水稻BAC在玉米有丝分裂染色体上FISH杂交体系的构建

 以水稻细菌人工染色体(BAC)为探针在玉米有丝分裂的细胞学制片上进行荧光原位杂交(FISH),探讨玉米基因组C_ot DNA对BAC探针重复序列的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化、水稻BAC探针的特异性重复序列的封阻对FISH杂交信号特异性的影响.初步形成了一套以水稻BAC探针在玉米有丝分裂染色体上进行BAC-FISH杂交的优化技术体系.研究结果表明：使用玉米基因组C_ot DNA来封阻水稻BAC探针的重复序列玉米基因组C _ot DNA的C_ot值应小于50,同时还需根据不同探针调整C_ot DNA的C_ot值及与探针的比例;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻BAC探针本身特异的重复序列的封阻对BAC-FISH杂交信号特异性的改善具有较好的效果.     

玉米有丝分裂染色体上玉米BAC探针的FISH杂交体系的构建

本研究分别探讨了玉米和水稻基因组c 0t DNA对探针的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化对BAC-FISH杂交的影响;探讨了玉米BAC探针中重复序列含量对FISH信号的影响.初步形成了一套以玉米BAC探针在玉米有丝分裂染色体上进行FISH杂交的优化技术体系.结果表明,玉米基因组c 0t DNA对探针封阻的c 0t值应小于50;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻基因组的c 0t 100 DNA封阻探针重复序列对BAC-FISH杂交信号特异性的改善具有明显的效果;同时,验证了选择重复序列含量较少的玉米BAC作为FISH杂交的探针也是获得特异性杂交信号的重要条件.     

Technological exploration of BAC-FISH on mitotic chromosomes of maize

The rice BAC-DNA was used as probes and fluorescence in situ hybridization (FISH) was applied to the interphase and metaphase mitotic chromosomes of maize. To optimize the BAC-FISH technique, we respect-ively assayed the effect of several factors, including maize or rice genomic Cot DNA used as blocking reagent of DNA, washing temperatures and FAD concentration in the washing buffer and in the hybrid solution. The results show that Cot DNA of maize genome blocked the repet-itive sequence of the rice BAC-DNA when the Cot value was below 50. Meanwhile, it was necessary to adjust the Cot value according to the different probes and their ratios. Decreasing the concentration of FAD in the hybridization mixtures, adjusting the washing rate after hybridization, and most especially, blocking the rice-specific repetitive sequences of BAC-DNA could improve the positive signals of BAC-FISH.     

High cell density and high expression of recombinant human ApoA-IMilano in Escherichia coli by twice temperature-shifted induction

The effect of temperature on the formation of recombinant protein, apolipoprotein A-I_Milano was investigated in the present study. The temperature of the initial growth phase was set at 30°C, while temperature variation in induction phase was arranged in three modes. High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out. Experimental results showed that ApoA-I_Milano reached 4.8 g/L with the final cell density of OD₆₀₀, 150. It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product. The present study provides a basic procedure for the production of recombinant ApoA-I_Milano. __________ Translated from Microbiology, 2005, 32(2): 54–59 [译自: 微生物学通报, 2005, 32(2): 54–59]     

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase II (EG II) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR. It was cloned into the expression vector pGAPZαA. The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion. The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG II were also explored. __________ Translated from Microbiology, 2006, 33(6): 68273 [译自: 微生物学 通报]     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyl1 and Pichia stipitis gene xyl2, encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively, were amplified by PCR. The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-P12. Subsequently the vector pYES2-P12 was transformed into S. cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12. The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%. __________ Translated from Microbiology, 2006, 33(3): 104–108 [译自:微生物学通报]     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866),DNA fingerprint technique, amplified ribosomal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes of Acinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity, and could be a useful tool in Acinetobacter genomic species identification. __________ Translated from Microbiology, 2007, 34(2): 303–306 [译自:微生物学通报]     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58) is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine. The Fourier-transform infrared (FT-IR) spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma). A bioassay shows that the LC₅₀ of a Bacillus thuringiensis (Bt) formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml, which is similar to that of the Bt formulation without UV treatment, however, it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58. The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation. This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation. Translated from Microbiology, 2006, 33(1): 42–45 [译自: 微生物学通报]     

Gill rays of primitive vertebrate Yunnanozoon from Early Cambrian: a first record

