Genetic identification of bucktooth parrotfish Sparisoma radians (Valenciennes, 1840) (Labridae,Scarinae) by chromosomal and molecular markers

Parrotfishes (Labridae, Scarinae) comprise a large marine fish group of difficult identification, particularly during juvenile phase when the typical morphology and coloration of adults are absent. Therefore, the goal of this study was to test cytogenetic markers and DNA barcoding in the identification of bucktooth parrtotfish Sparisoma radians from the northeastern coast of Brazil. Sequencing of cytochrome c oxidase subunit I (COI) confirmed all studied samples as S. radians, and all showed high similarity (99–100%) with Caribbean populations. The karyotype of this species was divergent from most marine Perciformes, being composed of 2n = 46 chromosomes. These consisted of a large number of metacentric and submetacentric pairs with small amounts of heterochromatin and GC-rich single nucleolar organizer regions (NORs) not syntenic to 5S rDNA clusters. These are the first data about DNA barcoding in parrotfish from the Brazilian province and the first refined chromosomal analysis in Scarinae, providing useful data to a reliable genetic identification of S. radians.     

Recombination between satellite phage P4 and its helper P2. II. In vitro construction of a helper-independent P4: :P2 hybrid phage

A helper-independent P4::P2 hybrid (Hy19), with the essential gene region of P4 linked to the late genes of P2, has been isolated by in vitro recombination techniques. This hybrid expresses a P4 Sid^? phenotype since it makes large heads. The int-C region of P2 is deleted from Hy19 and its DNA replication is independent of the host rep gene, indicating that it depends on the P4 replicon.     

限制酶介导整合技术：一种克隆新基因的新策略

限制酶介导整合（REMI）技术是近年发展起来的一种研究新基因的方法,已被用于盘基网柄菌、酵母和丝状真菌等微生物细胞与发育过程的研究。结合质粒拯救技术,可快速分离质粒两侧未知的基因组DNA序列,进而对该序列做进一步分析和研究。我们简要介绍了REMI技术的基本原理、影响因素、缺点及其应用。     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

Planarian mitochondria sequence heterogeneity: relationships between the type of cytochrome c oxidase subunit I gene sequence, karyotype and genital organ

Freshwater planarians Dugesia japonica from three localities were examined for cytochrome c oxidase subunit I (COI) gene sequence, karyotype and the presence of genital organ. The planarians from Mt Fujiwara in Japan were composed of two different groups; one revealed inter- and intraindividual COI gene heterogeneity, while another revealed no sequence heterogeneity. The sequence in planarians from Mt Alishan in Taiwan was homogeneous, while that from the Kenting National Park in Taiwan revealed a considerable heterogeneity. All the planarians having the homogeneous gene sequences carry the 2X karyotype and many of them had genital organs. These are assumed to belong to the sexual lineage. In contrast, almost all planarians having heterogeneous sequences carry the karyotype of either 3X plus 2X (mixoploid) or 3X, and all of them lack genital organs. These lineages are assumed to be asexual. The heterogeneity of COI gene sequences in the presumed asexual lineages would have resulted from an accumulation of mutations by repeated asexual reproduction.     

VIRS: A visual tool for identifying restriction sites in multiple DNA sequences

VIRS (A visual tool for identifying restriction sites in multiple DNA sequences) is an interactive web‐based program designed for restriction endonuclease cut sites prediction and visualization. It can afford to analyze multiple DNA sequences simultaneously and produce visual restriction maps with several useful options intended for users' customization. These options also perform in‐depth analysis of the restriction maps, such as providing virtual electrophoretic result for digested fragments. Different from other analytical tools, VIRS not only displays visual outputs but also provides the detailed properties of restriction endonucleases that are commercially available. All the information of these enzymes is stored in our internal database, which is updated monthly from the manufacturers' web pages. It is freely available online at http://bis.zju.edu.cn/virs/index.html . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

酶化学计量揭示5年氮添加加剧毛竹林土壤微生物碳磷限制

土壤胞外酶活性和酶化学计量比能很好地反映土壤养分有效性和微生物对养分的需求变化.然而,氮(N)沉降对亚热带森林土壤微生物养分相对限制情况的影响尚不清楚.通过在亚热带毛竹林进行N添加试验来模拟N沉降,并在试验满5年时进行取样,测定不同处理下土壤养分和与碳(C)、N、磷(P)循环相关的酶活性,利用酶化学计量比及矢量分析探究...     

