Genetic analysis of 21 cases of methylmalonic acidemia / 中华医学遗传学杂志

OBJECTIVE@#To carry out genetic analysis for 21 patients with methylmalonic acidemia (MMA) and provide genetic counseling for their families.@*METHODS@#Next generation sequencing (panel) was used to detect the pathogenic variants underlying the disease.@*RESULTS@#In total 29 variant sites of MMUT, MMAA, MMUT were identified in the 21 patients, with common variants including c.323G>A (10%), c.917C>T (10%), c.984delC (10%) of MMUT gene, and c.609G>A (45%), c.80A>G (10%) , c.567dupT (10%) of MMACHC gene. Among these, c.2000A>G of MMUT, c.298G>T of MMACHC and c.734-7A>G of MMAA gene were unreported previously.@*CONCLUSION@#Genetic testing for MMA patients can clarify the cause of the disease and provide a basis for the clinical diagnosis. Discovery of novel variants has enriched the mutational spectrum of MMA.

Pre-conception carrier screening for 21 inherited metabolic diseases in a Chinese population / 中华医学遗传学杂志

OBJECTIVE@#To determine the carrier rate for 21 inherited metabolic diseases among a Chinese population of childbearing age.@*METHODS@#A total of 897 unrelated healthy individuals (including 143 couples) were recruited, and DNA was extracted from their peripheral blood samples. Whole exome sequencing (WES) was carried out to screen potential variants among 54 genes associated with 21 inherited metabolic diseases. Pathogenic and likely pathogenic variants and unreported loss-of-function variants were analyzed.@*RESULTS@#One hundred fourty types of pathogenic/likely pathogenic variants (with an overall number of 183) and unreported loss-of-function variants were detected, which yield a frequency of 0.20 per capita. A husband and wife were both found to carry pathogenic variants of the SLC25A13 gene and have given birth to a healthy baby with the aid of preimplantation genetic diagnosis. The detected variants have involved 40 genes, with the most common ones including ATP7B, SLC25A13, PAH, CBS and MMACHC. Based on the Hardy-Weinberg equilibrium, the incidence of the 21 inherited metabolic diseases in the population was approximately 1/1100, with the five diseases with higher incidence including citrullinemia, methylmalonic acidemia, Wilson disease, glycogen storage disease, and phenylketonuria.@*CONCLUSION@#This study has preliminarily determined the carrier rate and incidence of 21 inherited metabolic diseases among a Chinese population of childbearing age, which has provided valuable information for the design of neonatal screening program for inherited metabolic diseases. Pre-conception carrier screening can provide an important measure for the prevention of transmission of Mendelian disorders in the population.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Over-expression of dehydroascorbate reductase enhances oxidative stress tolerance in tobacco

Background: Ascorbic acid (Asc) is one of the most abundant antioxidants and it serves as a major contributor to protect plants against oxidative damage. Plants use two enzymes that participate in the metabolic recycling of Asc. One of these two enzymes is dehydroascorbate reductase (DHAR). It directly regenerates Asc from its oxidized state and thus prevents Asc from being irreversibly hydrolyzed to 2, 3-diketogulonic acid. This study aimed to examine whether over-expression of DHAR leads to an enhanced oxidative stress tolerance in tobacco plants. Results: In this study, we functionally characterized a novel JcDHAR gene from Jatropha curcas and found via quantitative RT-PCR analysis that JcDHAR can be induced with H2O2, salt and PEG stresses. The DHAR activities of transgenic tobacco plants increased from 2.0 to 5.3 fold compared to wild-type plants. As a result, the transgenic plants displayed enhanced tolerance to oxidative stress. Conclusions: Our results indicate that JcDHAR expression can effectively enhance the tolerance to oxidative stress in plants.

GenoType MTBDR plus 1.0 ® para la detección de resistencia cruzada entre isoniacida y etionamida en aislamientos de Mycobacterium tuberculosis multirresistentes / GenoType MTBDR plus 1.0 ® for the detection of cross-resistance between isoniazide and ethionamide in isolates of multidrug-resistant Mycobacterium tuberculosis

