Variation in the nucleotide sequence of a prolamin gene family in wild rice

Variation in the DNA sequence of the 10 kDa prolamin gene family within the wild rice species Oryza rufipogon was probed using the direct sequencing of PCR-amplified genes. A comparison of the nucleotide and deduced amino-acid sequences of eight Asian strains of O. rufipogon and one strain of the related African species O. longistaminata is presented.     

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

Cloning and sequencing of a novel metallothionein gene in Mytilus galloprovincialis Lam

Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 5' promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http://www.unizh.ch/~mtpage/classif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

用于“野生小家鼠来源一号染色体替换系”构建的PCR-LDR分型系统

目的 建立用于“野生小家鼠来源一号染色体替换系”构建的PCR-LDR (polymerase chain reaction and ligase detection reaction,PCR-LDR)分型系统.方法 采用易于判断的二元性遗传标记单核苷酸多态性位点( single nuclear polymorphism...     

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages

The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.     

Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer

Abstract The 16S rRNA gene sequences of 19 strains covering 97% of the molecules were determined for the members of the family Rhizobiaceae and related bacteria by PCR and DNA sequencer. The three biovars of Agrobacterium were located separately, whereas Agrobacterium rubi clustered with A. tumefaciens . Phylogenetic locations for the species of the genera Rhizobium, Sinorhizobium, Agrobacterium, Phylobacterium, Mycoplana (M. dimorpha), Ochrobactrum, Brucella and Rochalimaea (a rickettsia) were intermingled with each other with the similarity values higher than 92%. The family Rhizobiaceae should be redefined including the above-mentioned genera despite the ability for plant association and nitrogen fixation. Bradyrhizobium japonicum and Mycoplana bullata were far remote from the other species and should be excluded from this family.     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

Phylogenetic relationships of the garter snakes based on DNA sequence and allozyme variation

We estimated phylogenetic relationships among 26 species of garter snakes (genus Thamnophis ) using allozyme and mitochondrial cytochrome b gene nucleotide sequence variation. Parsimony analyses of the two data sets give substantially different estimates of phylogeny. Several lines of evidence indicate that much of this conflict is due to error associated with the restricted number of characters in each data set. Such sampling error may be reduced by combining all the characters; we therefore present an estimate of phylogeny based on parsimony analysis of all the data combined. All our analyses support several conclusions in conflict with previous views: a very distant relationship between T.errans and T. elegans , non-monophyly of the elegans group (even excluding T: errans ), and nesting of the form validus (previously considered a member of the genus Nerodia ) within Thamnophis.
The combined analysis gives an almost fully resolved tree. However, bootstrapping indicates only weak support for many clades in this tree. Furthermore, paraphyly of the assemblages of cytochrome b gene lineages within T. elegans and T. radix indicate the potential for discordance between the mitochondrial DNA (mtDNA) and species phylogenies through the sorting of ancestral mtDNA polymorphisms. These problems suggest the need for assaying additional characters, especially ones likely to be independent of those used in the present study.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

Comparative Analysis of Mitochondrial DNA Diversity in Smelts

The polymerase chain reaction (PCR) was used to amplify a segment of the mitochondrial DNA coding for NADH-dehydrogenase subunits ND5/ND6 in five smelt species (family Osmeridae). Amplified DNA was screened for restriction fragment length polymorphism (RFLP). Nucleotide sequence divergence of mitochondrial DNA between species ranges from 11.9 (between Hypomesus nipponensis and H. japonicus) to 24.7% (between Osmerus mordax dentex and Mallotus villosus catervarius). The genetic divergence between populations of H. nipponensis, H. japonicus, and Osmerus mordax dentex was 0.32, 0.08 to 0.15, and 0.025%, respectively. The absence of common haplotypes enables differentiation of closely related smelt species and, therefore, can be used for solving current problems in the taxonomy and biogeography of this family.     

促髓细胞分化的锌指基因单向PCR扩增直接测序的研究

锌指基因是一种造血调节基因,编码锌指结构蛋白,主要在髓细胞中表达,促进髓细胞分化,在急性早幼粒白血病维甲酸治疗中,促使病情缓解。本文报道了我们从基因分子上研究锌指基因作用中,探索并建立了单向聚合酶链反应（ＰＣＲ）扩增特定单链ＤＮＡ,直接测序的新方法。它能产生质和量均佳的单链ＤＮＡ,无需纯化即可直接用于测序,使复杂的测序研究简便易行,可在２,３天内完成。这种单向ＰＣＲ扩增特定单链ＤＮＡ直接测序的方法,经对锌指基因的ｃＤＮＡ测序,得到验证。此法不仅适用于疾病研究中的ＤＮＡ测序,还可制各单链ＤＮＡ探针,更利于基因结构组成的研究。     

