圈卷产色链霉菌尼可霉素生物合成基因——sanH和sanI的研究

通过反向遗传学方法克隆到圈卷产色链霉菌尼可霉素生物合成基因簇中约 7 0kb的DNA片段。该片段除含有尼可霉素生物合成基因sanF外 ,对sanF上游约 2 2kb的BglⅡDNA片段进行序列测定及分析表明 ,还含有两个完整的开放阅读框 (ORF)。ORF1由 1 2 33个核苷酸组成 ,ORF2由 1 95个核苷酸组成 ,它们分别编码由 41 0个氨基酸残基和 64个氨基酸残基组成的蛋白质 ,依次命名为sanH和sanI。蛋白序列数据库比较结果表明 ,SanH和SanI与浅灰链霉菌 (Streptomycesgriseolus)中共转录的细胞色素P450 (cytochromeP450 )和铁氧还蛋白 (ferredoxin)有较高的同源性 ,一致性分别为 46%和 56% ,相似性分别为 62 %和70 %。基因功能研究表明 ,sanH基因的破坏虽不影响圈卷产色链霉菌产生的尼可霉素的生物活性 ,但该基因可能参与了尼可霉素羟基化反应的生物合成。

Autoantibodies and hepatitis C virus genotypes in chronic hepatitis C patients in Estonia

AIM: To determine the prevalence of several autoantibodies in chronic hepatitis C patients, and to find out whether the pattern of autoantibodies was associated with hepatitis C virus (HCV) genotypes. METHODS: Sera from 90 consecutive patients with chronic hepatitis C were investigated on the presence of anti-nuclear (ANA), anti-mitochondrial (AMA), anti-smooth muscle (SMA), anti-liver-kidney microsomal type 1 (LKMA1), anti-parietal cell (PCA), anti-thyroid microsomal (TMA), and anti-reticulin (ARA) autoantibodies. The autoantibodies were identified by indirect immunofluorescence. HCV genotypes were determined by a restriction fragment length polymorphism analysis of the amplified 5' noncoding genome region. RESULTS: Forty-six (51.1%) patients were positive for at least one autoantibody. Various antibodies were presented as follows: ANA in 13 (14.4%) patients, SMA in 39 (43.3%), TMA in 2 (2.2%), and ARA in 1 (1.1%) patients. In 9 cases, sera were positive for two autoantibodies (ANA and SMA). AMA, PCA and LKMAI were not detected in the observed sera. HCV genotypes were distributed as follows: 1b in 66 (73.3%) patients, 3a in 18 (20.0%), and 2a in 6 (6.7%) patients. CONCLUSION: A high prevalence of ANA and SMA can be found in chronic hepatitis C patients. Autoantibodies are present at low titre (1:10) in most of the cases. Distribution of the autoantibodies show no differences in the sex groups and between patients infected with different HCV genotypes.

Analysis of the Gene Polymorphism of O. aureus and O. niloticus by using RAPD

In the present study, RAPD technique was used to analyze the geneticrelationship between two cultured populations in china, oreochromis aureus and a niloticus,and the application of this technique in species identification was also investigated. Resultsrevealed that no DNA polymorphism was detected within the population of O.aureus andpolymorphism in varying degrees was observed among individuals. Similarity index andgenetic distance calculated have no remakable difference with data reported by others. RAPDanalysis using mixed samples was also tested in this experiment. In addition, someelectrophoregrams of amplification products also suggested the occurrence of geneticintrogression between the two cultured tilapia populations. Conclusively, RAPD technique isa rapid and efficient method for studying genetic polymorphism within and between differentpopulations, and RAPD markers can be used to identify different fish populations.

Molecular cloning and expression analysis of FTZ-F1 in the GIFT tilapia,Oreochromis niloticus

The FTZ-F1 genes encode orphan receptors of the nuclear receptor superfamily and in mammals have been found to play important roles in the proper development of the adrenal-gonadal axis and sex-determination.We isolated the homologue of FTZ-F1 in genetically improved farmed tilapia(gfFTZ-F1).The full-length cDNA was isolated from the ovary,which included an open reading frame encoding a predicted protein of 486 amino acids.Sequence,tissue distribution and phylogenic analysis of the FTZ-F1 showed that the gfFTZ-F1 belonged to SF-1/Ad4BP group and that gfFTZ-F1 transcripts were only expressed in the gonads and kidney but not in other tissues.Likewise our data suggests that the gfFTZ-F1 gene may share similar functions with other fish and mammalian counterparts,though further study is needed to make any definitive conclusions.

