Genetic structure of Chinese sucker population Myxocyprinus asiaticus in the Yangtze River based on mitochondrial DNA marker

The sequencing analysis of the mitochondrial DNA control region (mtCR DNA) was performed to assess the genetic divergence and population structure of the Chinese sucker Myxocyprinus asiaticus (Cypriniformes Catostomidae) using four sample lots from natural populations of the Yangtze River. The mtCR DNA sequences of approximately 920 base pairs were obtained. A total of 223 nucleotide positions were polymorphic, and these defined 39 haplotypes. Of the 39 haplotypes, 37 (90%) were not shared, and among the populations as a whole there was little sharing of haplotypes. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated a higher level of genetic diversity of Chinese sucker through the river. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P<0.001) among populations (60.29%), and within populations (39.71%). The topology according to the neighbor joining and maximum parsimony methods showed mosaic composition of the 39 haplotypes, suggesting that the populations wore not completely divergent. The pairwise F statistic values, however, indicated that the population structuring existed to some extent among the geographic populations. There was a positive relationship between the aquatic distance and the genetic distance (Fst) among the populations (P<0.05). Based on our data, it is suggested that genetic drift, gene flow, and stochastic events are the possible factors influencing the population structure and genetic variation.

Genetic heterogeneity analysis and RAPD marker detection among four forms of Atrina pectinata Linnaeus

Pen shell (Atrina pectinata Linnaeus) can be distinguished into four forms based on the morphololgic characteristics. Genetic similarity, and heterogeneity were analyzed among the four forms by random amplified polymorphic DNA (RAPD) technique using 24 10-nucleotide-long primers. Of these primers, 22 pruners produced well-identifiable RAPD band patterns. Significant differences in RAPD band patterns were revealed among the four forms. A total of 198 polymorphic fragments were scored from 22 pruners. and they are specific for one form, shared by two or three forms. Several pruners, such as S451, S453 S463 S464, S470. S473 and S474, produced abundant band patterns and provided sufficient information for reliable discrimination of the four forms. The average genetic distances and phylogenetic relationships were calculated and analyzed according to the distinguishable fragments. The data indicate that pen shells of form G and form Y are similar not only among individuals within the same form, but also between individuals from the two forms, and that shells of form T and form S are highly divergent. The constructed phylogenetic free matches the average genetic distances. Three clusters were clearly distinguishable, in which two were corresponding to form S and form T respectively and one included forms G and Y. This Study will be benefit to further studies oil the taxonomy and selective breeding of Pinnid species. It is suggested that the four forms of pen shell should be categorized to at least two species taxonomically.

Phylogeny of the East Asian cyprinids inferred from sequences of the mitochondrial DNA control region

With 210 genera and 2010 species, Cyprinidae is the largest freshwater fish family in the world. Several papers, based on morphological and molecular data, have been published and have led to some solid conclusions, such as the close relationships between North American phoxinins and European leuciscins. However, the relationships among major subgroups of this family are still not well resolved, especially for those East Asian groups. In the present paper, the mitochondrial DNA (mtDNA) control region, 896-956 base pairs, of 17 representative species of East Asian cyprinids was sequenced and compared with those of 21 other cyprinids to study their phylogenetic relationships. After alignment, there were 1051 sites. The comparison between pairwise substitutions and HKY distances showed that the mtDNA control region was suitable for phylogenetic study. Phylogenetic analysis indicated that there are two principal lineages in Cyprinidae: Cyprinine and Leuciscine. In Cyprinine, the relationships could be a basal Labeoinae, an intermediate Cyprininae, and a diversified Barbinae (including Schizothroaxinae). In Leuciscine, Rasborinae is at the basal position; Gobioninae and Leuciscinae are sister groups; the East Asian cultrin-xenocyprinin taxa form a large monophyletic group with some small affiliated groups; and the positions of Acheilognathinae and Tincinae are still uncertain.