Phylogenetic analysis of tnpR. genes in mercury resistant soil bacteria: the relationship between DNA sequencing and RFLP typing approaches

Abstract The diversity of resolvase ( tnpR ) genes carried by a number of mercury resistant soil bacteria has been investigated by DNA sequencing. The resulting DNA sequence information was compared to previously published tnp R. DNA sequences and to previously published restriction fragment length polymorphism (RFLP) data, permitting the relationships between DNA sequencing and RFLP approaches to be studied by the use of phylogenetic trees. DNA maximum likelihood and DNA parsimony were used to construct a variety of phylogenetic trees. DNA sequencing confirmed the validity of RFLP analysis and highlighted the importance of restriction endonuclease choice upon the resulting RFLP patterns and dendrogram topology. The tnp R genes of two previously uncharacterised mercury resistant bacteria, T2–7 and T2–12 were also studied. DNA sequence data placed T2–7 in a previously described gene class, tnp R-D and T2–12 in a new gene class, tnp R-F. The significance of this data with respect to the recombination and evolution events occurring within bacterial populations are discussed.     

Analysis of disease-causing genes and DNA-based drugs by capillary electrophoresis towards DNA diagnosis and gene therapy for human diseases

Rapid progress in the Human Genome Project has stimulated investigations for gene therapy and DNA diagnosis of human diseases through mutation or polymorphism analysis of disease-causing genes and has resulted in a new class of drugs, i.e., DNA-based drugs, including human gene, disease-causing gene, antisene DNA, DNA vaccine, triplex-forming oligonucleotide, protein-binding oligonucleotides, and ribozyme. The recent development of capillary electrophoresis technologies has facilitated the application of capillary electrophoresis to the analysis of DNA-based drugs and the detection of mutations and polymorphism on human genes towards DNA diagnosis and gene therapy for human diseases. In this article the present state of studies on the analysis of DNA-based drugs and disease-causing genes by capillary electrophoresis is reviewed. The paper gives an overview of recent progress in the Human Genome Project and the fundamental aspects of polymerase chain reaction-based technologies for the detection of mutations and polymorphism on human genes and capillary electrophoresis techniques. Attention is mainly paid to the application of capillary electrophoresis to polymerase chain reaction analysis, restriction fragment length polymorphism, single strand conformational polymorphism, variable number of tandem repeat, microsatellite analysis, hybridization technique, and monitoring of DNA-based drugs. Possible future trends are also discussed.     

Accessing genes in the tertiary gene pool of rice by direct introduction of total DNA fromZizania palustris (wild rice)

Transfer of useful genes from wild relatives of crop plants has relied upon successful conventional crossing or the availability of the cloned gene. Co-bombardment of rice callus with total genomic DNA from wild rice (Zizania palustris) and a plasmid containing a gene confirming hygromycin resistance allowed recovery under selection of transgenic plants with grain characteristics from wild rice. Amplified Fragment Length Polymorphism (AFLP) analysis suggested that a significant amount of DNA fromZizania was introduced by this procedure. One plant had 16 of a possible 122Zizania specific AFLP markers detected with the primers used. This approach may have potential for introgression of genes from wild relatives in other cases where highly efficient transformation methods are available.     

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.     

Sequence analysis of attachment gene of lumpy skin disease and sheep poxviruses

In Egypt, protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy. In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected. Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared. The causative agent in the collected samples was identified using immunoflourescence (IF) and immunoperoxidase techniques. Of the 15 skin nodules suspected of LSD, 10 showed a positive reaction and 3 out of 5 skin scabs suspected of sheeppox were found to be positive. An antigenic correlation between field skin isolate of LSDV, tissue culture adapted LSDV/Ismailyia88 strain, field skin isolate of SPV and SPV/Kenyan vaccinal strain was studied using prepared hyperimmune sera. Also, nucleotide sequence of the PCR amplified attachment gene fragments of field skin isolate of LSDV, tissue culture adapted LSDV/Ismailyia88 strain, field skin isolate of SPV and SPV /Kenyan vaccinal strain were compared. The results revealed that the four used viruses were antigenically identical. Sequence analysis indicated that field skin LSDV isolate is more related to tissue culture adapted LSDV/Ismailyia88 strain than to vaccinal SPV/ Kenyan strain and the skin isolate of SPV is more closely related to field skin isolate of LSDV than to SPV/Kenyan vaccinal strain. Thus, further study should be applied on the advantage of a LSD vaccine prepared from LSDV in protection of cattle against LSD compared to the commonly used sheep pox vaccine.     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

Sequence Analysis of Attachment Gene of Lumpy Skin Disease and Sheep Poxviruses

In Egypt,protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy.In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected.Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared.The causative agent in the collected samples was identified using immunoflourescence (IF) and immuno...