DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.     

Selectivity and affinity of triplex-forming oligonucleotides containing 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine for recognizing AT base pairs in duplex DNA

We have used DNase I footprinting, fluorescence and ultraviolet (UV) melting experiments and circular dichroism to demonstrate that, in the parallel triplex binding motif, 2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine (bis-amino-U, BAU) has very high affinity for AT relative to all other Watson–Crick base pairs in DNA. Complexes containing two or more substitutions with this nucleotide analogue are stable at pH 7.0, even though they contain several C.GC base triplets. These modified triplex-forming oligonucleotides retain exquisite sequence specificity, with enhanced discrimination against YR base pairs (especially CG). These properties make BAU a useful base analogue for the sequence-specific creation of stable triple helices at pH 7.0.     

Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells.

It is widely accepted that most cell types efficiently exclude oligonucleotides in vitro and require specific delivery systems, such as cationic lipids, to enhance uptake and subsequent antisense effects. Oligonucleotides are not readily transfected into leukaemia cell lines using cationic lipid systems and streptolysin O (SLO) is used to effect their delivery. We wished to investigate the optimal oligonucleotide composition for antisense efficacy and specificity following delivery into leukaemia cells using SLO. For this study the well characterised chronic myeloid leukaemia cell line KYO-1 was selected and oligonucleotides (20mers) were targeted to an empirically identified accessible site of c- myc mRNA. The efficiency and specificity of antisense effect was measured 4 and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne phosphodiester and phosphorothioate compounds were found to present substantial non-specific effects at 20 microM but were inactive at 0.2 microM. Indeed, no antisense-specific effect was noted at any concentration at either time. All of the other oligonucleotides tested induced some measurable antisense effect, except 7 (chimeric, all-phosphorothioate, 2'-methoxyethoxy termini) which was essentially inactive at 20 microM. The rank efficiency order of the remaining antisense compounds was 4 = 3 >> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects induced by the chimeric methylphosphonate-phosphodiester compounds were found to be highly specific. Increased phosphorothioate content in the oligonucleotide backbone correlated with reduced antisense activity (efficacy: 2'-methoxyethoxy series 9 >> 8 >> 7, 2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained for increased activity correlating with increased oligonucleotide-mRNA heteroduplex thermal stability. In conclusion, the chimeric methylphosphonate-phosphodiester oligodeoxynucleotides present the most favourable characteristics of the compounds tested, for efficient and specific antisense suppression of gene expression following SLO-mediated delivery.     

Association with litter size of new polymorphisms on ESR1 and ESR2 genes in a Chinese-European pig line

The objective of this study was to search for polymorphisms in the coding region of the estrogen receptors 1 and 2 (ESR1 and ESR2 )and to analyze the effects of these variants and the well known intronic ESR1 Pvu II polymorphism on litter size in a Chinese-European pig line. We identified five silent single nucleotide polymorphisms (SNP) in the ESR1 cDNA: c.669T > C (exon 3), c.1227C > T (exon 5), c.1452C > T (exon 7), c.1665T > C and c.1755A > G (exon 8). One pair of these SNP (c.1665T > C and c.1755A > G) co-segregated in the analyzed line, and the SNP c.669T > C showed the same segregation pattern as the Pvu II polymorphism. These polymorphisms were tested in this study, although the c.1452C > T SNP within exon 7 was not analyzed due to its low informativeness. In the ESR2 cDNA, one missense SNP was found within exon 5, which caused an amino acid substitution in the coded protein: "c.949G > A (p.Val317Met)" and was tested on sow litter size. Information on 1622 litter records from 408 genotyped sows was analyzed to determine whether these SNP influenced the total number of piglets born (TNB) or the number of born alive (NBA). The polymorphisms ESR1: [Pvu II; c.669T > C], ESR1: [c.1665T > C; c.1755A > G] and ESR2: c.949G > A showed no statistically significant association with litter size. However, the ESR1: c.1227T allele was significantly associated with TNB. The additive substitution effect was estimated to be 0.40 piglets born per litter (P < 0.03), and no dominance effects were observed. This SNP could be useful in assisted selection for litter size in some pig lines, as a new genetic marker in linkage disequilibrium with the causative mutation.     

Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.     

Comparison of antiparallel A.AT and T.AT triplets within an alternate strand DNA triple helix.