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIF158 and CaIF156, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IF160, IF158, IF156, IFI54 and their homologues in some other mammalian species. The transcripts of CaIF158 and CaIF156 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CalF158 and CalF156 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues. (C) 2003 Elsevier B.V All rights reserved.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

Sequence variations of the S7 ribosomal protein gene in primitive cyprinid fishes: Implication on phylogenetic analysis

Cyprinidae is the largest fish family in the world and contains about 210 genera and 2010 species. Appropriate DNA markers must be selected for the phylogenetic analyses of Cyprinidae. In present study, the 1st intron of the S7 ribosomal protein (r-protein) gene is first used to examine the relationships among cyprinid fishes. The length of the 1st intron obtained by PCR amplification ranges from 655 to 859 by in the 16 cyprinid species investigated, and is 602 by in Myxocyprinus asiaticus. Out of the alignment of 925 nucleotide sites obtained, the parsimony informative sites are 499 and occupy 54% of the total sites. The results indicate that the 1st intron sequences of the S7 r-protein gene in cyprinids are rich in informative sites and vary remarkably in sequence divergence from 2.3% between close species to 66.6% between distant species. The bootstrap values of the interior nodes in the NJ (neighbor-joining) and MP (most-parsimony) trees based on the present S7 r-protein gene data are higher than those based on cytochrome b and the d-loop region respectively. Therefore, the 1st intron sequences of the S7 r-protein gene in cyprinids are sensitive enough for phylogenetic analyses, and the 1st intron is an appropriate genetic marker for the phylogenetic reconstruction of the taxa in different cyprinid subfamilies. However, attempts to discuss whether the present S7 r-protein gene data can be applied to the phylogeny of the taxa at the level of the family or the higher categories in Cypriniformes need further studies.

Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.

Sequence variations in the mitochondrial DNA control region and their implications for the phylogeny of the Cypriniformes

The mitochondrial DNA control region of six cobitids and two catostomids was sequenced and compared with sequences of other cypriniforms to study their sequence variations. The extended termination associated sequence (ETAS) domain, central domain, and conserved sequence block (CSB) domain were partitioned and the ETAS sequence, CSB-D, CSB-E, ECSB-F, CSB1, CSB2, and CSB3 were identified. It is suggested that the "hairpin" TACAT-ATGTA is the key sequence of ETAS and GACATA is the symbol of CSB1. Phylogenetic analysis based on the CSB domain showed that all cyprinids evolved as one monophyletic group, while the non-cyprinid Cypriniformes could be another monophyly that is in accordance with the hypothesis proposed by Siebert. Further analysis of the phylogeny of the Cobitoidei was also conducted and it is tentatively suggested that their relationships are Catostomidae + (Gyrinocheilidae + (Botiinae + (Homalopteridae + (Cobitinae + Nemacheilinae)))).

Molecular analysis of silver crucian carp (Carassius auratus gibelio Bloch) clones by SCAR markers

Random amplified polymorphic DNA (RAPD) molecular markers specific for one, two or three clones have been identified from five gynogenetic clones of silver crucian carp (Carassius auratus gibelio Bloch) using RAPD markers developed earlier. In this study, three RAPD markers (RA1-PA, RA2-EF and RA4-D) produced by Opj-1, and two RAPD DNA fragments (RA3-PAD and RA5-D) produced by Opj-7, were selected for molecular cloning and sequencing. Sequence data indicated that there were identical 801-bp nucleotide sequences in the shared marker RA1-PA cloned respectively from clones P and A, and the shared marker RA2-EF (which was cloned from clones E and F), were also of identical 958-by nucleotide sequences. The nucleotide sequences of the shared marker RA3-PAD fragments were also similar for 1181 by among clones P, A and D. The specific fragment RA4-D was composed of 628 bp, and the fragment RA5-D from clone D contained 385 nucleotides. According to the nucleotide sequences, we designed and synthesized five pairs of sequence characterized amplified regions (SCAR) primers to identify the specific fragments in these gynogenetic clones of silver crucian carp. Only individuals from clones P and A amplified a specific band using a pair of SCI-PA primers synthesized according to the marker RA1-PA sequences, whereas no products were detected in individuals from clones D, E and F. The PCR products amplified using SC2-EF and SC3-PAD primers were as expected. Furthermore, the pair of SC4-D primers amplified specific bands only in individuals from clone D, although weak bands could be produced in all individuals of the five clones when lower annealing temperatures were used. However, an additional pair of SC5-D primers designed from the RA5-D marker sequences could amplify a DNA band in individuals from clones P, A and D, and the same weak band was produced in clone E, whereas no products were detected in individuals from clone F. Searches in GenBank revealed that the 385-bp DNA fragment from RA5-D was homologous to the 5' end of gonadotropin I beta subunit 2 gene and growth hormone gene. No homologous sequences were found for other markers in GenBank. The SCAR markers identified in this study will offer a powerful, easy, and rapid method for discrimination of different clones and for genetic analyses that examine their origins and unique reproductive modes in crucian carp. Furthermore, they will likely benefit future selective breeding programs as reliable and reproducible molecular markers. (C) 2001 Elsevier Science B.V. All rights reserved.

