Synergistic Deoxynucleoside and Gene Therapies for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2KI ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. METHODS: We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2KI , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides. RESULTS: AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan. INTERPRETATION: Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.

Exploring New Potential Anticancer Activities of the G-Quadruplexes Formed by [(GTG2T(G3T)3] and Its Derivatives with an Abasic Site Replacing Single Thymidine.

In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.

Mitochondrial Transcription Factor A Binds to and Promotes Mutagenic Transcriptional Bypass of O4-Alkylthymidine Lesions.

O2- and O4-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O2- and O4-n-butylthymidine (O2- and O4-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O2-nBudT- and O4-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O2-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O4-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O4-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O4-nBudT and O4-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O4-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.

Structural properties and anticoagulant/cytotoxic activities of heterochiral enantiomeric thrombin binding aptamer (TBA) derivatives.

The thrombin binding aptamer (TBA) possesses promising antiproliferative properties. However, its development as an anticancer agent is drastically impaired by its concomitant anticoagulant activity. Therefore, suitable chemical modifications in the TBA sequence would be required in order to preserve its antiproliferative over anticoagulant activity. In this paper, we report structural investigations, based on circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR), and biological evaluation of four pairs of enantiomeric heterochiral TBA analogues. The four TBA derivatives of the d-series are composed by d-residues except for one l-thymidine in the small TT loops, while their four enantiomers are composed by l-residues except for one d-thymidine in the same TT loop region. Apart from the left-handedness for the l-series TBA derivatives, CD and NMR measurements have shown that all TBA analogues are able to adopt the antiparallel, monomolecular, 'chair-like' G-quadruplex structure characteristic of the natural D-TBA. However, although all eight TBA derivatives are endowed with remarkable cytotoxic activities against colon and lung cancer cell lines, only TBA derivatives of the l-series show no anticoagulant activity and are considerably resistant in biological environments.

Engineering of high-precision base editors for site-specific single nucleotide replacement.

RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes.

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

T/T homozygosity of the tenascin-C gene polymorphism rs2104772 negatively influences exercise-induced angiogenesis.

BACKGROUND: Mechanical stress, including blood pressure related factors, up-regulate expression of the pro-angiogenic extracellular matrix protein tenascin-C in skeletal muscle. We hypothesized that increased capillarization of skeletal muscle with the repeated augmentation in perfusion during endurance training is associated with blood vessel-related expression of tenascin-C and would be affected by the single-nucleotide polymorphism (SNP) rs2104772, which characterizes the non-synonymous exchange of thymidine (T)-to-adenosine (A) in the amino acid codon 1677 of tenascin-C. METHODS: Sixty-one healthy, untrained, male white participants of Swiss descent performed thirty 30-min bouts of endurance exercise on consecutive weekdays using a cycling ergometer. Genotype and training interactions were called significant at Bonferroni-corrected p-value of 5% (repeated measures ANOVA). RESULTS: Endurance training increased capillary-to-fiber-ratio (+11%), capillary density (+7%), and mitochondrial volume density (+30%) in m. vastus lateralis. Tenascin-C protein expression in this muscle was confined to arterioles and venules (80% of cases) and increased after training in A-allele carriers. Prior to training, volume densities of subsarcolemmal and myofibrillar mitochondria in m. vastus lateralis muscle were 49% and 18%, respectively, higher in A/A homozygotes relative to T-nucleotide carriers (A/T and T/T). Training specifically increased capillary-to-fiber ratio in A-nucleotide carriers but not in T/T homozygotes. Genotype specific regulation of angiogenesis was reflected by the expression response of 8 angiogenesis-associated transcripts after exercise, and confirmed by training-induced alterations of the shear stress related factors, vimentin and VEGF A. CONCLUSION: Our findings provide evidence for a negative influence of T/T homozygosity in rs2104772 on capillary remodeling with endurance exercise.

Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions.

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.

Characterization of Staphylococcus aureus Strains Isolated from Czech Cystic Fibrosis Patients: High Rate of Ribosomal Mutation Conferring Resistance to MLS(B) Antibiotics as a Result of Long-Term and Low-Dose Azithromycin Treatment.

Staphylococcus aureus is one of the most frequent pathogens infecting the respiratory tract of patients with cystic fibrosis (CF). This study was the first to examine S. aureus isolates from CF patients in the Czech Republic. Among 100 S. aureus isolates from 92 of 107 observed patients, we found a high prevalence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics (56%). More than half of the resistant strains (29 of 56) carried a mutation in the MLS(B) target site. The emergence of MLS(B) resistance and mutations conferring resistance to MLS(B) antibiotics was associated with azithromycin treatment (p=0.000000184 and p=0.000681, respectively). Methicillin resistance was only detected in 3% of isolates and the rate of resistance to other antibiotics did not exceed 12%. The prevalence of small-colony variant (SCV) strains was relatively low (9%) and eight of nine isolates with the SCV phenotype were thymidine dependent. The study population of S. aureus was heterogeneous in structure and both the most prevalent community-associated and hospital-acquired clonal lineages were represented. Of the virulence genes, enterotoxin genes seg (n=52), sei (n=49), and sec (n=16) were the most frequently detected among the isolates. The PVL genes (lukS-PV and lukF-PV) have not been revealed in any of the isolates.

Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.

DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In this study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3 + 2] Huisgen cyclo-addition between an alkyne-functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI(-)-MS/MS sequencing of the extension products has revealed that post-lesion synthesis was highly error-prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.

Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii.

BACKGROUND: Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages. RESULTS: By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively). CONCLUSIONS: The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.

Detection and knockdown of microRNA-34a using thioacetamido nucleic acid.

Thioacetamido nucleic acids (TANA) contain a backbone modification of dinucleotides consisting of a 5-atom amide linker N3'-COCH2-S-CH2 at thymidine or thymidine-cytidine dimer blocks. Here, the chemical synthesis of the TANA linked 5-methyl-cytidine-cytidine ((Me)cc) dimer block and its incorporation into the DNA sequence, complementary to human microRNA 34 (miR-34) is described. Further, for the first time, we demonstrate the biological applications of TANA modified oligonucleotides in detection and intracellular knockdown of a cancer related microRNA in comparison with DNA containing locked nucleic acid (LNA) and 2'-O-methyl modifications. The human microRNA miR-34 is a pro-apoptotic microRNA under the transcriptional control of protein 53 (p53). It gets expressed in response to DNA damage and regulates several cell cycle and apoptosis related targets. Here, we show that the TANA modified antisense oligonucleotide binds specifically to miR-34a, allowing its detection using primer extension. We also show that, using the TANA modified antisense oligonucleotide against miR-34a, intracellular levels of miR-34 can be reduced, and consequently, the expression of its target oncogene V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN) is enhanced. Further, we assessed the toxicity and serum stability of the oligonucleotide to conclude that it is suitable for detection and modulation of the vital biomarker and tumor suppressor microRNA.

Structure of the oncoprotein Rcl bound to three nucleotide analogues.

Rcl is a novel N-glycoside hydrolase found in mammals that shows specificity for the hydrolysis of 5'-monophosphate nucleotides. Its role in nucleotide catabolism and the resulting production of 2-deoxyribose 5-phosphate has suggested that it might fuel cancer growth. Its expression is regulated by c-Myc, but its role as an oncoprotein remains to be clarified. In parallel, various nucleosides have been shown to acquire pro-apoptotic properties upon 5'-monophosphorylation in cells. These include triciribine, a tricyclic nucleoside analogue that is currently in clinical trials in combination with a farnesyltransferase inhibitor. Similarly, an N(6)-alkyl-AMP has been shown to be cytotoxic. Interestingly, Rcl has been shown to be inhibited by such compounds in vitro. In order to gain better insight into the precise ligand-recognition determinants, the crystallization of Rcl with these nucleotide analogues was attempted. The first crystal structure of Rcl was solved by molecular replacement using its NMR structure in combination with distantly related crystal structures. The structures of Rcl bound to two other nucleotides were then solved by molecular replacement using the previous crystal structure as a template. The resulting structures, solved at high resolution, led to a clear characterization of the protein-ligand interactions that will guide further rational drug design.

Poly thymidine polymorphism and cystic fibrosis in a non-Caucasian population.

BACKGROUND: Cystic fibrosis is a monogenic recessive disorder found predominantly in Caucasian population. This disease arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this study we consider poly T polymorphism c.1210-12T[5], c.1210-12T[7], c.1210-12T[9] (T{5}, T{7}, T{9}) in the intron 8 of CFTR gene in normal individuals and cystic fibrosis patients in the north of Iran. MATERIAL AND METHODS: 40 CF patients and 40 normal individuals were screened for poly T polymorphism in intron 8 of CFTR gene using Reverse Dot Blot method which was also used to detect p.Phe508del among CF patients. RESULTS: T{7} allele is the most prevalent in both normal and CF patients. Its abundance is approximately 75%. T{9} and T{5} represent approximately 20% and 5% of alleles respectively. T{7}/T{7} genotype is the most present in both normal and CF patients with 72.5% and 60% prevalence respectively. p.Phe508del was present in 13 CFTR alleles belonging to 7 patients with either homozygote T{9}/ T{9}, T{7}/ T{7} or compound heterozygote T{7}/ T{9} genotypes. CONCLUSION: Contrary to the Caucasians, T{7} allele is more frequent in Northern Iranian CF patients. The presence of p.Phe508del and T{7} allele in the same framework is reported for the first time in this part of the world. Further investigations of other populations will help to understand whether p.Phe508del arose by selection pressure in this part of the world or was imported from European countries. The abundance of T{5}, T{7}, T{9} alleles indicates that this polymorphism can be used as one of the informative markers for detection of normal and mutant alleles in prenatal diagnosis or carrier assessment in families with previous history of the disease in regions with high degree of CFTR mutation heterogeneity.

