for identifying and discriminating F. langsethiae from F. sibiricum. The paper also reports the first record of F. sibiricum from Iran, outside northern Asia and Norway, and the first isolation of F. langsethiae, an European pathogen, from western Siberia. Thus, the distribution of F. sibiricum and F. langsethiae may be much larger in Eurasia than what is currently known.

Multi-physiopathological consequences of the c.1392G>T CFTR mutation revealed by clinical and cellular investigations.

This study combines a clinical approach and multiple level cellular analyses to determine the physiopathological consequences of the c.1392G>T (p.Lys464Asn) CFTR exon 10 mutation, detected in a CF patient with a frameshift deletion in trans and a TG(11)T(5) in cis. Minigene experiment, with different TG(m)T(n) alleles, and nasal cell mRNA extracts were used to study the impact of c.1392G>T on splicing in both in cellulo and in vivo studies. The processing and localization of p.Lys464Asn protein were evaluated, in cellulo, by western blotting analyses and confocal microscopy. Clinical and channel exploration tests were performed on the patient to determine the exact CF phenotype profile and the CFTR chloride transport activity. c.1392G>T affects exon 10 splicing by inducing its complete deletion and encoding a frameshift transcript. The polymorphism TG(11)T(5) aggravates the effects of this mutation on aberrant splicing. Analysis of mRNA obtained from parental airway epithelial cells confirmed these in cellulo results. At the protein level the p.Lys464Asn protein showed neither maturated form nor membrane localization. Furthermore, the in vivo channel tests confirmed the absence of CFTR activity. Thus, the c.1392G>T mutation alone or in association with the TG repeats and the poly T tract revealed obvious impacts on splicing and CFTR protein processing and functionality. The c.[T(5); 1392G>T] complex allele contributes to the CF phenotype by affecting splicing and inducing a severe misprocessing defect. These results demonstrate that the classical CFTR mutations classification is not sufficient: in vivo and in cellulo studies of a possible complex allele in a patient are required to provide correct CFTR mutation classification, adequate medical counseling, and adapted therapeutic strategies.

Identification of Novel CELSR1 Mutations in Spina Bifida

Spina bifida is one of the most common neural tube defects (NTDs) with a complex etiology. Variants in planar cell polarity (PCP) genes have been associated with NTDs including spina bifida in both animal models and human cohorts. In this study, we sequenced all exons of CELSR1 in 192 spina bifida patients from a California population to determine the contribution of CELSR1 mutations in the studied population. Novel and rare variants identified in these patients were subsequently genotyped in 190 ethnically matched control individuals. Six missense mutations not found in controls were predicted to be deleterious by both SIFT and PolyPhen. Two TG dinucleotide repeat variants were individually detected in 2 spina bifida patients but not detected in controls. In vitro functional analysis showed that the two TG dinucleotide repeat variants not only changed subcellular localization of the CELSR1 protein, but also impaired the physical association between CELSR1 and VANGL2, and thus diminished the ability to recruit VANGL2 for cell-cell contact. In total, 3% of our spina bifida patients carry deleterious or predicted to be deleterious CELSR1 mutations. Our findings suggest that CELSR1 mutations contribute to the risk of spina bifida in a cohort of spina bifida patients from California.

Z-DNA-Forming TG Repeats are Dynamic Mechanical Switches Sensitive to Tension and Torsion

Although Z-DNA, the left-handed DNA, is an unstable state in comparison with B-DNA, ZDNA exists stably under certain conditions such as high salt concentrations or negative supercoiling. In biological systems, potential Z-DNA-forming sequences are located near the promoter region of many genes and are thought to play a role in transcription initiation. Recently we studied the B-Z transition in a short d(GC/GC)n repeats in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers[1]. In fact, another Z-DNA-forming sequence, d(CA/TG)n, is more frequently and widely found in eukaryotic genome and believed to have more important biological functions although the B-Z transition in that sequence is much less studied. Here, we examined the B-Z transition of the TG repeat sequence using the hybrid method from the mechanistic viewpoints. We found that negative supercoiling is more effective in inducing the Z-conformation than high salt concentrations. Even at room temperature, the sequence undergoes dynamic inter-conversion between the two states permitting direct determination of kinetic constants and implying smaller energy barrier between the states. Compared to the GC repeat, TG repeats required more torsional energy to trigger the transition and the repeat length dependence of the critical superhelicity provides quantitative information about the transition. In summary, this study provides the biophysical details of the transition and also demonstrates that physical factors such as tension and torsion play critical roles in biological phenomena