Association of Fas/Apo1 gene promoter (-670 A/G) polymorphism in Tunisian patients with IBD

AIM： To detect a possible association between the polymorphism of the （-670 A/G） Fas/Apol gene promoter and susceptibility to Crohn＇s disease （CD） and ulcerative colitis （UC） in the Tunisian population. METHODS： The （-670 A/G） Fas polymorphism was analyzed in 105 patients with CD, 59 patients with UC, and 100 controls using the polymerase chain reaction restriction fragment length polymorphism method. RESULTS： Significantly lower frequencies of the Fas -670 A allele and A/A homozygous individuals were observed in CD and UC patients when compared with controls. Analysis of （-670 A/G） Fas polymorphism with respect to sex in CD and UC showed a significant difference in A/A genotypes between female patients and controls （P corrected = 0.004 ＂in CD patients＂ and P corrected = 0.02 ＂in UC patients＂, respectively）. Analysis also showed a statistically significant association between genotype AA of the （-670 A/G） polymorphism and the ileum localization of the lesions （P corrected = 0.048） and between genotype GG and the colon localization （P corrected = 0.009）. The analysis of IBD patients according to clinical behavior revealed no difference. CONCLUSION： Fas-670 polymorphism was associated with the development of CD and UC in the Tunisian population.

Multi-location Evaluation of Turmeric Genotypes in Nigeria

Fifteen turmeric genotypes from the germplasm held at National Root Crops Research Institute Umudike, Nigeria, were evaluated during the season of 2012-2013 at four locations-Jos （8.3833°N, 7.1833°E, 1,200 m a.s.l.）, Otobi （7.11667°N and 8.08333°E）, Umudike （5.4758°N, 7.5489°E） and Igbariam （6.4°N and 6.93333°E）-in order to select high yielding and stable turmeric cultivars with good quality for release in Nigeria. At each location, the experiment was laid out in randomized complete block design （RCBD） in three replications. Plot size was 9 m2. Data were collected on sprout count, plant height, number of tillers, number of leaves, main pseudo stem girth, rhizome number and weight. Analysis of variance was carried out on the combined data using GenStat Discovery Edition software. Results based on the combined data from four locations indicate that turmeric genotypes did not vary in percentage emergence and number of leaves. However, they varied in height, main pseudo stem girth, tillering, number and yield of fresh rhizomes. The effect of location on all attributes was significant （P 〈 0.05） with Jos location giving consistently the least values for all attributes thus suggesting that this location may not be suitable for the commercial production of turmeric. Genotype by environment interaction for most attributes was not significant indicating that the genotypes responded the same way across the locations. Ten genotypes, viz., UT39, UT44, UT46, UT58, UT50, UTI4, UT41, UT6, UT38 and UT35, are identified as promising and require further evaluation as pre-condition for nomination for official release to farmers.

Stability Assessment of Some West African Okra （Abelmoschus caillel~ Genotypes in Nigerian Genebank

Stability among 50 accessions of West African okra （Abelmoschus caillei） was assessed under three diverse ecological environments at Abeokuta, Ibadan and Mokwa in Nigeria during 2005 and 2006 cropping season. The accessions were grown in a Randomized Complete Block Design with three replications; data were collected on 5-10 randomly selected plants from each plot. Only 20 accessions were subjected to stability analysis on the basis of yield across the three environments. The joint regression analysis, deviation means square were computed using Eberhart and Russell method and complemented with Francis and Kannenberg method. The regression coefficients of accessions mean yields on the environmental index resulted in regression coefficients ranging in values from 0.5549 to 1.6667. OAA/96/175-5328, NGAE-96-011 and NGAE-96-0060 were among the superior genotypes with high yield performance. The large variation in regression values indicated large differences in genotype response to different environments. It suggests that stability concept of Ebelhart and Russell could be modified to use any yield components that has strong correlation with yield for stability analysis. The two promising accessions ofA. caillei （NGAE-96-011 and NGAE-96-0060） needed to be further tested on farmers＇ field to obtain on-farm data, alter which it should be recommended for official registration and released by the National Committee on Naming, Registration and Release of Crop varieties in Nigeria.