Prometaphase chromosomes of the howler monkey (Aloutta caraya): G,C, NOR,and restriction enzyme (RES) banding

Prometaphase lymphocyte chromosomes from eight adult argentinian Alouatta caraya females were characterized using sequential G-C banding techniques, Ag-NOR bands and bands obtained with the restriction enzymes Hae III, Eco RI, Alu I and Sau 3A. The cytogenetic analysis showed 2n = 52, with four, five, or six NOR chromosomes. Digestion with Hae III and Eco RI produced G-like-bands. Centromere regions and two interstitial C-bands (in chromosomes number 16 and 21) showed intraindividual or interindividual heterochromatic polymorphisms. Alu I digestion produced C-like bands with gaps in the centromere regions, and Sau 3A produced C-like bands. The karyotypes and banding patterns of A. caraya, A. palliata, A. belzebul, and A. seniculus are compared, based on whole chromosome and whole arm homeologies. © 1994 Wiley-Liss, Inc.     

A general assay for restriction endonucleases and other DNA-modifying enzymes with plasmid substrates

A procedure for measuring the activities of enzymes that alter the covalent structure of DNA is described. The assay utilizes covalently closed circles of DNA as the substrate and yields quantitative data on the fraction of this DNA converted to both open-circle and linear forms.     

Identification of Cultured Brachionus Rotifers Based on RFLP and SSCP Screening

The marine finfish industry worldwide depends greatly on the mass culture of Brachionus rotifers. Recently, molecular data have revealed a more complicated view about the species status of Brachionus rotifers than previous mainly morphological assessments. Under this view, Brachionus rotifers are comprised of many morphologically similar, albeit genetically differentiated, cryptic members of larger groups. A redefinition of the cultured rotifer species/biotypes is therefore needed if aquaculture is to reach higher levels of standardization and predictability. In this work, restriction fragment length polymorphism (RFLP) and single-strand conformational polymorphism (SSCP) methods are applied to the COI and 16S rRNA mitochondrial genes. A detailed COI restriction map was constructed, using sequence data from all known representatives of Brachionus phylogroups. Therefore, it is the first time that such an extended restriction database has been produced. Several restriction endonucleases are proposed for the discrimination of the different Brachionus species/biotypes. Furthermore, eight different SSCP gel alleles are described for the 16S region. Using these data, five Brachionus species/biotypes were identified in 78 samples collected from laboratories and hatcheries around the world. Spiros Papakostas and Stefania Dooms contributed equally to this work.     

Inter-Strain Variability of Insertion/Deletion Events in the Encephalitozoon cuniculi Genome: A Comparative KARD-PFGE Analysis

ABSTRACT. We applied a two-dimensional pulsed-field gel electrophoresis procedure to the genomes of two karyotype variants assigned to two different strains of the microsporidian Encephalitozoon cuniculi , termed D (strain III) and F (strain II). Data obtained for Bss HII and Mlul restriction fragment length polymorphisms in each chromosome are compiled and compared to the reference strain I variant A. Six Insertion/Deletion (InDels) are found in subterminal position, some of these being characteristic of either D or F. Like in strain I, the terminal fragments extending between each telomere and rDNA locus are conserved in length for each chromosome. They are however smaller than in reference variant. This size reduction is estimated to be 2.5 kbp for the strain III isolate and 3.5 kbp for the strain II isolate. We hypothesize that for the three E. cuniculi strains, all chromosome extremities are prone to a constant process of sequence homogenization through mitolic recombination between conserved regions.     

Chip ligating human genomic DNA serves as storage material and template for polymerase chain reaction

A chip was developed to store DNA for medical research. The optional restriction site fixed on the chip can randomly ligate with whole human genomic DNA treated by the corresponding restriction enzyme. PCR can then use the chip as template DNA. Moreover, a chip fixing two restriction sites (e.g. EcoRI and HindIII) showed the amplification by PCR for any location of genomic DNA. Repetitive PCRs have confirmed that a DNA chip can be stored by at –4 °C for 2 years.     