New variant of CFTR gene in recurrent idiopathic pancreatitis: Potential role of IVS-8 polyt

Background: Acute and chronic pancreatitis in childhood cause occasional and significant morbidity, but our understanding of pancreatic inflammation is rudimentary. Recent advances in cell biology, genetics and imaging technology provide hope that improved understanding of pancreatitis will facilitate novel therapeutic strategies. Examples include the identification of genes predisposing patients to pancreatitis. Genetic pancreatitis (GP) is associated with mutations of cystic fibrosis transmembrane conductor regulator gene (CFTR), cationic trypsinogen (PRSS1) gene, and serine protease inhibitor Kazal type 1 (SPINK1). In 1988, two groups reported an association between idiopathic chronic pancreatitis (ICP) and CFTR mutations. Several mild pancreatic sufficient CFTR mutations were found to be associated with ICP. Those with a single mutation are carriers and those who compound heterozygotes with one severe and one mild mutation are at risk of developing pancreatitis. Whereas a diverse range of loss-of-function CFTR variants have been reported to be associated with idiopathic pancreatitis, their functional effects remain to be clarified in most cases. The 5T variant is a stretch of five contiguous thymidines at the 3′ of the intron 8 of CFTR gene that exacerbates skipping of exon 9, resulting in reduced levels of functional CFTR protein. This process seems to be influenced by the number of TG repeats immediately adjacent to 5T. Individuals carrying 5T adjacent to either 12 or 13TG repeats are more likely to exhibit an abnormal phenotype with a risk of ICP. Materials and methods: We have investigated the cases of 4 children (aged 12, 10, 6 and 3 years) affected by ICP. The diagnosis of pancreatitis was made by the presence of typical abdominal pain, serum amylase and/or lipase 3 times greater than the upper limit of normal and characteristic imaging findings detected by US e CT (volume increase of the pancreas, inhomogeneous structure and peripancreatic fluid collection). Sequence analysis of PRSS1, SPINK1 and CFTR genes was performed. Results: Sequence analysis showed the absence of mutations in PRSS1 and SPINK1 genes. CFTR gene sequencing in three boys showed the presence of following IVS-8 polyT polymorphisms: 5T/5T 12TG/12TG; 5T/5T 11TG/12TG; 5T/9T 13TG/11TG. The 3-years old girl with the most severe form of idiopathic pancreatitis showed 5T/7T 13TG/9TG. In this girl the 5T allele, in cis with 13TG, was associated in trans with F508del, a severe CFTR mutation. Conclusions: Our results even in a limited number of cases have shown the high prevalence of 5T carriers that makes the assessment of the TG repeat number of great interest as a reliable predictor of the IPA risk. The knowledge of TG repeat number in individuals with 5T can be of diagnostic value. The TG repeat number proves to be a reliable predictor of penetrance for 5T but further studies are needed to comparing the prevalence of the 5T-poly(TG) in the general population. IPA in children remain enigmatic with more questions, but it's possible that 5T-poly (TG) has both a diagnostic and predictive role.

5 Large scale, high throughput, cystic fibrosis screening using a new kit for CFTR mutation detection

Cystic fibrosis (CF, OMIM 219700) is an autosomal recessive disorder caused by the presence of mutations in both alleles of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Until now, more than 1,900 CFTR gene mutations have been described. The most widespread mutation found is the c.1521_1523delCTT (p.Phe508del, F508del), with a high relative frequency in different populations. The frequency of the DeltaF508 mutation is estimated to about 50% of all CFTR mutations in southern Europe and up to 80−90% in the Northern European Countries. Other mutations typically have a population frequency below 5 percent, or are specific in certain ethnic groups. This project aims to analyze high number of consecutive anonymous newborn blood spots, from Tuscany and Umbria, Italy, for the detection of the most frequent mutations in the CFTR gene as well as the polythymidine and TG repeats of intron 9. The qualitative, high throughput, screening is performed using the Devyser CFTR Core Kit, based on PCR allele specific technology and capillary electrophoresis. The method has been optimized for automated processing to ensure the accuracy and the reliability of the results while minimizing the time and costs.