Phenotypic and Genotypic Comparison of Vibrio in Seawater Fish from Batam and Mataram, Indonesia

Grouper and snapper are the potential fishery commodity in Indonesia with a high economic value, as well as an export commodity. A common disease in grouper and snapper aquaculture is vibriosis. Vibriosis is a disease caused by bacteria of the genus Vibrio. The aim of study was to compare between phenotypic and genotypic identification of Vibrio isolated from Batam and Mataram, Indonesia. Bacteria were isolated from anterior kidney and eye of fish, then grown in thiosulfate-citrate-bile salts-sucrose （TCBS） and incubated in room temperature （25-28 ~C） for 24 h, and identified using morphology and biochemical test. Bacterial isolates were extracted, amplified and sequenced on 16S rRNA region. Phylogenetic tree of bacteria was constructed using neighbor-joining and maximum-parsimony methods. The phenotypic identification was found six isolates of Vibrio from Batam, such as K alginolyticus, V. carchariae, K damselae, V. fluvialis, V. furnissii and K parahaemolyticus. Three isolates were found from Mataram, such as 1I. alginolyticus, V. carchariae and V. fluvialis. Blast analysis showed isolates of V. alginolyticus_btm and V. carchariae_btm homolog to V. parahaemolyticus strain DAHMV3; isolates of V. damselae_btm and K alginolyticus_mtr homolog to V. neocaledonicus strain MS1; isolates of V. parahaemolyticus__btm and V. furnisii_btm homolog with Photobacterium damselae subsp, damselae strain： 04Ya311 and isolate of K fluvialis_mtr homolog to V. azureus strain MMRF532, respectively. All phenotypic identification was not supported by molecular identification on 16S rRNA region. It was suggested that phenotypic identification should be supported by molecular examination, especially in identification of Vibrio species.

Polymorphism in the Regulatory Region of the Aromatase CYP19a Gene in Nile Tilapia

The aim of this study was to evaluate the polymorphism in a portion of the gene regulatory region for ovarian aromatase （CYP19a） in three strains of Tilapia, Oreochomis niloticus （Linnaeus） （GIFT--Genetically Improved Farmed Tilapia, Chitralada and Supreme）. A total of 90 animals per strain of Tilapia, Oreochromis niloticus （Linnaeus） were analysed. After DNA extraction, samples were subjected to PCR using primers designed to flank the region of interest encompassing the sites of transcription （WT1-KTS and SRY）. Samples were analyzed by PCR-SSCP and subsequently sequenced. Three polymorphisms were identified in this region, resulting in two different sequences, in the GIFT strain while no polymorphism was found in both Supreme and Chitralada strains. At the position - 1178 the substitution of a guanine for a cytosine, at the - 1081 the exchange of guanine for adenine and at the position -1 138 we found a SNP, possible site of heterozygosity. Even with polymorphisms in the target study area, when taking the three strains into account, one can assume that the portion of the regulatory region of the ovarian aromatase gene in the Supreme strain and Chitralada does not show polymorphism.

Phenotypic and Genotypic Comparison of Pseudomonas stutzeri in Freshwater Fish in Indonesia

Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, Jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehensively identify special phenotypic and genotypic characteristics of P. stutzeri isolated from several areas in Indonesia, including its morphometric and biochemical characteristics and molecular variation. Bacteria were isolated from internal organs （kidney, ulcer and eye） of fish. They were then identified using morphology and biochemical test. DNA isolates were entirely extracted, amplified and reversed on 16S rRNA region, and further then were sequenced. Phylogenetic trees of bacteria were constructed using neighbor-joining and maximum-parsimony methods. The colony were similar, such as rod shape （Jambi, Tanjung Pinang, Bali）, bacil shape （Luwuk Banggai）, transparant in tryptic soy agar （TSA） （Luwuk Banggai）, creamy beige in glutamate starch phenol red （GSP） （Bali）, gram negative, motile, no reaction in the oxidative-fermentative test, positive result in catalase and oxidase test, negative in lysine decarboxylase and ornithine decarboxylase test and positive result in indole test; gelatin was degraded （only Bali）, urea was not degraded, no color change in Methyl-red and Voges-proskaeur （MR-VP） test; acid not produce from glucose, inositol or sucrose. Citrate was utilized by some isolates： positive （Jambi, Tanjung Pinang） and negative （Bali, Luwuk Banggai）. Results showed us that isolates of Jambi, Bali and Tanjung Pinang were monophyletic species with P. stutzeri $8 and ZH-1 comparing to gen bank. However, merely phenotypic analysis among Pseudomonas sp. was confused compared to each other.

Effect of ABCB1 C3435T Polymorphism on Clinical Outcomes in Kenyan HIV Patients on Lopinavir-Based Regimens

ATP Binding Cassette sub-family B member 1 （ABCB1） affects disposition of many drugs and thus affects the pharmacokinetics of drugs and ultimately treatment response. Polymorphisms of ABCB 1 especially ABCB 1 C3435T polymorphism may thus affect pharmacokinetics of antiretroviral drugs and hence CD4 treatment response and other clinical outcomes of HIV patients. Methods： The study design was a historical cohort study and entailed collection of patient data. PureLink genomic DNA extraction mini kit was used for the extraction and purification of genomic DNA. TaqMan drug genotyping assay and protocol was used in the DNA amplification and genotyping. Data analysis was done using STATA software version 10. Results： Study participants with the CT genotype had lower creatinine levels after 6 months on lopinavir-based regimens compared with those with the CC genotype （p = 0.001）. In addition, the study participants with the CT genotype had consistently higher CD4 cell counts compared with those with the CC genotype from the time of ART switch but this was not statistically significant. However, there was no significant association between the ABCB 1 C3435T genotypes and haemoglobin and ALT levels. Conclusion： There was a significant association between ABCB1 C3435T polymorphism and creatinine levels 6 months after therapy on lopinavir-based regimens.

Wheat Stripe Rust Virulence and Varietal Resistance in the Foot Hill Himalayas of Nepal

Stripe rust, caused by Puccinia striiformis f. sp. tritici （Pst） is an important disease of wheat （Triticum aestivum） in Nepal, which is a part of the Himalayas stretching over the North of Nepal, India, Pakistan, Bhutan and beyond. Wheat production plays a crucial role in food security of the marginal hill farmers of Nepal. Frequent epidemics of the rust have caused huge loss in farmer＇s field. Periodic monitoring during 1980-2008 showed that changes in virulence occurred during this period. The objective of this study was to evaluate Pst resistance and its effective genes in wheat genotypes. For this, trap nurseries, wheat stripe rust differentials, commercial cultivars and advanced breeding lines were tested under artificial epiphytotic and natural hot spots conditions during 2005 to 2010. Four genes （Yr5, Yr10, Yr15 and YrSp） consistently showed resistance to the prevailing races. The gene Yr9 and Yr27 in combinations with Yrl8 were found effective. Other lines with combination of minor genes were also found effective. The genotypes Amadina, Kukuna, Tukuru, Kakatsi and Buck Buck widely used in breeding program were resistant. The cultivation of varieties WK1204, Gautam, Gaura and Dhaulagiri have ensured genetic diversity for the rust resistance and slowed down frequent occurrence of epidemics. The findings of these studies could help in developing effective varietal resistance program in the sub-continent.

