The Sequence of the Hartmannella vermiformis Small Subunit rRNA Coding Region

ABSTRACT The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.     

Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.     

Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Genetic Identification of Staphylococcus aureus by Polymerase Chain Reaction Using Single-Base-Pair Mismatch in 16S Ribosomal RNA Gene

Staphylococcus aureus is the most predominant and important pathogen in clinical microbiology. A DNA amplification assay using the polymerase chain reaction (PCR) was designed to identify S. aureus through a single-base-pair mismatch in the sequences of staphylococcal 16S ribosomal RNA (16S rRNA) genes. It was able to detect and identify S. aureus without requiring additional analytical techniques. Twenty-eight staphylococcal and non-staphylococcal strains were tested to verify the specificity of the assay, and only S. aureus strains gave a positive reaction. It may be possible to provide immediate and exact information for the identification of S. aureus.     

Phylogenetic Investigations on Ten Genera of Tintinnid Ciliates (Ciliophora: Spirotrichea: Tintinnida), Based on Small Subunit Ribosomal RNA Gene Sequences

Tintinnida is a diverse taxon that accommodates over 1,500 morphospecies, which is an important component of marine planktonic food webs. However, evolutionary relationships of tintinnids are poorly known because molecular data of most groups within this order are lacking. In our study, the small subunit (SSU) rRNA genes representing 10 genera, 5 families of Tintinnida were sequenced, including the first SSU rRNA gene sequences for Coxliella, Dadayiella, Epiplocyloides, and Protorhabdonella, and phylogenetic trees were constructed to assess their intergeneric relationships. Phylogenies inferred from different methods showed that (1) Three newly sequenced Eutintinnus species fell into Eutintinnus clade forming a sister group to the clade containing Amphorides, Steenstrupiella, Amphorellopsis, and Salpingella; (2) Surprisingly, the genetic distances between Amphorides amphora and Amphorellopsis acuta population 1 was even smaller than that between the two populations of Amphorellopsis acuta, casting doubt on the validity of Amphorides and Amphorellopsis as presently defined; (3) The SSU rRNA sequences of Dadayiella ganymedes and Parundella aculeata were almost identical. Therefore, Parundella ganymedes novel combination is proposed; (4) Coxliella, which is currently assigned within Metacylididae, branched instead with some Tintinnopsis species. Furthermore, the validation of Coxliella, which was considered to be a “questionable” genus, was confirmed based on evidences from morphology, ecology, and molecular data; (5) Protorhabdonella and Rhabdonella showed rather low intergeneric distance and grouped together with strong support suggesting that Rhabdonellidae is a well‐defined taxon; and (6) Epiplocyloides branched with species in Cyttarocylididae indicating their close relationship.     

Simple Identification of Transgenic Arabidopsis Plants Carrying a Single Copy of the Integrated Gene

Transgenic Arabidopsis thaliana plants carrying a single copy of integrated DNA can be identified by single-step genomic polymerase chain reaction. The reaction employs two sets of primer pairs with the same melting temperature that amplify the amplicons derived from the integrated T-DNA together with those from an endogenous single-copy gene as a reference. When the band intensity ratio is one, this means that the transgenic plants are carrying a single copy of the integrated gene per haploid.     

鸡大肝大脾病毒RT—PCR检测方法的建立及其检测

鸡大肝大脾病(Big liver and spleen disease,BLS)是发生于商品代肉用产蛋母鸡的一种传染性疾病,1980年最先在澳大利亚发现,1988年Handlinger等对该病进行了描述.感染鸡直到性成熟后才出现临床症状,仅表现为精神沉郁、产蛋下降或达不到产蛋高峰.死亡率为1%.剖检患鸡或死亡鸡见肝脏、脾脏肿大,呈现腹膜炎和肠炎.     

基于PIPE现象的质粒克隆位点序列设计与功能评价

分子克隆是现代生物学研究的核心技术之一,是基因工程、蛋白质工程中的重要手段。为提高分子克隆实验的操作效率,本研究设计并合成基于聚合酶引物不完全延伸（polymerase incomplete primer extension,PIPE）现象的质粒克隆位点序列。并以此为基础统一相关引物的设计方案,避免传统酶切--连接法中需针对不同载体MCS序列设计不同引物的缺点。该方案利用13 bp定长接头序列,在同一体系中使用2对引物、2种线性化模板同时扩增载体和插入片段,通过20个循环,在1次PCR过程中即合成可供转化使用的带缺口质粒产物。在NEB Q5酶系统中,利用此法将3种荧光素酶序列插入pET-15b及pET-21b(+)载体,均获得成功。且利用商品化感受态细胞（转化效率 > 5×108 cfu/μg）转化后所获得转化子数量均在300个以上,其中含插入片段的阳性克隆比例可达85%以上。基于本方案的设计及作用原理,可将其应用于10 kb以内载体和插入片段的快速重组。且具有通用性强、耗时少、阳性克隆得率高和成本低等优点,是传统DNA重组方法的有益补充,可作为各实验室的常规分子克隆手段之一。     