Cell fate control gene therapy based on engineered variants of human deoxycytidine kinase.

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

To investigate the biological effects of the O(2)-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), we have replicated a plasmid containing O(2)-methylthymidine (O(2)-Me-dT) or O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine (O(2)-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2-5% in most strains but increased 2-4-fold with SOS. In the SOS-induced wild type E. coli, O(2)-Me-dT and O(2)-POB-dT induced 21% and 56% mutations, respectively. For O(2)-POB-dT, the major type of mutation was T → G followed by T → A, whereas for O(2)-Me-dT, T → G and T → A occurred in equal frequency. For both lesions, T → C also was detected in low frequency. The T → G mutation was reduced in strains with deficiency in any of the three SOS polymerases. By contrast, T → A was abolished in the pol V(-) strain, while its frequency in other strains remained unaltered. This suggests that pol V was responsible for the T → A mutations. The potent mutagenicity of these lesions may be related to NNK mutagenesis and carcinogenesis.

[Comparison of the promoter activity of metabolism-related pabB and lpdA genes between Mycobacterium tuberculosis strains H37Rv and H37Ra].

OBJECTIVE: Comparative genomic study revealed mutations at the promoter regions of pabB and lpdA in avirulent Mycobacterium tuberculosis strain H37Ra compared to its virulent counterpart strain H37Rv. We used LacZ reporter system to test whether those mutations will affect promoter activity and its potential relationship to virulence attenuation of H37Ra. METHODS: Promoter regions of pabB and lpdA were predicted by the "Neural Network Promoter Prediction" method (http://www. fruitfly. org/seq_tools/promoter. html). Promoter sequences were PCR amplified and cloned into mycobacterial promoterless probe vector pMC210. Resultant recombinant plasmids were transformed into M. smegmatis by electroporation. The transcription activity of lacZ under the control of cloned promoters were monitored by Quantitative Real-Time RT-PCR. RESULTS: Quantitative Real Time PCR results showed that the promoter activity of H37Ra pabB was six times more than that of H37Rv pabB (p < 0.05), while the promoter activity of the H37Rv lpdA was two fold of that of H37Ra lpdA (p < 0.05). CONCLUSION: The mutations in pabB and lpdA promoters affect its expression activity. The T-A mutation in lpdA promoter may be related to virulence attenuation of H37Ra.

Role of TGF-beta1 (-509C>T) promoter polymorphism in susceptibility to cervical cancer.

Transforming growth factor-beta (TGF-beta) family members are multifunctional cytokines that play a key role in cellular growth, proliferation, and differentiation. The aim of study was to evaluate the association of TGF-beta1 -509 C>T gene polymorphism with risk of cervical cancer. The study was carried out in 150 histopathology confirmed patients with cervical cancer and 162 cervical-cytology negative females. Polymorphisms for TGF-beta1 -509C>T gene was genotyped by polymerase chain reaction and restriction enzyme digestion. Frequencies of individuals with -509TT genotype and T allele of TGF-beta1 gene polymorphisms did not differ significantly in patients with cervical cancer and controls (p = 0.328, OR = 1.37 and p = 0.605, OR = 1.09). Cervical cancer patients with -509TT had marginal low risk for stage I (p = 0.04, OR = 0.95, 95% CI = 0.91-0.99) but -509TT genotype of TGF-beta1 was associated with increased risk of stage II of cancer (p = 0.07, OR = 3.13, 95% CI = 0.87-11.14). In gene-environment interaction, carriers of TGF-beta1 -509TT genotype with tobacco usage were at higher risk of cervical cancer (OR = 3.67, 95% CI = 0.38-35.1). In conclusion, our data suggest that TGF-beta1 -509T allele confers marginal protection for early stage 1B but risk for stage II of cervical cancer.

A novel frameshift mutation in the EYA1 gene in a Korean family with branchio-oto-renal syndrome.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial cleft fistulae or cysts, preauricular pits, ear malformations, hearing loss, and renal anomalies. Mutations in the human homologue of the Drosophilia eyes absent gene (EYA1) are the most common cause of BOR syndrome. In this study, we found a Korean family showing clinical features of the disease. Mutation analysis of the EYA1 gene revealed a novel one-base-pair deletion resulting in truncated protein (c.321delT; p.Ala107fs). This is the first report of BOR syndrome caused by deletion mutation of the EYA1 gene in Korea.