Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

The structural transition of DNA-Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol-water solution

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when e is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)(3)(3+) in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B-A conformation transition takes place at more than 60% ethanol solution.

Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Mitochondrial DNA variation in cattle of South China: origin and introgression

Ten restriction endonucleases were used to investigate the mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) of 11 native cattle breeds and one cultivated cattle breed in South China. Twenty-three restriction morphs were detected, which can be sorted into five haplotypes, A phylogenetic tree of the haplotypes was constructed by using the 'upgma' method. Our study showed that haplotype I and II are identical to the zebu (Bos indicus) and taurine (Bos taurus) haplotypes, respectively. Zebu and taurine were the two major origins of cattle populations in South China, and the zebu probably had more influence on the native cattle population than taurine did. Haplotype III is identical to haplotype I of yak (Bos grunniens), which was only detected in the Diqing cattle breed. Haplotype IV was detected for the first time. This haplotype, found only in Dehong cattle, might be from an independent domestication event, probably from another Bos indicus population. Divergence of haplotypes I and IV occurred about 268,000-535 000 years ago, much earlier than the 10,000-year history of cattle husbandry. Our results also suggest a secondary introgression of mtDNA from yak to Diqing cattle.

Molecular phylogeny of Sinocyclocheilus (Cypriniformes : Cyprinidae) inferred from mitochondrial DNA sequences

More than 10 species within the freshwater fish genus Sinoncyclocbeilus adapt to caves and show different degrees of degeneration of eyes and pigmentation. Therefore, this genus can be useful for studying evolutionary developmental mechanisms, role of natural selection and adaptation in cave animals. To better understand these processes, it is indispensable to have background knowledge about phylogenetic relationships of surface and cave species within this genus. To investigate phylogenetic relationships among species within this genus, we determined nucleotide sequences of complete mitochondrial cytochrome b gene (1140 bp) and partial ND4 gene (1032 bp) of 31 recognized ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony. Bayesian, and maximum likelihood analyses. Our phylogenetic results showed that all species except for two surface species S. jii and S. macrolepis clustered as five major monophyletic clades (I, II, III, IV, and V) with strong supports. S. jii was the most basal species in all analyses, but the position of S. macrolepis was not resolved. The cave species were polyphyletic and occurred in these five major clades. Our results indicate that adaptation to cave environments has occurred multiple times during the evolutionary history of Sinocyclocheilus. The branching orders among the clades I, II, III, and IV were not resolved, and this might be due to early rapid radiation in Sinocyclocheilus. All species distributed in Yunnan except for S. rhinocerous and S. hyalinus formed a strongly supported monophyletic group (clade V), probably reflecting their common origins. This result suggested that the diversification of Sinocyclocheilus in Yunnan may correlate with the uplifting of Yunnan Plateau. © 2005 Published by Elsevier Inc.

A molecular model of braid-like DNA structure

The three-dimensional molecular models of DNA triple helices and triple-stranded brain-like structure were built up by molecular architecture, and their structural features and energy decomposition were examined. The results showed: (i) The base triplet is the element forming braid-like and triple helix DNA; (ii) Under specified conditions, DNA could form the triplet-stranded braid-like structure; (iii) DNA stability of the braid-like structure is less than that of the triple helix structure. (C) 1995 Academic Press Limited.

云南龙陵黄山羊线粒体DNA限制性酶切分析

采用碱性变性法提取来自于龙陵县不同地区的18只黄山羊个体的线粒体DNA(mtDNA),并用Apa I、Ava I、BamH I、Bcl I、Bgl I、Bgl II、Cla I、Dra I、EcoR I、EcoR V、Hae I、Hind III、Kpn I、Pst I、Pvu II、Sac I、Sal I、Sma I、Stu I和Xba I等20种限制性内切酶进行酶切分析。 结果发现龙陵黄山羊线粒体DNA的分子量大小约为15.8Kb;不同酶的酶切位点分别为:Dra I有7个酶切位点,Ava II有6个酶切位点,EcoR V和Stu I共有5个酶切位点,Hind III和Hea II有4个酶切位点,BamH I、Bgl II、Pst I和Pvu II有3个酶切位点,Apa I、Cla I有2个酶切位点,其余有1个酶切位点。

DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

Objective To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. Methods Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. Results Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. Conclusion Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

Comparative studies on parallel and antiparallel duplex and triplex DNA

Parallel strand models for base sequences d(A)(10). d(T)(10), d(AT)(5) . d(TA)(5), d(G(5)C(5)). d(C(5)G(5)), d(GC)(5) . d(CG)(5) and d(CTATAGGGAT). d(GATATCCCTA), where reverse Watson-Crick A-T pairing with two H-bonds and reverse Watson-Crick G-C pairing with one H-bond or with two H-bonds were adopted, and three models of d(T)(14). d(A)(14). d(T)(14) triple helix with different strand orientations were built up by molecular architecture and energy minimization. Comparisons of parallel duplex models with their corresponding B-DNA models and comparisons among the three triple helices showed: (i) conformational energies of parallel AT duplex models were a little lower, while for GC duplex models they were about 8% higher than that of their corresponding B-DNA models; (ii) the energy differences between parallel and B-type duplex models and among the three triple helices arose mainly from base stacking energies, especially for GC base pairing; (iii) the parallel duplexes with one H-bond G-C pairs were less stable than those with two H-bonds G-C pairs. The present paper includes a brief discussion about the effect of base stacking and base sequences on DNA conformations. (C) 1997 Academic Press Limited.

鲤鱼3个亚种线粒体DNA ND5/6区段的PCR-RFLP分析及亚种间分子标记的确立

从鲤鱼3个亚种(Cyprinus carpio carpio,Cyprinus carpio haematopterus和Cyprinus carpiorubrofuscus)中选出具代表性的品系共10个,运用PCR方法,扩增出2.4 kb的mtDNA ND5/6区段.共用9种限制性内切酶进行酶切分析,结果表明,每个亚种拥有一种单倍型,有4种限制性内切酶(Dde I,Hae III,Taq I和Mbo I)酶切后产生了能将3个亚种区分开来的诊断性限制性酶切位点.可利用其作为鉴定3个亚种的遗传标记和遗传育种

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6 - 99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide Ligation assay on DNA chips

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.