We have examined the formation of alternate strand triple-helices at the target sequence A11(TC)6.(GA)6T11 using the oligonucleotides T11(AG)6 and T11(TG)6, by DNase I footprinting. These third strands were designed so as to form parallel T.AT triplets together with antiparallel G.GC and A.AT or T.AT triplets. We find that, although both oligonucleotides yield clear footprints at similar concentrations (0.3 microM) in the presence of manganese, only T11(TG)6 forms a stable complex in magnesium-containing buffers, albeit at a higher concentration (10-30 microM). Examination of the interaction of (AG)6 and (TG)6 with half the target site confirmed that the complex containing A.AT triplets was only stable in the presence of manganese. In contrast no binding of (TG)6 was detected in the presence of either metal ion, suggesting that the reverse-Hoogsteen T.AT triplet is less stable that G.GC. We suggest that, within the context of G.GC triplets, the rank order of antiparallel triplet stability is A.AT (Mn2+) > T.AT (Mn2+) > T.AT (Mg2+) > A.AT (Mg2+). Third strands containing a single base substitution in the centre of either the parallel or antiparallel portion showed a (10-fold) weaker interaction in manganese-containing buffers, and no interaction in the presence of magnesium.     

Thermodynamic stability of base pairs between 2-hydroxyadenine and incoming nucleotides as a determinant of nucleotide incorporation specificity during replication

We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5′-GA*C-3′ and 5′-TA*A-3′, A* represents 2-OH-Ade). When an A*–N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*–N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5′-GA*C-3′ sequences and T > A > C > G in the 5′-TA*A-3′ sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5′-GA*C-3′ and 5′-TA*A-3′ sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*–N base pair may be an important factor for the mutation spectra of 2-OH-Ade.     

Full Genome Characterization of the Culicoides-Borne Marsupial Orbiviruses: Wallal Virus,Mudjinbarry Virus and Warrego Viruses

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.     

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.     

Effect of CYP2B6 Gene Polymorphisms on Efavirenz Plasma Concentrations in Chinese Patients with HIV Infection

Objectives

The main aim of this study was to investigate the effect of CYP2B6 gene polymorphisms on efavirenz (EFV) plasma concentrations in Han Chinese patients with human immunodeficiency virus (HIV) infection.

Methods

In total, 322 patients were recruited for study. EFV plasma concentrations at steady-state were determined using high-performance liquid chromatography. Genotyping for seven single nucleotide polymorphisms (SNPs), including 171+967C>A, 171+3212C>T, 171+4335T>C, 516G>T, 785A>G, 1295-913G>A, and *1355A>G of CYP2B6, was performed using ligase detection reaction (LDR). SPSS 18.0 and Haploview 4.2 were applied for statistical analyses.

Results

The average EFV concentration of patients was 2.35±2.09 μg/mL. Overall, 22% patients displayed EFV concentrations out of the therapeutic range of 1–4 μg/mL (13.1% < 1 μg/mL, 9.3% > 4 μg/mL). We observed significant association of 171+967C>A, 171+4335T>C, 516G>T, 785A>G and *1355A>G with high plasma EFV levels (p<.01). The predictive accuracy values of 171+4335CC, 516TT and 785GG for EFV concentrations > 4 μg/mL were 56.7%, 56.7% and 60%, respectively. We observed strong linkage disequilibrium for 171+967C>A, 171+4335T>C, 516G>T and 785A>G, resulting in five haplotypes. The frequencies of the five haplotypes (high to low) were as follows: CCTG (0.328), ACTG (0.280), ACCT (0.189), ATTG (0.186) and ACCG (0.017). The frequency of CCTG (0.524) in patients with EFV plasma concentrations < 1 μg/mL was significantly higher than that in other patient groups, while that of ACCT (0.733) was significantly higher in patients with EFV concentrations > 4 μg/mL, relative to other patient groups. Average EFV concentrations of patients carrying ACTG (1.78 μg/mL), ACCT (7.50 μg/mL), and ATTG (1.92 μg/mL) haplotypes were markedly higher than those of patients carrying the CCTG haplotype. The predictive accuracy of ACCT for EFV > 4 μg/mL was 81%.

Conclusions

Chinese patients administered standard doses of EFV require therapeutic drug monitoring or personalized medication management. Based on the current findings, we propose that 171+4335T>C, 516G>T, 785A>G and haplotype ACCT may be effectively used as genomic markers for EFV, which should aid in improving the efficacy of EFV-containing treatments and reduce the incidence of adverse reactions.     