Yunnanozoans (including Yunnanozoon and Haikouella) are important representatives of the primitive vertebrates in the Early Cambrian Chengjiang fauna. For Yunnanozoans, we know less about Yunnanozoon than about Haikouella due to the poor preservation of Yunnanozoon. Up to now, there have been some reports that Haikouella had developed gill rays, while there have been no reports on Yunnanozoon. In this paper, we described our new findings of the distinct gill rays of Yunnanozoon lividum based on new well-preserved material collected from the Lower Cambrian Maotianshan Shale in Xiaolantian of Yunnan Province, China. This study provides new data on the evolutionary relationship of the primitive vertebrates and their early evolution. __________ Translated from Acta Palaeontologica Sinica, 2006, 45(3): 345–350 [译自: 古生物学报]     

DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations. The method is based on the formation and deformation of double-stranded DNA (dsDNA) membranes on a gold surface. It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface. In these systems, Fe(CN)₆ ³⁻ was used as the reporter. As the temperature increases, a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears. At a special temperature, the method gives 100:1 selectivity for the perfect complement and single base mutation target. Thus, the system provides a simple and sensitive method to detect DNA point mutations without labeling targets. __________ Translated from Acta Biophysica Sinica 2005, 21 (2) [译 自: 生物物理学报, 2005,21(2)]     

Interactive effects of elevated CO2 and temperature on the anatomical characteristics of leaves in eleven species

The anatomical features of leaves in 11 species of plants grown in a temperature gradient and a temperature + CO₂ gradient were studied. The palisade parenchyma thickness, the spongy parenchyma thickness and the total leaf thickness were measured and analyzed to investigate the effects of elevated temperature and CO₂ on the anatomical characteristics of the leaves. Our results show that with the increase of temperature, the leaf thickness of C₄ species increased while the leaf thickness of C₃ species showed no constant changes. With increased CO₂, seven out of nine C₃ species exhibited increased total leaf thickness. In C₄ species, leaf thickness decreased. As for the trend on the multi-grades, the plants exhibited linear or non-linear changes. With the increase of temperature or both temperature and CO₂ for the 11 species investigated, leaf thickness varied greatly in different plants (species) and even in different branches on the same plant. These results demonstrated that the effect of increasing CO₂ and temperature on the anatomical features of the leaves were species-specific. Since plant structures are correlated with plant functions, the changes in leaf anatomical characteristics in elevated temperature and CO₂ may lead to functional differences. Translated from Acta Ecologica Sinica, 2006, 26(2): 326–333 [译自: 生态学报]     

Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138

L-cysteine desulfhydrase (CD) plays an important role in L-cysteine decomposition. To identify the CD gene in Pseudomonas sp. TS1138 and investigate its effect on the L-cysteine biosynthetic pathway, the CD gene was cloned from Pseudomonas sp. TS1138 by polymerase chain reaction (PCR) method. The nucleotide sequence of CD gene was determined to be 1,215 bp, and its homology with other sequences encoding CD was analyzed. Then the CD gene was subcloned into pET-21a(+) vector and expressed in Escherichia coli (E. coli) by isopropyl-β-D-thiogalactopyranoside (IPTG) inducement. The recombinant CD was purified by Ni-NTA His-Bind resin, and its activity was identified by the CD activity staining. The enzymatic properties of the recombinant CD were characterized and its critical role involved in the L-cysteine biosynthetic pathway was also discussed. __________ Translated from Microbiology, 2006, 33(4): 21–26 [译自: 微生物学通报]     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) from β-hydroxyacetophenone without any additive to regenerate NAD⁺ from NADH. __________ Translated from Microbiology, 2006, 33(4): 112–118 [译自: 微生物学通报]     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris, we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique, which was composed of frequently used codons in the highly expressed Pichia pastoris genes. Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing. The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2. The plasmid was transformed into GS115 cells of the methylotrophic yeast, Pichia pastoris by electroporation, and we got the expression cell through phenotype selection and induction with methanol. Separation, purification, and bioactivity analysis of the expressed products were performed. __________ Translated from Microbiology, 2006, 33(1): 1–6 [译自: 微生物学通报]     

Effects of heavy metals Cd2+, Pb2+ and Zn2+ on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd²⁺, Pb²⁺ and Zn²⁺ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1–35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar body size (5.17–7.99 g; 11.79–13.21 cm) were selected and kept in aquaria with dechlorinated water at (22±1)°C and fed a commercial diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value, the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%) of damaged DNA was shown in 5.0 mg/L Zn²⁺ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd²⁺, Pb²⁺ or Zn²⁺ revealed much complexity, but it generally displayed that the presence of Cd²⁺ could enhance the genotoxicity of Pb²⁺ or Zn²⁺. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms. __________ Translated from Acta Hydrobiologica Sinica, 2006, 30(4): 399–403 [译自: 水生生物学报]     