Salt Tolerance of 15 Rice （Oryza sativa L.） Varieties in the Office du Niger Zone of Mali

From 2003 to 2005, in pot and field experiments, rice response to salinity stress of 15 rice varieties was studied at germination and young seedling stages using salt affected soils collected from rice production areas in the Office du Niger zone of Mali. The rice varieties were composed of 10 rice genotypes from the breeding program of Mali and five from West African Rice Development Association （WARDA） program （Saint Louis, Senegal）. Soil samples were collected from the visually affected soils which were characterized by the appearance of white or black efflorescence on the soil surface. In pot experiments, the genotypes were allowed to germinate in both affected soil types （white efflorescence and black efflorescence） and salt effects on plant seedling growth were observed. Results showed that all varieties were significantly sensitive to salinity stress based on germination, young seedling shoot and root dry weights. Among the rice varieties, the most salt tolerant variety was BG90-2 （a high yielding genotype from the Institut d＇Economie Rurale （IER） breeding program） while the most sensitive variety was Telimani （also from the breeding program of IER）. All other varieties were intermediary between these two genotypes. A three year field experiment conducted in a highly affected area near Niono confirmed the results of the pot experiment. The relatively salt tolerant genotypes were found in both Malian （BG90-2, Kogoni91-1, SK51-5-2） and WARDA （Was30-11-1-1-4-6-1B） rice breeding programs.

Release of Stem Rust Resistant Wheat Varieties for Commercial Production in Kenya

Detection in 1999 of a new stem rust （Puccinia graminis f. sp. tritici） race Ug99 in Uganda with broad virulence including the virulence for Sr31 and its migration to Kenya and Ethiopia has been recognized as a significant threat to local and world wheat production. All the Current Kenyan commercial varieties are susceptible to this race. This study was aimed at identifying suitable wheat varieties with resistance to Ug99 and replacing the susceptible commercial varieties through multi-locational testing and variety release. Thirty three lines were identified from a prescreen population of 104 lines and tested in 3 wheat growing regions in Kenya for two seasons in 2006 and 2007. The resulting four superior lines were evaluated under the National Performance Trial （NPT） where two lines which out-performed the best check variety were released as for commercial production. ＇Robin＇ was the best line and out yielded the commercial variety by 27%. ＂Eaglel0＂ was the second best and was better significantly than the check variety. These two lines which combined both adult plant resistant gene Sr2 complex and other major genes are expected to have some durable resistance and may be used to replace the current susceptible commercial varieties grown in Kenya.

F8基因倒位检测及其在甲型血友病基因诊断中的应用

目的建立尼基因第22内含子倒位突变检测新方法，应用于甲型血友病（hemophilia A）基因诊断。方法应用长距离PCR（long distance-polymerase chain reaction，LD-PCR）、倒位PCR（inversion-PCR，I-PCR）技术检测31例甲型血友病患者尼基因22内含子倒位；对于倒位突变阳性患者的母亲应用上述两种方法进行携带者诊断；而对倒位携带者孕妇于孕中期抽取羊水，进行产前基因诊断。结果31例甲型血友病患者中查出7例存在倒位突变；4例倒位突变阳性患者的母亲有3。例为倒位携带者；对1例倒位携带者孕妇进行了产前诊断，确定其胎儿无倒位突变。结论LD-PCR、I-PCR技术可快速检测尼基因22内含子倒位突变，可应用于患者及携带者基因诊断；I-PCR可用于尼基因22内含子倒位的产前基因诊断。

Cloning Envelope Gene of Dengue Virus Type 3 （Indonesia D3-1703 Strain） into pYES2/CT Vector

The global burden caused by dengue has increased dramatically in recent decades. Dengue Fever and its severe clinical manifestation, Dengue Hemorrhagic Fever and Dengue Shock Syndrome, have become international public health problem endemic in more than 100 countries, risking 2.5 to 3 billion world populations in tropical and subtropical region. Envelope （E） protein of dengue virus has been proposed as the most important antigen that enables it as vaccine candidate or diagnostic materials. Recombinant protein E production is desirable for dengue vaccine and diagnostic development, especially in Indonesia, where dengue is epidemic. Cloning E gene in an expression vector is essential as an initial method to produce dengue E antigens. The purpose of this research was to clone E gene of dengue virus type 3 （Indonesia D3-1703 strain） into Saccharomyces cerevisiae expression vector pYES2/CT. The cloning method used was the in vitro ligation protocol. First, the cDNA from dengue virus type 3 strain （D3-1703） was generated. Then the polymerase chain reaction （PCR） amplification product of E gene cassette from this cDNA was obtained. The E gene cassette was ligated into linearized pYES2/CT resulting a recombinant vector named pYES2/CT-E. The cloned E gene was verified by restriction enzyme digestion and PCR. Sequencing analysis at its 5＇ end showed that the E gene was inserted at the right open reading frame. In conclusion, the results showed that for the first time the E gene originated from an Indonesian dengue virus type 3 strain was successfully cloned within the yeast expression vector pYES2/CT. In the future, this clone could be expressed and provided as materials for dengue vaccine and diagnostic kit, specific for Indonesian dengue virus strain.