Thymol antifungal mode of action involves telomerase inhibition

The antifungal mode of action of thymol was investigated by a chemical-genetic profile analysis. Growth of each of ~4700 haploid Saccharomyces cerevisiae gene deletion mutants was monitored on medium with a subinhibitory concentration (50 μg/ml) of thymol and compared to growth on non-thymol control medium. This analysis revealed that, of the 76 deletion mutants with the greatest degree of susceptibility to thymol, 29% had deletions in genes involved in telomere length maintenance. A telomere restriction fragment (TRF) length assay showed that yeast exposed to a subinhibitory concentration of thymol for 15 days had telomere size reductions of 13-20% compared to non-thymol controls. By accelerating telomere shortening, thymol may increase the rate of cell senescence and apoptosis. Furthermore, real-time RT-PCR analysis revealed approximately two-fold reductions in EST2 mRNA but no change in TLC1 RNA in thymol-treated S. cerevisiae relative to untreated cells. EST2 encodes the essential reverse transcriptase subunit of telomerase that uses TLC1 RNA as a template during addition of TG(1-3) repeats to maintain telomere ends. This study provides compelling evidence that a primary mode of thymol antifungal activity is through inhibition of transcription of EST2 and thus telomerase activity.

Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG(1-3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase-mediated telomere repeat addition is negatively modulated by the levels of telomere-bound Rap1-Rif1-Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere-binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady-state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C-terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere-bound Est2 is increased, while telomere-bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.

Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species

The kelp or longtooth grouper (Epinephelus bruneus), which inhabits Eastern Asia, is the most economically important of 11 grouper species that inhabit the Southern Sea near Jeju Island in Korea. This species is listed as vulnerable by the International Union for the Conservation of Nature and Natural Resources because of a rapid decrease in its resources. We developed microsatellite markers for E. bruneus using the pyrosequencing technique for applications in resource management and aquaculture. In addition, we tested the cross-species transferability of the microsatellite markers in four species belonging to the Epinephelus genus. Among 66,452 simple sequence repeats, 64 loci containing more than eight CA or TG repeats were randomly selected for primer synthesis; 45 primer sets (75.0%) produced polymerase chain reaction (PCR) products of 100-300 bp and were selected as candidates. After primary testing with four E. bruneus fish, 28 polymorphic loci were selected as the final microsatellite markers, and 23 sets showing clear amplification of polymorphic loci were used to analyze 71 fish. These loci have allele numbers ranging from 2 to 23. Null alleles were detected at three loci, and three loci showed an excess of homozygotes in the Hardy-Weinberg equilibrium test. Of the three species used for cross-species transfer of these markers, Epinephelus moara showed the highest transferability (92.9%) and polymorphism (67.9%), followed by Epinephelus fuscoguttatus (75.0 and 67.9%, respectively) and Epinephelus septemfasciatus (57.1 and 46.4%, respectively). These results suggested that these microsatellite loci should be valuable tools for population genetic studies of the species Epinephelus.

CFTR polymorphisms of healthy individuals in two Chinese cities--Changchun and Nanjing.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel, cause cystic fibrosis. In order to investigate the polymorphic backgrounds of CFTR genes of healthy populations in different Chinese cities (Changchun and Nanjing), we analyzed 119 blood samples (Changchun 64, Nanjing 55) of randomly selected healthy individuals for poly T, TG-repeats and M470V polymorphisms. We analyzed the differences of CFTR polymorphic distributions between the two Chinese cities from the south and the north. Methods Genomic DNA was extracted from whole blood. DNA fragments of CFTR gene were amplified by polymerase chain reaction (PCR). Poly-T and TG repeats were directly sequenced by auto sequencer (ABI 310). M470V was detected by a HphI restriction enzyme. The T7 allele was the most common haplotype in Changchun (0.938) and Nanjing (0.927) populations. The T5 allele was present in only 7 Changchun and 3 Nanjing subjects. The TG11 and TG12 alleles were dominant haplotypes in Changchun (TG11 0.500, TG12 0.453) and Nanjing (TG11 0.345, TG12 0.609). The frequency of the V470 allele was 0.633 in Changchun, which was higher than that in Nanjing (0.500) (p < 0.05). There were three major haplotypes: T7-TG11-V470, T7-TG12-M470 and T7-TG12-V470. The T7-TG11-V470 was the most common haplotype in Changchun (0.514), while T7-TG12-M470 was the most common haplotype in Nanjing (0.500). Though Changchun and Nanjing are in the same country, their polymorphic backgrounds of CFTR gene are very different. Most of the two populations have genotypes that cause lower CFTR function.