Introducción. Una parte de los aislamientos de Mycobacterium tuberculosis multirresistente también presenta resistencia a la etionamida. Es importante determinar si la resistencia a la isoniacida es independiente o se cruza con la resistencia a la etionamida, ya que si sucede lo segundo habría que reevaluar el tratamiento antituberculoso de segunda línea. La prueba molecular GenoType MTBDR plus ® detecta las mutaciones asociadas con la resistencia a isoniacida y podría detectar la resistencia cruzada a la etionamida. Objetivo. Evaluar la prueba GenoType MTBDR plus ® y comparar su desempeño con el de la secuenciación, en la detección de mutaciones en el gen katG y en el promotor inhA en aislamientos clínicos de M. tuberculosis multirresistente. Materiales y métodos. Se utilizaron el estuche comercial GenoType MTBDR plus 1.0 ® y la secuenciación para evaluar mutaciones en el gen katG y en el promotor inhA en 30 aislamientos de M. tuberculosis multirresistente con resistencia a la etionamida. La cepa de laboratorio H37Rv y tres aislamientos sensibles a los medicamentos de primera línea, sirvieron de control. Resultados. Al comparar los resultados de la secuenciación y de la prueba GenoType MTBDR plus ® , el índice kappa fue de 1. Todos los aislamientos resistentes a la isoniacida y la etionamida tenían las mutaciones detectadas con la prueba GenoTypeMTBDR plus ® en el gen katG, y 40 % de ellos, las detectadas en el promotor inhA. Mediante secuenciación se encontraron, además, mutaciones en katG en posiciones diferentes a las detectadas por la prueba GenoType MTBDR plus ® . Conclusión. La prueba GenoTypeMTBDR plus ® tiene la capacidad de detectar rápidamente la resistencia a isoniacida. Además, los resultados del estudio sugieren que también podría utilizarse como prueba de tamización para detectar la resistencia cruzada a etionamida.

Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide. Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis . Materials and methods: The GenoType MTBDR plus 1.0 ® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls. Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus ® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus ® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus ® . Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.

Photo-fermentational hydrogen production of Rhodobacter sp. KKU-PS1 isolated from an UASB reactor

Background In this study, the detection of nifH and nifD by a polymerase chain reaction assay was used to screen the potential photosynthetic bacteria capable of producing hydrogen from five different environmental sources. Efficiency of photo-hydrogen production is highly dependent on the culture conditions. Initial pH, temperature and illumination intensity were optimized for maximal hydrogen production using response surface methodology with central composite design. Results Rhodobacter sp. KKU-PS1 (GenBank Accession No. KC478552) was isolated from the methane fermentation broth of an UASB reactor. Malic acid was the favored carbon source while Na-glutamate was the best nitrogen source. The optimum conditions for simultaneously maximizing the cumulative hydrogen production (Hmax) and hydrogen production rate (Rm) from malic acid were an initial of pH 7.0, a temperature of 25.6°C, and an illumination intensity of 2500 lx. Hmax and Rm levels of 1264 ml H2/l and 6.8 ml H2/L-h were obtained, respectively. The optimum initial pH and temperature were further used to optimize the illumination intensity for hydrogen production. An illumination intensity of 7500 lx gave the highest values of Hmax (1339 ml H2/l) and Rm (12.0 ml H2/L-h) with a hydrogen yield and substrate conversion efficiency of 3.88 mol H2/mol malate and 64.7%, respectively. Conclusions KKU-PS1 can produce hydrogen from at least 8 types of organic acids. By optimizing pH and temperature, a maximal hydrogen production by this strain was obtained. Additionally, by optimizing the light intensity, Rm was increased by approximately two fold and the lag phase of hydrogen production was shortened.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Characterization of mercury-resistant clinical Aeromonas species

Mercury-resistant Aeromonas strains isolated from diarrhea were studied. Resistance occurs via mercuric ion reduction but merA and merR genes were only detected in some strains using PCR and dot hybridization. Results indicate a high variability in mer operons in Aeromonas. To our knowledge, this is the first report of mercury-resistant clinical Aeromonas strains.

Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants

Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed.

Detection of genomic mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis isolates using polymerase chain reaction and multiplex allele-specific polymerase chain reaction

OBJECTIVE: Isoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance. METHODS: We characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method. RESULTS: The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7 percent of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15 percent) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526). CONCLUSION: The results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases.

Clinical data and molecular analysis of Mycobacterium tuberculosi isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.

Pharmacogenetics of glucocorticoid replacement could optimize the treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

INTRODUCTION: 21-hydroxylase deficiency is an autosomal recessive disorder that causes glucocorticoid deficiency and increased androgen production. Treatment is based on glucocorticoid replacement; however, interindividual variability in the glucocorticoid dose required to achieve adequate hormonal control has been observed. OBJECTIVE: The present study aimed to evaluate the association between polymorphic variants involved inglucocorticoid action and/or metabolism and the mean daily glucocorticoid dose in 21-hydroxylase deficiency patients. METHODS: We evaluated 53 patients with classical forms of 21-hydroxylase deficiency who were receiving cortisone acetate. All patients were between four and six years of age and had normal androgen levels. RESULTS: The P450 oxidoreductase A503V, HSD11B1 rs12086634, and CYP3A7*1C variants were found in 19 percent, 11.3 percent and 3.8 percent of the patients, respectively. The mean ± SD glucocorticoid dose in patients with the CYP3A7*1C and wild-type alleles was 13.9 ± 0.8 and 19.5 ± 3.2 mg/m²/d, respectively. We did not identify an association between the P450 oxidoreductase or HSD11B1 allelic variants and the mean glucocorticoid dose. CONCLUSION: Patients carrying the CYP3A7*1C variant required a significantly lower mean glucocorticoid dose. Indeed, the CYP3A7*1C allele accounted for 20 percent of the variability in the cortisone acetate dose. The analysis of genes involved in glucocorticoid metabolism may be useful in the optimization of treatment of 21-hydroxylase deficiency.