Expression of hHR21^(sp) gene by peripheral blood and hematopoietic cells of normal subjects and Fanconi anemia patients

Objective The radiation sensitive gene rad 21 of Schizosaccharomyces pombe is involved in the repair of double-stranded breaks in DNA and is essential for mitotic growth. The hHR21 sp gene is its human homologue. In an attempt to investigate the role of hHR21 sp in DNA repair, we studied the effects of UV and γ-ray irradiation on hHR21 sp gene expression in normal human peripheral blood cells, and non-iradiated peripheral and bone marrow cells from Fanconi anemia (FA) patients who have shown DNA repair deficiency.Methods Total steady state RNA was extracted from peripheral blood cells and bone marrow. RNA transcripts were quantified after RT-PCR and Southern blot, phosphoimmage and autoradiogram analysis. The results were compared with control groups. Results hHR21 sp expression was significantly increased from 3?h to 9?h after UV irradiation in peripheral blood cells from normal subjects at doses of 40-80?j/m 2 (P<0.05). hHR21 sp was also up-regulated by γ-ray irradiation at 6?h to 9?h at dose of 1 to 5?Gy (P<0.01), which was more significant than the UV irradiation. In the non-irradiated FA patient group, hHR21 sp expression was decreased in bone marrow hematopoietic cells (P<0.05). After activation by PHA and IL-2, there was still a significant depression in expression by the FA patients peripheral blood cells compared with control groups (P<0.05). Conclusion hHR21 sp was up-regulated at doses and times irradiated at the range tested in normal peripheral blood cells, and is more affected by γ-ray irradiation than UV irradiation. FA patient bone marrow hematopoietic cells and peripheral blood mononuclear cells showed down-regulation of hHR21 sp expression. The results imply that defects in DNA repair via hHR21 sp expression may play an important role in the pathogenesis of FA syndrome.

Coordination of glycerol utilization and clavulanic acid biosynthesis to improve clavulanic acid production in Streptomyces clavuligerus

The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyl operon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaR expressed from Pgyl (the gyl promoter) was integrated into the chromosome of S. clavuligerus NRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyl operon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaR controlled by its own promoter (PccaR). Consistent with enhanced CA production, the transcription levels of ccaR, ceas2 and claR were significantly up-regulated in the transformants containing Pgyl-controlled ccaR.

Duck egg drop syndrome virus:an emerging Tembusu-related flavivirus in China

Duck egg drop syndrome virus(DEDSV) is a newly emerging pathogenic flavivirus isolated from ducks in China.DEDSV infection mainly results in severe egg drop syndrome in domestic poultry,which leads to huge economic losses.Thus,the discovery of ways and means to combat DEDSV is urgent.Since 2010,a remarkable amount of progress concerning DEDSV research has been achieved.Here,we review current knowledge on the epidemiology,symptomatology,and pathology of DEDSV.A detailed dissection of the viral genome and polyprotein sequences,comparative analysis of viral antigenicity and the corresponding potential immunity against the virus are also summarized.Current findings indicate that DEDSV should be a distinct species from Tembusu virus.Moreover,the adaption of DEDSV in wildlife and its high homology to pathogenic flaviviruses(e.g.,West Nile virus,Japanese encephalitis virus,and dengue virus),illustrate its reemergence and potential to become a zoonotic pathogen that should not be overlooked.Detailed insight into the antigenicity and corresponding immunity against the virus is of clear significance for the development of vaccines and antiviral drugs specific for DEDSV.