用干血纸片扩增人雄激素受体基因

雄激素不敏感综合征(AIS)为一类主要与雄激素受体(AR)基因缺陷密切相关的X-连锁隐性遗传病.为进一步阐明AIS的发病机制,建立了用干血纸片直接PCR扩增或将干血纸片中的血细胞洗脱裂解后进行PCR扩增雄激素受体(AR)基因的方法,结合已建立的SSCP分析及DNA直接测序等方法,可对AR基因进行突变分析.干血纸片取样及保存容易,便于邮寄,适用于外地,特别是边远地区患者的取样.该法不仅为AIS患者的AR基因突变分析和家系调查提供简便易行的方法,也适用于PCR基础上的其他各种基因的突变分析.     

Universal Primers for Amplification of Mitochondrial Small Subunit Ribosomal RNA-Encoding Gene in Scleractinian Corals

We describe the construction of polymerase chain reaction primers designed to amplify a portion of the mitochondrial (mt) small subunit ribosomal (SSU) RNA-encoding genes in scleractinian corals. Combinations of cloning and sequencing show that the amplified fragments are between 694 and 896 bp in length. Alignment of the amplified DNA sequences to the published mt SSU rRNA genes of Metridium senile and Sarcophyton glaucum indicates several conserved regions among actiniarian, corallimorpharian, octocorallian, and scleractinians, suggesting this primer set can successfully amplify over 80% of the mt SSU rDNA region of scleractinian corals. Surveys of sequence variation and estimation of the rate of evolution show an extremely slow divergence of the SSU rRNA gene in the family Acroporidae. Received June 11, 1999; accepted October 4, 1999.     

The Small Subunit Ribosomal RNA Gene Sequence of Pleistophora anguillarum and The Use of PCR Primers for Diagnostic Detection of the Parasite

Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA.) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels ( Anguilla japonica ) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum , enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post-infection. No PCR product was obtained with templates prepared from clinically healthy control animals.     

纳米金对PCR扩增效率影响的机制探讨

近年来,纳米金粒子对聚合酶链式反应（polymerasechainreaction,PCR）的增效作用倍受关注,但是其具体的机制仍未明确提出。研究发现在PCR反应中,纳米粒子的增效作用是存在最佳浓度的,增加DNA聚合酶或者小牛血清蛋白（BSA）可以消除纳米金粒子导致的抑制。我们认为,纳米金粒子可能起到了类似于聚合酶B亚基的作用,提高了DNA聚合酶的延伸能力;而过量纳米金对PCR的抑制作用可能与纳米金结合单链DNA产生的位阻效应有关。     

柑橘衰退病毒柚类分离株的分子鉴定

应用限制性片段长度多态性(RFLP)、双向逆转录-聚合酶链式反应(BD-PCR)、序列分析等技术对我国部分地区柑橘衰退病毒(Citrus tristeza virus,CTV)柚类分离株进行了鉴定分析。结果表明:柚类以受相对单一的CTV株系侵染为主;柚类上的优势流行株系属于p25/HinfⅠRFLP 6组群(占79.4%)和p23/BD-PCRⅢ组群(占84.1%);在对柚类CTV分离株S051~S058及国外CTV分离株PB61、T30、T36、T385、SY568、VT、NUAGA的p23、p25基因序列分析中,S051与弱毒株PB61的同源性最高;S052与弱毒株T30、T385的同源性最高;S053、S054、S055、S056、S057、S058与其它CTV分离株的同源关系均较远,并在系统发生树上形成了独立的分枝。     

Molecular methods for environmental monitoring and containment of genetically engineered microorganisms

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

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．     

Comparison of a Reversed Passive Latex Agglutination and a Polymerase Chain Reaction for Identification of Cholera Toxin Producing Vibrio cholerae O1

Production of cholera toxin (CT) in AKI medium and conservation of CT gene (ctx) of 49 strains of Vibrio cholerae O1 were compared by reversed passive latex agglutination (RPLA) and polymerase chain reaction (PCR). The production of CT agreed with conservation of the ctx in 48 out of the 49 strains. Ten strains were positive, and 38 strains were negative by both methods. Only one strain was negative in RPLA and positive in PCR. This suggested that the combination of AKI-SW and RPLA is comparable to PCR to identify CT-producing V. cholerae O1.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.