腾冲热海眼镜泉菌藻席细菌ARDRA指纹图谱分析

对腾冲热海眼镜泉中粉红色丝状菌藻席进行ARDRA指纹图谱分析,获得其细菌多样性的部分信息。直接提取菌藻席总DNA,以之为模板PCR扩增出细菌的16S rDNA,将其克隆、转化进入大肠杆菌,获得120个克隆子。用限制性内切酶(RsaⅠ、HhaⅠ、HapⅡ)筛选出了29个16S rDNA插入片段酶切带型差异明显的克隆,表明眼镜泉菌藻席细菌组成丰富。聚类分析显示这29个克隆聚为A、B两个大类,表明该菌藻席可能主要由两个遗传多样性丰富的分类群组成。     

DNA-induced conformational changes in type II restriction endonucleases: the structure of unliganded HincII

The 2.1A crystal structure of the unliganded type II restriction endonuclease, HincII, is described. Although the asymmetric unit contains only a single monomer, crystal lattice contacts bring two monomers together to form a dimer very similar to that found in the DNA bound form. Comparison with the published DNA bound structure reveals that the DNA binding pocket is expanded in the unliganded structure. Comparison of the unliganded and DNA liganded structures reveals a simple rotation of subunits by 11 degrees each, or 22 degrees total, to a more closed structure around the bound DNA. Comparison of this conformational change to that observed in the other type II restriction endonucleases where DNA bound and unliganded forms have both been characterized, shows considerable variation in the types of conformational changes that can occur. The conformational changes in three can be described by a simple rotation of subunits, while in two others both rotation and translation of subunits relative to one another occurs. In addition, the endonucleases having subunits that undergo the greatest amount of rotation upon DNA binding are found to be those that distort the bound DNA the least, suggesting that DNA bending may be less facile in dimers possessing greater flexibility.     

Molecular epidemiological survey of Listeria monocytogenes in broilers and poultry products

AIMS: To investigate the prevalence of Listeria monocytogenes in poultry products, and to elucidate whether poultry products may be linked to listeriosis cases. A further goal was to identify contamination routes for L. monocytogenes to broiler carcasses. METHODS AND RESULTS: Poultry products (385 samples) were screened for L. monocytogenes. The recovered isolates and 19 patient isolates were characterized by multilocus enzyme electrophoresis and restriction enzyme analysis. The poultry isolates showed great genetic diversity, but no identical subclones were identified from poultry sources and patients. One slaughterhouse was examined in detail during a 16-month period. The contamination rates increased along the processing line, and one subclone was found during the whole period. Only low prevalence of the bacteria was revealed from broiler faeces. CONCLUSIONS: The prevalence of L. monocytogenes in poultry products was high, but no listeriosis cases was linked to poultry products. Broilers seem to be contaminated during the slaughter process, and specific strains may persist in the processing environment. Broiler faeces does not seem to be an important source of L. monocytogenes in poultry products. SIGNIFICANCE AND IMPACT OF THE STUDY: Preventive measures to avoid contamination of poultry products by L. monocytogenes must be taken in the processing plants.     

The type I restriction endonuclease EcoR124I, couples ATP hydrolysis to bidirectional DNA translocation

Type I restriction endonuclease holoenzymes contain methylase (M), restriction (R) and specificity (S) subunits, present in an M2:R2:S1 stoichiometry. These enzymes bind to specific DNA sequences and translocate dsDNA in an ATP-dependent manner toward the holoenzyme anchored at the recognition sequence. Once translocation is impeded, DNA restriction, which functions to protect the host cell from invading DNA, takes place. Translocation and DNA cleavage are afforded by the two diametrically opposed R-subunits. To gain insight into the mechanism of translocation, a detailed characterization of the ATPase activity of EcoR124I was done. Results show that following recognition sequence binding, ATP hydrolysis-coupled, bidirectional DNA translocation by EcoR124I ensues, with the R-subunits transiently disengaging, on average, every 515 bp. Macroscopic processivity of 2031(+/-184)bp is maintained, as the R-subunits remain in close proximity to the DNA through association with the methyltransferase. Transient uncoupling of ATP hydrolysis from translocation results in 3.1(+/-0.4) ATP molecules being hydrolyzed per base-pair translocated per R-subunit. This is the first clear demonstration of the coupling of ATP hydrolysis to dsDNA translocation, albeit inefficient. Once translocation is impeded on supercoiled DNA, the DNA is cleaved. DNA cleavage inactivates the EcoR124I holoenzyme partially and reversibly, which explains the stoichiometric behaviour of type I restriction enzymes. Inactivated holoenzyme remains bound to the DNA at the recognition sequence and immediately releases the nascent ends. The release of nascent ends was demonstrated using a novel, fluorescence-based, real-time assay that takes advantage of the ability of the Escherichia coli RecBCD enzyme to unwind restricted dsDNA. The resulting unwinding of EcoR124I-restricted DNA by RecBCD reveals coordination between the restriction-modification and recombination systems that functions to destroy invading DNA efficiently. In addition, we demonstrate the displacement of EcoR124I following DNA cleavage by the translocating RecBCD enzyme, resulting in the restoration of catalytic function to EcoR124I.     

Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.