RAPD and AFLP techniques for the analysis of genetic relationships in two genera of Decapoda

RAPD was used fur analysing three (sub-)species of mitten crabs (Eriocheir sinensis, E. japonicus, and E. japonicus hepuensis) and three populations of E. sinensis. The results show that their relationships on DNA level are similar to the classical taxonomic hypotheses (Dai, 1991). No diagnostic RAPD marker could be found, but there were statistically significant genetic differences among these taxa (P < 0.001) or populations (P < 0.001). That is, the intraspecific similarities were larger than the interspecific similarities; the intrasubspecific similarities were larger than the intraspecific similarities; and the intrapopulational similarities were larger than the interpopulational similarities. In AFLP analysis, no significant genetic difference has been found between E. sinensis and E. japonicus, but AFLP markers among four species of Macrobrachium (M. rosenbergii. M. nipponense, M. hainanense, and M. asperulum) were found. The DNA similarities among these four species of Macrobrachium are in accordance with morphological similarities.

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

Cancer subtypes recognition and its gene expression profiles analysis based on geometrical learning

The accurate recognition of cancer subtypes is very significant in clinic. Especially, the DNA microarray gene expression technology is applied to diagnosing and recognizing cancer types. This paper proposed a method of that recognized cancer subtypes based on geometrical learning. Firstly, the cancer genes expression profiles data was pretreated and selected feature genes by conventional method; then the expression data of feature genes in the training samples was construed each convex hull in the high-dimensional space using training algorithm of geometrical learning, while the independent test set was tested by the recognition algorithm of geometrical learning. The method was applied to the human acute leukemia gene expression data. The accuracy rate reached to 100%. The experiments have proved its efficiency and feasibility.