Detection of Isoniazid and Rifampicin Resistance by Sequencing of katG, inhA, and rpoB Genes in Korea / 대한진단검사의학회지

BACKGROUND: In Korea, tuberculosis is resistant to isoniazid (INH) and/or rifampicin (RIF) in more than 10% of cases. To prevent the spread of resistant Mycobacterium tuberculosis strains, it is crucial to develop more rapid resistance detection methods. METHODS: To determine the feasibility of using direct sequencing for detecting INH- and RIF-resistant strains, the katG gene, the regulatory region of the inhA gene, and the 81-bp hot-spot region of the rpoB gene from 95 culture isolates and 46 respiratory specimens were sequenced. Total 141 culture isolates were classified by conventional drug susceptibility testing (DST) as INH(R)/RIF(R) (N=30), INH(R)/RIF(S) (N=23), INH(S)/RIF(R) (N=15), and INH(S)/RIF(S) (N=73). RESULTS: Compared with phenotypic DST, the overall sensitivity and specificity of sequencing were 83.0% (44/53) and 96.6% (85/88), respectively, for INH resistance, and 93.3% (42/45) and 100% (96/96), respectively, for RIF resistance. The rates were similar between culture isolates and respiratory specimens. Interestingly, three specimens with inhA -15C>T mutation were susceptible to INH by conventional DST. CONCLUSIONS: Detection of mutations in the katG codon 315, the inhA regulatory region, and the hot-spot region of rpoB would be useful for rapid detection of INH and RIF resistance in Korea.

[Enhansment of Biodesulfurization activity via the over expression of the oxidoreductase gene from R.erytheropolis IGTS8 using pet 21 a vector]

Biological desulforezation is a microbial procedure which uses manipulated bacterial strains for increasing of desulforezation from petroleum products. The method is being tested by several big oil companies for making a safe fuel and for preventing air pollution one of the methods for enhancement of desulforezation of microbial oil strains is increasing of copy number of oxidoreductase gene, this gene is able to supply the required FMNH2 for desulforezation genes of Rhodococcus erythropolis IGTS8, which is used as a model micro organism in desulforezation researches, was amplified with PCR and was cloned in to the PTZ57R vector. Then the oxidoreductase gene was extracted by agarose gel DNA extraction kit as a 600 bp fragment and was inserted in to the pkk223-3 expression vector. After induction, the desired protein was weakly detected as a 25 KD band in SDS-PAGE gel. However using another expression vector [PET 21a], a sharp band was detected in the same area. At present, the studies was focused on increasing the production in National Instutute of genetic Engineering and bio technology we are trying to apply genetic Engineering tools to increase gene dosage of oxidoreductase gene to enhancement of desulforezation gene expression

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da via em resposta à relação NADP/NADPH, é codificada por um número de genes por genoma haplóide e é induzida até 46-vezes por peróxido de hidrogênio em trypomastigotas metacíclicos. Os genes que codificam 6-fosfogluconolactonase, 6-fosfogluconato desidrogenase, transaldolase e transcetolase estão presentes no clone CL Brener como cópia única por genoma haplóide. 6-fosfogluconato desidrogenase é muito instável, mas foi estabilizada introduzindo duas pontes salinas por mutagênese sítio-dirigida. A Ribose-5-fosfato isomerase pertence ao Tipo B; genes que codificam enzimas Tipo A, presentes em mamíferos estão ausentes. A Ribulose-5-fosfato epimerase é codificada por dois genes. As enzimas da via têm um componente citosólico principal, embora várias delas tenham uma localização glicosomal secundária e também, localizações em menor número em outras organelas.

Molecular studies of E. coli mercuric reductase gene (merA) and its impact on human health.