Expression of hSef in various human tissues and cell lines

Sef is a transmembrane protein inhibiting FGF signaling. To determine the correlation of Sef with human diseases, Sef expression patterns were observed in cell lines and human cancer tissues. Western blot using anti-hSef antibodies showed that hSef, when expressed in Cos7 cells gave a molecular mass of 100 KD as compared with 80 KD in an in vitro translation assay suggesting occurrence of glycosylation at the potential N-linked glycosylation sites in the extracellular domain. Northern blot showed that hSef was mainly expressed in human kidney and testis. RT-PCR analysis showed a widely spread expression pattern in several cell lines. Immunohistochemical analysis revealed a high expression level of hSef in kidney, testis, and the corresponding carcinoma tissues. Results demonstrated that Sef might be up-regulated in the cancer tissues suggesting a possible role of Sef in pathophysiology of human diseases. __________ Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21 (2) [译自: 中国生物化学与分子生物学报, 2005,21(2)]     

The Shanita fauna (Permian foraminifera) from Baoshan area, western Yunnan Province, China

Studies in the past decade have proven the Shanita fauna to be an excellent marker of the northern peri-Gondwana tectonic blocks. Thus, a study of the Shanita fauna from the Baoshan area in western Yunnan Province, China, could provide pivotal paleontological evidence for the paleogeographic reconstruction of the Baoshan block. We systematically analyzed the composition and the age of the Shanita fauna from the Permian Da’aozi Formation in Woniusi Section of the Baoshan area. Results suggested that the characteristic genera Shanita and Hemigordiopsis in this fauna comprised eight species (including two new species), and ten genera of other nonfusulinid foraminifera were also recognized from this fauna. Further comparative study showed that the Shanita fauna from the Baoshan area were probably late Maokouan (Lengwuan) to Wuchiapingian in age. In general composition, this fauna is comparable to those Shanita faunas from Shan State of Burma, Peninsular Thailand, and Nagri of Tibet, China. However, the relatively low generic diversity and occurrence of some endemic species, as well as the absence of fusulinids, indicate certain regional features of the Shanita fauna from the Baoshan area. Translated from Acta Palaeontologica Sinica, 2005, 44(4): 545–555 [译自: 古生物学报]     

Molecular phylogenetic relationship of Epinephelus based on sequences of mtDNA Cty b

The mtDNA Cyt b gene was sequenced partially for Variola louti of Serranidae, Epinephelinae and seven endemic species of groupers—Epinephelus awoara, E. brunneus, E. coioides, E. longispinis, E. sexfasciatus, E. spilotoceps and E. tauvina in China. The seven endemic species and other seven foreign species of groupers—E. aeneus, E. caninus, E. drummondhayi, E. haifensis, E. labriformis, E. marginatus and E. multinotatus from the GenBank were combined and analysed as ingroup, while Variola louti was used as outgroup. We compared the 420 bp sequences of Cyt b among the 15 species and constructed two types of molecular phylogenetic trees with maximum parsimony method (MP) and neighbor-joining method (NJ) respectively. The results were as follows: (1) As to the base composition of mtDNA Cyt b sequence (402 bp) of 14 species of Epinephelus, the content of (A + T) was 53.6%, higher than that of (G + C) (46.4%). The transition/transversion ratio was 4.78 with no mutation saturation. (2) The cluster relationships between E. awoara and E. sexfasciatus, E. coioides and E. tauvina, E. longispinis and E. spilotoceps were consistent with phenotypes in taxonomy. (3) In the phylogenetic tree, the species in the Atlantic Ocean were associated closely with those in the Pacific Ocean, which suggested that the Cyt b sequences of Epinephelus were highly conserved. This may be attributed to the coordinate evolution. (4) In wel1-bred mating or heredity management, mating Epinephelus of the same branch should be avoided. It is likely to be an effective way to mate the species of the Atlantic Ocean with those of the Pacific Ocean to improve the inheritance species. __________ Translated from Acta Hydrobiologica Sinica, 2006, 30(4): 432–438 [译自: 水生生物学报]     

Effects of Arg26 and Lys27 mutation on the bioactivity of HNTX-IV

Hainantoxin-IV (HNTX-IV) was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive (TTX-S) sodium channels. As revealed by the solution structure of HNTX-IV solved by two-dimensional nuclear magnetic resonance (2D-NMR), HNTX-IV adopts an inhibitor cystine knot motif. To check the role of basic residues during HNTX-IV’s interaction with TTX-S sodium channels, R26A and K27A mutants of HNTX-IV were constructed by solid-phase chemical synthesis. The synthesized peptides were purified and refolded under optimized oxidation conditions. Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy, respectively. Using the whole-cell patch-clamp technique, Lys27 but not Arg26 was identified as a key residue for HNTX-IV’s bioactivity against TTX-S sodium channels, because R26A-HNTX-IV showed slightly reduced activity and K27A-HNTX-IV showed almost no inhibition. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(4): 499–503 [译自: 中国生物化学与分子生物学报]