大赖草总DNA转化小麦引起的HMW-GS基因变化及大赖草HMW-GS基因的克隆

应用花粉管通道技术将新疆大赖草总DNA导入小麦，用高重序列分析方法，已为大赖草总DNA转入小麦提供了初步的分子证据。在转 化后代中选育出稳定遗传的大穗变异株系，分析表明，这些转化株中蛋白质含量明显增高(13%-17%)。对基因供体新疆大赖草、受 体春麦761、转化株的高分子量谷蛋白亚基(HMW-GS)进行了SDS-PAGE分析，发现这些转化株中HMW-GS发生了很大变化，并在此基础 上，用来自小麦基因组的四对特异引物，以PCR方法扩增供体、受体以及转化株的1Ax、1Bx、1Dx及1Ay、1By、1Dy型HMW-GS全基因 ，比较他们扩增产物的差异，结果表明，受体与转化株在HMW-GS基因1Ax、1Bx位点上的扩增产物差异不大，在HMW-GS基因位点1Dx 和y型基因上的扩增产物有较大差异，说明了受体在基因位点1Dx、1Ay、1By和1Dy上可能发生了多位点插入，可能由于这些基因位 点上的插入引起了转化株的高分子量谷蛋白亚基(HMW-GS)的变化，这就再一次为大赖草总DNA导入提供了直接的分子证据。虽然大 赖草总DNA导入提高了小麦蛋白质的含量，改变了HMW-GS的组成，部分改变了品质评分，但我们感到这些转化株在品质改良方面仍 有很大余地，如何更好地利用新疆大赖草蛋白质的优良特性及避免总DNA导入给转化株带来的不良性状，一个大赖草HMW-GS基因正 被分离及克隆，并准备将其利用于未来的品质育种当中。Total DNA of Leymus racemosus had been transformed into wheat through pollen tube pathway. Analysis of the repeated gene sequence had provided an elementary proof. Some variant cultivars with big ear were screened from their offsprings, and their protein content increased greatly from 13% (receptor)to 17%(transformed). The result from SDS-PAGE analysis of high-molecular-weight glutenin subunits(HMW- GS) respectively in donor(Xinjiang Leymus racemosus), receptor(spring wheat cultivar 761)and transformed wheats, showed the HMW-GS composition changed in the transformed plants. On the basis of the research, Four special pairs primers from wheat(T.aestivum L.) genome were used to amplify complete coding regions of HMW-GS genes on 1Ax、1Bx 、1Dx and 1Ay、1By、1Dy loci of donor、receptor and the big ear transformed cultivars. By comparing amplified PCR products. Faint differences were found among receptor and transformed cultivar's 1Ax、1Bx PCR amplifed products and apparent differences on those of 1Dx、y-typePCR product. We gueseed that there may be some DNA inserts in 1Dx 、1By、1Dy loci resulted in the changes of the HMW-GS among transformed cultivars. This provides second direct molecular witness to the exogenous DNA introduction. Even though the transformed plants have higher protein content, changed HMW-GS composition, partially improved process quality, there still leave much work to improve quality. In order to make full use of the excellent property of Leymus racemosus protein and avoid the disadvantages introducced by total DNA transformation, a HMW-GS gene of Leymus racemosus was being isolated and cloned.

Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Urinary 8-hydroxydeoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. This article studied oxidative DNA damage in patients with diabetic nephropathy and in healthy control subjects by urinary 8-OHdG evaluations. Contents of 8-OHdG in urine were analyzed by capillary electrophoresis with end-column amperometric detection (CE-AD) after a single-step solid-phase extraction (SPE). Levels of urinary 8-OHdG in diabetic nephropathy patients with macroalbuminuria was significant higher than in control subjects (5.72 +/- 6.89 mumol/mol creatinine versus 2.33 +/- 2.83 mumol/mol creatinine, P = 0.018). A significant difference of 24 h urinary 8-OHdG excretions exists between the patients with macroalbuminuria and the patients with nonnoalbuminuria (19.2 +/- 16.8 mug/24 h versus 8.1 +/- 1.7 mug/24 h, P = 0.015). There was a positive correlation between urinary excretion of 8-OHdG and glycosylated hemoglobin (HbA(1)c) (r = 0.287, P = 0.022). A weak correlation exists between the levels of 8-OHdG and triglyceride (r = 0.230, P = 0.074). However, the urinary 8-OHdG contents are not correlated with blood pressure and total cholesterol. The increased excretion of urinary 8-OHdG is seen as indicating an increased systemic level of oxidative DNA damage in diabetic nephropathy patients. (C) 2004 Elsevier B.V. All rights reserved.

DNA diagnosis by capillary electrophoresis and microfabricated electrophoretic devices

DNA diagnosis is experiencing an impressive progression towards the development of novel technology to identity various clinically relevant categories of genetic changes and to meet the exponential growth of genomics. The introduction of capillary electrophoresis has dramatically accelerated the completion of the first draft of the human DNA sequence in the Human Genome Project, and thus, has become the method of choice for analysis of various genetic variants. The recent development of microfabricated electrophoretic devices has led to the possibility of integrating multiple sample handling with the actual measurement steps required for automation of molecular diagnostics. This review highlights the most recent progress in capillary electrophoresis and electrophoretic microdevices for DNA-based diagnostics, including the important areas of genotyping for point mutation, single nucleotide polymorphisms, short tandem repeats and organism identification. The application of these techniques for infectious and genetic disease diagnosis, as well as forensic identification purpose, are covered. The promising development and the challenges for techinical problems are also discussed.