Bacterial plasmids encode resistance systems for toxic metal ions including Hg2+ functioning by energy-dependent efflux of toxic ions. The inducible mercury resistance (mer) operon encodes both a mercuric ion uptake and a detoxification enzymes. In Gram-negative bacteria especially in E. coli, a periplasmic protein, MerP, an inner- membrane transport protein, MerT, and a cytoplasmic enzyme, mercuric reductase (the MerA protein), are responsible for the transport of mercuric ions into cell and their reduction to elemental mercury, Hg0. Phytoremediation involves the use of plants to extract, detoxify and/or sequester environmental pollutants from soil and water. Transgenic plants cleave mercury ions from methyl-mercury complexes; reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve less toxic elemental mercury. PCR were performed to detect 1695 bp of mercuric reductase gene (merA), which is mainly responsible for the conversion of mercuric (Hg+2) and mercurous (Hg+1) ions into non-toxic elemental mercury. PCR products of putative merA genes from environmental E. coli strains were purified and cloned into a plant expression vector pRT100. The construct will be transformed in calli of Nicotiana plants.

Multiple mutations in katG and inhA identified in Thai isoniazid-resistant Mycobacterium tuberculosis isolates.

A total of 29 Thai multi-drug-resistant/isoniazid-resistant Mycobacterium tuberculosis isolates were analyzed for mutations in katG from codons 254 to 549, inhA promoter and inhA open reading frame by DNA sequencing and single strand conformation polymorphism. Twenty-five multi-drug resistant isolates exhibited single point mutations (17 isolates at Ser315Thr plus Arg463Leu, 1 at Thr308Pro plus Arg463Leu, 7 at either Ser315Thr or Arg463Leu) while the other 4 isoniazid-resistant isolates had single point mutation only at Arg463Leu. Seven of 25 multi-drug-resistant isolates [4 at C(-15)T, 1 at T(-8)C; 1 at C(-15)T plus Ser94Ala and 1 at Ile21Val] and 2 of 4 isoniazid-resistant isolates [1 at C(-15)T, 1 at C (-15)T plus Ile21Thr] had mutations in inhA promoter and open reading frame, while the other 20 isolates had no mutation at any position. No frame shift mutation was observed in any tested isolates. This is the first report of two mutations, Trp308Pro of katG and T (-8)C of inhA in Mycobacterium tuberculosis isolates.

Genetic diversity in some perennial plant species with-in short distances.

Distinct morphophysiological variations observed for over 2 years with-in short distances among four perennial plants indicated genetic diversity among the lines growing at three places. The isozyme and SDS polyacrylamide gel banding patterns as genetic markers were used to investigate four perennial species, namely Dalbergia sissoo Roxb., Delonix regia (Boj.) Refin., Cassia fistula L. and Calotropis procera R. Br. Plant materials collected from three locations (Agra, Gwalior and Lucknow) differing in climo-edaphic variables were examined for 4 enzyme systems, viz., esterase, polyphenol oxidase, peroxidase and superoxide dismutase (EST, PPO, PRX and SOD). Among the four isozymes SOD and PRX revealed best discriminating power. Protein banding patterns as well as zymogram revealed that Dalbergia sissoo growing at Gwalior was closer to that of Agra; Delonix regia depicted highest similarity between Lucknow and Agra and Calotropis procera of Lucknow location was more closer to Gwalior than Agra. The results confirm genetic diversity in the species as a means of adaptation to differing climo-edaphic variables.

Deletion and mutation of WWOX exons 6-8 in human non-small cell lung cancer / 华中科技大学学报（医学）（英德文版）

To examine the deletion and point mutation of WWOX (WW domain containing oxidoreductase) exons 6-8 in human non-small cell lung cancer and their possible relationship with pathological stages, tumor tissues and the corresponding normal tissues were obtained from 44 Chinese patients who had undergone surgery for non-small cell lung cancer. RNA was extracted from each sample and deletion and mutation of WWOX exons 6-8 were analyzed by RT-PCR and DNA sequencing. Our results showed that 28 of 44 (63.6%) lung cancer samples showed loss of WWOX exons 6-8 transcript and the deletion was detected in only 3 of 44 (6.8%) corresponding adjacent normal tissues (P < 0.05). The transcript sequencing analyses of the 16 lung cancer samples without transcript loss of WWOX exons 6-8 revealed no difference from the sequence of GenBank. Moreover, the deletion of WWOX exons 6-8 was significantly higher in the smokers when compared with the non-smokers. It is also higher in the men and squamous carcinomas than in women and adenocarcinomas (P < 0. 05). The deletion, however, was not found to be associated with pathological stages of the tumors. Our study documented a high incidence of deletion of WWOX exons 6-8 in non-small cell lung cancer in Chinese patients and suggested that the frequent loss of WWOX exons 6-8 might play an important role in the tumorigenesis of non-small cell lung cancer in Chinese. WWOX exons 6-8 may serves as a candidate molecular target of smoking carcinogenesis, and point mutation is not a predominant way of alteration of WWOX exons 6-8.