Real-time closed tube single nucleotide polymorphism (SNP) quantification in pooled samples by quencher extension (QEXT)

Quencher extension (QEXT) is a novel single step closed tube real-time method to quantify SNPs using reporters and quenchers in combination with primer extension. A probe with a 5′-reporter dye is single base extended with a dideoxy nucleotide containing a quencher dye if the target SNP allele is present. The extension is recorded from the quenching (reduced fluorescence) of the reporter dye. This avoids the influence of the unincorporated dye-labeled nucleotides, resulting in high accuracy and a high signal-to-noise ratio. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermo-cycling reaction. We tested the QEXT assay using five SNPs in the Listeria monocytogenes inlA gene as a model system. The presence of the target SNP alleles was determined with high statistical confidence (P < 0.0005). The quantitative detection limits were between 0 and 5% for the targeted SNP alleles on a background of other SNP alleles (P < 0.05). The QEXT method is directly adaptable to current real-time PCR equipment and is thus suited for high throughput and a wide application range.     

Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro.

Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.     

Actions of a Proline Analogue,L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas'' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC₅₀ = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.     

Glycerin supplementation strategies for three or seven days affects oxidative stress,follicle dynamics and ovulatory response in Morada Nova sheep

This study examined the effect of glycerin supply strategies in different short-term protocols on follicular dynamics and ovulatory rate in Morada Nova sheep. Eighteen Morada Nova ewes with body condition > 2.9 had their estrus and follicular waves synchronized using three injections of prostaglandin analogue at seven-day intervals. All animals received the same diet during 21 days, which consisted of a total mixed ration (TMR) based on chopped elephant grass and concentrate twice daily. In the control group (n=9), ewes were fed the TMR diet. In the other four groups, ewes received 150 mL of glycerol daily, supplied as an oral drench or mixed in the TMR during three or seven days prior to the application of the third PGF2 alfa analogue. These groups were named as follows: Drench3d (n=10), Drench7d (n=8), TMR3d (n=9) and TMR7d (n=9). Follicle dynamics were monitored by ultrasonography, and plasma glucose and glutathione peroxidase levels were measured at the third prostaglandin administration. Six days after the final PGF2 alfa analogue dose, ovulatory rate was measured by laparoscopy. Glucose was higher (P< 0.001) in the glycerin-treated groups than in control group (83.7 ± 1.7 vs. 68.4 ± 4.5 mg. dL^-1; P < 0.001). Ewes in the TMR3d, Drench7d and TMR7d groups had a greater (P < 0.001) number of large follicles (≥ 3 < 5 mm), and the presence of follicles larger than 5 mm was observed. In the same groups, at the third PGF2 alfa analogue dose, a greater (P < 0.001) number of growing follicles (> 3 mm) and a larger size of the largest follicle (P < 0.001) were also recorded. Ovulation rate was 30% higher in the groups that received glycerin for seven days (1.6 ± 0.1 53 vs. 1.1 ± 0.1; P < 0.05), and they also exhibited a 38% reduction in glutathione peroxidase. Thus, the use of glycerin in Morada Nova sheep as a source of energy in short-term supplementation for increase ovulation rate is an efficient strategy when provided for seven days, either orally or in the feed.     

A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5′ ‘HEAD’ region and a 3′ region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1–2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5′ nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.     

Mutation Screening of 1,237 Cancer Genes across Six Model Cell Lines of Basal-Like Breast Cancer