The CFTR polymorphisms poly-T, TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.

Anticheckpoint pathways at telomeres in yeast

Telomeres hide (or 'cap') chromosome ends from DNA-damage surveillance mechanisms that arrest the cell cycle and promote repair, but the checkpoint status of telomeres is not well understood. Here we characterize the response in Saccharomyces cerevisiae to DNA double-strand breaks (DSBs) flanked by varying amounts of telomeric repeat sequences (TG(1-3)). We show that even short arrays of TG(1-3) repeats do not induce G2/M arrest. Both Rif1 and Rif2 are required for capping at short, rapidly elongating ends, yet are largely dispensable for protection of longer telomeric arrays. Rif1 and Rif2 act through parallel pathways to block accumulation of both RPA and Rad24, activators of checkpoint kinase Mec1 (ATR). Finally, we show that Rif function is correlated with an 'anticheckpoint' effect, in which checkpoint recovery at an adjacent unprotected end is stimulated, and we provide insight into the molecular mechanism of this phenomenon.

SWI/SNF-mediated chromatin remodeling induces Z-DNA formation on a nucleosome

BackgroundZ-DNA is a higher-energy, left-handed form of the double helix. A primary function of Z-DNA formation is to facilitate transcriptional initiation and activation. Sequences favoring Z-DNA formation are frequently located in promoter regions and Z-DNA is stabilized by torsional strain resulting from negative supercoiling, such as that generated by an actively transcribing polymerase or by a nucleosome remodeling event. We previously have shown that activation of the CSF1 gene by a chromatin remodeling event in the promoter results in Z-DNA formation at TG repeats within the promoter.ResultsWe show that remodeling of a mononucleosome by the human SWI/SNF complex results in Z-DNA formation when the DNA within the mononucleosome contains Z-DNA favoring sequence. Nuclease accessibility patterns of nucleosome core particle consisting of Z-DNA are quite different from counterpart nucleosomes containing classic B-DNA. Z-nucleosomes represent a novel mononucleosome structure.ConclusionsWe present evidence that Z-DNA can form on nucleosomes though previous observations indicate the occlusion of nucleosome formation from Z-DNA.

Subtelomere-binding protein Tbf1 and telomere-binding protein Rap1 collaborate to inhibit localization of the Mre11 complex to DNA ends in budding yeast

Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks that activate DNA damage checkpoints. In budding yeast, the Mre11-Rad50-Xrs2 (MRX) complex associates with DNA ends and promotes checkpoint activation. Rap1 binds to double-stranded telomeric regions and recruits Rif1 and Rif2 to telomeres. Rap1 collaborates with Rif1 and Rif2 and inhibits MRX localization to DNA ends. This Rap1-Rif1-Rif2 function becomes attenuated at shortened telomeres. Here we show that Rap1 acts together with the subtelomere-binding protein Tbf1 and inhibits MRX localization to DNA ends. The placement of a subtelomeric sequence or TTAGGG repeats together with a short telomeric TG repeat sequence inhibits MRX accumulation at nearby DNA ends in a Tbf1-dependent manner. Moreover, tethering of both Tbf1 and Rap1 proteins decreases MRX and Tel1 accumulation at nearby DNA ends. This Tbf1- and Rap1-dependent pathway operates independently of Rif1 or Rif2 function. Depletion of Tbf1 protein stimulates checkpoint activation in cells containing short telomeres but not in cells containing normal-length telomeres. These data support a model in which Tbf1 and Rap1 collaborate to maintain genomic stability of short telomeres.