Basal-like breast cancer is an aggressive subtype generally characterized as poor prognosis and lacking the expression of the three most important clinical biomarkers, estrogen receptor, progesterone receptor, and HER2. Cell lines serve as useful model systems to study cancer biology in vitro and in vivo. We performed mutational profiling of six basal-like breast cancer cell lines (HCC38, HCC1143, HCC1187, HCC1395, HCC1954, and HCC1937) and their matched normal lymphocyte DNA using targeted capture and next-generation sequencing of 1,237 cancer-associated genes, including all exons, UTRs and upstream flanking regions. In total, 658 somatic variants were identified, of which 378 were non-silent (average 63 per cell line, range 37–146) and 315 were novel (not present in the Catalogue of Somatic Mutations in Cancer database; COSMIC). 125 novel mutations were confirmed by Sanger sequencing (59 exonic, 48 3’UTR and 10 5’UTR, 1 splicing), with a validation rate of 94% of high confidence variants. Of 36 mutations previously reported for these cell lines but not detected in our exome data, 36% could not be detected by Sanger sequencing. The base replacements C/G>A/T, C/G>G/C, C/G>T/A and A/T>G/C were significantly more frequent in the coding regions compared to the non-coding regions (OR 3.2, 95% CI 2.0–5.3, P<0.0001; OR 4.3, 95% CI 2.9–6.6, P<0.0001; OR 2.4, 95% CI 1.8–3.1, P<0.0001; OR 1.8, 95% CI 1.2–2.7, P = 0.024, respectively). The single nucleotide variants within the context of T[C]T/A[G]A and T[C]A/T[G]A were more frequent in the coding than in the non-coding regions (OR 3.7, 95% CI 2.2–6.1, P<0.0001; OR 3.8, 95% CI 2.0–7.2, P = 0.001, respectively). Copy number estimations were derived from the targeted regions and correlated well to Affymetrix SNP array copy number data (Pearson correlation 0.82 to 0.96 for all compared cell lines; P<0.0001). These mutation calls across 1,237 cancer-associated genes and identification of novel variants will aid in the design and interpretation of biological experiments using these six basal-like breast cancer cell lines.     

Identification of 5-formyluracil DNA glycosylase activity of human hNTH1 protein

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The elucidation of repair mechanisms for 5-foU will yield important insights into the biological consequences of the lesion. Recently, we reported that 5-foU is recognized and removed from DNA by Escherichia coli enzymes Nth (endonuclease III), Nei (endonuclease VIII) and MutM (formamidopyrimidine DNA glycosylase). Human cells have been shown to have enzymatic activities that release 5-foU from X-ray-irradiated DNA, but the molecular identities of these activities are not yet known. In this study, we demonstrate that human hNTH1 (endonuclease III homolog) has a DNA glycosylase/AP lyase activity that recognizes 5-foU in DNA and removes it. hNTH1 cleaved 5-foU-containing duplex oligonucleotides via a β-elimination reaction. It formed Schiff base intermediates with 5-foU-containing oligonucleotides. Furthermore, hNTH1 cleaved duplex oligonucleotides containing all of the 5-foU/N pairs (N = G, A, T or C). The specific activities of hNTH1 for cleavage of oligonucleotides containing 5-foU and thymine glycol were 0.011 and 0.045 nM/min/ng protein, respectively. These results indicate that hNTH1 has DNA glycosylase activity with the potential to recognize 5-foU in DNA and remove it in human cells.     

Role of the DLGAP2 Gene Encoding the SAP90/PSD-95-Associated Protein 2 in Schizophrenia

Aberrant synaptic dysfunction is implicated in the pathogenesis of schizophrenia. The DLGAP2 gene encoding the SAP90/PSD-95-associated protein 2 (SAPAP2) located at the post-synaptic density of neuronal cells is involved in the neuronal synaptic function. This study aimed to investigate whether the DLGAP2 gene is associated with schizophrenia. We resequenced the putative promoter region and all the exons of the DLGAP2 gene in 523 patients with schizophrenia and 596 non-psychotic controls from Taiwan and conducted a case-control association analysis. We identified 19 known SNPs in this sample. Association analysis of 9 SNPs with minor allele frequency greater than 5% showed no association with schizophrenia. However, we found a haplotype (CCACCAACT) significantly associated with schizophrenia (odds ratio:2.5, p<0.001). We also detected 16 missense mutations and 1 amino acid-insertion mutation in this sample. Bioinformatic analysis showed some of these mutations were damaging or pathological to the protein function, but we did not find increased burden of these mutations in the patient group. Notably, we identified 5 private rare variants in 5 unrelated patients, respectively, including c.−69+9C>T, c.−69+13C>T, c.−69+47C>T, c.−69+55C>T at intron 1 and c.−32A>G at untranslated exon 2 of the DLGAP2 gene. These rare variants were not detected in 559 control subjects. Further reporter gene assay of these rare variants except c.−69+13C>T showed significantly elevated promoter activity than the wild type, suggesting increased DLGAP2 gene expression may contribute to the pathogenesis of schizophrenia. Our results indicate that DLGAP2 is a susceptible gene of schizophrenia.