A role for TDP-43 in tau gene expression regulation

Like in AD, abnormal tau protein aggregation and transcriptional deregulation plays an important role in a significant number of front temporal dementia (FTD) cases. A sub-group of FTD cases was shown to be associated with abnormal clumping of a seemingly unrelated protein called TDP-43. Mutations in TDP-43 gene are found in familial FTD and amyotrophic lateral sclerosis. TDP-43 is a member of hnRNP proteins and is directly involved in the expression process of several thousands genes. Any defect in TDP-43 could lead to alterations in the level of these proteins whose homeostasis is usually precisely regulated for healthy functioning of neurons. In a search for transcription factors acting as putative regulators of MAPT, we identified multiple putative binding sites for the DNA/RNA binding protein TDP-43 within the human tau gene. We over expressed Flag-TDP-43 in human neuroblastoma cells, both undifferentiated or differentiated. Total protein extracts from TDP-43 or mock-transfected cells were analyzed by Western blot for the total tau level, normalized to beta-actin. To look for a potential role of TDP-43 as a co-transcriptional regulator, we used luciferase reporter vectors for the MAPT core promoter regions containing the putative TDP-43 binding motif, alone or co-transfected together with Flag-TDP-43 or siRNA targeting TDP-43. Interestingly, we observed a bimodal effect of TDP-43 on tau gene expression. In undifferentiated cells, TDP-43 overexpression resulted in increased levels of tau protein whereas in retinoic acid differentiated cells we noticed the opposite effect. The appearance of tau higher molecular weight isoforms would also suggest that TDP-43 may affect splicing of the alternatively spliced MAPT exons. Using luciferase reporter vectors for the MAPT promoter regions containing the putative TDP-43 binding site, we show that overexpression of TDP-43 significantly represses MAPT transcription for all three MAPT haplotypes H1b, H1c and H2, whereas an opposite effect is observed knocking-down TDP-43. Our results suggest a crucial role for TDP-43 in tau gene expression regulation at both RNA and protein level, which might be dependent on the cellular differentiation state. Scheme of the human MAPT promoter proximal region, the arrow indicates the transcription starting site (TSS) associated to RefSeq gene annotations. The position of the putative binding site for TDP-43, which comprises a polymorphic 22x TG repeat, is highlighted by the black bar.Non-coding exon (-1) representing the MAPT core promoter is indicated by the bold blue lines. Luciferase reporter constructs used in our preliminary assays. Two conserved elements of sequences spanning the core promoter including the non-coding exon (-1) and proximal intronic sequences of human MAPT were cloned as previously reported (REF) upstream to a luciferase Luc2 reporter gene. The elements B1 and B2 were screened either separately (B1, B2) or together (B3). Effect of TDP-43 overexpression on MAPT promoter/luciferase activity: SK-N-F1 neuroblastoma cells were transfected in triplicate with the luciferase reporter vectors containing domains B1, B2 or B3 alone or in combination with a plasmid vector for expressing Flag-TDP-43 with a CMV promoter. Bars indicate relative firefly luciferase intensity normalised against Renilla luciferase from a co-transfected control plasmid. A pMIR plasmid was used as positive control. A t-test statistical analysis shows that in most of the cases, co-expression of pFlag-TDP-43 results in a significant repression of the transcriptional activity of the MAPT core promoter proximal elements. Western blot of total protein extracts from SK-N-F1 neuroblastoma cells undifferentiated or differentiated after 7 days of retinoic acid (RA) treatment, and transfected with increasing amounts of pFlag-TDP-43 (0.25, 0.5, 1, 2 and 3 μg of vector) or mock transfected with empty vector (0). Blots were probed with: (A) mouse anti-FLAG antibody (B) rabbit anti-tau (C) mouse anti-Beta actin (D) long exposure of blot shown in (B). Densitometric analysis total tau on Western blot: The vertical bar indicates the amounts (μg) of p-Flag-TDP-43 vector used for transfection of SK-N-F1 cells that were either differentiated (red) or not (blue) with retinoic acid. The ×-axis indicates total tau level normalised to beta-actin in arbitrary units. Gels were acquired as TIFF high-resolution images and processed by ImageJ Gel Analysis function. We observe that TDP-43 overexpression affects tau levels in a bi-modal manner depending on differentiation.

Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae

Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups.

Factors that influence telomeric oxidative base damage and repair by DNA glycosylase OGG1

Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non-telomeric double-stranded substrates. In addition, telomere repeat binding factors TRF1 and TRF2 do not impair OGG1 incision activity. Yet, 8-oxodG in some telomere structures ( e.g., fork-opening, 3′-overhang, and D-loop) were less effectively excised by OGG1, depending upon its position in these substrates. Collectively, our data indicate that the sequence context of telomere repeats and certain telomere configurations may contribute to telomere vulnerability to oxidative DNA damage processing.